Quantitative imaging with fluorescent biosensors.

PubMed

Okumoto, Sakiko; Jones, Alexander; Frommer, Wolf B

2012-01-01

Molecular activities are highly dynamic and can occur locally in subcellular domains or compartments. Neighboring cells in the same tissue can exist in different states. Therefore, quantitative information on the cellular and subcellular dynamics of ions, signaling molecules, and metabolites is critical for functional understanding of organisms. Mass spectrometry is generally used for monitoring ions and metabolites; however, its temporal and spatial resolution are limited. Fluorescent proteins have revolutionized many areas of biology-e.g., fluorescent proteins can report on gene expression or protein localization in real time-yet promoter-based reporters are often slow to report physiologically relevant changes such as calcium oscillations. Therefore, novel tools are required that can be deployed in specific cells and targeted to subcellular compartments in order to quantify target molecule dynamics directly. We require tools that can measure enzyme activities, protein dynamics, and biophysical processes (e.g., membrane potential or molecular tension) with subcellular resolution. Today, we have an extensive suite of tools at our disposal to address these challenges, including translocation sensors, fluorescence-intensity sensors, and Förster resonance energy transfer sensors. This review summarizes sensor design principles, provides a database of sensors for more than 70 different analytes/processes, and gives examples of applications in quantitative live cell imaging.

Visualization of metallodrugs in single cells by secondary ion mass spectrometry imaging.

PubMed

Wu, Kui; Jia, Feifei; Zheng, Wei; Luo, Qun; Zhao, Yao; Wang, Fuyi

2017-07-01

Secondary ion mass spectrometry, including nanoscale secondary ion mass spectrometry (NanoSIMS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), has emerged as a powerful tool for biological imaging, especially for single cell imaging. SIMS imaging can provide information on subcellular distribution of endogenous and exogenous chemicals, including metallodrugs, from membrane through to cytoplasm and nucleus without labeling, and with high spatial resolution and chemical specificity. In this mini-review, we summarize recent progress in the field of SIMS imaging, particularly in the characterization of the subcellular distribution of metallodrugs. We anticipate that the SIMS imaging method will be widely applied to visualize subcellular distributions of drugs and drug candidates in single cells, exerting significant influence on early drug evaluation and metabolism in medicinal and pharmaceutical chemistry. Recent progress of SIMS applications in characterizing the subcellular distributions of metallodrugs was summarized.

Imaging live cells at high spatiotemporal resolution for lab-on-a-chip applications.

PubMed

Chin, Lip Ket; Lee, Chau-Hwang; Chen, Bi-Chang

2016-05-24

Conventional optical imaging techniques are limited by the diffraction limit and difficult-to-image biomolecular and sub-cellular processes in living specimens. Novel optical imaging techniques are constantly evolving with the desire to innovate an imaging tool that is capable of seeing sub-cellular processes in a biological system, especially in three dimensions (3D) over time, i.e. 4D imaging. For fluorescence imaging on live cells, the trade-offs among imaging depth, spatial resolution, temporal resolution and photo-damage are constrained based on the limited photons of the emitters. The fundamental solution to solve this dilemma is to enlarge the photon bank such as the development of photostable and bright fluorophores, leading to the innovation in optical imaging techniques such as super-resolution microscopy and light sheet microscopy. With the synergy of microfluidic technology that is capable of manipulating biological cells and controlling their microenvironments to mimic in vivo physiological environments, studies of sub-cellular processes in various biological systems can be simplified and investigated systematically. In this review, we provide an overview of current state-of-the-art super-resolution and 3D live cell imaging techniques and their lab-on-a-chip applications, and finally discuss future research trends in new and breakthrough research areas of live specimen 4D imaging in controlled 3D microenvironments.

High-Speed and Scalable Whole-Brain Imaging in Rodents and Primates.

PubMed

Seiriki, Kaoru; Kasai, Atsushi; Hashimoto, Takeshi; Schulze, Wiebke; Niu, Misaki; Yamaguchi, Shun; Nakazawa, Takanobu; Inoue, Ken-Ichi; Uezono, Shiori; Takada, Masahiko; Naka, Yuichiro; Igarashi, Hisato; Tanuma, Masato; Waschek, James A; Ago, Yukio; Tanaka, Kenji F; Hayata-Takano, Atsuko; Nagayasu, Kazuki; Shintani, Norihito; Hashimoto, Ryota; Kunii, Yasuto; Hino, Mizuki; Matsumoto, Junya; Yabe, Hirooki; Nagai, Takeharu; Fujita, Katsumasa; Matsuda, Toshio; Takuma, Kazuhiro; Baba, Akemichi; Hashimoto, Hitoshi

2017-06-21

Subcellular resolution imaging of the whole brain and subsequent image analysis are prerequisites for understanding anatomical and functional brain networks. Here, we have developed a very high-speed serial-sectioning imaging system named FAST (block-face serial microscopy tomography), which acquires high-resolution images of a whole mouse brain in a speed range comparable to that of light-sheet fluorescence microscopy. FAST enables complete visualization of the brain at a resolution sufficient to resolve all cells and their subcellular structures. FAST renders unbiased quantitative group comparisons of normal and disease model brain cells for the whole brain at a high spatial resolution. Furthermore, FAST is highly scalable to non-human primate brains and human postmortem brain tissues, and can visualize neuronal projections in a whole adult marmoset brain. Thus, FAST provides new opportunities for global approaches that will allow for a better understanding of brain systems in multiple animal models and in human diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Super-resolution imaging of subcortical white matter using stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI)

PubMed Central

Hainsworth, A. H.; Lee, S.; Patel, A.; Poon, W. W.; Knight, A. E.

2018-01-01

Aims The spatial resolution of light microscopy is limited by the wavelength of visible light (the ‘diffraction limit’, approximately 250 nm). Resolution of sub-cellular structures, smaller than this limit, is possible with super resolution methods such as stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI). We aimed to resolve subcellular structures (axons, myelin sheaths and astrocytic processes) within intact white matter, using STORM and SOFI. Methods Standard cryostat-cut sections of subcortical white matter from donated human brain tissue and from adult rat and mouse brain were labelled, using standard immunohistochemical markers (neurofilament-H, myelin-associated glycoprotein, glial fibrillary acidic protein, GFAP). Image sequences were processed for STORM (effective pixel size 8–32 nm) and for SOFI (effective pixel size 80 nm). Results In human, rat and mouse, subcortical white matter high-quality images for axonal neurofilaments, myelin sheaths and filamentous astrocytic processes were obtained. In quantitative measurements, STORM consistently underestimated width of axons and astrocyte processes (compared with electron microscopy measurements). SOFI provided more accurate width measurements, though with somewhat lower spatial resolution than STORM. Conclusions Super resolution imaging of intact cryo-cut human brain tissue is feasible. For quantitation, STORM can under-estimate diameters of thin fluorescent objects. SOFI is more robust. The greatest limitation for super-resolution imaging in brain sections is imposed by sample preparation. We anticipate that improved strategies to reduce autofluorescence and to enhance fluorophore performance will enable rapid expansion of this approach. PMID:28696566

Super-resolution imaging of subcortical white matter using stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI).

PubMed

Hainsworth, A H; Lee, S; Foot, P; Patel, A; Poon, W W; Knight, A E

2018-06-01

The spatial resolution of light microscopy is limited by the wavelength of visible light (the 'diffraction limit', approximately 250 nm). Resolution of sub-cellular structures, smaller than this limit, is possible with super resolution methods such as stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI). We aimed to resolve subcellular structures (axons, myelin sheaths and astrocytic processes) within intact white matter, using STORM and SOFI. Standard cryostat-cut sections of subcortical white matter from donated human brain tissue and from adult rat and mouse brain were labelled, using standard immunohistochemical markers (neurofilament-H, myelin-associated glycoprotein, glial fibrillary acidic protein, GFAP). Image sequences were processed for STORM (effective pixel size 8-32 nm) and for SOFI (effective pixel size 80 nm). In human, rat and mouse, subcortical white matter high-quality images for axonal neurofilaments, myelin sheaths and filamentous astrocytic processes were obtained. In quantitative measurements, STORM consistently underestimated width of axons and astrocyte processes (compared with electron microscopy measurements). SOFI provided more accurate width measurements, though with somewhat lower spatial resolution than STORM. Super resolution imaging of intact cryo-cut human brain tissue is feasible. For quantitation, STORM can under-estimate diameters of thin fluorescent objects. SOFI is more robust. The greatest limitation for super-resolution imaging in brain sections is imposed by sample preparation. We anticipate that improved strategies to reduce autofluorescence and to enhance fluorophore performance will enable rapid expansion of this approach. © 2017 British Neuropathological Society.

Techniques for the Cellular and Subcellular Localization of Endocannabinoid Receptors and Enzymes in the Mammalian Brain.

PubMed

Cristino, Luigia; Imperatore, Roberta; Di Marzo, Vincenzo

2017-01-01

This chapter attempts to piece together knowledge about new advanced microscopy techniques to study the neuroanatomical distribution of endocannabinoid receptors and enzymes at the level of cellular and subcellular structures and organelles in the brain. Techniques ranging from light to electron microscopy up to the new advanced LBM, PALM, and STORM super-resolution microscopy will be discussed in the context of their contribution to define the spatial distribution and organization of receptors and enzymes of the endocannabinoid system (ECS), and to better understand ECS brain functions. © 2017 Elsevier Inc. All rights reserved.

Visible light-sensitive APTES-bound ZnO nanowire toward a potent nanoinjector sensing biomolecules in a living cell

NASA Astrophysics Data System (ADS)

Lee, Jooran; Choi, Sunyoung; Bae, Seon Joo; Yoon, Seok Min; Choi, Joon Sig; Yoon, Minjoong

2013-10-01

Nanoscale cell injection techniques combined with nanoscopic photoluminescence (PL) spectroscopy have been important issues in high-resolution optical biosensing, gene and drug delivery and single-cell endoscopy for medical diagnostics and therapeutics. However, the current nanoinjectors remain limited for optical biosensing and communication at the subwavelength level, requiring an optical probe such as semiconductor quantum dots, separately. Here, we show that waveguided red emission is observed at the tip of a single visible light-sensitive APTES-modified ZnO nanowire (APTES-ZnO NW) and it exhibits great enhancement upon interaction with a complementary sequence-based double stranded (ds) DNA, whereas it is not significantly affected by non-complementary ds DNA. Further, the tip of a single APTES-ZnO NW can be inserted into the subcellular region of living HEK 293 cells without significant toxicity, and it can also detect the enhancement of the tip emission from subcellular regions with high spatial resolution. These results indicate that the single APTES-ZnO NW would be useful as a potent nanoinjector which can guide visible light into intracellular compartments of mammalian cells, and can also detect nanoscopic optical signal changes induced by interaction with the subcellular specific target biomolecules without separate optical probes.Nanoscale cell injection techniques combined with nanoscopic photoluminescence (PL) spectroscopy have been important issues in high-resolution optical biosensing, gene and drug delivery and single-cell endoscopy for medical diagnostics and therapeutics. However, the current nanoinjectors remain limited for optical biosensing and communication at the subwavelength level, requiring an optical probe such as semiconductor quantum dots, separately. Here, we show that waveguided red emission is observed at the tip of a single visible light-sensitive APTES-modified ZnO nanowire (APTES-ZnO NW) and it exhibits great enhancement upon interaction with a complementary sequence-based double stranded (ds) DNA, whereas it is not significantly affected by non-complementary ds DNA. Further, the tip of a single APTES-ZnO NW can be inserted into the subcellular region of living HEK 293 cells without significant toxicity, and it can also detect the enhancement of the tip emission from subcellular regions with high spatial resolution. These results indicate that the single APTES-ZnO NW would be useful as a potent nanoinjector which can guide visible light into intracellular compartments of mammalian cells, and can also detect nanoscopic optical signal changes induced by interaction with the subcellular specific target biomolecules without separate optical probes. Electronic supplementary information (ESI) available: Synthesis of APTES-modified ZnO nanowires, DNA functionalization and spectroscopic measurements with additional fluorescence image ad fluorescence decay times, cell culture, injection of a single nanowire into living cells, subcellular imaging and determination of cytotoxicity. See DOI: 10.1039/c3nr03042c

Biomechanics of subcellular structures by non-invasive Brillouin microscopy

NASA Astrophysics Data System (ADS)

Antonacci, Giuseppe; Braakman, Sietse

2016-11-01

Cellular biomechanics play a pivotal role in the pathophysiology of several diseases. Unfortunately, current methods to measure biomechanical properties are invasive and mostly limited to the surface of a cell. As a result, the mechanical behaviour of subcellular structures and organelles remains poorly characterised. Here, we show three-dimensional biomechanical images of single cells obtained with non-invasive, non-destructive Brillouin microscopy with an unprecedented spatial resolution. Our results quantify the longitudinal elastic modulus of subcellular structures. In particular, we found the nucleoli to be stiffer than both the nuclear envelope (p < 0.0001) and the surrounding cytoplasm (p < 0.0001). Moreover, we demonstrate the mechanical response of cells to Latrunculin-A, a drug that reduces cell stiffness by preventing cytoskeletal assembly. Our technique can therefore generate valuable insights into cellular biomechanics and its role in pathophysiology.

RESOLFT nanoscopy with photoswitchable organic fluorophores

NASA Astrophysics Data System (ADS)

Kwon, Jiwoong; Hwang, Jihee; Park, Jaewan; Han, Gi Rim; Han, Kyu Young; Kim, Seong Keun

2015-12-01

Far-field optical nanoscopy has been widely used to image small objects with sub-diffraction-limit spatial resolution. Particularly, reversible saturable optical fluorescence transition (RESOLFT) nanoscopy with photoswitchable fluorescent proteins is a powerful method for super-resolution imaging of living cells with low light intensity. Here we demonstrate for the first time the implementation of RESOLFT nanoscopy for a biological system using organic fluorophores, which are smaller in size and easier to be chemically modified. With a covalently-linked dye pair of Cy3 and Alexa647 to label subcellular structures in fixed cells and by optimizing the imaging buffer and optical parameters, our RESOLFT nanoscopy achieved a spatial resolution of ~74 nm in the focal plane. This method provides a powerful alternative for low light intensity RESOLFT nanoscopy, which enables biological imaging with small organic probes at nanoscale resolution.

Multimodal imaging of human cerebellum - merging X-ray phase microtomography, magnetic resonance microscopy and histology

NASA Astrophysics Data System (ADS)

Schulz, Georg; Waschkies, Conny; Pfeiffer, Franz; Zanette, Irene; Weitkamp, Timm; David, Christian; Müller, Bert

2012-11-01

Imaging modalities including magnetic resonance imaging and X-ray computed tomography are established methods in daily clinical diagnosis of human brain. Clinical equipment does not provide sufficient spatial resolution to obtain morphological information on the cellular level, essential for applying minimally or non-invasive surgical interventions. Therefore, generic data with lateral sub-micrometer resolution have been generated from histological slices post mortem. Sub-cellular spatial resolution, lost in the third dimension as a result of sectioning, is obtained using magnetic resonance microscopy and micro computed tomography. We demonstrate that for human cerebellum grating-based X-ray phase tomography shows complementary contrast to magnetic resonance microscopy and histology. In this study, the contrast-to-noise values of magnetic resonance microscopy and phase tomography were comparable whereas the spatial resolution in phase tomography is an order of magnitude better. The registered data with their complementary information permit the distinct segmentation of tissues within the human cerebellum.

Whole-animal imaging with high spatio-temporal resolution

NASA Astrophysics Data System (ADS)

Chhetri, Raghav; Amat, Fernando; Wan, Yinan; Höckendorf, Burkhard; Lemon, William C.; Keller, Philipp J.

2016-03-01

We developed isotropic multiview (IsoView) light-sheet microscopy in order to image fast cellular dynamics, such as cell movements in an entire developing embryo or neuronal activity throughput an entire brain or nervous system, with high resolution in all dimensions, high imaging speeds, good physical coverage and low photo-damage. To achieve high temporal resolution and high spatial resolution at the same time, IsoView microscopy rapidly images large specimens via simultaneous light-sheet illumination and fluorescence detection along four orthogonal directions. In a post-processing step, these four views are then combined by means of high-throughput multiview deconvolution to yield images with a system resolution of ≤ 450 nm in all three dimensions. Using IsoView microscopy, we performed whole-animal functional imaging of Drosophila embryos and larvae at a spatial resolution of 1.1-2.5 μm and at a temporal resolution of 2 Hz for up to 9 hours. We also performed whole-brain functional imaging in larval zebrafish and multicolor imaging of fast cellular dynamics across entire, gastrulating Drosophila embryos with isotropic, sub-cellular resolution. Compared with conventional (spatially anisotropic) light-sheet microscopy, IsoView microscopy improves spatial resolution at least sevenfold and decreases resolution anisotropy at least threefold. Compared with existing high-resolution light-sheet techniques, such as lattice lightsheet microscopy or diSPIM, IsoView microscopy effectively doubles the penetration depth and provides subsecond temporal resolution for specimens 400-fold larger than could previously be imaged.

Studying dyadic structure-function relationships: a review of current modeling approaches and new insights into Ca2+ (mis)handling.

PubMed

Maleckar, Mary M; Edwards, Andrew G; Louch, William E; Lines, Glenn T

2017-01-01

Excitation-contraction coupling in cardiac myocytes requires calcium influx through L-type calcium channels in the sarcolemma, which gates calcium release through sarcoplasmic reticulum ryanodine receptors in a process known as calcium-induced calcium release, producing a myoplasmic calcium transient and enabling cardiomyocyte contraction. The spatio-temporal dynamics of calcium release, buffering, and reuptake into the sarcoplasmic reticulum play a central role in excitation-contraction coupling in both normal and diseased cardiac myocytes. However, further quantitative understanding of these cells' calcium machinery and the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease requires accurate knowledge of cardiac ultrastructure, protein distribution and subcellular function. As current imaging techniques are limited in spatial resolution, limiting insight into changes in calcium handling, computational models of excitation-contraction coupling have been increasingly employed to probe these structure-function relationships. This review will focus on the development of structural models of cardiac calcium dynamics at the subcellular level, orienting the reader broadly towards the development of models of subcellular calcium handling in cardiomyocytes. Specific focus will be given to progress in recent years in terms of multi-scale modeling employing resolved spatial models of subcellular calcium machinery. A review of the state-of-the-art will be followed by a review of emergent insights into calcium-dependent etiologies in heart disease and, finally, we will offer a perspective on future directions for related computational modeling and simulation efforts.

Nanowire-based single-cell endoscopy

NASA Astrophysics Data System (ADS)

Yan, Ruoxue; Park, Ji-Ho; Choi, Yeonho; Heo, Chul-Joon; Yang, Seung-Man; Lee, Luke P.; Yang, Peidong

2012-03-01

One-dimensional smart probes based on nanowires and nanotubes that can safely penetrate the plasma membrane and enter biological cells are potentially useful in high-resolution and high-throughput gene and drug delivery, biosensing and single-cell electrophysiology. However, using such probes for optical communication across the cellular membrane at the subwavelength level remains limited. Here, we show that a nanowire waveguide attached to the tapered tip of an optical fibre can guide visible light into intracellular compartments of a living mammalian cell, and can also detect optical signals from subcellular regions with high spatial resolution. Furthermore, we show that through light-activated mechanisms the endoscope can deliver payloads into cells with spatial and temporal specificity. Moreover, insertion of the endoscope into cells and illumination of the guided laser did not induce any significant toxicity in the cells.

Five Micron High Resolution MALDI Mass Spectrometry Imaging with Simple, Interchangeable, Multi-Resolution Optical System

DOE PAGES

Feenstra, Adam D.; Dueñas, Maria Emilia; Lee, Young Jin

2017-01-03

High-spatial resolution mass spectrometry imaging (MSI) is crucial for the mapping of chemical distributions at the cellular and subcellular level. Here in this work, we improved our previous laser optical system for matrix-assisted laser desorption ionization (MALDI)-MSI, from ~9 μm practical laser spot size to a practical laser spot size of ~4 μm, thereby allowing for 5 μm resolution imaging without oversampling. This is accomplished through a combination of spatial filtering, beam expansion, and reduction of the final focal length. Most importantly, the new laser optics system allows for simple modification of the spot size solely through the interchanging ofmore » the beam expander component. Using 10×, 5×, and no beam expander, we could routinely change between ~4, ~7, and ~45 μm laser spot size, in less than 5 min. We applied this multi-resolution MALDI-MSI system to a single maize root tissue section with three different spatial resolutions of 5, 10, and 50 μm and compared the differences in imaging quality and signal sensitivity. Lastly, we also demonstrated the difference in depth of focus between the optical systems with 10× and 5× beam expanders.« less

Five Micron High Resolution MALDI Mass Spectrometry Imaging with Simple, Interchangeable, Multi-Resolution Optical System

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feenstra, Adam D.; Dueñas, Maria Emilia; Lee, Young Jin

High-spatial resolution mass spectrometry imaging (MSI) is crucial for the mapping of chemical distributions at the cellular and subcellular level. Here in this work, we improved our previous laser optical system for matrix-assisted laser desorption ionization (MALDI)-MSI, from ~9 μm practical laser spot size to a practical laser spot size of ~4 μm, thereby allowing for 5 μm resolution imaging without oversampling. This is accomplished through a combination of spatial filtering, beam expansion, and reduction of the final focal length. Most importantly, the new laser optics system allows for simple modification of the spot size solely through the interchanging ofmore » the beam expander component. Using 10×, 5×, and no beam expander, we could routinely change between ~4, ~7, and ~45 μm laser spot size, in less than 5 min. We applied this multi-resolution MALDI-MSI system to a single maize root tissue section with three different spatial resolutions of 5, 10, and 50 μm and compared the differences in imaging quality and signal sensitivity. Lastly, we also demonstrated the difference in depth of focus between the optical systems with 10× and 5× beam expanders.« less

Elemental and isotopic imaging of biological samples using NanoSIMS.

PubMed

Kilburn, Matt R; Clode, Peta L

2014-01-01

With its low detection limits and the ability to analyze most of the elements in the periodic table, secondary ion mass spectrometry (SIMS) represents one of the most versatile in situ analytical techniques available, and recent developments have resulted in significant advantages for the use of imaging mass spectrometry in biological and biomedical research. Increases in spatial resolution and sensitivity allow detailed interrogation of samples at relevant scales and chemical concentrations. Advances in dynamic SIMS, specifically with the advent of NanoSIMS, now allow the tracking of stable isotopes within biological systems at subcellular length scales, while static SIMS combines subcellular imaging with molecular identification. In this chapter, we present an introduction to the SIMS technique, with particular reference to NanoSIMS, and discuss its application in biological and biomedical research.

Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells

PubMed Central

Miragoli, Michele; Moshkov, Alexey; Novak, Pavel; Shevchuk, Andrew; Nikolaev, Viacheslav O.; El-Hamamsy, Ismail; Potter, Claire M. F.; Wright, Peter; Kadir, S.H. Sheikh Abdul; Lyon, Alexander R.; Mitchell, Jane A.; Chester, Adrian H.; Klenerman, David; Lab, Max J.; Korchev, Yuri E.; Harding, Sian E.; Gorelik, Julia

2011-01-01

Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters (cell volume, membrane potentials, cellular contraction, single ion-channel currents and some parameters of intracellular signalling) in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization (tissue, cellular and subcellular levels). Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies. PMID:21325316

Local structure of subcellular input retinotopy in an identified visual interneuron

NASA Astrophysics Data System (ADS)

Zhu, Ying; Gabbiani, Fabrizio; Fabrizio Gabbiani's lab Team

2015-03-01

How does the spatial layout of the projections that a neuron receives impact its synaptic integration and computation? What is the mapping topography of subcellular wiring at the single neuron level? The LGMD (lobula giant movement detector) neuron in the locust is an identified neuron that responds preferentially to objects approaching on a collision course. It receives excitatory inputs from the entire visual hemifield through calcium-permeable nicotinic acetylcholine receptors. Previous work showed that the projection from the locust compound eye to the LGMD preserved retinotopy down to the level of a single ommatidium (facet) by employing in vivo widefield calcium imaging. Because widefield imaging relies on global excitation of the preparation and has a relatively low resolution, previous work could not investigate this retinotopic mapping at the level of individual thin dendritic branches. Our current work employs a custom-built two-photon microscope with sub-micron resolution in conjunction with a single-facet stimulation setup that provides visual stimuli to the single ommatidium of locust adequate to explore the local structure of this retinotopy at a finer level. We would thank NIMH for funding this research.

Effect of small-molecule modification on single-cell pharmacokinetics of PARP inhibitors.

PubMed

Thurber, Greg M; Reiner, Thomas; Yang, Katherine S; Kohler, Rainer H; Weissleder, Ralph

2014-04-01

The heterogeneous delivery of drugs in tumors is an established process contributing to variability in treatment outcome. Despite the general acceptance of variable delivery, the study of the underlying causes is challenging, given the complex tumor microenvironment including intra- and intertumor heterogeneity. The difficulty in studying this distribution is even more significant for small-molecule drugs where radiolabeled compounds or mass spectrometry detection lack the spatial and temporal resolution required to quantify the kinetics of drug distribution in vivo. In this work, we take advantage of the synthesis of fluorescent drug conjugates that retain their target binding but are designed with different physiochemical and thus pharmacokinetic properties. Using these probes, we followed the drug distribution in cell culture and tumor xenografts with temporal resolution of seconds and subcellular spatial resolution. These measurements, including in vivo permeability of small-molecule drugs, can be used directly in predictive pharmacokinetic models for the design of therapeutics and companion imaging agents as demonstrated by a finite element model.

Effect of Small Molecule Modification on Single Cell Pharmacokinetics of PARP Inhibitors

PubMed Central

Thurber, Greg M.; Reiner, Thomas; Yang, Katherine S; Kohler, Rainer; Weissleder, Ralph

2014-01-01

The heterogeneous delivery of drugs in tumors is an established process contributing to variability in treatment outcome. Despite the general acceptance of variable delivery, the study of the underlying causes is challenging given the complex tumor microenvironment including intra- and inter-tumor heterogeneity. The difficulty in studying this distribution is even more significant for small molecule drugs where radiolabeled compounds or mass spectrometry detection lack the spatial and temporal resolution required to quantify the kinetics of drug distribution in vivo. In this work, we take advantage of the synthesis of fluorescent drug conjugates that retain their target binding but are designed with different physiochemical and thus pharmacokinetic properties. Using these probes, we followed the drug distribution in cell culture and tumor xenografts with temporal resolution of seconds and subcellular spatial resolution. These measurements, including in vivo permeability of small molecule drugs, can be used directly in predictive pharmacokinetic models for the design of therapeutics and companion imaging agents as demonstrated by a finite element model. PMID:24552776

Functional imaging of hippocampal place cells at cellular resolution during virtual navigation

PubMed Central

Dombeck, Daniel A.; Harvey, Christopher D.; Tian, Lin; Looger, Loren L.; Tank, David W.

2010-01-01

Spatial navigation is a widely employed behavior in rodent studies of neuronal circuits underlying cognition, learning and memory. In vivo microscopy combined with genetically-encoded indicators provides important new tools to study neuronal circuits, but has been technically difficult to apply during navigation. We describe methods to image the activity of hippocampal CA1 neurons with sub-cellular resolution in behaving mice. Neurons expressing the genetically encoded calcium indicator GCaMP3 were imaged through a chronic hippocampal window. Head-fixed mice performed spatial behaviors within a setup combining a virtual reality system and a custom built two-photon microscope. Populations of place cells were optically identified, and the correlation between the location of their place fields in the virtual environment and their anatomical location in the local circuit was measured. The combination of virtual reality and high-resolution functional imaging should allow for a new generation of studies to probe neuronal circuit dynamics during behavior. PMID:20890294

Physiological and structural differences in spatially distinct subpopulations of cardiac mitochondria: influence of cardiac pathologies

PubMed Central

Thapa, Dharendra; Shepherd, Danielle L.

2014-01-01

Cardiac tissue contains discrete pools of mitochondria that are characterized by their subcellular spatial arrangement. Subsarcolemmal mitochondria (SSM) exist below the cell membrane, interfibrillar mitochondria (IFM) reside in rows between the myofibrils, and perinuclear mitochondria are situated at the nuclear poles. Microstructural imaging of heart tissue coupled with the development of differential isolation techniques designed to sequentially separate spatially distinct mitochondrial subpopulations have revealed differences in morphological features including shape, absolute size, and internal cristae arrangement. These findings have been complemented by functional studies indicating differences in biochemical parameters and, potentially, functional roles for the ATP generated, based upon subcellular location. Consequently, mitochondrial subpopulations appear to be influenced differently during cardiac pathologies including ischemia/reperfusion, heart failure, aging, exercise, and diabetes mellitus. These influences may be the result of specific structural and functional disparities between mitochondrial subpopulations such that the stress elicited by a given cardiac insult differentially impacts subcellular locales and the mitochondria contained within. The goal of this review is to highlight some of the inherent structural and functional differences that exist between spatially distinct cardiac mitochondrial subpopulations as well as provide an overview of the differential impact of various cardiac pathologies on spatially distinct mitochondrial subpopulations. As an outcome, we will instill a basis for incorporating subcellular spatial location when evaluating the impact of cardiac pathologies on the mitochondrion. Incorporation of subcellular spatial location may offer the greatest potential for delineating the influence of cardiac pathology on this critical organelle. PMID:24778166

Subcellular-level resolution MALDI-MS imaging of maize leaf metabolites by MALDI-linear ion trap-Orbitrap mass spectrometer

DOE PAGES

Korte, Andrew R.; Yandeau-Nelson, Marna D.; Nikolau, Basil J.; ...

2015-01-25

A significant limiting factor in achieving high spatial resolution for matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) imaging is the size of the laser spot at the sample surface. We present modifications to the beam-delivery optics of a commercial MALDI-linear ion trap-Orbitrap instrument, incorporating an external Nd:YAG laser, beam-shaping optics, and an aspheric focusing lens, to reduce the minimum laser spot size from ~50 μm for the commercial configuration down to ~9 μm for the modified configuration. This improved system was applied for MALDI-MS imaging of cross sections of juvenile maize leaves at 5-μm spatial resolution using an oversampling method. Theremore » are a variety of different metabolites including amino acids, glycerolipids, and defense-related compounds were imaged at a spatial resolution well below the size of a single cell. Such images provide unprecedented insights into the metabolism associated with the different tissue types of the maize leaf, which is known to asymmetrically distribute the reactions of C4 photosynthesis among the mesophyll and bundle sheath cell types. The metabolite ion images correlate with the optical images that reveal the structures of the different tissues, and previously known and newly revealed asymmetric metabolic features are observed.« less

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

5D-intravital tomography as a novel tool for non-invasive in-vivo analysis of human skin

NASA Astrophysics Data System (ADS)

König, Karsten; Weinigel, Martin; Breunig, Hans G.; Gregory, Axel; Fischer, Peter; Kellner-Höfer, Marcel; Bückle, Rainer; Schwarz, Martin; Riemann, Iris; Stracke, Frank; Huck, Volker; Gorzelanny, Christian; Schneider, Stefan W.

2010-02-01

Some years ago, CE-marked clinical multiphoton systems for 3D imaging of human skin with subcellular resolution have been launched. These tomographs provide optical biopsies with submicron resolution based on two-photon excited autofluorescence (NAD(P)H, flavoproteins, keratin, elastin, melanin, porphyrins) and second harmonic generation by collagen. The 3D tomograph was now transferred into a 5D imaging system by the additional detection of the emission spectrum and the fluorescence lifetime based on spatially and spectrally resolved time-resolved single photon counting. The novel 5D intravital tomograph (5D-IVT) was employed for the early detection of atopic dermatitis and the analysis of treatment effects.

Lights, Camera, Action! Antimicrobial Peptide Mechanisms Imaged in Space and Time

PubMed Central

Choi, Heejun; Rangarajan, Nambirajan; Weisshaar, James C.

2015-01-01

Deeper understanding of the bacteriostatic and bactericidal mechanisms of antimicrobial peptides (AMPs) should help in the design of new antibacterial agents. Over several decades, a variety of biochemical assays have been applied to bulk bacterial cultures. While some of these bulk assays provide time resolution on the order of 1 min, they do not capture faster mechanistic events. Nor can they provide subcellular spatial information or discern cell-to-cell heterogeneity within the bacterial population. Single-cell, time-resolved imaging assays bring a completely new spatiotemporal dimension to AMP mechanistic studies. We review recent work that provides new insights into the timing, sequence, and spatial distribution of AMP-induced effects on bacterial cells. PMID:26691950

Smartphone-based imaging of the corneal endothelium at sub-cellular resolution

NASA Astrophysics Data System (ADS)

Toslak, Devrim; Thapa, Damber; Erol, Muhammet Kazim; Chen, Yanjun; Yao, Xincheng

2017-07-01

This aim of this study was to test the feasibility of smartphone-based specular microscopy of the corneal endothelium at a sub-cellular resolution. Quantitative examination of endothelial cells is essential for evaluating corneal disease such as determining a diagnosis, monitoring progression and assessing treatment. Smartphone-based technology promises a new opportunity to develop affordable devices to foster quantitative examination of endothelial cells in rural and underserved areas. In our study, we incorporated an iPhone 6 and a slit lamp to demonstrate the feasibility of smartphone-based microscopy of the corneal endothelium at a sub-cellular resolution. The sub-cellular resolution images allowed quantitative calculation of the endothelial cell density. Comparative measurements revealed a normal endothelial cell density of 2978 cells/mm2 in the healthy cornea, and a significantly reduced cell density of 1466 cells/mm2 in the diseased cornea with Fuchs' dystrophy. Our ultimate goal is to develop a smartphone-based telemedicine device for low-cost examination of the corneal endothelium, which can benefit patients in rural areas and underdeveloped countries to reduce health care disparities.

Determination of elemental distribution in green micro-algae using synchrotron radiation nano X-ray fluorescence (SR-nXRF) and electron microscopy techniques--subcellular localization and quantitative imaging of silver and cobalt uptake by Coccomyxa actinabiotis.

PubMed

Leonardo, T; Farhi, E; Boisson, A-M; Vial, J; Cloetens, P; Bohic, S; Rivasseau, C

2014-02-01

The newly discovered unicellular micro-alga Coccomyxa actinabiotis proves to be highly radio-tolerant and strongly concentrates radionuclides, as well as large amounts of toxic metals. This study helps in the understanding of the mechanisms involved in the accumulation and detoxification of silver and cobalt. Elemental distribution inside Coccomyxa actinabiotis cells was determined using synchrotron nano X-ray fluorescence spectroscopy at the ID22 nano fluorescence imaging beamline of the European Synchrotron Radiation Facility. The high resolution and high sensitivity of this technique enabled the assessment of elemental associations and exclusions in subcellular micro-algae compartments. A quantitative treatment of the scans was implemented to yield absolute concentrations of each endogenous and exogenous element with a spatial resolution of 100 nm and compared to the macroscopic content in cobalt and silver determined using inductively coupled plasma-mass spectrometry. The nano X-ray fluorescence imaging was complemented by transmission electron microscopy coupled to X-ray microanalysis (TEM-EDS), yielding differential silver distribution in the cell wall, cytosol, nucleus, chloroplast and mitochondria with unique resolution. The analysis of endogenous elements in control cells revealed that iron had a unique distribution; zinc, potassium, manganese, molybdenum, and phosphate had their maxima co-localized in the same area; and sulfur, copper and chlorine were almost homogeneously distributed among the whole cell. The subcellular distribution and quantification of cobalt and silver in micro-alga, assessed after controlled exposure to various concentrations, revealed that exogenous metals were mainly sequestered inside the cell rather than on mucilage or the cell wall, with preferential compartmentalization. Cobalt was homogeneously distributed outside of the chloroplast. Silver was localized in the cytosol at low concentration and in the whole cell excluding the nucleus at high concentration. Exposure to low concentrations of cobalt or silver did not alter the localization nor the concentration of endogenous elements within the cells. To our knowledge, this is the first report on element co-localization and segregation at the sub-cellular level in micro-algae by means of synchrotron nano X-ray fluorescence spectroscopy.

Subcellular Investigation of Photosynthesis-Driven Carbon Assimilation in the Symbiotic Reef Coral Pocillopora damicornis

PubMed Central

Domart-Coulon, Isabelle; Escrig, Stephane; Humbel, Bruno M.; Hignette, Michel

2015-01-01

ABSTRACT  Reef-building corals form essential, mutualistic endosymbiotic associations with photosynthetic Symbiodinium dinoflagellates, providing their animal host partner with photosynthetically derived nutrients that allow the coral to thrive in oligotrophic waters. However, little is known about the dynamics of these nutritional interactions at the (sub)cellular level. Here, we visualize with submicrometer spatial resolution the carbon and nitrogen fluxes in the intact coral-dinoflagellate association from the reef coral Pocillopora damicornis by combining nanoscale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy with pulse-chase isotopic labeling using [13C]bicarbonate and [15N]nitrate. This allows us to observe that (i) through light-driven photosynthesis, dinoflagellates rapidly assimilate inorganic bicarbonate and nitrate, temporarily storing carbon within lipid droplets and starch granules for remobilization in nighttime, along with carbon and nitrogen incorporation into other subcellular compartments for dinoflagellate growth and maintenance, (ii) carbon-containing photosynthates are translocated to all four coral tissue layers, where they accumulate after only 15 min in coral lipid droplets from the oral gastroderm and within 6 h in glycogen granules from the oral epiderm, and (iii) the translocation of nitrogen-containing photosynthates is delayed by 3 h. PMID:25670779

Three-dimensional coherent X-ray diffractive imaging of whole frozen-hydrated cells

PubMed Central

Rodriguez, Jose A.; Xu, Rui; Chen, Chien-Chun; Huang, Zhifeng; Jiang, Huaidong; Chen, Allan L.; Raines, Kevin S.; Pryor Jr, Alan; Nam, Daewoong; Wiegart, Lutz; Song, Changyong; Madsen, Anders; Chushkin, Yuriy; Zontone, Federico; Bradley, Peter J.; Miao, Jianwei

2015-01-01

A structural understanding of whole cells in three dimensions at high spatial resolution remains a significant challenge and, in the case of X-rays, has been limited by radiation damage. By alleviating this limitation, cryogenic coherent diffractive imaging (cryo-CDI) can in principle be used to bridge the important resolution gap between optical and electron microscopy in bio-imaging. Here, the first experimental demonstration of cryo-CDI for quantitative three-dimensional imaging of whole frozen-hydrated cells using 8 keV X-rays is reported. As a proof of principle, a tilt series of 72 diffraction patterns was collected from a frozen-hydrated Neospora caninum cell and the three-dimensional mass density of the cell was reconstructed and quantified based on its natural contrast. This three-dimensional reconstruction reveals the surface and internal morphology of the cell, including its complex polarized sub-cellular structure. It is believed that this work represents an experimental milestone towards routine quantitative three-dimensional imaging of whole cells in their natural state with spatial resolutions in the tens of nanometres. PMID:26306199

Three-dimensional coherent X-ray diffractive imaging of whole frozen-hydrated cells

DOE PAGES

Rodriguez, Jose A.; Xu, Rui; Chen, Chien -Chun; ...

2015-09-01

Here, a structural understanding of whole cells in three dimensions at high spatial resolution remains a significant challenge and, in the case of X-rays, has been limited by radiation damage. By alleviating this limitation, cryogenic coherent diffractive imaging (cryo-CDI) can in principle be used to bridge the important resolution gap between optical and electron microscopy in bio-imaging. Here, the first experimental demonstration of cryo-CDI for quantitative three-dimensional imaging of whole frozen-hydrated cells using 8 Kev X-rays is reported. As a proof of principle, a tilt series of 72 diffraction patterns was collected from a frozen-hydrated Neospora caninum cell and themore » three-dimensional mass density of the cell was reconstructed and quantified based on its natural contrast. This three-dimensional reconstruction reveals the surface and internal morphology of the cell, including its complex polarized sub-cellular structure. Finally, it is believed that this work represents an experimental milestone towards routine quantitative three-dimensional imaging of whole cells in their natural state with spatial resolutions in the tens of nanometres.« less

Three-dimensional coherent X-ray diffractive imaging of whole frozen-hydrated cells.

PubMed

Rodriguez, Jose A; Xu, Rui; Chen, Chien-Chun; Huang, Zhifeng; Jiang, Huaidong; Chen, Allan L; Raines, Kevin S; Pryor, Alan; Nam, Daewoong; Wiegart, Lutz; Song, Changyong; Madsen, Anders; Chushkin, Yuriy; Zontone, Federico; Bradley, Peter J; Miao, Jianwei

2015-09-01

A structural understanding of whole cells in three dimensions at high spatial resolution remains a significant challenge and, in the case of X-rays, has been limited by radiation damage. By alleviating this limitation, cryogenic coherent diffractive imaging (cryo-CDI) can in principle be used to bridge the important resolution gap between optical and electron microscopy in bio-imaging. Here, the first experimental demonstration of cryo-CDI for quantitative three-dimensional imaging of whole frozen-hydrated cells using 8 keV X-rays is reported. As a proof of principle, a tilt series of 72 diffraction patterns was collected from a frozen-hydrated Neospora caninum cell and the three-dimensional mass density of the cell was reconstructed and quantified based on its natural contrast. This three-dimensional reconstruction reveals the surface and internal morphology of the cell, including its complex polarized sub-cellular structure. It is believed that this work represents an experimental milestone towards routine quantitative three-dimensional imaging of whole cells in their natural state with spatial resolutions in the tens of nanometres.

Imaging ion and molecular transport at subcellular resolution by secondary ion mass spectrometry

NASA Astrophysics Data System (ADS)

Chandra, Subhash; Morrison, George H.

1995-05-01

The transport of K+, Na+, and Ca2+ were imaged in individual cells with a Cameca IMS-3f ion microscope. Strict cryogenic frozen freeze-dry sample preparations were employed. Ion redistribution artifacts in conventional chemical preparations are discussed. Cryogenically prepared freeze-fractured freeze-dried cultured cells allowed the three-dimensional ion microscopic imaging of elements. As smaller structures in calcium images can be resolved with the 0.5 [mu]m spatial resolution, correlative techniques are needed to confirm their identity. The potentials of reflected light microscopy, scanning electron microscopy and laser scanning confocal microscopy are discussed for microfeature recognition in freeze-fractured freeze-dried cells. The feasibility of using frozen freeze-dried cells for imaging molecular transport at subcellular resolution was tested. Ion microscopy successfully imaged the transport of the isotopically tagged (13C, 15N) amino acid, -arginine. The labeled amino acid was imaged at mass 28 with a Cs+ primary ion beam as the 28(13C15N)- species. After a 4 h exposure of LLC-PK1 kidney cells to 4 mM labeled arginine, the amino acid was localized throughout the cell with a preferential incorporation into the nucleus and nucleolus. An example is also shown of the ion microscopic imaging of sodium borocaptate, an experimental therapeutic drug for brain tumors, in cryogenically prepared frozen freeze-dried Swiss 3T3 cells.

Matrix assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) for direct visualization of plant metabolites in situ

DOE PAGES

Sturtevant, Drew; Lee, Young -Jin; Chapman, Kent D.

2015-11-22

Direct visualization of plant tissues by matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) has revealed key insights into the localization of metabolites in situ. Recent efforts have determined the spatial distribution of primary and secondary metabolites in plant tissues and cells. Strategies have been applied in many areas of metabolism including isotope flux analyses, plant interactions, and transcriptional regulation of metabolite accumulation. Technological advances have pushed achievable spatial resolution to subcellular levels and increased instrument sensitivity by several orders of magnitude. Furthermore, it is anticipated that MALDI-MSI and other MSI approaches will bring a new level of understanding tomore » metabolomics as scientists will be encouraged to consider spatial heterogeneity of metabolites in descriptions of metabolic pathway regulation.« less

Matrix assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) for direct visualization of plant metabolites in situ

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sturtevant, Drew; Lee, Young -Jin; Chapman, Kent D.

Direct visualization of plant tissues by matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) has revealed key insights into the localization of metabolites in situ. Recent efforts have determined the spatial distribution of primary and secondary metabolites in plant tissues and cells. Strategies have been applied in many areas of metabolism including isotope flux analyses, plant interactions, and transcriptional regulation of metabolite accumulation. Technological advances have pushed achievable spatial resolution to subcellular levels and increased instrument sensitivity by several orders of magnitude. Furthermore, it is anticipated that MALDI-MSI and other MSI approaches will bring a new level of understanding tomore » metabolomics as scientists will be encouraged to consider spatial heterogeneity of metabolites in descriptions of metabolic pathway regulation.« less

Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors.

PubMed

Salomé, R; Kremer, Y; Dieudonné, S; Léger, J-F; Krichevsky, O; Wyart, C; Chatenay, D; Bourdieu, L

2006-06-30

Two-photon scanning microscopy (TPSM) is a powerful tool for imaging deep inside living tissues with sub-cellular resolution. The temporal resolution of TPSM is however strongly limited by the galvanometric mirrors used to steer the laser beam. Fast physiological events can therefore only be followed by scanning repeatedly a single line within the field of view. Because acousto-optic deflectors (AODs) are non-mechanical devices, they allow access at any point within the field of view on a microsecond time scale and are therefore excellent candidates to improve the temporal resolution of TPSM. However, the use of AOD-based scanners with femtosecond pulses raises several technical difficulties. In this paper, we describe an all-digital TPSM setup based on two crossed AODs. It includes in particular an acousto-optic modulator (AOM) placed at 45 degrees with respect to the AODs to pre-compensate for the large spatial distortions of femtosecond pulses occurring in the AODs, in order to optimize the spatial resolution and the fluorescence excitation. Our setup allows recording from freely selectable point-of-interest at high speed (1kHz). By maximizing the time spent on points of interest, random-access TPSM (RA-TPSM) constitutes a promising method for multiunit recordings with millisecond resolution in biological tissues.

Highly multiplexed subcellular RNA sequencing in situ

PubMed Central

Lee, Je Hyuk; Daugharthy, Evan R.; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas C.; Yang, Joyce L.; Terry, Richard; Jeanty, Sauveur S. F.; Li, Chao; Amamoto, Ryoji; Peters, Derek T.; Turczyk, Brian M.; Marblestone, Adam H.; Inverso, Samuel A.; Bernard, Amy; Mali, Prashant; Rios, Xavier; Aach, John; Church, George M.

2014-01-01

Understanding the spatial organization of gene expression with single nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked cDNA amplicons are sequenced within a biological sample. Using 30-base reads from 8,742 genes in situ, we examined RNA expression and localization in human primary fibroblasts using a simulated wound healing assay. FISSEQ is compatible with tissue sections and whole mount embryos, and reduces the limitations of optical resolution and noisy signals on single molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ. PMID:24578530

LITE microscopy: Tilted light-sheet excitation of model organisms offers high resolution and low photobleaching

PubMed Central

Gerbich, Therese M.; Rana, Kishan; Suzuki, Aussie; Schaefer, Kristina N.; Heppert, Jennifer K.; Boothby, Thomas C.; Allbritton, Nancy L.; Gladfelter, Amy S.; Maddox, Amy S.

2018-01-01

Fluorescence microscopy is a powerful approach for studying subcellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light-sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective by using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel live-cell LSFM method, lateral interference tilted excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, and coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution. PMID:29490939

Automated processing of label-free Raman microscope images of macrophage cells with standardized regression for high-throughput analysis.

PubMed

Milewski, Robert J; Kumagai, Yutaro; Fujita, Katsumasa; Standley, Daron M; Smith, Nicholas I

2010-11-19

Macrophages represent the front lines of our immune system; they recognize and engulf pathogens or foreign particles thus initiating the immune response. Imaging macrophages presents unique challenges, as most optical techniques require labeling or staining of the cellular compartments in order to resolve organelles, and such stains or labels have the potential to perturb the cell, particularly in cases where incomplete information exists regarding the precise cellular reaction under observation. Label-free imaging techniques such as Raman microscopy are thus valuable tools for studying the transformations that occur in immune cells upon activation, both on the molecular and organelle levels. Due to extremely low signal levels, however, Raman microscopy requires sophisticated image processing techniques for noise reduction and signal extraction. To date, efficient, automated algorithms for resolving sub-cellular features in noisy, multi-dimensional image sets have not been explored extensively. We show that hybrid z-score normalization and standard regression (Z-LSR) can highlight the spectral differences within the cell and provide image contrast dependent on spectral content. In contrast to typical Raman imaging processing methods using multivariate analysis, such as single value decomposition (SVD), our implementation of the Z-LSR method can operate nearly in real-time. In spite of its computational simplicity, Z-LSR can automatically remove background and bias in the signal, improve the resolution of spatially distributed spectral differences and enable sub-cellular features to be resolved in Raman microscopy images of mouse macrophage cells. Significantly, the Z-LSR processed images automatically exhibited subcellular architectures whereas SVD, in general, requires human assistance in selecting the components of interest. The computational efficiency of Z-LSR enables automated resolution of sub-cellular features in large Raman microscopy data sets without compromise in image quality or information loss in associated spectra. These results motivate further use of label free microscopy techniques in real-time imaging of live immune cells.

Infrared and Raman Microscopy in Cell Biology

PubMed Central

Matthäus, Christian; Bird, Benjamin; Miljković, Miloš; Chernenko, Tatyana; Romeo, Melissa; Diem, Max

2009-01-01

This chapter presents novel microscopic methods to monitor cell biological processes of live or fixed cells without the use of any dye, stains, or other contrast agent. These methods are based on spectral techniques that detect inherent spectroscopic properties of biochemical constituents of cells, or parts thereof. Two different modalities have been developed for this task. One of them is infrared micro-spectroscopy, in which an average snapshot of a cell’s biochemical composition is collected at a spatial resolution of typically 25 mm. This technique, which is extremely sensitive and can collect such a snapshot in fractions of a second, is particularly suited for studying gross biochemical changes. The other technique, Raman microscopy (also known as Raman micro-spectroscopy), is ideally suited to study variations of cellular composition on the scale of subcellular organelles, since its spatial resolution is as good as that of fluorescence microscopy. Both techniques exhibit the fingerprint sensitivity of vibrational spectroscopy toward biochemical composition, and can be used to follow a variety of cellular processes. PMID:19118679

Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy

PubMed Central

Wei, Lu; Yu, Yong; Shen, Yihui; Wang, Meng C.; Min, Wei

2013-01-01

Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial–temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon–deuterium bonds (C–D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo. PMID:23798434

Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

NASA Astrophysics Data System (ADS)

Chandra, Subhash

2008-12-01

Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O 2+) revealed local secondary ion signal enhancements correlated with the water image signals of 19(H 3O) +. A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K + and Na + in physiologically relevant concentrations. The high K-low Na signature in individual cells provided a rule-of-thumb criterion for the validation of sample preparation. The fractured freeze-dried cells allowed 3-D SIMS imaging and localization of 13C 15N labeled molecules and therapeutic drugs containing an elemental tag. Examples are shown to demonstrate that both diffusible elements and molecules are prone to artifact-induced relocation at subcellular scale if the sample preparation is compromised. The sample preparation is problem dependent and may vary widely between the diverse sample types of biological systems and the type of instrument used for SIMS analysis. The sample preparation, however, must be validated so that SIMS can be applied with confidence in biology and medicine.

Calcium homeostasis in crustaceans: subcellular Ca dynamics.

PubMed

Wheatly, M G; Zanotto, F P; Hubbard, M G

2002-05-01

The molting cycle of crustaceans, associated with renewal and remineralization of the cuticle, has emerged as a model system to study regulation of genes that code for Ca(2+)-transporting proteins, common to all eukaryotic cells. This article reviews state-of-the-art knowledge about how crustacean transporting epithelia (gills, hepatopancreas and antennal gland) effect mass transcellular movement of Ca(2+) while preventing cytotoxicity. The current model proposed is based on in vitro research on the intermolt stage with extrapolation to other molting stages. Plasma membrane proteins involved in apical and basolateral Ca(2+) movement (NCX, PMCA) are contrasted between aquatic species of different osmotic origin and among transporting epithelia of an individual species. Their roles are assessed in the context of epithelial Ca(2+) flux derived from organismic approaches. Exchange with extracellular environments is integrated with Ca(2+) sequestration mechanisms across endomembranes of the ER/SR and mitochondria. Finally, the review postulates how new Ca(2+) imaging techniques will allow spatial and temporal resolution of Ca(2+) concentration in subcellular domains.

Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution

PubMed Central

Lavenant, Gwendoline Thiery; Zavalin, Andrey I.; Caprioli, Richard M.

2013-01-01

Targeted multiplex Imaging Mass Spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This manuscript describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet. PMID:23397138

A graphene-based physiometer array for the analysis of single biological cells

NASA Astrophysics Data System (ADS)

Paulus, Geraldine L. C.; Nelson, Justin T.; Lee, Katherine Y.; Wang, Qing Hua; Reuel, Nigel F.; Grassbaugh, Brittany R.; Kruss, Sebastian; Landry, Markita P.; Kang, Jeon Woong; Vander Ende, Emma; Zhang, Jingqing; Mu, Bin; Dasari, Ramachandra R.; Opel, Cary F.; Wittrup, K. Dane; Strano, Michael S.

2014-10-01

A significant advantage of a graphene biosensor is that it inherently represents a continuum of independent and aligned sensor-units. We demonstrate a nanoscale version of a micro-physiometer - a device that measures cellular metabolic activity from the local acidification rate. Graphene functions as a matrix of independent pH sensors enabling subcellular detection of proton excretion. Raman spectroscopy shows that aqueous protons p-dope graphene - in agreement with established doping trajectories, and that graphene displays two distinct pKa values (2.9 and 14.2), corresponding to dopants physi- and chemisorbing to graphene respectively. The graphene physiometer allows micron spatial resolution and can differentiate immunoglobulin (IgG)-producing human embryonic kidney (HEK) cells from non-IgG-producing control cells. Population-based analyses allow mapping of phenotypic diversity, variances in metabolic activity, and cellular adhesion. Finally we show this platform can be extended to the detection of other analytes, e.g. dopamine. This work motivates the application of graphene as a unique biosensor for (sub)cellular interrogation.

Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators.

PubMed

Chamberland, Simon; Yang, Helen H; Pan, Michael M; Evans, Stephen W; Guan, Sihui; Chavarha, Mariya; Yang, Ying; Salesse, Charleen; Wu, Haodi; Wu, Joseph C; Clandinin, Thomas R; Toth, Katalin; Lin, Michael Z; St-Pierre, François

2017-07-27

Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and in Drosophila . These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.

Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution

NASA Astrophysics Data System (ADS)

Thiery-Lavenant, Gwendoline; Zavalin, Andre I.; Caprioli, Richard M.

2013-04-01

Targeted multiplex imaging mass spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This article describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet.

In vivo ROS and redox potential fluorescent detection in plants: Present approaches and future perspectives.

PubMed

Ortega-Villasante, Cristina; Burén, Stefan; Barón-Sola, Ángel; Martínez, Flor; Hernández, Luis E

2016-10-15

Reactive oxygen species (ROS) are metabolic by-products in aerobic organisms including plants. Endogenously produced ROS act as cellular messengers and redox regulators involved in several plant biological processes, but excessive accumulation of ROS cause oxidative stress and cell damage. Understanding ROS signalling and stress responses requires precise imaging and quantification of local, subcellular and global ROS dynamics with high selectivity, sensitivity, and spatiotemporal resolution. Several fluorescent vital dyes have been tested so far, which helped to provide relevant spatially resolved information of oxidative stress dynamics in plants subjected to harmful environmental conditions. However, certain plant characteristics, such as high background fluorescence of plant tissues in vivo and antioxidant mechanisms, can interfere with ROS detection. The development of improved small-molecule fluorescent dyes and protein-based ROS sensors targeted to subcellular compartments will enable in vivo monitoring of ROS and redox changes in photosynthetic organisms. Copyright © 2016 Elsevier Inc. All rights reserved.

Direct mapping of 19F in 19FDG-6P in brain tissue at subcellular resolution using soft X-ray fluorescence

NASA Astrophysics Data System (ADS)

Poitry-Yamate, C.; Gianoncelli, A.; Kourousias, G.; Kaulich, B.; Lepore, M.; Gruetter, R.; Kiskinova, M.

2013-10-01

Low energy x-ray fluorescence (LEXRF) detection was optimized for imaging cerebral glucose metabolism by mapping the fluorine LEXRF signal of 19F in 19FDG, trapped as intracellular 19F-deoxyglucose-6-phosphate (19FDG-6P) at 1μm spatial resolution from 3μm thick brain slices. 19FDG metabolism was evaluated in brain structures closely resembling the general cerebral cytoarchitecture following formalin fixation of brain slices and their inclusion in an epon matrix. 2-dimensional distribution maps of 19FDG-6P were placed in a cytoarchitectural and morphological context by simultaneous LEXRF mapping of N and O, and scanning transmission x-ray (STXM) imaging. A disproportionately high uptake and metabolism of glucose was found in neuropil relative to intracellular domains of the cell body of hypothalamic neurons, showing directly that neurons, like glial cells, also metabolize glucose. As 19F-deoxyglucose-6P is structurally identical to 18F-deoxyglucose-6P, LEXRF of subcellular 19F provides a link to in vivo 18FDG PET, forming a novel basis for understanding the physiological mechanisms underlying the 18FDG PET image, and the contribution of neurons and glia to the PET signal.

Efficient Cisplatin Pro-Drug Delivery Visualized with Sub-100 nm Resolution: Interfacing Engineered Thermosensitive Magnetomicelles with a Living System

DOE PAGES

Vitol, Elina A.; Rozhkova, Elena A.; Rose, Volker; ...

2014-06-06

Temperature-responsive magnetic nanomicelles can serve as thermal energy and cargo carriers with controlled drug release functionality. In view of their potential biomedical applications, understanding the modes of interaction between nanomaterials and living systems and evaluation of efficiency of cargo delivery is of the utmost importance. In this paper, we investigate the interaction between the hybrid magnetic nanomicelles engineered for controlled platinum complex drug delivery and a biological system at three fundamental levels: subcellular compartments, a single cell and whole living animal. Nanomicelles with polymeric P(NIPAAm-co-AAm)-b-PCL core-shell were loaded with a hydrophobic Pt(IV) complex and Fe 3O 4 nanoparticles though self-assembly.more » The distribution of a platinum complex on subcellular level is visualized using hard X-ray fluorescence microscopy with unprecedented level of detail at sub-100 nm spatial resolution. We then study the cytotoxic effects of platinum complex-loaded micelles in vitro on a head and neck cancer cell culture model SQ20B. In conclusion, by employing the magnetic functionality of the micelles and additionally loading them with a near infrared fluorescent dye, we magnetically target them to a tumor site in a live animal xenografted model which allows to visualize their biodistribution in vivo.« less

X-ray microscopy of human malaria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Magowan, C.; Brown, J.T.; Mohandas, N.

Associations between intracellular organisms and host cells are complex and particularly difficult to examine. X-ray microscopy provides transmission images of subcellular structures in intact cells at resolutions superior to available methodologies. The spatial resolution is 50-60nm with a 1 micron depth of focus, superior to anything achievable with light microscopy. Image contrast is generated by differences in photoelectric absorption by the atoms in different areas (i.e. subcellular structures) throughout the full thickness of the sample. Absorption due to carbon dominates among all the elements in the sample at 2.4 nm x-ray wavelength. Thus images show features or structures, in amore » way not usually seen by other types of microscopy. The authors used soft x-ray microscopy to investigate structural development of Plasmodium falciparum malaria parasites in normal and genetically abnormal erythrocytes, and in infected erythrocytes treated with compounds that have anti-malarial effects. X-ray microscopy showed newly elaborated structures in the cytosol of unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. Better understanding of the process of intracellular parasite maturation and the interactions between the parasite and its host erythrocyte can help define new approaches to the control of this deadly disease.« less

Submicron-resolution photoacoustic microscopy of endogenous light-absorbing biomolecules

NASA Astrophysics Data System (ADS)

Zhang, Chi

Photoacoustic imaging in biomedicine has the unique advantage of probing endogenous light absorbers at various length scales with a 100% relative sensitivity. Among the several modalities of photoacoustic imaging, optical-resolution photoacoustic microscopy (OR-PAM) can achieve high spatial resolution, on the order of optical wavelength, at <1 mm depth in biological tissue (the optical ballistic regime). OR-PAM has been applied successfully to structural and functional imaging of blood vasculature and red blood cells in vivo. Any molecules which absorb sufficient light at certain wavelengths can potentially be imaged by PAM. Compared with pure optical imaging, which typically targets fluorescent markers, label-free PAM avoids the major concerns that the fluorescent labeling probes may disturb the function of biomolecules and may have an insufficient density. This dissertation aims to advance label-free OR-PAM to the subcellular scale. The first part of this dissertation describes the technological advancement of PAM yielding high spatial resolution in 3D. The lateral resolution was improved by using optical objectives with high numerical apertures for optical focusing. The axial resolution was improved by using broadband ultrasonic transducers for ultrasound detection. We achieved 220 nm lateral resolution in transmission mode, 0.43 microm lateral resolution in reflection mode, 7.6 microm axial resolution in normal tissue, and 5.8 microm axial resolution with silicone oil immersion/injection. The achieved lateral resolution and axial resolution were the finest reported at the time. With high-resolution in 3D, PAM was demonstrated to resolve cellular and subcellular structures in vivo, such as red blood cells and melanosomes in melanoma cells. Compared with previous PAM systems, our high-resolution PAM could resolve capillaries in mouse ears more clearly. As an example application, we demonstrated intracellular temperature imaging, assisted by fluorescence signal detection, with sub-degree temperature resolution and sub-micron lateral resolution. The second part of this dissertation describes the exploration of endogenous light-absorbing biomolecules for PAM. We demonstrated cytochromes and myoglobin as new absorption contrasts for PAM and identified the corresponding optimal wavelengths for imaging. Fixed fibroblasts on slides and mouse ear sections were imaged by PAM at 422 nm and 250 nm wavelengths to reveal cytoplasms and nuclei, respectively, as confirmed by standard hematoxylin and eosin (H&E) histology. By imaging a blood-perfused mouse heart at 532 nm down to 150 microm in depth, we derived the myocardial sheet thickness and the cleavage height from an undehydrated heart for the first time. The findings promote PAM at new wavelengths and open up new possibilities for characterizing biological tissue. Of particular interest, dual-wavelength PAM around 250 nm and 420 nm wavelengths is analogous to H&E histology. The last part of this dissertation describes the development of sectioning photoacoustic microscopy (SPAM), based on the advancement in spatial resolution and new contrasts for PAM, with applications in brain histology. Label-free SPAM, assisted by a microtome, acquires serial distortion-free images of a specimen on the surface. By exciting cell nuclei at 266 nm wavelength with high resolution, SPAM could pinpoint cell nuclei sensitively and specifically in the mouse brain section, as confirmed by H&E histology. SPAM was demonstrated to generate high-resolution 3D images, highlighting cell nuclei, of formalin-fixed paraffin-embedded mouse brains without tissue staining or clearing. SPAM can potentially serve as a high-throughput and minimal-artifact substitute for histology, probe many other biomolecules and cells, and become a universal tool for animal or human whole-organ microscopy, with diverse applications in life sciences.

Characterization of Si p-i-n diode for scanning transmission ion microanalysis of biological samples

NASA Astrophysics Data System (ADS)

Devès, G.; Matsuyama, S.; Barbotteau, Y.; Ishii, K.; Ortega, R.

2006-05-01

The performance of a silicon p-i-n diode (Hamamatsu S1223-01) for the detection of charged particles was investigated and compared with the response of a standard passivated implanted planar silicon (PIPS) detector. The photodiode was characterized by ion beam induced charge collection with a micrometer spatial resolution using proton and alpha particle beams in the 1-3MeV energy range. Results indicate that homogeneity, energy resolution, and reproducibility of detection of charged particles enable the use of the low cost silicon p-i-n device as a replacement of conventional PIPS detector during scanning transmission ion microanalysis experiments. The Si p-i-n diode detection setup was successfully applied to scanning transmission ion microscopy determination of subcellular compartments on human cancer cultured cells.

Protein localization as a principal feature of the etiology and comorbidity of genetic diseases

PubMed Central

Park, Solip; Yang, Jae-Seong; Shin, Young-Eun; Park, Juyong; Jang, Sung Key; Kim, Sanguk

2011-01-01

Proteins targeting the same subcellular localization tend to participate in mutual protein–protein interactions (PPIs) and are often functionally associated. Here, we investigated the relationship between disease-associated proteins and their subcellular localizations, based on the assumption that protein pairs associated with phenotypically similar diseases are more likely to be connected via subcellular localization. The spatial constraints from subcellular localization significantly strengthened the disease associations of the proteins connected by subcellular localizations. In particular, certain disease types were more prevalent in specific subcellular localizations. We analyzed the enrichment of disease phenotypes within subcellular localizations, and found that there exists a significant correlation between disease classes and subcellular localizations. Furthermore, we found that two diseases displayed high comorbidity when disease-associated proteins were connected via subcellular localization. We newly explained 7584 disease pairs by using the context of protein subcellular localization, which had not been identified using shared genes or PPIs only. Our result establishes a direct correlation between protein subcellular localization and disease association, and helps to understand the mechanism of human disease progression. PMID:21613983

Subcellular investigation of photosynthesis-driven carbon assimilation in the symbiotic reef coral Pocillopora damicornis.

PubMed

Kopp, Christophe; Domart-Coulon, Isabelle; Escrig, Stephane; Humbel, Bruno M; Hignette, Michel; Meibom, Anders

2015-02-10

Reef-building corals form essential, mutualistic endosymbiotic associations with photosynthetic Symbiodinium dinoflagellates, providing their animal host partner with photosynthetically derived nutrients that allow the coral to thrive in oligotrophic waters. However, little is known about the dynamics of these nutritional interactions at the (sub)cellular level. Here, we visualize with submicrometer spatial resolution the carbon and nitrogen fluxes in the intact coral-dinoflagellate association from the reef coral Pocillopora damicornis by combining nanoscale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy with pulse-chase isotopic labeling using [(13)C]bicarbonate and [(15)N]nitrate. This allows us to observe that (i) through light-driven photosynthesis, dinoflagellates rapidly assimilate inorganic bicarbonate and nitrate, temporarily storing carbon within lipid droplets and starch granules for remobilization in nighttime, along with carbon and nitrogen incorporation into other subcellular compartments for dinoflagellate growth and maintenance, (ii) carbon-containing photosynthates are translocated to all four coral tissue layers, where they accumulate after only 15 min in coral lipid droplets from the oral gastroderm and within 6 h in glycogen granules from the oral epiderm, and (iii) the translocation of nitrogen-containing photosynthates is delayed by 3 h. Our results provide detailed in situ subcellular visualization of the fate of photosynthesis-derived carbon and nitrogen in the coral-dinoflagellate endosymbiosis. We directly demonstrate that lipid droplets and glycogen granules in the coral tissue are sinks for translocated carbon photosynthates by dinoflagellates and confirm their key role in the trophic interactions within the coral-dinoflagellate association. Copyright © 2015 Kopp et al.

Determination of the Subcellular Distribution of Liposomes Using Confocal Microscopy.

PubMed

Solomon, Melani A

2017-01-01

It is being increasingly recognized that therapeutics need to be delivered to specific organelle targets within cells. Liposomes are versatile lipid-based drug delivery vehicles that can be surface-modified to deliver the loaded cargo to specific subcellular locations within the cell. Hence, the development of such technology requires a means of measuring the subcellular distribution possibly by utilizing imaging techniques that can visualize and quantitate the extent of this subcellular localization. The apparent increase of resolution along the Z-axis offered by confocal microscopy makes this technique suitable for such studies. In this chapter, we describe the application of confocal laser scanning microscopy (CLSM) to determine the subcellular distribution of fluorescently labeled mitochondriotropic liposomes.

MALDI-MS and NanoSIMS imaging techniques to study cnidarian-dinoflagellate symbioses.

PubMed

Kopp, C; Wisztorski, M; Revel, J; Mehiri, M; Dani, V; Capron, L; Carette, D; Fournier, I; Massi, L; Mouajjah, D; Pagnotta, S; Priouzeau, F; Salzet, M; Meibom, A; Sabourault, C

2015-04-01

Cnidarian-dinoflagellate photosynthetic symbioses are fundamental to biologically diverse and productive coral reef ecosystems. The hallmark of this symbiotic relationship is the ability of dinoflagellate symbionts to supply their cnidarian host with a wide range of nutrients. Many aspects of this association nevertheless remain poorly characterized, including the exact identity of the transferred metabolic compounds, the mechanisms that control their exchange across the host-symbiont interface, and the precise subcellular fate of the translocated materials in cnidarian tissues. This lack of knowledge is mainly attributed to difficulties in investigating such metabolic interactions both in situ, i.e. on intact symbiotic associations, and at high spatial resolution. To address these issues, we illustrate the application of two in situ and high spatial resolution molecular and ion imaging techniques-matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and the nano-scale secondary-ion mass spectrometry (NanoSIMS) ion microprobe. These imaging techniques provide important new opportunities for the detailed investigation of many aspects of cnidarian-dinoflagellate associations, including the dynamics of cellular interactions. Copyright © 2014 Elsevier GmbH. All rights reserved.

Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy

PubMed Central

Fu, Qinyi; Martin, Benjamin L.; Matus, David Q.; Gao, Liang

2016-01-01

Despite the progress made in selective plane illumination microscopy, high-resolution 3D live imaging of multicellular specimens remains challenging. Tiling light-sheet selective plane illumination microscopy (TLS-SPIM) with real-time light-sheet optimization was developed to respond to the challenge. It improves the 3D imaging ability of SPIM in resolving complex structures and optimizes SPIM live imaging performance by using a real-time adjustable tiling light sheet and creating a flexible compromise between spatial and temporal resolution. We demonstrate the 3D live imaging ability of TLS-SPIM by imaging cellular and subcellular behaviours in live C. elegans and zebrafish embryos, and show how TLS-SPIM can facilitate cell biology research in multicellular specimens by studying left-right symmetry breaking behaviour of C. elegans embryos. PMID:27004937

Ultra-spatial synchrotron radiation for imaging molecular chemical structure: Applications in plant and animal studies

DOE PAGES

Yu, Peiqiang

2007-01-01

Synchrotron-based Fourier transform infrared microspectroscopy (S-FTIR) has been developed as a rapid, direct, non-destructive, bioanalytical technique. This technique takes advantage of synchrotron light brightness and small effective source size and is capable of exploring the molecular chemical features and make-up within microstructures of a biological tissue without destruction of inherent structures at ultra-spatial resolutions within cellular dimension. To date there has been very little application of this advanced synchrotron technique to the study of plant and animal tissues' inherent structure at a cellular or subcellular level. In this article, a novel approach was introduced to show the potential of themore » newly developed, advanced synchrotron-based analytical technology, which can be used to reveal molecular structural-chemical features of various plant and animal tissues.« less

Towards a magnetoresistive platform for neural signal recording

NASA Astrophysics Data System (ADS)

Sharma, P. P.; Gervasoni, G.; Albisetti, E.; D'Ercoli, F.; Monticelli, M.; Moretti, D.; Forte, N.; Rocchi, A.; Ferrari, G.; Baldelli, P.; Sampietro, M.; Benfenati, F.; Bertacco, R.; Petti, D.

2017-05-01

A promising strategy to get deeper insight on brain functionalities relies on the investigation of neural activities at the cellular and sub-cellular level. In this framework, methods for recording neuron electrical activity have gained interest over the years. Main technological challenges are associated to finding highly sensitive detection schemes, providing considerable spatial and temporal resolution. Moreover, the possibility to perform non-invasive assays would constitute a noteworthy benefit. In this work, we present a magnetoresistive platform for the detection of the action potential propagation in neural cells. Such platform allows, in perspective, the in vitro recording of neural signals arising from single neurons, neural networks and brain slices.

BODIPY-Based Two-Photon Fluorescent Probe for Real-Time Monitoring of Lysosomal Viscosity with Fluorescence Lifetime Imaging Microscopy.

PubMed

Li, Ling-Ling; Li, Kun; Li, Meng-Yang; Shi, Lei; Liu, Yan-Hong; Zhang, Hong; Pan, Sheng-Lin; Wang, Nan; Zhou, Qian; Yu, Xiao-Qi

2018-05-01

The viscosity of lysosome is reported to be a key indicator of lysosomal functionality. However, the existing mechanical methods of viscosity measurement can hardly be applied at the cellular or subcellular level. Herein, a BODIPY-based two-photon fluorescent probe was presented for monitoring lysosomal viscosity with high spatial and temporal resolution. By installing two morpholine moieties to the fluorophore as target and rotational groups, the TICT effect between the two morpholine rings and the main fluorophore scaffold endowed the probe with excellent viscosity sensitivity. Moreover, Lyso-B succeeded in showing the impact of dexamethasone on lysosomal viscosity in real time.

Intrinsic optical signal imaging of glucose-stimulated physiological responses in the insulin secreting INS-1 β-cell line

NASA Astrophysics Data System (ADS)

Li, Yi-Chao; Cui, Wan-Xing; Wang, Xu-Jing; Amthor, Franklin; Yao, Xin-Cheng

2011-03-01

Intrinsic optical signal (IOS) imaging has been established for noninvasive monitoring of stimulus-evoked physiological responses in the retina and other neural tissues. Recently, we extended the IOS imaging technology for functional evaluation of insulin secreting INS-1 cells. INS-1 cells provide a popular model for investigating β-cell dysfunction and diabetes. Our experiments indicate that IOS imaging allows simultaneous monitoring of glucose-stimulated physiological responses in multiple cells with high spatial (sub-cellular) and temporal (sub-second) resolution. Rapid image sequences reveal transient optical responses that have time courses comparable to glucose-evoked β-cell electrical activities.

Zone plate lenses for X-ray microscopy

NASA Astrophysics Data System (ADS)

Vladimirsky, Y.; Kern, D. P.; Chang, T. H. P.; Attwood, D. T.; Iskander, N.; Rothman, S.; McQuaide, K.; Kirz, J.; Ade, H.; McNulty, I.; Rarback, H.; Shu, D.

1988-04-01

Fresnel zone plate lenses with feature sizes as small as 50 nm have been constructed and used in the Stony Brook/NSLS scanning X-ray microscope with 3.1 nm radiation from Brookhaven's X-17 mini-undulator. The zone plates were fabricated at IBM using electron beam writing techniques, moiré pattern techniques to monitor ellipticity, and a double development/double plating technique to provide additional thickness in the central region. A spatial resolution down to 75 nm was measured in the microscope. Using these zone plates, biological images were obtained of unaltered subcellular components. The images highlight protein concentration in unsectioned, unfixed, and unstained enzymatic granules in an aqueous environment.

A graphene-based physiometer array for the analysis of single biological cells

PubMed Central

Paulus, Geraldine L. C.; Nelson, Justin T.; Lee, Katherine Y.; Wang, Qing Hua; Reuel, Nigel F.; Grassbaugh, Brittany R.; Kruss, Sebastian; Landry, Markita P.; Kang, Jeon Woong; Vander Ende, Emma; Zhang, Jingqing; Mu, Bin; Dasari, Ramachandra R.; Opel, Cary F.; Wittrup, K. Dane; Strano, Michael S.

2014-01-01

A significant advantage of a graphene biosensor is that it inherently represents a continuum of independent and aligned sensor-units. We demonstrate a nanoscale version of a micro-physiometer – a device that measures cellular metabolic activity from the local acidification rate. Graphene functions as a matrix of independent pH sensors enabling subcellular detection of proton excretion. Raman spectroscopy shows that aqueous protons p-dope graphene – in agreement with established doping trajectories, and that graphene displays two distinct pKa values (2.9 and 14.2), corresponding to dopants physi- and chemisorbing to graphene respectively. The graphene physiometer allows micron spatial resolution and can differentiate immunoglobulin (IgG)-producing human embryonic kidney (HEK) cells from non-IgG-producing control cells. Population-based analyses allow mapping of phenotypic diversity, variances in metabolic activity, and cellular adhesion. Finally we show this platform can be extended to the detection of other analytes, e.g. dopamine. This work motivates the application of graphene as a unique biosensor for (sub)cellular interrogation. PMID:25359450

Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators

PubMed Central

Chamberland, Simon; Yang, Helen H; Pan, Michael M; Evans, Stephen W; Guan, Sihui; Chavarha, Mariya; Yang, Ying; Salesse, Charleen; Wu, Haodi; Wu, Joseph C; Clandinin, Thomas R; Toth, Katalin; Lin, Michael Z; St-Pierre, François

2017-01-01

Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and in Drosophila. These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision. DOI: http://dx.doi.org/10.7554/eLife.25690.001 PMID:28749338

Design and characterization of a handheld multimodal imaging device for the assessment of oral epithelial lesions

NASA Astrophysics Data System (ADS)

Higgins, Laura M.; Pierce, Mark C.

2014-08-01

A compact handpiece combining high resolution fluorescence (HRF) imaging with optical coherence tomography (OCT) was developed to provide real-time assessment of oral lesions. This multimodal imaging device simultaneously captures coregistered en face images with subcellular detail alongside cross-sectional images of tissue microstructure. The HRF imaging acquires a 712×594 μm2 field-of-view at the sample with a spatial resolution of 3.5 μm. The OCT images were acquired to a depth of 1.5 mm with axial and lateral resolutions of 9.3 and 8.0 μm, respectively. HRF and OCT images are simultaneously displayed at 25 fps. The handheld device was used to image a healthy volunteer, demonstrating the potential for in vivo assessment of the epithelial surface for dysplastic and neoplastic changes at the cellular level, while simultaneously evaluating submucosal involvement. We anticipate potential applications in real-time assessment of oral lesions for improved surveillance and surgical guidance.

Sub-cellular mRNA localization modulates the regulation of gene expression by small RNAs in bacteria

NASA Astrophysics Data System (ADS)

Teimouri, Hamid; Korkmazhan, Elgin; Stavans, Joel; Levine, Erel

2017-10-01

Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellular distributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

Self-assembled dual-modality contrast agents for non-invasive stem cell tracking via near-infrared fluorescence and magnetic resonance imaging.

PubMed

Liu, Hong; Tan, Yan; Xie, Lisi; Yang, Lei; Zhao, Jing; Bai, Jingxuan; Huang, Ping; Zhan, Wugen; Wan, Qian; Zou, Chao; Han, Yali; Wang, Zhiyong

2016-09-15

Stem cells hold great promise for treating various diseases. However, one of the main drawbacks of stem cell therapy is the lack of non-invasive image-tracking technologies. Although magnetic resonance imaging (MRI) and near-infrared fluorescence (NIRF) imaging have been employed to analyse cellular and subcellular events via the assistance of contrast agents, the sensitivity and temporal resolution of MRI and the spatial resolution of NIRF are still shortcomings. In this study, superparamagnetic iron oxide nanocrystals and IR-780 dyes were co-encapsulated in stearic acid-modified polyethylenimine to form a dual-modality contrast agent with nano-size and positive charge. These resulting agents efficiently labelled stem cells and did not influence the cellular viability and differentiation. Moreover, the labelled cells showed the advantages of dual-modality imaging in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Cell Signaling Experiments Driven by Optical Manipulation

PubMed Central

Difato, Francesco; Pinato, Giulietta; Cojoc, Dan

2013-01-01

Cell signaling involves complex transduction mechanisms in which information released by nearby cells or extracellular cues are transmitted to the cell, regulating fundamental cellular activities. Understanding such mechanisms requires cell stimulation with precise control of low numbers of active molecules at high spatial and temporal resolution under physiological conditions. Optical manipulation techniques, such as optical tweezing, mechanical stress probing or nano-ablation, allow handling of probes and sub-cellular elements with nanometric and millisecond resolution. PicoNewton forces, such as those involved in cell motility or intracellular activity, can be measured with femtoNewton sensitivity while controlling the biochemical environment. Recent technical achievements in optical manipulation have new potentials, such as exploring the actions of individual molecules within living cells. Here, we review the progress in optical manipulation techniques for single-cell experiments, with a focus on force probing, cell mechanical stimulation and the local delivery of active molecules using optically manipulated micro-vectors and laser dissection. PMID:23698758

An Integrative Platform for Three-dimensional Quantitative Analysis of Spatially Heterogeneous Metastasis Landscapes

NASA Astrophysics Data System (ADS)

Guldner, Ian H.; Yang, Lin; Cowdrick, Kyle R.; Wang, Qingfei; Alvarez Barrios, Wendy V.; Zellmer, Victoria R.; Zhang, Yizhe; Host, Misha; Liu, Fang; Chen, Danny Z.; Zhang, Siyuan

2016-04-01

Metastatic microenvironments are spatially and compositionally heterogeneous. This seemingly stochastic heterogeneity provides researchers great challenges in elucidating factors that determine metastatic outgrowth. Herein, we develop and implement an integrative platform that will enable researchers to obtain novel insights from intricate metastatic landscapes. Our two-segment platform begins with whole tissue clearing, staining, and imaging to globally delineate metastatic landscape heterogeneity with spatial and molecular resolution. The second segment of our platform applies our custom-developed SMART 3D (Spatial filtering-based background removal and Multi-chAnnel forest classifiers-based 3D ReconsTruction), a multi-faceted image analysis pipeline, permitting quantitative interrogation of functional implications of heterogeneous metastatic landscape constituents, from subcellular features to multicellular structures, within our large three-dimensional (3D) image datasets. Coupling whole tissue imaging of brain metastasis animal models with SMART 3D, we demonstrate the capability of our integrative pipeline to reveal and quantify volumetric and spatial aspects of brain metastasis landscapes, including diverse tumor morphology, heterogeneous proliferative indices, metastasis-associated astrogliosis, and vasculature spatial distribution. Collectively, our study demonstrates the utility of our novel integrative platform to reveal and quantify the global spatial and volumetric characteristics of the 3D metastatic landscape with unparalleled accuracy, opening new opportunities for unbiased investigation of novel biological phenomena in situ.

Inner membrane fusion mediates spatial distribution of axonal mitochondria

PubMed Central

Yu, Yiyi; Lee, Hao-Chih; Chen, Kuan-Chieh; Suhan, Joseph; Qiu, Minhua; Ba, Qinle; Yang, Ge

2016-01-01

In eukaryotic cells, mitochondria form a dynamic interconnected network to respond to changing needs at different subcellular locations. A fundamental yet unanswered question regarding this network is whether, and if so how, local fusion and fission of individual mitochondria affect their global distribution. To address this question, we developed high-resolution computational image analysis techniques to examine the relations between mitochondrial fusion/fission and spatial distribution within the axon of Drosophila larval neurons. We found that stationary and moving mitochondria underwent fusion and fission regularly but followed different spatial distribution patterns and exhibited different morphology. Disruption of inner membrane fusion by knockdown of dOpa1, Drosophila Optic Atrophy 1, not only increased the spatial density of stationary and moving mitochondria but also changed their spatial distributions and morphology differentially. Knockdown of dOpa1 also impaired axonal transport of mitochondria. But the changed spatial distributions of mitochondria resulted primarily from disruption of inner membrane fusion because knockdown of Milton, a mitochondrial kinesin-1 adapter, caused similar transport velocity impairment but different spatial distributions. Together, our data reveals that stationary mitochondria within the axon interconnect with moving mitochondria through fusion and fission and that local inner membrane fusion between individual mitochondria mediates their global distribution. PMID:26742817

Development of high-spatial and high-mass resolution mass spectrometric imaging (MSI) and its application to the study of small metabolites and endogenous molecules of plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jun, Ji Hyun

High-spatial and high-mass resolution laser desorption ionization (LDI) mass spectrometric (MS) imaging technology was developed for the attainment of MS images of higher quality containing more information on the relevant cellular and molecular biology in unprecedented depth. The distribution of plant metabolites is asymmetric throughout the cells and tissues, and therefore the increase in the spatial resolution was pursued to reveal the localization of plant metabolites at the cellular level by MS imaging. For achieving high-spatial resolution, the laser beam size was reduced by utilizing an optical fiber with small core diameter (25 μm) in a vacuum matrix-assisted laser desorptionmore » ionization-linear ion trap (vMALDI-LTQ) mass spectrometer. Matrix application was greatly improved using oscillating capillary nebulizer. As a result, single cell level spatial resolution of ~ 12 μm was achieved. MS imaging at this high spatial resolution was directly applied to a whole Arabidopsis flower and the substructures of an anther and single pollen grains at the stigma and anther were successfully visualized. MS imaging of high spatial resolution was also demonstrated to the secondary roots of Arabidopsis thaliana and a high degree of localization of detected metabolites was successfully unveiled. This was the first MS imaging on the root for molecular species. MS imaging with high mass resolution was also achieved by utilizing the LTQ-Orbitrap mass spectrometer for the direct identification of the surface metabolites on the Arabidopsis stem and root and differentiation of isobaric ions having the same nominal mass with no need of tandem mass spectrometry (MS/MS). MS imaging at high-spatial and high-mass resolution was also applied to cer1 mutant of the model system Arabidopsis thaliana to demonstrate its usefulness in biological studies and reveal associated metabolite changes in terms of spatial distribution and/or abundances compared to those of wild-type. The spatial distribution of targeted metabolites, mainly waxes and flavonoids, was systematically explored on various organs, including flowers, leaves, stems, and roots at high spatial resolution of ~ 12-50 μm and the changes in the abundance level of these metabolites were monitored on the cer1 mutant with respect to the wild-type. This study revealed the metabolic biology of CER1 gene on each individual organ level with very detailed high spatial resolution. The separate MS images of isobaric metabolites, i.e. C29 alkane vs. C28 aldehyde could be constructed on both genotypes from MS imaging at high mass resolution. This allows tracking of abundance changes for those compounds along with the genetic mutation, which is not achievable with low mass resolution mass spectrometry. This study supported previous hypothesis of molecular function of CER1 gene as aldehyde decarbonylase, especially by displaying hyper accumulation of aldehydes and C30 fatty acid and decrease in abundance of alkanes and ketones in several plant organs of cer1 mutant. The scope of analytes was further directed toward internal cell metabolites from the surface metabolites of the plant. MS profiling and imaging of internal cell metabolites were performed on the vibratome section of Arabidopsis leaf. Vibratome sectioning of the leaf was first conducted to remove the surface cuticle layer and it was followed by enzymatic treatment of the section to induce the digestion of primary cell walls, middle lamella, and expose the internal cells underneath to the surface for detection with the laser by LDI-MS. The subsequent MS imaging onto the enzymatically treated vibratome section allowed us to map the distribution of the metabolites in the internal cell layers, linolenic acid (C18:3 FA) and linoleic acid (C18:2 FA). The development of an assay for relative quantification of analytes at the single subcellular/organelle level by LDI-MS imaging was attempted and both plausibility and significant obstacles were seen. As a test system, native plant organelle, chloroplasts isolated from the spinach leaves were used and the localization of isolated chloroplasts dispersed on the target plate in low density was monitored by detecting the ion signal of chlorophyll a (Chl a) degradation products such as pheophytin a and pheophobide a by LDI-MS imaging in combination with fluorescence microscopy. The number of chloroplasts and their localization visualized in the MS image exactly matched those in the fluorescence image especially at low density, which first shows the plausibility of single-organelle level quantification of analytes by LDI-MS. The accumulation level of Chl a within a single chloroplast detected by LDI-MS was compared to the fluorescence signal on a pixel-to-pixel basis to further confirm the correlations of the accumulation levels measured by two methods. The proportional correlation was observed only for the chloroplasts which do not show the significant leakage of chlorophyll indicated by MS ion signal of Chl a degradation products and fluorescence signal, which was presumably caused by the prior fluorescence measurement before MS imaging. Further investigation is necessary to make this method more complete and develop LDI-MS imaging as an effective analytical tool to evaluate a relative accumulation of analytes of interest at the single subcellular/organelle level.« less

Multiplexed multi-scale imaging: novel roles for the scaffold protein IQGAP1 in epithelial cell development (Conference Presentation)

NASA Astrophysics Data System (ADS)

Schweikhard, Volker

2016-02-01

The precise sub-cellular spatial localization of multi-protein complexes is increasingly recognized as a key mechanism governing the organization of mammalian cells. Consequently, there is a need for novel microscopy techniques capable of investigating such sub-cellular architectures in comprehensive detail. Here, we applied a novel multiplexed STORM super-resolution microscopy technique, in combination with high-throughput immunofluorescence microscopy and live-cell imaging, to investigate the roles of the scaffold protein IQGAP1 in epithelial cells. IQGAP1 is known to orchestrate a wide range of biological processes, including intracellular signaling, cytoskeletal regulation, cell-cell adhesion, and protein trafficking, by forming distinct complexes with a number of known interaction partners, and recruiting these complexes to specific subcellular locations. Our results demonstrate that, in addition to supporting epithelial adherens junctions by associating with specialized cortical actin structures, IQGAP1 plays a second role in which it controls the confinement of a unique, previously undocumented class of membranous compartments to the basal actin cortex. These largely immotile yet highly dynamic structures appear transiently as cells merge into clusters and establish of apical-basolateral (epithelial) polarity, and are identified as an intermediate compartment in the endocytic recycling pathways for cell junction complexes and cell surface receptors. Although these two functions of IQGAP1 occur in parallel and largely independently of each other, they both support the maturation and maintenance of polarized epithelial cell architectures.

An algorithm for automated detection, localization and measurement of local calcium signals from camera-based imaging.

PubMed

Ellefsen, Kyle L; Settle, Brett; Parker, Ian; Smith, Ian F

2014-09-01

Local Ca(2+) transients such as puffs and sparks form the building blocks of cellular Ca(2+) signaling in numerous cell types. They have traditionally been studied by linescan confocal microscopy, but advances in TIRF microscopy together with improved electron-multiplied CCD (EMCCD) cameras now enable rapid (>500 frames s(-1)) imaging of subcellular Ca(2+) signals with high spatial resolution in two dimensions. This approach yields vastly more information (ca. 1 Gb min(-1)) than linescan imaging, rendering visual identification and analysis of local events imaged both laborious and subject to user bias. Here we describe a routine to rapidly automate identification and analysis of local Ca(2+) events. This features an intuitive graphical user-interfaces and runs under Matlab and the open-source Python software. The underlying algorithm features spatial and temporal noise filtering to reliably detect even small events in the presence of noisy and fluctuating baselines; localizes sites of Ca(2+) release with sub-pixel resolution; facilitates user review and editing of data; and outputs time-sequences of fluorescence ratio signals for identified event sites along with Excel-compatible tables listing amplitudes and kinetics of events. Copyright © 2014 Elsevier Ltd. All rights reserved.

In vivo cardiac nano-imaging: A new technology for high-precision analyses of sarcomere dynamics in the heart.

PubMed

Shimozawa, Togo; Hirokawa, Erisa; Kobirumaki-Shimozawa, Fuyu; Oyama, Kotaro; Shintani, Seine A; Terui, Takako; Kushida, Yasuharu; Tsukamoto, Seiichi; Fujii, Teruyuki; Ishiwata, Shin'ichi; Fukuda, Norio

2017-03-01

The cardiac pump function is a result of a rise in intracellular Ca 2+ and the ensuing sarcomeric contractions [i.e., excitation-contraction (EC) coupling] in myocytes in various locations of the heart. In order to elucidate the heart's mechanical properties under various settings, cardiac imaging is widely performed in today's clinical as well as experimental cardiology by using echocardiogram, magnetic resonance imaging and computed tomography. However, because these common techniques detect local myocardial movements at a spatial resolution of ∼100 μm, our knowledge on the sub-cellular mechanisms of the physiology and pathophysiology of the heart in vivo is limited. This is because (1) EC coupling occurs in the μm partition in a myocyte and (2) cardiac sarcomeres generate active force upon a length change of ∼100 nm on a beat-to-beat basis. Recent advances in optical technologies have enabled measurements of intracellular Ca 2+ dynamics and sarcomere length displacements at high spatial and temporal resolution in the beating heart of living rodents. Future studies with these technologies are warranted to open a new era in cardiac research. Copyright © 2016 Elsevier Ltd. All rights reserved.

In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy

NASA Astrophysics Data System (ADS)

Megens, Remco T. A.; Reitsma, Sietze; Prinzen, Lenneke; Oude Egbrink, Mirjam G. A.; Engels, Wim; Leenders, Peter J. A.; Brunenberg, Ellen J. L.; Reesink, Koen D.; Janssen, Ben J. A.; Ter Haar Romeny, Bart M.; Slaaf, Dick W.; van Zandvoort, Marc A. M. J.

2010-01-01

In vivo (molecular) imaging of the vessel wall of large arteries at subcellular resolution is crucial for unraveling vascular pathophysiology. We previously showed the applicability of two-photon laser scanning microscopy (TPLSM) in mounted arteries ex vivo. However, in vivo TPLSM has thus far suffered from in-frame and between-frame motion artifacts due to arterial movement with cardiac and respiratory activity. Now, motion artifacts are suppressed by accelerated image acquisition triggered on cardiac and respiratory activity. In vivo TPLSM is performed on rat renal and mouse carotid arteries, both surgically exposed and labeled fluorescently (cell nuclei, elastin, and collagen). The use of short acquisition times consistently limit in-frame motion artifacts. Additionally, triggered imaging reduces between-frame artifacts. Indeed, structures in the vessel wall (cell nuclei, elastic laminae) can be imaged at subcellular resolution. In mechanically damaged carotid arteries, even the subendothelial collagen sheet (~1 μm) is visualized using collagen-targeted quantum dots. We demonstrate stable in vivo imaging of large arteries at subcellular resolution using TPLSM triggered on cardiac and respiratory cycles. This creates great opportunities for studying (diseased) arteries in vivo or immediate validation of in vivo molecular imaging techniques such as magnetic resonance imaging (MRI), ultrasound, and positron emission tomography (PET).

Micro axial tomography: A miniaturized, versatile stage device to overcome resolution anisotropy in fluorescence light microscopy

NASA Astrophysics Data System (ADS)

Staier, Florian; Eipel, Heinz; Matula, Petr; Evsikov, Alexei V.; Kozubek, Michal; Cremer, Christoph; Hausmann, Michael

2011-09-01

With the development of novel fluorescence techniques, high resolution light microscopy has become a challenging technique for investigations of the three-dimensional (3D) micro-cosmos in cells and sub-cellular components. So far, all fluorescence microscopes applied for 3D imaging in biosciences show a spatially anisotropic point spread function resulting in an anisotropic optical resolution or point localization precision. To overcome this shortcoming, micro axial tomography was suggested which allows object tilting on the microscopic stage and leads to an improvement in localization precision and spatial resolution. Here, we present a miniaturized device which can be implemented in a motor driven microscope stage. The footprint of this device corresponds to a standard microscope slide. A special glass fiber can manually be adjusted in the object space of the microscope lens. A stepwise fiber rotation can be controlled by a miniaturized stepping motor incorporated into the device. By means of a special mounting device, test particles were fixed onto glass fibers, optically localized with high precision, and automatically rotated to obtain views from different perspective angles under which distances of corresponding pairs of objects were determined. From these angle dependent distance values, the real 3D distance was calculated with a precision in the ten nanometer range (corresponding here to an optical resolution of 10-30 nm) using standard microscopic equipment. As a proof of concept, the spindle apparatus of a mature mouse oocyte was imaged during metaphase II meiotic arrest under different perspectives. Only very few images registered under different rotation angles are sufficient for full 3D reconstruction. The results indicate the principal advantage of the micro axial tomography approach for many microscopic setups therein and also those of improved resolutions as obtained by high precision localization determination.

SamuROI, a Python-Based Software Tool for Visualization and Analysis of Dynamic Time Series Imaging at Multiple Spatial Scales.

PubMed

Rueckl, Martin; Lenzi, Stephen C; Moreno-Velasquez, Laura; Parthier, Daniel; Schmitz, Dietmar; Ruediger, Sten; Johenning, Friedrich W

2017-01-01

The measurement of activity in vivo and in vitro has shifted from electrical to optical methods. While the indicators for imaging activity have improved significantly over the last decade, tools for analysing optical data have not kept pace. Most available analysis tools are limited in their flexibility and applicability to datasets obtained at different spatial scales. Here, we present SamuROI (Structured analysis of multiple user-defined ROIs), an open source Python-based analysis environment for imaging data. SamuROI simplifies exploratory analysis and visualization of image series of fluorescence changes in complex structures over time and is readily applicable at different spatial scales. In this paper, we show the utility of SamuROI in Ca 2+ -imaging based applications at three spatial scales: the micro-scale (i.e., sub-cellular compartments including cell bodies, dendrites and spines); the meso-scale, (i.e., whole cell and population imaging with single-cell resolution); and the macro-scale (i.e., imaging of changes in bulk fluorescence in large brain areas, without cellular resolution). The software described here provides a graphical user interface for intuitive data exploration and region of interest (ROI) management that can be used interactively within Jupyter Notebook: a publicly available interactive Python platform that allows simple integration of our software with existing tools for automated ROI generation and post-processing, as well as custom analysis pipelines. SamuROI software, source code and installation instructions are publicly available on GitHub and documentation is available online. SamuROI reduces the energy barrier for manual exploration and semi-automated analysis of spatially complex Ca 2+ imaging datasets, particularly when these have been acquired at different spatial scales.

SamuROI, a Python-Based Software Tool for Visualization and Analysis of Dynamic Time Series Imaging at Multiple Spatial Scales

PubMed Central

Rueckl, Martin; Lenzi, Stephen C.; Moreno-Velasquez, Laura; Parthier, Daniel; Schmitz, Dietmar; Ruediger, Sten; Johenning, Friedrich W.

2017-01-01

The measurement of activity in vivo and in vitro has shifted from electrical to optical methods. While the indicators for imaging activity have improved significantly over the last decade, tools for analysing optical data have not kept pace. Most available analysis tools are limited in their flexibility and applicability to datasets obtained at different spatial scales. Here, we present SamuROI (Structured analysis of multiple user-defined ROIs), an open source Python-based analysis environment for imaging data. SamuROI simplifies exploratory analysis and visualization of image series of fluorescence changes in complex structures over time and is readily applicable at different spatial scales. In this paper, we show the utility of SamuROI in Ca2+-imaging based applications at three spatial scales: the micro-scale (i.e., sub-cellular compartments including cell bodies, dendrites and spines); the meso-scale, (i.e., whole cell and population imaging with single-cell resolution); and the macro-scale (i.e., imaging of changes in bulk fluorescence in large brain areas, without cellular resolution). The software described here provides a graphical user interface for intuitive data exploration and region of interest (ROI) management that can be used interactively within Jupyter Notebook: a publicly available interactive Python platform that allows simple integration of our software with existing tools for automated ROI generation and post-processing, as well as custom analysis pipelines. SamuROI software, source code and installation instructions are publicly available on GitHub and documentation is available online. SamuROI reduces the energy barrier for manual exploration and semi-automated analysis of spatially complex Ca2+ imaging datasets, particularly when these have been acquired at different spatial scales. PMID:28706482

Dual-channel (green and red) fluorescence microendoscope with subcellular resolution

NASA Astrophysics Data System (ADS)

de Paula D'Almeida, Camila; Fortunato, Thereza Cury; Teixeira Rosa, Ramon Gabriel; Romano, Renan Arnon; Moriyama, Lilian Tan; Pratavieira, Sebastião.

2018-02-01

Usually, tissue images at cellular level need biopsies to be done. Considering this, diagnostic devices, such as microendoscopes, have been developed with the purpose of do not be invasive. This study goal is the development of a dual-channel microendoscope, using two fluorescent labels: proflavine and protoporphyrin IX (PpIX), both approved by Food and Drug Administration. This system, with the potential to perform a microscopic diagnosis and to monitor a photodynamic therapy (PDT) session, uses a halogen lamp and an image fiber bundle to perform subcellular image. Proflavine fluorescence indicates the nuclei of the cell, which is the reference for PpIX localization on image tissue. Preliminary results indicate the efficacy of this optical technique to detect abnormal tissues and to improve the PDT dosimetry. This was the first time, up to our knowledge, that PpIX fluorescence was microscopically observed in vivo, in real time, combined to other fluorescent marker (Proflavine), which allowed to simultaneously observe the spatial localization of the PpIX in the mucosal tissue. We believe this system is very promising tool to monitor PDT in mucosa as it happens. Further experiments have to be performed in order to validate the system for PDT monitoring.

Photoacoustic imaging of single circulating melanoma cells in vivo

NASA Astrophysics Data System (ADS)

Wang, Lidai; Yao, Junjie; Zhang, Ruiying; Xu, Song; Li, Guo; Zou, Jun; Wang, Lihong V.

2015-03-01

Melanoma, one of the most common types of skin cancer, has a high mortality rate, mainly due to a high propensity for tumor metastasis. The presence of circulating tumor cells (CTCs) is a potential predictor for metastasis. Label-free imaging of single circulating melanoma cells in vivo provides rich information on tumor progress. Here we present photoacoustic microscopy of single melanoma cells in living animals. We used a fast-scanning optical-resolution photoacoustic microscope to image the microvasculature in mouse ears. The imaging system has sub-cellular spatial resolution and works in reflection mode. A fast-scanning mirror allows the system to acquire fast volumetric images over a large field of view. A 500-kHz pulsed laser was used to image blood and CTCs. Single circulating melanoma cells were imaged in both capillaries and trunk vessels in living animals. These high-resolution images may be used in early detection of CTCs with potentially high sensitivity. In addition, this technique enables in vivo study of tumor cell extravasation from a primary tumor, which addresses an urgent pre-clinical need.

Whole mouse cryo-imaging

NASA Astrophysics Data System (ADS)

Wilson, David; Roy, Debashish; Steyer, Grant; Gargesha, Madhusudhana; Stone, Meredith; McKinley, Eliot

2008-03-01

The Case cryo-imaging system is a section and image system which allows one to acquire micron-scale, information rich, whole mouse color bright field and molecular fluorescence images of an entire mouse. Cryo-imaging is used in a variety of applications, including mouse and embryo anatomical phenotyping, drug delivery, imaging agents, metastastic cancer, stem cells, and very high resolution vascular imaging, among many. Cryo-imaging fills the gap between whole animal in vivo imaging and histology, allowing one to image a mouse along the continuum from the mouse -> organ -> tissue structure -> cell -> sub-cellular domains. In this overview, we describe the technology and a variety of exciting applications. Enhancements to the system now enable tiled acquisition of high resolution images to cover an entire mouse. High resolution fluorescence imaging, aided by a novel subtraction processing algorithm to remove sub-surface fluorescence, makes it possible to detect fluorescently-labeled single cells. Multi-modality experiments in Magnetic Resonance Imaging and Cryo-imaging of a whole mouse demonstrate superior resolution of cryo-images and efficiency of registration techniques. The 3D results demonstrate the novel true-color volume visualization tools we have developed and the inherent advantage of cryo-imaging in providing unlimited depth of field and spatial resolution. The recent results continue to demonstrate the value cryo-imaging provides in the field of small animal imaging research.

Intravital microscopy: a novel tool to study cell biology in living animals.

PubMed

Weigert, Roberto; Sramkova, Monika; Parente, Laura; Amornphimoltham, Panomwat; Masedunskas, Andrius

2010-05-01

Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criterion. Indeed, first we will focus on those studies in which organs were imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures.

Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

NASA Astrophysics Data System (ADS)

Chandra, Subhash

2004-06-01

Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes 13C and 15N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK 1 kidney cells at mass 28 ( 13C15N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of 39K, 23Na and 40Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

Tomographic brain imaging with nucleolar detail and automatic cell counting

NASA Astrophysics Data System (ADS)

Hieber, Simone E.; Bikis, Christos; Khimchenko, Anna; Schweighauser, Gabriel; Hench, Jürgen; Chicherova, Natalia; Schulz, Georg; Müller, Bert

2016-09-01

Brain tissue evaluation is essential for gaining in-depth insight into its diseases and disorders. Imaging the human brain in three dimensions has always been a challenge on the cell level. In vivo methods lack spatial resolution, and optical microscopy has a limited penetration depth. Herein, we show that hard X-ray phase tomography can visualise a volume of up to 43 mm3 of human post mortem or biopsy brain samples, by demonstrating the method on the cerebellum. We automatically identified 5,000 Purkinje cells with an error of less than 5% at their layer and determined the local surface density to 165 cells per mm2 on average. Moreover, we highlight that three-dimensional data allows for the segmentation of sub-cellular structures, including dendritic tree and Purkinje cell nucleoli, without dedicated staining. The method suggests that automatic cell feature quantification of human tissues is feasible in phase tomograms obtained with isotropic resolution in a label-free manner.

In vivo biochemistry: quantifying ion and metabolite levels in individual cells or cultures of yeast.

PubMed

Bermejo, Clara; Ewald, Jennifer C; Lanquar, Viviane; Jones, Alexander M; Frommer, Wolf B

2011-08-15

Over the past decade, we have learned that cellular processes, including signalling and metabolism, are highly compartmentalized, and that relevant changes in metabolic state can occur at sub-second timescales. Moreover, we have learned that individual cells in populations, or as part of a tissue, exist in different states. If we want to understand metabolic processes and signalling better, it will be necessary to measure biochemical and biophysical responses of individual cells with high temporal and spatial resolution. Fluorescence imaging has revolutionized all aspects of biology since it has the potential to provide information on the cellular and subcellular distribution of ions and metabolites with sub-second time resolution. In the present review we summarize recent progress in quantifying ions and metabolites in populations of yeast cells as well as in individual yeast cells with the help of quantitative fluorescent indicators, namely FRET metabolite sensors. We discuss the opportunities and potential pitfalls and the controls that help preclude misinterpretation. © The Authors Journal compilation © 2011 Biochemical Society

Electrical Identification and Selective Microstimulation of Neuronal Compartments Based on Features of Extracellular Action Potentials

NASA Astrophysics Data System (ADS)

Radivojevic, Milos; Jäckel, David; Altermatt, Michael; Müller, Jan; Viswam, Vijay; Hierlemann, Andreas; Bakkum, Douglas J.

2016-08-01

A detailed, high-spatiotemporal-resolution characterization of neuronal responses to local electrical fields and the capability of precise extracellular microstimulation of selected neurons are pivotal for studying and manipulating neuronal activity and circuits in networks and for developing neural prosthetics. Here, we studied cultured neocortical neurons by using high-density microelectrode arrays and optical imaging, complemented by the patch-clamp technique, and with the aim to correlate morphological and electrical features of neuronal compartments with their responsiveness to extracellular stimulation. We developed strategies to electrically identify any neuron in the network, while subcellular spatial resolution recording of extracellular action potential (AP) traces enabled their assignment to the axon initial segment (AIS), axonal arbor and proximal somatodendritic compartments. Stimulation at the AIS required low voltages and provided immediate, selective and reliable neuronal activation, whereas stimulation at the soma required high voltages and produced delayed and unreliable responses. Subthreshold stimulation at the soma depolarized the somatic membrane potential without eliciting APs.

Dual-view plane illumination microscopy for rapid and spatially isotropic imaging

PubMed Central

Kumar, Abhishek; Wu, Yicong; Christensen, Ryan; Chandris, Panagiotis; Gandler, William; McCreedy, Evan; Bokinsky, Alexandra; Colón-Ramos, Daniel A; Bao, Zhirong; McAuliffe, Matthew; Rondeau, Gary; Shroff, Hari

2015-01-01

We describe the construction and use of a compact dual-view inverted selective plane illumination microscope (diSPIM) for time-lapse volumetric (4D) imaging of living samples at subcellular resolution. Our protocol enables a biologist with some prior microscopy experience to assemble a diSPIM from commercially available parts, to align optics and test system performance, to prepare samples, and to control hardware and data processing with our software. Unlike existing light sheet microscopy protocols, our method does not require the sample to be embedded in agarose; instead, samples are prepared conventionally on glass coverslips. Tissue culture cells and Caenorhabditis elegans embryos are used as examples in this protocol; successful implementation of the protocol results in isotropic resolution and acquisition speeds up to several volumes per s on these samples. Assembling and verifying diSPIM performance takes ~6 d, sample preparation and data acquisition take up to 5 d and postprocessing takes 3–8 h, depending on the size of the data. PMID:25299154

X-ray micro-modulated luminescence tomography (XMLT)

PubMed Central

Cong, Wenxiang; Liu, Fenglin; Wang, Chao; Wang, Ge

2014-01-01

Imaging depth of optical microscopy has been fundamentally limited to millimeter or sub-millimeter due to strong scattering of light in a biological sample. X-ray microscopy can resolve spatial details of few microns deep inside a sample but contrast resolution is inadequate to depict heterogeneous features at cellular or sub-cellular levels. To enhance and enrich biological contrast at large imaging depth, various nanoparticles are introduced and become essential to basic research and molecular medicine. Nanoparticles can be functionalized as imaging probes, similar to fluorescent and bioluminescent proteins. LiGa5O8:Cr3+ nanoparticles were recently synthesized to facilitate luminescence energy storage with x-ray pre-excitation and subsequently stimulated luminescence emission by visible/near-infrared (NIR) light. In this paper, we propose an x-ray micro-modulated luminescence tomography (XMLT, or MLT to be more general) approach to quantify a nanophosphor distribution in a thick biological sample with high resolution. Our numerical simulation studies demonstrate the feasibility of the proposed approach. PMID:24663898

Investigation of the subcellular architecture of L7 neurons of Aplysia californica using magnetic resonance microscopy (MRM) at 7.8 microns.

PubMed

Lee, Choong H; Flint, Jeremy J; Hansen, Brian; Blackband, Stephen J

2015-06-10

Magnetic resonance microscopy (MRM) is a non-invasive diagnostic tool which is well-suited to directly resolve cellular structures in ex vivo and in vitro tissues without use of exogenous contrast agents. Recent advances in its capability to visualize mammalian cellular structure in intact tissues have reinvigorated analytical interest in aquatic cell models whose previous findings warrant up-to-date validation of subcellular components. Even if the sensitivity of MRM is less than other microscopic technologies, its strength lies in that it relies on the same image contrast mechanisms as clinical MRI which make it a unique tool for improving our ability to interpret human diagnostic imaging through high resolution studies of well-controlled biological model systems. Here, we investigate the subcellular MR signal characteristics of isolated cells of Aplysia californica at an in-plane resolution of 7.8 μm. In addition, direct correlation and positive identification of subcellular architecture in the cells is achieved through well-established histology. We hope this methodology will serve as the groundwork for studying pathophysiological changes through perturbation studies and allow for development of disease-specific cellular modeling tools. Such an approach promises to reveal the MR contrast changes underlying cellular mechanisms in various human diseases, for example in ischemic stroke.

Multiscale Imaging of the Mouse Cortex Using Two-Photon Microscopy and Wide-Field Illumination

NASA Astrophysics Data System (ADS)

Bumstead, Jonathan R.

The mouse brain can be studied over vast spatial scales ranging from microscopic imaging of single neurons to macroscopic measurements of hemodynamics acquired over the majority of the mouse cortex. However, most neuroimaging modalities are limited by a fundamental trade-off between the spatial resolution and the field-of-view (FOV) over which the brain can be imaged, making it difficult to fully understand the functional and structural architecture of the healthy mouse brain and its disruption in disease. My dissertation has focused on developing multiscale optical systems capable of imaging the mouse brain at both microscopic and mesoscopic spatial scales, specifically addressing the difference in spatial scales imaged with two-photon microscopy (TPM) and optical intrinsic signal imaging (OISI). Central to this work has been the formulation of a principled design strategy for extending the FOV of the two-photon microscope. Using this design approach, we constructed a TPM system with subcellular resolution and a FOV area 100 times greater than a conventional two-photon microscope. To image the ellipsoidal shape of the mouse cortex, we also developed the microscope to image arbitrary surfaces within a single frame using an electrically tunable lens. Finally, to address the speed limitations of the TPM systems developed during my dissertation, I also conducted research in large-scale neural phenomena occurring in the mouse brain imaged with high-speed OISI. The work conducted during my dissertation addresses some of the fundamental principles in designing and applying optical systems for multiscale imaging of the mouse brain.

Correlative cryogenic tomography of cells using light and soft x-rays

PubMed Central

Smith, Elizabeth A.; Cinquin, Bertrand P.; Do, Myan; McDermott, Gerry; Le Gros, Mark A.; Larabell, Carolyn A.

2013-01-01

Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryo-preserved state. The resultant images are, therefore, highly representative of both the cellular architecture and molecular organization in vivo. SXT quantitatively visualizes the cell and sub-cellular structures; FCM images the spatial distribution of fluorescently labeled molecules. Here, we review the characteristics of SXT-FCM, and briefly discuss how this method compares with existing correlative imaging techniques. We also describe how the incorporation of a cryo-rotation stage into a cryogenic fluorescence microscope allows acquisition of fluorescence cryogenic tomography (FCT) data. FCT is optimally suited to correlation with SXT, since both techniques image the specimen in 3-D, potentially with similar, isotropic spatial resolution. PMID:24355261

High resolution IVEM tomography of biological specimens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sedat, J.W.; Agard, D.A.

Electron tomography is a powerful tool for elucidating the three-dimensional architecture of large biological complexes and subcellular organelles. The introduction of intermediate voltage electron microscopes further extended the technique by providing the means to examine very large and non-symmetrical subcellular organelles, at resolutions beyond what would be possible using light microscopy. Recent studies using electron tomography on a variety of cellular organelles and assemblies such as centrosomes, kinetochores, and chromatin have clearly demonstrated the power of this technique for obtaining 3D structural information on non-symmetric cell components. When combined with biochemical and molecular observations, these 3D reconstructions have provided significantmore » new insights into biological function.« less

Multi-scale imaging and informatics pipeline for in situ pluripotent stem cell analysis.

PubMed

Gorman, Bryan R; Lu, Junjie; Baccei, Anna; Lowry, Nathan C; Purvis, Jeremy E; Mangoubi, Rami S; Lerou, Paul H

2014-01-01

Human pluripotent stem (hPS) cells are a potential source of cells for medical therapy and an ideal system to study fate decisions in early development. However, hPS cells cultured in vitro exhibit a high degree of heterogeneity, presenting an obstacle to clinical translation. hPS cells grow in spatially patterned colony structures, necessitating quantitative single-cell image analysis. We offer a tool for analyzing the spatial population context of hPS cells that integrates automated fluorescent microscopy with an analysis pipeline. It enables high-throughput detection of colonies at low resolution, with single-cellular and sub-cellular analysis at high resolutions, generating seamless in situ maps of single-cellular data organized by colony. We demonstrate the tool's utility by analyzing inter- and intra-colony heterogeneity of hPS cell cycle regulation and pluripotency marker expression. We measured the heterogeneity within individual colonies by analyzing cell cycle as a function of distance. Cells loosely associated with the outside of the colony are more likely to be in G1, reflecting a less pluripotent state, while cells within the first pluripotent layer are more likely to be in G2, possibly reflecting a G2/M block. Our multi-scale analysis tool groups colony regions into density classes, and cells belonging to those classes have distinct distributions of pluripotency markers and respond differently to DNA damage induction. Lastly, we demonstrate that our pipeline can robustly handle high-content, high-resolution single molecular mRNA FISH data by using novel image processing techniques. Overall, the imaging informatics pipeline presented offers a novel approach to the analysis of hPS cells that includes not only single cell features but also colony wide, and more generally, multi-scale spatial configuration.

Scanning superlens microscopy for non-invasive large field-of-view visible light nanoscale imaging

NASA Astrophysics Data System (ADS)

Wang, Feifei; Liu, Lianqing; Yu, Haibo; Wen, Yangdong; Yu, Peng; Liu, Zhu; Wang, Yuechao; Li, Wen Jung

2016-12-01

Nanoscale correlation of structural information acquisition with specific-molecule identification provides new insight for studying rare subcellular events. To achieve this correlation, scanning electron microscopy has been combined with super-resolution fluorescent microscopy, despite its destructivity when acquiring biological structure information. Here we propose time-efficient non-invasive microsphere-based scanning superlens microscopy that enables the large-area observation of live-cell morphology or sub-membrane structures with sub-diffraction-limited resolution and is demonstrated by observing biological and non-biological objects. This microscopy operates in both non-invasive and contact modes with ~200 times the acquisition efficiency of atomic force microscopy, which is achieved by replacing the point of an atomic force microscope tip with an imaging area of microspheres and stitching the areas recorded during scanning, enabling sub-diffraction-limited resolution. Our method marks a possible path to non-invasive cell imaging and simultaneous tracking of specific molecules with nanoscale resolution, facilitating the study of subcellular events over a total cell period.

Membrane Potential and Calcium Dynamics in Beta Cells from Mouse Pancreas Tissue Slices: Theory, Experimentation, and Analysis.

PubMed

Dolenšek, Jurij; Špelič, Denis; Klemen, Maša Skelin; Žalik, Borut; Gosak, Marko; Rupnik, Marjan Slak; Stožer, Andraž

2015-10-28

Beta cells in the pancreatic islets of Langerhans are precise biological sensors for glucose and play a central role in balancing the organism between catabolic and anabolic needs. A hallmark of the beta cell response to glucose are oscillatory changes of membrane potential that are tightly coupled with oscillatory changes in intracellular calcium concentration which, in turn, elicit oscillations of insulin secretion. Both membrane potential and calcium changes spread from one beta cell to the other in a wave-like manner. In order to assess the properties of the abovementioned responses to physiological and pathological stimuli, the main challenge remains how to effectively measure membrane potential and calcium changes at the same time with high spatial and temporal resolution, and also in as many cells as possible. To date, the most wide-spread approach has employed the electrophysiological patch-clamp method to monitor membrane potential changes. Inherently, this technique has many advantages, such as a direct contact with the cell and a high temporal resolution. However, it allows one to assess information from a single cell only. In some instances, this technique has been used in conjunction with CCD camera-based imaging, offering the opportunity to simultaneously monitor membrane potential and calcium changes, but not in the same cells and not with a reliable cellular or subcellular spatial resolution. Recently, a novel family of highly-sensitive membrane potential reporter dyes in combination with high temporal and spatial confocal calcium imaging allows for simultaneously detecting membrane potential and calcium changes in many cells at a time. Since the signals yielded from both types of reporter dyes are inherently noisy, we have developed complex methods of data denoising that permit for visualization and pixel-wise analysis of signals. Combining the experimental approach of high-resolution imaging with the advanced analysis of noisy data enables novel physiological insights and reassessment of current concepts in unprecedented detail.

Nuclear Cytoplasmic Trafficking of Proteins is a Major Response of Human Fibroblasts to Oxidative Stress

PubMed Central

Baqader, Noor O.; Radulovic, Marko; Crawford, Mark; Stoeber, Kai; Godovac-Zimmermann, Jasminka

2014-01-01

We have used a subcellular spatial razor approach based on LC–MS/MS-based proteomics with SILAC isotope labeling to determine changes in protein abundances in the nuclear and cytoplasmic compartments of human IMR90 fibroblasts subjected to mild oxidative stress. We show that response to mild tert-butyl hydrogen peroxide treatment includes redistribution between the nucleus and cytoplasm of numerous proteins not previously associated with oxidative stress. The 121 proteins with the most significant changes encompass proteins with known functions in a wide variety of subcellular locations and of cellular functional processes (transcription, signal transduction, autophagy, iron metabolism, TCA cycle, ATP synthesis) and are consistent with functional networks that are spatially dispersed across the cell. Both nuclear respiratory factor 2 and the proline regulatory axis appear to contribute to the cellular metabolic response. Proteins involved in iron metabolism or with iron/heme as a cofactor as well as mitochondrial proteins are prominent in the response. Evidence suggesting that nuclear import/export and vesicle-mediated protein transport contribute to the cellular response was obtained. We suggest that measurements of global changes in total cellular protein abundances need to be complemented with measurements of the dynamic subcellular spatial redistribution of proteins to obtain comprehensive pictures of cellular function. PMID:25133973

Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization

PubMed Central

Ursell, Tristan S.; Nguyen, Jeffrey; Monds, Russell D.; Colavin, Alexandre; Billings, Gabriel; Ouzounov, Nikolay; Gitai, Zemer; Shaevitz, Joshua W.; Huang, Kerwyn Casey

2014-01-01

Cells typically maintain characteristic shapes, but the mechanisms of self-organization for robust morphological maintenance remain unclear in most systems. Precise regulation of rod-like shape in Escherichia coli cells requires the MreB actin-like cytoskeleton, but the mechanism by which MreB maintains rod-like shape is unknown. Here, we use time-lapse and 3D imaging coupled with computational analysis to map the growth, geometry, and cytoskeletal organization of single bacterial cells at subcellular resolution. Our results demonstrate that feedback between cell geometry and MreB localization maintains rod-like cell shape by targeting cell wall growth to regions of negative cell wall curvature. Pulse-chase labeling indicates that growth is heterogeneous and correlates spatially and temporally with MreB localization, whereas MreB inhibition results in more homogeneous growth, including growth in polar regions previously thought to be inert. Biophysical simulations establish that curvature feedback on the localization of cell wall growth is an effective mechanism for cell straightening and suggest that surface deformations caused by cell wall insertion could direct circumferential motion of MreB. Our work shows that MreB orchestrates persistent, heterogeneous growth at the subcellular scale, enabling robust, uniform growth at the cellular scale without requiring global organization. PMID:24550515

3D membrane segmentation and quantification of intact thick cells using cryo soft X-ray transmission microscopy: A pilot study

PubMed Central

Klementieva, Oxana; Werner, Stephan; Guttmann, Peter; Pratsch, Christoph; Cladera, Josep

2017-01-01

Structural analysis of biological membranes is important for understanding cell and sub-cellular organelle function as well as their interaction with the surrounding environment. Imaging of whole cells in three dimension at high spatial resolution remains a significant challenge, particularly for thick cells. Cryo-transmission soft X-ray microscopy (cryo-TXM) has recently gained popularity to image, in 3D, intact thick cells (∼10μm) with details of sub-cellular architecture and organization in near-native state. This paper reports a new tool to segment and quantify structural changes of biological membranes in 3D from cryo-TXM images by tracking an initial 2D contour along the third axis of the microscope, through a multi-scale ridge detection followed by an active contours-based model, with a subsequent refinement along the other two axes. A quantitative metric that assesses the grayscale profiles perpendicular to the membrane surfaces is introduced and shown to be linearly related to the membrane thickness. Our methodology has been validated on synthetic phantoms using realistic microscope properties and structure dimensions, as well as on real cryo-TXM data. Results demonstrate the validity of our algorithms for cryo-TXM data analysis. PMID:28376110

New techniques for motion-artifact-free in vivo cardiac microscopy

PubMed Central

Vinegoni, Claudio; Lee, Sungon; Aguirre, Aaron D.; Weissleder, Ralph

2015-01-01

Intravital imaging microscopy (i.e., imaging in live animals at microscopic resolution) has become an indispensable tool for studying the cellular micro-dynamics in cancer, immunology and neurobiology. High spatial and temporal resolution, combined with large penetration depth and multi-reporter visualization capability make fluorescence intravital microscopy compelling for heart imaging. However, tissue motion caused by cardiac contraction and respiration critically limits its use. As a result, in vitro cell preparations or non-contracting explanted heart models are more commonly employed. Unfortunately, these approaches fall short of understanding the more complex host physiology that may be dynamic and occur over longer periods of time. In this review, we report on novel technologies, which have been recently developed by our group and others, aimed at overcoming motion-induced artifacts and capable of providing in vivo subcellular resolution imaging in the beating mouse heart. The methods are based on mechanical stabilization, image processing algorithms, gated/triggered acquisition schemes or a combination of both. We expect that in the immediate future all these methodologies will have considerable applications in expanding our understanding of the cardiac biology, elucidating cardiomyocyte function and interactions within the organism in vivo, and ultimately improving the treatment of cardiac diseases. PMID:26029116

Long-Term Growth of Moss in Microfluidic Devices Enables Subcellular Studies in Development.

PubMed

Bascom, Carlisle S; Wu, Shu-Zon; Nelson, Katherine; Oakey, John; Bezanilla, Magdalena

2016-09-01

Key developmental processes that occur on the subcellular and cellular level or occur in occluded tissues are difficult to access, let alone image and analyze. Recently, culturing living samples within polydimethylsiloxane (PDMS) microfluidic devices has facilitated the study of hard-to-reach developmental events. Here, we show that an early diverging land plant, Physcomitrella patens, can be continuously cultured within PDMS microfluidic chambers. Because the PDMS chambers are bonded to a coverslip, it is possible to image P. patens development at high resolution over long time periods. Using PDMS chambers, we report that wild-type protonemal tissue grows at the same rate as previously reported for growth on solid medium. Using long-term imaging, we highlight key developmental events, demonstrate compatibility with high-resolution confocal microscopy, and obtain growth rates for a slow-growing mutant. By coupling the powerful genetic tools available to P. patens with long-term growth and imaging provided by PDMS microfluidic chambers, we demonstrate the capability to study cellular and subcellular developmental events in plants directly and in real time. © 2016 American Society of Plant Biologists. All rights reserved.

Redox Indicator Mice Stably Expressing Genetically Encoded Neuronal roGFP: Versatile Tools to Decipher Subcellular Redox Dynamics in Neuropathophysiology.

PubMed

Wagener, Kerstin C; Kolbrink, Benedikt; Dietrich, Katharina; Kizina, Kathrin M; Terwitte, Lukas S; Kempkes, Belinda; Bao, Guobin; Müller, Michael

2016-07-01

Reactive oxygen species (ROS) and downstream redox alterations not only mediate physiological signaling but also neuropathology. For long, ROS/redox imaging was hampered by a lack of reliable probes. Genetically encoded redox sensors overcame this gap and revolutionized (sub)cellular redox imaging. Yet, the successful delivery of sensor-coding DNA, which demands transfection/transduction of cultured preparations or stereotaxic microinjections of each subject, remains challenging. By generating transgenic mice, we aimed to overcome limiting cultured preparations, circumvent surgical interventions, and to extend effectively redox imaging to complex and adult preparations. Our redox indicator mice widely express Thy1-driven roGFP1 (reduction-oxidation-sensitive green fluorescent protein 1) in neuronal cytosol or mitochondria. Negative phenotypic effects of roGFP1 were excluded and its proper targeting and functionality confirmed. Redox mapping by ratiometric wide-field imaging reveals most oxidizing conditions in CA3 neurons. Furthermore, mitochondria are more oxidized than cytosol. Cytosolic and mitochondrial roGFP1s reliably report cell endogenous redox dynamics upon metabolic challenge or stimulation. Fluorescence lifetime imaging yields stable, but marginal, response ranges. We therefore developed automated excitation ratiometric 2-photon imaging. It offers superior sensitivity, spatial resolution, and response dynamics. Redox indicator mice enable quantitative analyses of subcellular redox dynamics in a multitude of preparations and at all postnatal stages. This will uncover cell- and compartment-specific cerebral redox signals and their defined alterations during development, maturation, and aging. Cross-breeding with other disease models will reveal molecular details on compartmental redox homeostasis in neuropathology. Combined with ratiometric 2-photon imaging, this will foster our mechanistic understanding of cellular redox signals in their full complexity. Antioxid. Redox Signal. 25, 41-58.

Redox Indicator Mice Stably Expressing Genetically Encoded Neuronal roGFP: Versatile Tools to Decipher Subcellular Redox Dynamics in Neuropathophysiology

PubMed Central

Wagener, Kerstin C.; Kolbrink, Benedikt; Dietrich, Katharina; Kizina, Kathrin M.; Terwitte, Lukas S.; Kempkes, Belinda; Bao, Guobin

2016-01-01

Abstract Aims: Reactive oxygen species (ROS) and downstream redox alterations not only mediate physiological signaling but also neuropathology. For long, ROS/redox imaging was hampered by a lack of reliable probes. Genetically encoded redox sensors overcame this gap and revolutionized (sub)cellular redox imaging. Yet, the successful delivery of sensor-coding DNA, which demands transfection/transduction of cultured preparations or stereotaxic microinjections of each subject, remains challenging. By generating transgenic mice, we aimed to overcome limiting cultured preparations, circumvent surgical interventions, and to extend effectively redox imaging to complex and adult preparations. Results: Our redox indicator mice widely express Thy1-driven roGFP1 (reduction–oxidation-sensitive green fluorescent protein 1) in neuronal cytosol or mitochondria. Negative phenotypic effects of roGFP1 were excluded and its proper targeting and functionality confirmed. Redox mapping by ratiometric wide-field imaging reveals most oxidizing conditions in CA3 neurons. Furthermore, mitochondria are more oxidized than cytosol. Cytosolic and mitochondrial roGFP1s reliably report cell endogenous redox dynamics upon metabolic challenge or stimulation. Fluorescence lifetime imaging yields stable, but marginal, response ranges. We therefore developed automated excitation ratiometric 2-photon imaging. It offers superior sensitivity, spatial resolution, and response dynamics. Innovation and Conclusion: Redox indicator mice enable quantitative analyses of subcellular redox dynamics in a multitude of preparations and at all postnatal stages. This will uncover cell- and compartment-specific cerebral redox signals and their defined alterations during development, maturation, and aging. Cross-breeding with other disease models will reveal molecular details on compartmental redox homeostasis in neuropathology. Combined with ratiometric 2-photon imaging, this will foster our mechanistic understanding of cellular redox signals in their full complexity. Antioxid. Redox Signal. 25, 41–58. PMID:27059697

Correlative super-resolution fluorescence microscopy combined with optical coherence microscopy

NASA Astrophysics Data System (ADS)

Kim, Sungho; Kim, Gyeong Tae; Jang, Soohyun; Shim, Sang-Hee; Bae, Sung Chul

2015-03-01

Recent development of super-resolution fluorescence imaging technique such as stochastic optical reconstruction microscopy (STORM) and photoactived localization microscope (PALM) has brought us beyond the diffraction limits. It allows numerous opportunities in biology because vast amount of formerly obscured molecular structures, due to lack of spatial resolution, now can be directly observed. A drawback of fluorescence imaging, however, is that it lacks complete structural information. For this reason, we have developed a super-resolution multimodal imaging system based on STORM and full-field optical coherence microscopy (FF-OCM). FF-OCM is a type of interferometry systems based on a broadband light source and a bulk Michelson interferometer, which provides label-free and non-invasive visualization of biological samples. The integration between the two systems is simple because both systems use a wide-field illumination scheme and a conventional microscope. This combined imaging system gives us both functional information at a molecular level (~20nm) and structural information at the sub-cellular level (~1μm). For thick samples such as tissue slices, while FF-OCM is readily capable of imaging the 3D architecture, STORM suffer from aberrations and high background fluorescence that substantially degrade the resolution. In order to correct the aberrations in thick tissues, we employed an adaptive optics system in the detection path of the STORM microscope. We used our multimodal system to obtain images on brain tissue samples with structural and functional information.

Biochemical label-free tissue imaging with subcellular-resolution synchrotron FTIR with focal plane array detector.

PubMed

Kastyak-Ibrahim, M Z; Nasse, M J; Rak, M; Hirschmugl, C; Del Bigio, M R; Albensi, B C; Gough, K M

2012-03-01

The critical questions into the cause of neural degeneration, in Alzheimer disease and other neurodegenerative disorders, are closely related to the question of why certain neurons survive. Answers require detailed understanding of biochemical changes in single cells. Fourier transform infrared microspectroscopy is an excellent tool for biomolecular imaging in situ, but resolution is limited. The mid-infrared beamline IRENI (InfraRed ENvironmental Imaging) at the Synchrotron Radiation Center, University of Wisconsin-Madison, enables label-free subcellular imaging and biochemical analysis of neurons with an increase of two orders of magnitude in pixel spacing over current systems. With IRENI's capabilities, it is now possible to study changes in individual neurons in situ, and to characterize their surroundings, using only the biochemical signatures of naturally-occurring components in unstained, unfixed tissue. We present examples of analyses of brain from two transgenic mouse models of Alzheimer disease (TgCRND8 and 3xTg) that exhibit different features of pathogenesis. Data processing on spectral features for nuclei reveals individual hippocampal neurons, and neurons located in the proximity of amyloid plaque in TgCRND8 mouse. Elevated lipids are detected surrounding and, for the first time, within the dense core of amyloid plaques, offering support for inflammatory and aggregation roles. Analysis of saturated and unsaturated fatty acid ester content in retina allows characterization of neuronal layers. IRENI images also reveal spatially-resolved data with unprecedented clarity and distinct spectral variation, from sub-regions including photoreceptors, neuronal cell bodies and synapses in sections of mouse retina. Biochemical composition of retinal layers can be used to study changes related to disease processes and dietary modification. Copyright © 2011 Elsevier Inc. All rights reserved.

Quantitative protein localization signatures reveal an association between spatial and functional divergences of proteins.

PubMed

Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

2014-03-01

Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein functions and how these functions were acquired in cells from different organisms or species. A public web interface of PLAST is available at http://plast.bii.a-star.edu.sg.

Quantitative Protein Localization Signatures Reveal an Association between Spatial and Functional Divergences of Proteins

PubMed Central

Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

2014-01-01

Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein functions and how these functions were acquired in cells from different organisms or species. A public web interface of PLAST is available at http://plast.bii.a-star.edu.sg. PMID:24603469

Imaging of single cells and tissue using MeV ions

NASA Astrophysics Data System (ADS)

Watt, F.; Bettiol, A. A.; van Kan, J. A.; Ynsa, M. D.; Minqin, Ren; Rajendran, R.; Huifang, Cui; Fwu-Shen, Sheu; Jenner, A. M.

2009-06-01

With the attainment of sub-100 nm high energy (MeV) ion beams, comes the opportunity to image cells and tissue at nano-dimensions. The advantage of MeV ion imaging is that the ions will penetrate whole cells, or relatively thick tissue sections, without any significant loss of resolution. In this paper, we demonstrate that whole cells (cultured N2A neuroblastoma cells ATCC) and tissue sections (rabbit pancreas tissue) can be imaged at sub-100 nm resolutions using scanning transmission ion microscopy (STIM), and that sub-cellular structural details can be identified. In addition to STIM imaging we have also demonstrated for the first time, that sub-cellular proton induced fluorescence imaging (on cultured N2A neuroblastoma cells ATCC) can also be carried out at resolutions of 200 nm, compared with 300-400 nm resolutions achieved by conventional optical fluorescence imaging. The combination of both techniques offers a potentially powerful tool in the quest for elucidating cell function, particularly when it should be possible in the near future to image down to sub-50 nm.

Intracellular probes for imaging oxygen concentration: how good are they?

NASA Astrophysics Data System (ADS)

Dmitriev, Ruslan I.; Papkovsky, Dmitri B.

2015-09-01

In the last decade a number of cell-permeable phosphorescence based probes for imaging of (intra)cellular oxygen (icO2) have been described. These small molecule, supramolecular and nanoparticle structures, although allowing analysis of hypoxia, local gradients and fluctuations in O2, responses to stimulation and drug treatment at sub-cellular level with high spatial and temporal resolution, differ significantly in their operational performance and applicability to different cell and tissue models. Here we discuss and compare these probes with respect to their staining efficiency, brightness, photostability, toxicity, cell specificity, compatibility with different cell and tissue models, and analytical performance. Merits and limitations of particular probes are highlighted and strategies for development of new high-performance O2 imaging probes defined. Key application areas in hypoxia research, stem cells, cancer biology and tissue physiology are also discussed.

Hard X-ray submicrometer tomography of human brain tissue at Diamond Light Source

NASA Astrophysics Data System (ADS)

Khimchenko, A.; Bikis, C.; Schulz, G.; Zdora, M.-C.; Zanette, I.; Vila-Comamala, J.; Schweighauser, G.; Hench, J.; Hieber, S. E.; Deyhle, H.; Thalmann, P.; Müller, B.

2017-06-01

There is a lack of the necessary methodology for three-dimensional (3D) investigation of soft tissues with cellular resolution without staining or tissue transformation. Synchrotron radiation based hard X-ray in-line phase contrast tomography using single-distance phase reconstruction (SDPR) provides high spatial resolution and density contrast for the visualization of individual cells using a standard specimen preparation and data reconstruction. In this study, we demonstrate the 3D characterization of a formalin-fixed paraffin-embedded (FFPE) human cerebellum specimen by SDPR at the Diamond-Manchester Imaging Branchline I13-2 (Diamond Light Source, UK) at pixel sizes down to 0.45 μm. The approach enables visualization of cerebellar layers (Stratum moleculare and Stratum granulosum), the 3D characterization of individual cells (Purkinje, stellate and granule cells) and can even resolve some subcellular structures (nucleus and nucleolus of Purkinje cells). The tomographic results are qualitatively compared to hematoxylin and eosin (H&E) stained histological sections. We demonstrate the potential benefits of hard X-ray microtomography for the investigations of biological tissues in comparison to conventional histology.

Two-photon imaging of neuronal activity in motor cortex of marmosets during upper-limb movement tasks.

PubMed

Ebina, Teppei; Masamizu, Yoshito; Tanaka, Yasuhiro R; Watakabe, Akiya; Hirakawa, Reiko; Hirayama, Yuka; Hira, Riichiro; Terada, Shin-Ichiro; Koketsu, Daisuke; Hikosaka, Kazuo; Mizukami, Hiroaki; Nambu, Atsushi; Sasaki, Erika; Yamamori, Tetsuo; Matsuzaki, Masanori

2018-05-14

Two-photon imaging in behaving animals has revealed neuronal activities related to behavioral and cognitive function at single-cell resolution. However, marmosets have posed a challenge due to limited success in training on motor tasks. Here we report the development of protocols to train head-fixed common marmosets to perform upper-limb movement tasks and simultaneously perform two-photon imaging. After 2-5 months of training sessions, head-fixed marmosets can control a manipulandum to move a cursor to a target on a screen. We conduct two-photon calcium imaging of layer 2/3 neurons in the motor cortex during this motor task performance, and detect task-relevant activity from multiple neurons at cellular and subcellular resolutions. In a two-target reaching task, some neurons show direction-selective activity over the training days. In a short-term force-field adaptation task, some neurons change their activity when the force field is on. Two-photon calcium imaging in behaving marmosets may become a fundamental technique for determining the spatial organization of the cortical dynamics underlying action and cognition.

Correlative cryogenic tomography of cells using light and soft x-rays.

PubMed

Smith, Elizabeth A; Cinquin, Bertrand P; Do, Myan; McDermott, Gerry; Le Gros, Mark A; Larabell, Carolyn A

2014-08-01

Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryopreserved. The resultant images are, therefore, highly representative of both the cellular architecture and molecular organization in vivo. SXT quantitatively visualizes the cell and sub-cellular structures; FCM images the spatial distribution of fluorescently labeled molecules. Here, we review the characteristics of SXT-FCM, and briefly discuss how this method compares with existing correlative imaging techniques. We also describe how the incorporation of a cryo-rotation stage into a cryogenic fluorescence microscope allows acquisition of fluorescence cryogenic tomography (FCT) data. FCT is optimally suited for correlation with SXT, since both techniques image the specimen in 3-D, potentially with similar, isotropic spatial resolution. © 2013 Elsevier B.V. All rights reserved.

Differential subcellular distribution of ion channels and the diversity of neuronal function.

PubMed

Nusser, Zoltan

2012-06-01

Following the astonishing molecular diversity of voltage-gated ion channels that was revealed in the past few decades, the ion channel repertoire expressed by neurons has been implicated as the major factor governing their functional heterogeneity. Although the molecular structure of ion channels is a key determinant of their biophysical properties, their subcellular distribution and densities on the surface of nerve cells are just as important for fulfilling functional requirements. Recent results obtained with high resolution quantitative localization techniques revealed complex, subcellular compartment-specific distribution patterns of distinct ion channels. Here I suggest that within a given neuron type every ion channel has a unique cell surface distribution pattern, with the functional consequence that this dramatically increases the computational power of nerve cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

Improving axial resolution in confocal microscopy with new high refractive index mounting media.

PubMed

Fouquet, Coralie; Gilles, Jean-François; Heck, Nicolas; Dos Santos, Marc; Schwartzmann, Richard; Cannaya, Vidjeacoumary; Morel, Marie-Pierre; Davidson, Robert Stephen; Trembleau, Alain; Bolte, Susanne

2015-01-01

Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.

Sub-diffraction Limit Localization of Proteins in Volumetric Space Using Bayesian Restoration of Fluorescence Images from Ultrathin Specimens

PubMed Central

Wang, Gordon; Smith, Stephen J.

2012-01-01

Photon diffraction limits the resolution of conventional light microscopy at the lateral focal plane to 0.61λ/NA (λ = wavelength of light, NA = numerical aperture of the objective) and at the axial plane to 1.4nλ/NA2 (n = refractive index of the imaging medium, 1.51 for oil immersion), which with visible wavelengths and a 1.4NA oil immersion objective is ∼220 nm and ∼600 nm in the lateral plane and axial plane respectively. This volumetric resolution is too large for the proper localization of protein clustering in subcellular structures. Here we combine the newly developed proteomic imaging technique, Array Tomography (AT), with its native 50–100 nm axial resolution achieved by physical sectioning of resin embedded tissue, and a 2D maximum likelihood deconvolution method, based on Bayes' rule, which significantly improves the resolution of protein puncta in the lateral plane to allow accurate and fast computational segmentation and analysis of labeled proteins. The physical sectioning of AT allows tissue specimens to be imaged at the physical optimum of modern high NA plan-apochormatic objectives. This translates to images that have little out of focus light, minimal aberrations and wave-front distortions. Thus, AT is able to provide images with truly invariant point spread functions (PSF), a property critical for accurate deconvolution. We show that AT with deconvolution increases the volumetric analytical fidelity of protein localization by significantly improving the modulation of high spatial frequencies up to and potentially beyond the spatial frequency cut-off of the objective. Moreover, we are able to achieve this improvement with no noticeable introduction of noise or artifacts and arrive at object segmentation and localization accuracies on par with image volumes captured using commercial implementations of super-resolution microscopes. PMID:22956902

Sub-diffraction limit localization of proteins in volumetric space using Bayesian restoration of fluorescence images from ultrathin specimens.

PubMed

Wang, Gordon; Smith, Stephen J

2012-01-01

Photon diffraction limits the resolution of conventional light microscopy at the lateral focal plane to 0.61λ/NA (λ = wavelength of light, NA = numerical aperture of the objective) and at the axial plane to 1.4nλ/NA(2) (n = refractive index of the imaging medium, 1.51 for oil immersion), which with visible wavelengths and a 1.4NA oil immersion objective is -220 nm and -600 nm in the lateral plane and axial plane respectively. This volumetric resolution is too large for the proper localization of protein clustering in subcellular structures. Here we combine the newly developed proteomic imaging technique, Array Tomography (AT), with its native 50-100 nm axial resolution achieved by physical sectioning of resin embedded tissue, and a 2D maximum likelihood deconvolution method, based on Bayes' rule, which significantly improves the resolution of protein puncta in the lateral plane to allow accurate and fast computational segmentation and analysis of labeled proteins. The physical sectioning of AT allows tissue specimens to be imaged at the physical optimum of modern high NA plan-apochormatic objectives. This translates to images that have little out of focus light, minimal aberrations and wave-front distortions. Thus, AT is able to provide images with truly invariant point spread functions (PSF), a property critical for accurate deconvolution. We show that AT with deconvolution increases the volumetric analytical fidelity of protein localization by significantly improving the modulation of high spatial frequencies up to and potentially beyond the spatial frequency cut-off of the objective. Moreover, we are able to achieve this improvement with no noticeable introduction of noise or artifacts and arrive at object segmentation and localization accuracies on par with image volumes captured using commercial implementations of super-resolution microscopes.

Infrared Microspectroscopy: A Multiple-Screening Platform for Investigating Single-Cell Biochemical Perturbations upon Prion Infection

PubMed Central

2011-01-01

Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of prions in the central nervous system. The pathogenic prion (PrPSc) possesses the capability to convert the host-encoded cellular isoform of the prion protein, PrPC, into nascent PrPSc. The present work aims at providing novel insight into cellular response upon prion infection evidenced by synchrotron radiation infrared microspectroscopy (SR-IRMS). This non-invasive, label-free analytical technique was employed to investigate the biochemical perturbations undergone by prion infected mouse hypothalamic GT1-1 cells at the cellular and subcellular level. A decrement in total cellular protein content upon prion infection was identified by infrared (IR) whole-cell spectra and validated by bicinchoninic acid assay and single-cell volume analysis by atomic force microscopy (AFM). Hierarchical cluster analysis (HCA) of IR data discriminated between infected and uninfected cells and allowed to deduce an increment of lysosomal bodies within the cytoplasm of infected GT1-1 cells, a hypothesis further confirmed by SR-IRMS at subcellular spatial resolution and fluorescent microscopy. The purpose of this work, therefore, consists of proposing IRMS as a powerful multiscreening platform, drawing on the synergy with conventional biological assays and microscopy techniques in order to increase the accuracy of investigations performed at the single-cell level. PMID:22778865

Geometric approach to segmentation and protein localization in cell culture assays.

PubMed

Raman, S; Maxwell, C A; Barcellos-Hoff, M H; Parvin, B

2007-01-01

Cell-based fluorescence imaging assays are heterogeneous and require the collection of a large number of images for detailed quantitative analysis. Complexities arise as a result of variation in spatial nonuniformity, shape, overlapping compartments and scale (size). A new technique and methodology has been developed and tested for delineating subcellular morphology and partitioning overlapping compartments at multiple scales. This system is packaged as an integrated software platform for quantifying images that are obtained through fluorescence microscopy. Proposed methods are model based, leveraging geometric shape properties of subcellular compartments and corresponding protein localization. From the morphological perspective, convexity constraint is imposed to delineate and partition nuclear compartments. From the protein localization perspective, radial symmetry is imposed to localize punctate protein events at submicron resolution. Convexity constraint is imposed against boundary information, which are extracted through a combination of zero-crossing and gradient operator. If the convexity constraint fails for the boundary then positive curvature maxima are localized along the contour and the entire blob is partitioned into disjointed convex objects representing individual nuclear compartment, by enforcing geometric constraints. Nuclear compartments provide the context for protein localization, which may be diffuse or punctate. Punctate signal are localized through iterative voting and radial symmetries for improved reliability and robustness. The technique has been tested against 196 images that were generated to study centrosome abnormalities. Corresponding computed representations are compared against manual counts for validation.

Fluorescent sensors of protein kinases: from basics to biomedical applications.

PubMed

Nhu Ngoc Van, Thi; Morris, May C

2013-01-01

Protein kinases constitute a major class of enzymes underlying essentially all biological processes. These enzymes present similar structural folds, yet their mechanism of action and of regulation vary largely, as well as their substrate specificity and their subcellular localization. Classical approaches to study the function/activity of protein kinases rely on radioactive endpoint assays, which do not allow for characterization of their dynamic activity in their native environment. The development of fluorescent biosensors has provided a whole new avenue for studying protein kinase behavior and regulation in living cells in real time with high spatial and temporal resolution. Two major classes of biosensors have been developed: genetically encoded single-chain fluorescence resonance energy transfer biosensors and peptide/protein biosensors coupled to small synthetic fluorophores which are sensitive to changes in their environment. In this review, we discuss the developments in fluorescent biosensor technology related to protein kinase sensing and the different strategies employed to monitor protein kinase activity, conformation, or relative abundance, as well as kinase regulation and subcellular dynamics in living cells. Moreover, we discuss their application in biomedical settings, for diagnostics and therapeutics, to image disease progression and monitor response to therapeutics, in drug discovery programs, for high-throughput screening assays, for postscreen characterization of drug candidates, and for clinical evaluation of novel drugs. Copyright © 2013 Elsevier Inc. All rights reserved.

Infrared microspectroscopy: a multiple-screening platform for investigating single-cell biochemical perturbations upon prion infection.

PubMed

Didonna, Alessandro; Vaccari, Lisa; Bek, Alpan; Legname, Giuseppe

2011-03-16

Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of prions in the central nervous system. The pathogenic prion (PrP(Sc)) possesses the capability to convert the host-encoded cellular isoform of the prion protein, PrP(C), into nascent PrP(Sc). The present work aims at providing novel insight into cellular response upon prion infection evidenced by synchrotron radiation infrared microspectroscopy (SR-IRMS). This non-invasive, label-free analytical technique was employed to investigate the biochemical perturbations undergone by prion infected mouse hypothalamic GT1-1 cells at the cellular and subcellular level. A decrement in total cellular protein content upon prion infection was identified by infrared (IR) whole-cell spectra and validated by bicinchoninic acid assay and single-cell volume analysis by atomic force microscopy (AFM). Hierarchical cluster analysis (HCA) of IR data discriminated between infected and uninfected cells and allowed to deduce an increment of lysosomal bodies within the cytoplasm of infected GT1-1 cells, a hypothesis further confirmed by SR-IRMS at subcellular spatial resolution and fluorescent microscopy. The purpose of this work, therefore, consists of proposing IRMS as a powerful multiscreening platform, drawing on the synergy with conventional biological assays and microscopy techniques in order to increase the accuracy of investigations performed at the single-cell level.

High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography

NASA Astrophysics Data System (ADS)

Kim, Kyoohyun; Yoon, HyeOk; Diez-Silva, Monica; Dao, Ming; Dasari, Ramachandra R.; Park, YongKeun

2014-01-01

We present high-resolution optical tomographic images of human red blood cells (RBC) parasitized by malaria-inducing Plasmodium falciparum (Pf)-RBCs. Three-dimensional (3-D) refractive index (RI) tomograms are reconstructed by recourse to a diffraction algorithm from multiple two-dimensional holograms with various angles of illumination. These 3-D RI tomograms of Pf-RBCs show cellular and subcellular structures of host RBCs and invaded parasites in fine detail. Full asexual intraerythrocytic stages of parasite maturation (ring to trophozoite to schizont stages) are then systematically investigated using optical diffraction tomography algorithms. These analyses provide quantitative information on the structural and chemical characteristics of individual host Pf-RBCs, parasitophorous vacuole, and cytoplasm. The in situ structural evolution and chemical characteristics of subcellular hemozoin crystals are also elucidated.

High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography

PubMed Central

Kim, Kyoohyun; Yoon, HyeOk; Diez-Silva, Monica; Dao, Ming; Dasari, Ramachandra R.

2013-01-01

Abstract. We present high-resolution optical tomographic images of human red blood cells (RBC) parasitized by malaria-inducing Plasmodium falciparum (Pf)-RBCs. Three-dimensional (3-D) refractive index (RI) tomograms are reconstructed by recourse to a diffraction algorithm from multiple two-dimensional holograms with various angles of illumination. These 3-D RI tomograms of Pf-RBCs show cellular and subcellular structures of host RBCs and invaded parasites in fine detail. Full asexual intraerythrocytic stages of parasite maturation (ring to trophozoite to schizont stages) are then systematically investigated using optical diffraction tomography algorithms. These analyses provide quantitative information on the structural and chemical characteristics of individual host Pf-RBCs, parasitophorous vacuole, and cytoplasm. The in situ structural evolution and chemical characteristics of subcellular hemozoin crystals are also elucidated. PMID:23797986

Preclinical Whole-body Fluorescence Imaging: Review of Instruments, Methods and Applications

PubMed Central

Leblond, Frederic; Davis, Scott C.; Valdés, Pablo A.; Pogue, Brain W.

2013-01-01

Fluorescence sampling of cellular function is widely used in all aspects of biology, allowing the visualization of cellular and sub-cellular biological processes with spatial resolutions in the range from nanometers up to centimeters. Imaging of fluorescence in vivo has become the most commonly used radiological tool in all pre-clinical work. In the last decade, full-body pre-clinical imaging systems have emerged with a wide range of utilities and niche application areas. The range of fluorescent probes that can be excited in the visible to near-infrared part of the electromagnetic spectrum continues to expand, with the most value for in vivo use being beyond the 630 nm wavelength, because the absorption of light sharply decreases. Whole-body in vivo fluorescence imaging has not yet reached a state of maturity that allows its routine use in the scope of large-scale pre-clinical studies. This is in part due to an incomplete understanding of what the actual fundamental capabilities and limitations of this imaging modality are. However, progress is continuously being made in research laboratories pushing the limits of the approach to consistently improve its performance in terms of spatial resolution, sensitivity and quantification. This paper reviews this imaging technology with a particular emphasis on its potential uses and limitations, the required instrumentation, and the possible imaging geometries and applications. A detailed account of the main commercially available systems is provided as well as some perspective relating to the future of the technology development. Although the vast majority of applications of in vivo small animal imaging are based on epi-illumination planar imaging, the future success of the method relies heavily on the design of novel imaging systems based on state-of-the-art optical technology used in conjunction with high spatial resolution structural modalities such as MRI, CT or ultra-sound. PMID:20031443

Mass Spectrometry Analysis of Spatial Protein Networks by Colocalization Analysis (COLA).

PubMed

Mardakheh, Faraz K

2017-01-01

A major challenge in systems biology is comprehensive mapping of protein interaction networks. Crucially, such interactions are often dynamic in nature, necessitating methods that can rapidly mine the interactome across varied conditions and treatments to reveal change in the interaction networks. Recently, we described a fast mass spectrometry-based method to reveal functional interactions in mammalian cells on a global scale, by revealing spatial colocalizations between proteins (COLA) (Mardakheh et al., Mol Biosyst 13:92-105, 2017). As protein localization and function are inherently linked, significant colocalization between two proteins is a strong indication for their functional interaction. COLA uses rapid complete subcellular fractionation, coupled with quantitative proteomics to generate a subcellular localization profile for each protein quantified by the mass spectrometer. Robust clustering is then applied to reveal significant similarities in protein localization profiles, indicative of colocalization.

A Quantitative Spatial Proteomics Analysis of Proteome Turnover in Human Cells*

PubMed Central

Boisvert, François-Michel; Ahmad, Yasmeen; Gierliński, Marek; Charrière, Fabien; Lamont, Douglas; Scott, Michelle; Barton, Geoff; Lamond, Angus I.

2012-01-01

Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ∼20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function. PMID:21937730

Unsupervised Clustering of Subcellular Protein Expression Patterns in High-Throughput Microscopy Images Reveals Protein Complexes and Functional Relationships between Proteins

PubMed Central

Handfield, Louis-François; Chong, Yolanda T.; Simmons, Jibril; Andrews, Brenda J.; Moses, Alan M.

2013-01-01

Protein subcellular localization has been systematically characterized in budding yeast using fluorescently tagged proteins. Based on the fluorescence microscopy images, subcellular localization of many proteins can be classified automatically using supervised machine learning approaches that have been trained to recognize predefined image classes based on statistical features. Here, we present an unsupervised analysis of protein expression patterns in a set of high-resolution, high-throughput microscope images. Our analysis is based on 7 biologically interpretable features which are evaluated on automatically identified cells, and whose cell-stage dependency is captured by a continuous model for cell growth. We show that it is possible to identify most previously identified localization patterns in a cluster analysis based on these features and that similarities between the inferred expression patterns contain more information about protein function than can be explained by a previous manual categorization of subcellular localization. Furthermore, the inferred cell-stage associated to each fluorescence measurement allows us to visualize large groups of proteins entering the bud at specific stages of bud growth. These correspond to proteins localized to organelles, revealing that the organelles must be entering the bud in a stereotypical order. We also identify and organize a smaller group of proteins that show subtle differences in the way they move around the bud during growth. Our results suggest that biologically interpretable features based on explicit models of cell morphology will yield unprecedented power for pattern discovery in high-resolution, high-throughput microscopy images. PMID:23785265

Resonance Raman spectroscopy in malaria research.

PubMed

Wood, Bayden R; McNaughton, Don

2006-10-01

In recent years, the field of Raman spectroscopy has witnessed a surge in technological development, with the incorporation of ultrasensitive, charge-coupled devices, improved laser sources and precision Rayleigh-filter systems. This has led to the development of sensitive confocal micro-Raman spectrometers and imaging spectrometers that are capable of obtaining high spatial-resolution spectra and images of subcellular components within single living cells. This review reports on the application of resonance micro-Raman spectroscopy to the study of malaria pigment (hemozoin), a by-product of hemoglobin catabolization by the malaria parasite, which is an important target site for antimalarial drugs. The review aims to briefly describe recent studies on the application of this technology, elucidate molecular and electronic properties of the malaria pigment and its synthetic analog beta-hematin, provide insight into the mechanism of hemozoin formation within the food vacuole of the parasite, and comment on developing strategies for using this technology in drug-screening protocols.

The role of nanotechnology in single-cell detection: a review.

PubMed

Wang, Changling; Zhang, Yuxiang; Xia, Mingdian; Zhu, Xingxi; Qi, Shitao; Shen, Huaqiang; Liu, Tiebing; Tang, Liming

2014-10-01

Biological processes in single cells, such as signal transduction, DNA duplication, and protein synthesis and trafficking, occur in subcellular compartments at nanoscale level. Achieving high spatial-temporal resolution, high sensitivity, and high specificity in single-cell detection poses a great challenge. Nanotechnology, which has been widely applied in the fields of medicine, electronics, biomaterials, and energy production, has the potential to provide solutions for single-cell detection. Here we present a review of the use of nanotechnology in single-cell detection over the past two decades. First, we review the main areas of scientific interest, including morphology, ion concentration, DNA, RNA, protein, intracellular temperature, elements, and mechanical properties. Second, four categories of application of nanotechnology to single-cell detection are described: nanomanipulation, nanodevices, nanomaterials as labels, and nano Secondary ion mass spectrometry. Finally, the prospects and future trends in single-cell detection and analysis are discussed.

3D nanoscale imaging of the yeast, Schizosaccharomyces pombe, by full-field transmission X-ray microscopy at 5.4 keV.

PubMed

Chen, Jie; Yang, Yunhao; Zhang, Xiaobo; Andrews, Joy C; Pianetta, Piero; Guan, Yong; Liu, Gang; Xiong, Ying; Wu, Ziyu; Tian, Yangchao

2010-07-01

Three-dimensional (3D) nanoscale structures of the fission yeast, Schizosaccharomyces pombe, can be obtained by full-field transmission hard X-ray microscopy with 30 nm resolution using synchrotron radiation sources. Sample preparation is relatively simple and the samples are portable across various imaging environments, allowing for high-throughput sample screening. The yeast cells were fixed and double-stained with Reynold's lead citrate and uranyl acetate. We performed both absorption contrast and Zernike phase contrast imaging on these cells in order to test this method. The membranes, nucleus, and subcellular organelles of the cells were clearly visualized using absorption contrast mode. The X-ray images of the cells could be used to study the spatial distributions of the organelles in the cells. These results show unique structural information, demonstrating that hard X-ray microscopy is a complementary method for imaging and analyzing biological samples.

Improving Axial Resolution in Confocal Microscopy with New High Refractive Index Mounting Media

PubMed Central

Fouquet, Coralie; Gilles, Jean-François; Heck, Nicolas; Dos Santos, Marc; Schwartzmann, Richard; Cannaya, Vidjeacoumary; Morel, Marie-Pierre; Davidson, Robert Stephen; Trembleau, Alain; Bolte, Susanne

2015-01-01

Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required. PMID:25822785

From 2D slices to 3D volumes: image based reconstruction and morphological characterization of hippocampal cells on charged and uncharged surfaces using FIB/SEM serial sectioning.

PubMed

Schmidt, Franziska; Kühbacher, Markus; Gross, Ulrich; Kyriakopoulos, Antonius; Schubert, Helmut; Zehbe, Rolf

2011-03-01

3D imaging at a subcellular resolution is a powerful tool in the life sciences to investigate cells and their interactions with native tissues or artificial objects. While a tomographic experimental setup achieving a sufficient structural resolution can be established with either X-rays or electrons, the use of electrons is usually limited to very thin samples in transmission electron microscopy due to the poor penetration depths of electrons. The combination of a serial sectioning approach and scanning electron microscopy in state of the art dual beam experimental setups therefore offers a means to image highly resolved spatial details using a focused ion beam for slicing and an electron beam for imaging. The advantage of this technique over X-ray μCT or X-ray microscopy attributes to the fact that absorption is not a limiting factor in imaging and therefore even strong absorbing structures can be spatially reconstructed with a much higher possible resolution. This approach was used in this study to elucidate the effect of an electric potential on the morphology of cells from a hippocampal cell line (HT22) deposited on gold microelectrodes. While cells cultivated on two different controls (gold and polymer substrates) did show the expected stretched morphology, cells on both the anode and the cathode differed significantly. Cells deposited on the anode part of the electrode exhibited the most extreme deviation, being almost spherical and showed signs of chromatin condensation possibly indicating cell death. Furthermore, EDX was used as supplemental methodology for combined chemical and structural analyses. Copyright © 2010 Elsevier B.V. All rights reserved.

Dual-axis confocal microscope for high-resolution in vivo imaging

PubMed Central

Wang, Thomas D.; Mandella, Michael J.; Contag, Christopher H.; Kino, Gordon S.

2007-01-01

We describe a novel confocal microscope that uses separate low-numerical-aperture objectives with the illumination and collection axes crossed at angle θ from the midline. This architecture collects images in scattering media with high transverse and axial resolution, long working distance, large field of view, and reduced noise from scattered light. We measured transverse and axial (FWHM) resolution of 1.3 and 2.1 μm, respectively, in free space, and confirm subcellular resolution in excised esophageal mucosa. The optics may be scaled to millimeter dimensions and fiber coupled for collection of high-resolution images in vivo. PMID:12659264

Spectro-microscopy of living plant cells.

PubMed

Harter, Klaus; Meixner, Alfred J; Schleifenbaum, Frank

2012-01-01

Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techniques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on recent developments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context. The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassinosteroide receptor BRI1 in the plasma membrane of living plant cells. Moreover, the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime (FLT). Furthermore, a new spectro-microscopic technique, fluorescence intensity decay shape analysis microscopy (FIDSAM), has been developed. FIDSAM is capable of imaging low-expressed fluorophore-tagged proteins at high spatial resolution and precludes the misinterpretation of autofluorescence artifacts. In addition, FIDSAM provides a very effective and sensitive tool on the basis of Förster resonance energy transfer (FRET) for the qualitative and quantitative determination of protein-protein interaction. Finally, we report on the quantitative analysis of the photosystem I and II (PSI/PSII) ratio in the chloroplasts of living Arabidopsis plants at room temperature, using high-resolution, spatially resolved fluorescence spectroscopy. With this technique, it was not only possible to measure PSI/PSII ratios, but also to demonstrate the differential competence of wild-type and carbohydrate-deficient plants to adapt the PSI/PSII ratio to different light conditions. In summary, the information content of standard microscopic images is extended by several dimensions by the use of spectro-microscopic approaches. Therefore, novel cell physiological and molecular topics can be addressed and valuable insights into molecular and subcellular processes can be obtained in living plants.

The sub-cellular fate of mercury in the liver of wild mullets (Liza aurata)--Contribution to the understanding of metal-induced cellular toxicity.

PubMed

Araújo, Olinda; Pereira, Patrícia; Cesário, Rute; Pacheco, Mário; Raimundo, Joana

2015-06-15

Mercury is a recognized harmful pollutant in aquatic systems but still little is known about its sub-cellular partitioning in wild fish. Mercury concentrations in liver homogenate (whole organ load) and in six sub-cellular compartments were determined in wild Liza aurata from two areas - contaminated (LAR) and reference. Water and sediment contamination was also assessed. Fish from LAR displayed higher total mercury (tHg) organ load as well as in sub-cellular compartments than those from the reference area, reflecting environmental differences. However, spatial differences in percentage of tHg were only observed for mitochondria (Mit) and lysosomes plus microsomes (Lys+Mic). At LAR, Lys+Mic exhibited higher levels of tHg than the other fractions. Interestingly, tHg in Mit, granules (Gran) and heat-denaturable proteins was linearly correlated with the whole organ. Low tHg concentrations in heat stable proteins and Gran suggests that accumulated levels might be below the physiological threshold to activate those detoxification fractions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modular Detection of GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and Organisms.

PubMed

Ariotti, Nicholas; Hall, Thomas E; Rae, James; Ferguson, Charles; McMahon, Kerrie-Ann; Martel, Nick; Webb, Robyn E; Webb, Richard I; Teasdale, Rohan D; Parton, Robert G

2015-11-23

Reliable and quantifiable high-resolution protein localization is critical for understanding protein function. However, the time required to clone and characterize any protein of interest is a significant bottleneck, especially for electron microscopy (EM). We present a modular system for enzyme-based protein tagging that allows for improved speed and sampling for analysis of subcellular protein distributions using existing clone libraries to EM-resolution. We demonstrate that we can target a modified soybean ascorbate peroxidase (APEX) to any GFP-tagged protein of interest by engineering a GFP-binding peptide (GBP) directly to the APEX-tag. We demonstrate that APEX-GBP (1) significantly reduces the time required to characterize subcellular protein distributions of whole libraries to less than 3 days, (2) provides remarkable high-resolution localization of proteins to organelle subdomains, and (3) allows EM localization of GFP-tagged proteins, including proteins expressed at endogenous levels, in vivo by crossing existing GFP-tagged transgenic zebrafish lines with APEX-GBP transgenic lines. Copyright © 2015 Elsevier Inc. All rights reserved.

Fluorescent-Protein Stabilization and High-Resolution Imaging of Cleared, Intact Mouse Brains

PubMed Central

Schwarz, Martin K.; Scherbarth, Annemarie; Sprengel, Rolf; Engelhardt, Johann; Theer, Patrick; Giese, Guenter

2015-01-01

In order to observe and quantify long-range neuronal connections in intact mouse brain by light microscopy, it is first necessary to clear the brain, thus suppressing refractive-index variations. Here we describe a method that clears the brain and preserves the signal from proteinaceous fluorophores using a pH-adjusted non-aqueous index-matching medium. Successful clearing is enabled through the use of either 1-propanol or tert-butanol during dehydration whilst maintaining a basic pH. We show that high-resolution fluorescence imaging of entire, structurally intact juvenile and adult mouse brains is possible at subcellular resolution, even following many months in clearing solution. We also show that axonal long-range projections that are EGFP-labelled by modified Rabies virus can be imaged throughout the brain using a purpose-built light-sheet fluorescence microscope. To demonstrate the viability of the technique, we determined a detailed map of the monosynaptic projections onto a target cell population in the lateral entorhinal cortex. This example demonstrates that our method permits the quantification of whole-brain connectivity patterns at the subcellular level in the uncut brain. PMID:25993380

Dynamic neuroanatomy at subcellular resolution in the zebrafish.

PubMed

Faucherre, Adèle; López-Schier, Hernán

2014-01-01

Genetic means to visualize and manipulate neuronal circuits in the intact animal have revolutionized neurobiology. "Dynamic neuroanatomy" defines a range of approaches aimed at quantifying the architecture or subcellular organization of neurons over time during their development, regeneration, or degeneration. A general feature of these approaches is their reliance on the optical isolation of defined neurons in toto by genetically expressing markers in one or few cells. Here we use the afferent neurons of the lateral line as an example to describe a simple method for the dynamic neuroanatomical study of axon terminals in the zebrafish by laser-scanning confocal microscopy.

Spatial Distribution of Cellular Function: The Partitioning of Proteins between Mitochondria and the Nucleus in MCF7 Breast Cancer Cells

PubMed Central

Qattan, Amal T.; Radulovic, Marko; Crawford, Mark; Godovac-Zimmermann, Jasminka

2014-01-01

Concurrent proteomics analysis of the nuclei and mitochondria of MCF7 breast cancer cells identified 985 proteins (40% of all detected proteins) present in both organelles. Numerous proteins from all five complexes involved in oxidative phosphorylation (e.g., NDUFA5, NDUFB10, NDUFS1, NDUF2, SDHA, UQRB, UQRC2, UQCRH, COX5A, COX5B, MT-CO2, ATP5A1, ATP5B, ATP5H, etc.), from the TCA-cycle (DLST, IDH2, IDH3A, OGDH, SUCLAG2, etc.), and from glycolysis (ALDOA, ENO1, FBP1, GPI, PGK1, TALDO1, etc.) were distributed to both the nucleus and mitochondria. In contrast, proteins involved in nuclear/mitochondrial RNA processing/translation and Ras/Rab signaling showed different partitioning patterns. The identity of the OxPhos, TCA-cycle, and glycolysis proteins distributed to both the nucleus and mitochondria provides evidence for spatio-functional integration of these processes over the two different subcellular organelles. We suggest that there are unrecognized aspects of functional coordination between the nucleus and mitochondria, that integration of core functional processes via wide subcellular distribution of constituent proteins is a common characteristic of cells, and that subcellular spatial integration of function may be a vital aspect of cancer. PMID:23051583

Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms.

PubMed

Liu, Tsung-Li; Upadhyayula, Srigokul; Milkie, Daniel E; Singh, Ved; Wang, Kai; Swinburne, Ian A; Mosaliganti, Kishore R; Collins, Zach M; Hiscock, Tom W; Shea, Jamien; Kohrman, Abraham Q; Medwig, Taylor N; Dambournet, Daphne; Forster, Ryan; Cunniff, Brian; Ruan, Yuan; Yashiro, Hanako; Scholpp, Steffen; Meyerowitz, Elliot M; Hockemeyer, Dirk; Drubin, David G; Martin, Benjamin L; Matus, David Q; Koyama, Minoru; Megason, Sean G; Kirchhausen, Tom; Betzig, Eric

2018-04-20

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Coherence and diffraction limited resolution in microscopic OCT by a unified approach for the correction of dispersion and aberrations

NASA Astrophysics Data System (ADS)

Schulz-Hildebrandt, H.; Münter, Michael; Ahrens, M.; Spahr, H.; Hillmann, D.; König, P.; Hüttmann, G.

2018-03-01

Optical coherence tomography (OCT) images scattering tissues with 5 to 15 μm resolution. This is usually not sufficient for a distinction of cellular and subcellular structures. Increasing axial and lateral resolution and compensation of artifacts caused by dispersion and aberrations is required to achieve cellular and subcellular resolution. This includes defocus which limit the usable depth of field at high lateral resolution. OCT gives access the phase of the scattered light and hence correction of dispersion and aberrations is possible by numerical algorithms. Here we present a unified dispersion/aberration correction which is based on a polynomial parameterization of the phase error and an optimization of the image quality using Shannon's entropy. For validation, a supercontinuum light sources and a costume-made spectrometer with 400 nm bandwidth were combined with a high NA microscope objective in a setup for tissue and small animal imaging. Using this setup and computation corrections, volumetric imaging at 1.5 μm resolution is possible. Cellular and near cellular resolution is demonstrated in porcine cornea and the drosophila larva, when computational correction of dispersion and aberrations is used. Due to the excellent correction of the used microscope objective, defocus was the main contribution to the aberrations. In addition, higher aberrations caused by the sample itself were successfully corrected. Dispersion and aberrations are closely related artifacts in microscopic OCT imaging. Hence they can be corrected in the same way by optimization of the image quality. This way microscopic resolution is easily achieved in OCT imaging of static biological tissues.

High spatial resolution mass spectrometry imaging reveals the genetically programmed, developmental modification of the distribution of thylakoid membrane lipids among individual cells of maize leaf

DOE PAGES

Duenas, Maria Emilia; Klein, Adam T.; Alexander, Liza E.; ...

2016-11-17

Metabolism in plants is compartmentalized among different tissues, cells and subcellular organelles. Mass spectrometry imaging (MSI) with matrix-assisted laser desorption ionization (MALDI) has recently advanced to allow for the visualization of metabolites at single-cell resolution. Here we applied 5- and 10 μm high spatial resolution MALDI-MSI to the asymmetric Kranz anatomy of Zea mays (maize) leaves to study the differential localization of two major anionic lipids in thylakoid membranes, sulfoquinovosyldiacylglycerols (SQDG) and phosphatidylglycerols (PG). The quantification and localization of SQDG and PG molecular species, among mesophyll (M) and bundle sheath (BS) cells, are compared across the leaf developmental gradient frommore » four maize genotypes (the inbreds B73 and Mo17, and the reciprocal hybrids B73 × Mo17 and Mo17 × B73). SQDG species are uniformly distributed in both photosynthetic cell types, regardless of leaf development or genotype; however, PG shows photosynthetic cell-specific differential localization depending on the genotype and the fatty acyl chain constituent. Overall, 16:1-containing PGs primarily contribute to the thylakoid membranes of M cells, whereas BS chloroplasts are mostly composed of 16:0-containing PGs. Furthermore, PG 32:0 shows genotype-specific differences in cellular distribution, with preferential localization in BS cells for B73, but more uniform distribution between BS and M cells in Mo17. Maternal inheritance is exhibited within the hybrids, such that the localization of PG 32:0 in B73 × Mo17 is similar to the distribution in the B73 parental inbred, whereas that of Mo17 × B73 resembles the Mo17 parent. As a result, this study demonstrates the power of MALDI-MSI to reveal unprecedented insights on metabolic outcomes in multicellular organisms at single-cell resolution.« less

Bio-metals imaging and speciation in cells using proton and synchrotron radiation X-ray microspectroscopy

PubMed Central

Ortega, Richard; Devès, Guillaume; Carmona, Asunción

2009-01-01

The direct detection of biologically relevant metals in single cells and of their speciation is a challenging task that requires sophisticated analytical developments. The aim of this article is to present the recent achievements in the field of cellular chemical element imaging, and direct speciation analysis, using proton and synchrotron radiation X-ray micro- and nano-analysis. The recent improvements in focusing optics for MeV-accelerated particles and keV X-rays allow application to chemical element analysis in subcellular compartments. The imaging and quantification of trace elements in single cells can be obtained using particle-induced X-ray emission (PIXE). The combination of PIXE with backscattering spectrometry and scanning transmission ion microscopy provides a high accuracy in elemental quantification of cellular organelles. On the other hand, synchrotron radiation X-ray fluorescence provides chemical element imaging with less than 100 nm spatial resolution. Moreover, synchrotron radiation offers the unique capability of spatially resolved chemical speciation using micro-X-ray absorption spectroscopy. The potential of these methods in biomedical investigations will be illustrated with examples of application in the fields of cellular toxicology, and pharmacology, bio-metals and metal-based nano-particles. PMID:19605403

In vivo multiphoton tomography and fluorescence lifetime imaging of human brain tumor tissue.

PubMed

Kantelhardt, Sven R; Kalasauskas, Darius; König, Karsten; Kim, Ella; Weinigel, Martin; Uchugonova, Aisada; Giese, Alf

2016-05-01

High resolution multiphoton tomography and fluorescence lifetime imaging differentiates glioma from adjacent brain in native tissue samples ex vivo. Presently, multiphoton tomography is applied in clinical dermatology and experimentally. We here present the first application of multiphoton and fluorescence lifetime imaging for in vivo imaging on humans during a neurosurgical procedure. We used a MPTflex™ Multiphoton Laser Tomograph (JenLab, Germany). We examined cultured glioma cells in an orthotopic mouse tumor model and native human tissue samples. Finally the multiphoton tomograph was applied to provide optical biopsies during resection of a clinical case of glioblastoma. All tissues imaged by multiphoton tomography were sampled and processed for conventional histopathology. The multiphoton tomograph allowed fluorescence intensity- and fluorescence lifetime imaging with submicron spatial resolution and 200 picosecond temporal resolution. Morphological fluorescence intensity imaging and fluorescence lifetime imaging of tumor-bearing mouse brains and native human tissue samples clearly differentiated tumor and adjacent brain tissue. Intraoperative imaging was found to be technically feasible. Intraoperative image quality was comparable to ex vivo examinations. To our knowledge we here present the first intraoperative application of high resolution multiphoton tomography and fluorescence lifetime imaging of human brain tumors in situ. It allowed in vivo identification and determination of cell density of tumor tissue on a cellular and subcellular level within seconds. The technology shows the potential of rapid intraoperative identification of native glioma tissue without need for tissue processing or staining.

Organelle-targeting surface-enhanced Raman scattering (SERS) nanosensors for subcellular pH sensing.

PubMed

Shen, Yanting; Liang, Lijia; Zhang, Shuqin; Huang, Dianshuai; Zhang, Jing; Xu, Shuping; Liang, Chongyang; Xu, Weiqing

2018-01-25

The pH value of subcellular organelles in living cells is a significant parameter in the physiological activities of cells. Its abnormal fluctuations are commonly believed to be associated with cancers and other diseases. Herein, a series of surface-enhanced Raman scattering (SERS) nanosensors with high sensitivity and targeting function was prepared for the quantification and monitoring of pH values in mitochondria, nucleus, and lysosome. The nanosensors were composed of gold nanorods (AuNRs) functionalized with a pH-responsive molecule (4-mercaptopyridine, MPy) and peptides that could specifically deliver the AuNRs to the targeting subcellular organelles. The localization of our prepared nanoprobes in specific organelles was confirmed by super-high resolution fluorescence imaging and bio-transmission electron microscopy (TEM) methods. By the targeting ability, the pH values of the specific organelles can be determined by monitoring the vibrational spectral changes of MPy with different pH values. Compared to the cases of reported lysosome and cytoplasm SERS pH sensors, more accurate pH values of mitochondria and nucleus, which could be two additional intracellular tracers for subcellular microenvironments, were disclosed by this SERS approach, further improving the accuracy of discrimination of related diseases. Our sensitive SERS strategy can also be employed to explore crucial physiological and biological processes that are related to subcellular pH fluctuations.

First multiphoton tomography of brain in man

NASA Astrophysics Data System (ADS)

König, Karsten; Kantelhardt, Sven R.; Kalasauskas, Darius; Kim, Ella; Giese, Alf

2016-03-01

We report on the first two-photon in vivo brain tissue imaging study in man. High resolution in vivo histology by multiphoton tomography (MPT) including two-photon FLIM was performed in the operation theatre during neurosurgery to evaluate the feasibility to detect label-free tumor borders with subcellular resolution. This feasibility study demonstrates, that MPT has the potential to identify tumor borders on a cellular level in nearly real-time.

Vital-dye enhanced fluorescence imaging of gastrointestinal mucosa: metaplasia, neoplasia, inflammation

PubMed Central

Muldoon, Timothy J; Polydorides, Alexandros D; Maru, Dipen M; Harpaz, Noam; Harris, Michael T; Hofstettor, Wayne; Hiotis, Spiros P; Kim, Sanghyun A; Ky, Alex J; Anandasabapathy, Sharmila; Richards-Kortum, Rebecca

2012-01-01

Background Confocal endomicroscopy has revolutionized endoscopy by offering sub-cellular images of gastrointestinal epithelium; however, field-of-view is limited. There is a need for multi-scale endoscopy platforms that use widefield imaging to better direct placement of high-resolution probes. Design Feasibility Study Objective This study evaluates the feasibility of a single agent, proflavine hemisulfate, as a contrast medium during both widefield and high resolution imaging to characterize morphologic changes associated with a variety of gastrointestinal conditions. Setting U.T. M.D. Anderson Cancer Center (Houston, TX) and Mount Sinai Medical Center (New York, NY) Patients, Interventions, and Main Outcome Measurements Surgical specimens were obtained from 15 patients undergoing esophagectomy/colectomy. Proflavine, a vital fluorescent dye, was applied topically. Specimens were imaged with a widefield multispectral microscope and a high-resolution microendoscope. Images were compared to histopathology. Results Widefield-fluorescence imaging enhanced visualization of morphology, including the presence and spatial distribution of glands, glandular distortion, atrophy and crowding. High-resolution imaging of widefield-abnormal areas revealed that neoplastic progression corresponded to glandular heterogeneity and nuclear crowding in dysplasia, with glandular effacement in carcinoma. These widefield and high-resolution image features correlated well with histopathology. Limitations This imaging approach must be validated in vivo with a larger sample size. Conclusions Multi-scale proflavine-enhanced fluorescence imaging can delineate epithelial changes in a variety of gastrointestinal conditions. Distorted glandular features seen with widefield imaging could serve as a critical ‘bridge’ to high-resolution probe placement. An endoscopic platform combining the two modalities with a single vital-dye may facilitate point-of-care decision-making by providing real-time, in vivo diagnoses. PMID:22301343

Vital-dye enhanced fluorescence imaging of GI mucosa: metaplasia, neoplasia, inflammation.

PubMed

Thekkek, Nadhi; Muldoon, Timothy; Polydorides, Alexandros D; Maru, Dipen M; Harpaz, Noam; Harris, Michael T; Hofstettor, Wayne; Hiotis, Spiros P; Kim, Sanghyun A; Ky, Alex Jenny; Anandasabapathy, Sharmila; Richards-Kortum, Rebecca

2012-04-01

Confocal endomicroscopy has revolutionized endoscopy by offering subcellular images of the GI epithelium; however, the field of view is limited. Multiscale endoscopy platforms that use widefield imaging are needed to better direct the placement of high-resolution probes. Feasibility study. This study evaluated the feasibility of a single agent, proflavine hemisulfate, as a contrast medium during both widefield and high-resolution imaging to characterize the morphologic changes associated with a variety of GI conditions. The University of Texas MD Anderson Cancer Center, Houston, Texas, and Mount Sinai Medical Center, New York, New York. PATIENTS, INTERVENTIONS, AND MAIN OUTCOME MEASUREMENTS: Resected specimens were obtained from 15 patients undergoing EMR, esophagectomy, or colectomy. Proflavine hemisulfate, a vital fluorescent dye, was applied topically. The specimens were imaged with a widefield multispectral microscope and a high-resolution microendoscope. The images were compared with histopathologic examination. Widefield fluorescence imaging enhanced visualization of morphology, including the presence and spatial distribution of glands, glandular distortion, atrophy, and crowding. High-resolution imaging of widefield abnormal areas revealed that neoplastic progression corresponded to glandular heterogeneity and nuclear crowding in dysplasia, with glandular effacement in carcinoma. These widefield and high-resolution image features correlated well with the histopathologic features. This imaging approach must be validated in vivo with a larger sample size. Multiscale proflavine-enhanced fluorescence imaging can delineate epithelial changes in a variety of GI conditions. Distorted glandular features seen with widefield imaging could serve as a critical bridge to high-resolution probe placement. An endoscopic platform combining the two modalities with a single vital dye may facilitate point-of-care decision making by providing real-time, in vivo diagnoses. Copyright © 2012 American Society for Gastrointestinal Endoscopy. Published by Mosby, Inc. All rights reserved.

Spatially patterned matrix elasticity directs stem cell fate

NASA Astrophysics Data System (ADS)

Yang, Chun; DelRio, Frank W.; Ma, Hao; Killaars, Anouk R.; Basta, Lena P.; Kyburz, Kyle A.; Anseth, Kristi S.

2016-08-01

There is a growing appreciation for the functional role of matrix mechanics in regulating stem cell self-renewal and differentiation processes. However, it is largely unknown how subcellular, spatial mechanical variations in the local extracellular environment mediate intracellular signal transduction and direct cell fate. Here, the effect of spatial distribution, magnitude, and organization of subcellular matrix mechanical properties on human mesenchymal stem cell (hMSCs) function was investigated. Exploiting a photodegradation reaction, a hydrogel cell culture substrate was fabricated with regions of spatially varied and distinct mechanical properties, which were subsequently mapped and quantified by atomic force microscopy (AFM). The variations in the underlying matrix mechanics were found to regulate cellular adhesion and transcriptional events. Highly spread, elongated morphologies and higher Yes-associated protein (YAP) activation were observed in hMSCs seeded on hydrogels with higher concentrations of stiff regions in a dose-dependent manner. However, when the spatial organization of the mechanically stiff regions was altered from a regular to randomized pattern, lower levels of YAP activation with smaller and more rounded cell morphologies were induced in hMSCs. We infer from these results that irregular, disorganized variations in matrix mechanics, compared with regular patterns, appear to disrupt actin organization, and lead to different cell fates; this was verified by observations of lower alkaline phosphatase (ALP) activity and higher expression of CD105, a stem cell marker, in hMSCs in random versus regular patterns of mechanical properties. Collectively, this material platform has allowed innovative experiments to elucidate a novel spatial mechanical dosing mechanism that correlates to both the magnitude and organization of spatial stiffness.

Remote Control of Gene Function by Local Translation

PubMed Central

Jung, Hosung; Gkogkas, Christos G.; Sonenberg, Nahum; Holt, Christine E.

2014-01-01

The subcellular position of a protein is a key determinant of its function. Mounting evidence indicates that RNA localization, where specific mRNAs are transported subcellularly and subsequently translated in response to localized signals, is an evolutionarily conserved mechanism to control protein localization. On-site synthesis confers novel signaling properties to a protein and helps to maintain local proteome homeostasis. Local translation plays particularly important roles in distal neuronal compartments, and dysregulated RNA localization and translation cause defects in neuronal wiring and survival. Here, we discuss key findings in this area and possible implications of this adaptable and swift mechanism for spatial control of gene function. PMID:24679524

Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes

PubMed Central

Barlag, Britta; Beutel, Oliver; Janning, Dennis; Czarniak, Frederik; Richter, Christian P.; Kommnick, Carina; Göser, Vera; Kurre, Rainer; Fabiani, Florian; Erhardt, Marc; Piehler, Jacob; Hensel, Michael

2016-01-01

The investigation of the subcellular localization, dynamics and interaction of proteins and protein complexes in prokaryotes is complicated by the small size of the cells. Super-resolution microscopy (SRM) comprise various new techniques that allow light microscopy with a resolution that can be up to ten-fold higher than conventional light microscopy. Application of SRM techniques to living prokaryotes demands the introduction of suitable fluorescent probes, usually by fusion of proteins of interest to fluorescent proteins with properties compatible to SRM. Here we describe an approach that is based on the genetically encoded self-labelling enzymes HaloTag and SNAP-tag. Proteins of interest are fused to HaloTag or SNAP-tag and cell permeable substrates can be labelled with various SRM-compatible fluorochromes. Fusions of the enzyme tags to subunits of a type I secretion system (T1SS), a T3SS, the flagellar rotor and a transcription factor were generated and analysed in living Salmonella enterica. The new approach is versatile in tagging proteins of interest in bacterial cells and allows to determine the number, relative subcellular localization and dynamics of protein complexes in living cells. PMID:27534893

High-numerical-aperture cryogenic light microscopy for increased precision of superresolution reconstructions

PubMed Central

Nahmani, Marc; Lanahan, Conor; DeRosier, David; Turrigiano, Gina G.

2017-01-01

Superresolution microscopy has fundamentally altered our ability to resolve subcellular proteins, but improving on these techniques to study dense structures composed of single-molecule-sized elements has been a challenge. One possible approach to enhance superresolution precision is to use cryogenic fluorescent imaging, reported to reduce fluorescent protein bleaching rates, thereby increasing the precision of superresolution imaging. Here, we describe an approach to cryogenic photoactivated localization microscopy (cPALM) that permits the use of a room-temperature high-numerical-aperture objective lens to image frozen samples in their native state. We find that cPALM increases photon yields and show that this approach can be used to enhance the effective resolution of two photoactivatable/switchable fluorophore-labeled structures in the same frozen sample. This higher resolution, two-color extension of the cPALM technique will expand the accessibility of this approach to a range of laboratories interested in more precise reconstructions of complex subcellular targets. PMID:28348224

Estimating the magnitude of near-membrane PDE4 activity in living cells.

PubMed

Xin, Wenkuan; Feinstein, Wei P; Britain, Andrea L; Ochoa, Cristhiaan D; Zhu, Bing; Richter, Wito; Leavesley, Silas J; Rich, Thomas C

2015-09-15

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments. Copyright © 2015 the American Physiological Society.

Estimating the magnitude of near-membrane PDE4 activity in living cells

PubMed Central

Xin, Wenkuan; Feinstein, Wei P.; Britain, Andrea L.; Ochoa, Cristhiaan D.; Zhu, Bing; Richter, Wito; Leavesley, Silas J.

2015-01-01

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments. PMID:26201952

Selective Detection of Neurotransmitters by Fluorescence and Chemiluminescence Imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ziqiang Wang; Edward S. Yeung

In recent years, luminescence imaging has been widely employed in neurochemical analysis. It has a number of advantages for the study of neuronal and other biological cells: (1) a particular molecular species or cellular constituent can be selectively visualized in the presence of a large excess of other species in a heterogeneous environment; (2) low concentration detection limits can be achieved because of the inherent sensitivity associated with fluorescence and chemiluminescence; (3) low excitation intensities can be used so that long-term observation can be realized while the viability of the specimen is preserved; and (4) excellent spatial resolution can bemore » obtained with the light microscope so subcellular compartments can be identified. With good sensitivity, temporal and spatial resolution, the flux of ions and molecules and the distribution and dynamics of intracellular species can be measured in real time with specific luminescence probes, substrates, or with native fluorescence. A noninvasive detection scheme based on glutamate dehydrogenase (GDH) enzymatic assay combined with microscopy was developed to measure the glutamate release in cultured cells from the central nervous system (CNS). The enzyme reaction is very specific and sensitive. The detection limit with CCD imaging is down to {micro}M levels of glutamate with reasonable response time. They also found that chemiluminescence associated with the ATP-dependent reaction between luciferase and luciferin can be used to image ATP at levels down to 10 nM in the millisecond time scale. Similar imaging experiments should be feasible in a broad spectrum of biological systems.« less

Prion subcellular fractionation reveals infectivity spectrum, with a high titre-low PrPres level disparity

PubMed Central

2012-01-01

Background Prion disease transmission and pathogenesis are linked to misfolded, typically protease resistant (PrPres) conformers of the normal cellular prion protein (PrPC), with the former posited to be the principal constituent of the infectious 'prion'. Unexplained discrepancies observed between detectable PrPres and infectivity levels exemplify the complexity in deciphering the exact biophysical nature of prions and those host cell factors, if any, which contribute to transmission efficiency. In order to improve our understanding of these important issues, this study utilized a bioassay validated cell culture model of prion infection to investigate discordance between PrPres levels and infectivity titres at a subcellular resolution. Findings Subcellular fractions enriched in lipid rafts or endoplasmic reticulum/mitochondrial marker proteins were equally highly efficient at prion transmission, despite lipid raft fractions containing up to eight times the levels of detectable PrPres. Brain homogenate infectivity was not differentially enhanced by subcellular fraction-specific co-factors, and proteinase K pre-treatment of selected fractions modestly, but equally reduced infectivity. Only lipid raft associated infectivity was enhanced by sonication. Conclusions This study authenticates a subcellular disparity in PrPres and infectivity levels, and eliminates simultaneous divergence of prion strains as the explanation for this phenomenon. On balance, the results align best with the concept that transmission efficiency is influenced more by intrinsic characteristics of the infectious prion, rather than cellular microenvironment conditions or absolute PrPres levels. PMID:22534096

High-throughput microscopy must re-invent the microscope rather than speed up its functions

PubMed Central

Oheim, M

2007-01-01

Knowledge gained from the revolutions in genomics and proteomics has helped to identify many of the key molecules involved in cellular signalling. Researchers, both in academia and in the pharmaceutical industry, now screen, at a sub-cellular level, where and when these proteins interact. Fluorescence imaging and molecular labelling combine to provide a powerful tool for real-time functional biochemistry with molecular resolution. However, they traditionally have been work-intensive, required trained personnel, and suffered from low through-put due to sample preparation, loading and handling. The need for speeding up microscopy is apparent from the tremendous complexity of cellular signalling pathways, the inherent biological variability, as well as the possibility that the same molecule plays different roles in different sub-cellular compartments. Research institutes and companies have teamed up to develop imaging cytometers of ever-increasing complexity. However, to truly go high-speed, sub-cellular imaging must free itself from the rigid framework of current microscopes. PMID:17603553

Kibra and Merlin Activate the Hippo Pathway Spatially Distinct from and Independent of Expanded.

PubMed

Su, Ting; Ludwig, Michael Z; Xu, Jiajie; Fehon, Richard G

2017-03-13

The Hippo pathway is emerging as a key evolutionarily conserved signaling mechanism that controls organ size. Three membrane-associated proteins, Kibra, Merlin, and Expanded, regulate pathway activity, but the precise molecular mechanism by which they function is still poorly understood. Here we provide evidence that Merlin and Kibra activate Hippo signaling in parallel to Expanded at a spatially distinct cellular domain, the medial apical cortex. Merlin and Kibra together recruit the adapter protein Salvador, which in turn recruits the core kinase Hippo. In addition, we show that Crumbs has a dual effect on Hippo signaling. Crumbs promotes the ability of Expanded to activate the pathway but also sequesters Kibra to downregulate Hippo signaling. Together, our findings elucidate the mechanism of Hippo pathway activation by Merlin and Kibra, identify a subcellular domain for Hippo pathway regulation, and demonstrate differential activity of upstream regulators in different subcellular domains. Copyright © 2017 Elsevier Inc. All rights reserved.

Spectroscopic studies of anthracyclines: Structural characterization and in vitro tracking

NASA Astrophysics Data System (ADS)

Szafraniec, Ewelina; Majzner, Katarzyna; Farhane, Zeineb; Byrne, Hugh J.; Lukawska, Malgorzata; Oszczapowicz, Irena; Chlopicki, Stefan; Baranska, Malgorzata

2016-12-01

A broad spectroscopic characterization, using ultraviolet-visible (UV-vis) and Fourier transform infrared absorption as well as Raman scattering, of two commonly used anthracyclines antibiotics (DOX) daunorubicin (DNR), their epimers (EDOX, EDNR) and ten selected analogs is presented. The paper serves as a comprehensive spectral library of UV-vis, IR and Raman spectra of anthracyclines in the solid state and in solution. The particular advantage of Raman spectroscopy for the measurement and analysis of individual antibiotics is demonstrated. Raman spectroscopy can be used to monitor the in vitro uptake and distribution of the drug in cells, using both 488 nm and 785 nm as source wavelengths, with submicrometer spatial resolution, although the cellular accumulation of the drug is different in each case. The high information content of Raman spectra allows studies of the drug-cell interactions, and so the method seems very suitable for monitoring drug uptake and mechanisms of interaction with cellular compartments at the subcellular level.

Visualization and characterization of individual type III protein secretion machines in live bacteria

PubMed Central

Lara-Tejero, María; Bewersdorf, Jörg; Galán, Jorge E.

2017-01-01

Type III protein secretion machines have evolved to deliver bacterially encoded effector proteins into eukaryotic cells. Although electron microscopy has provided a detailed view of these machines in isolation or fixed samples, little is known about their organization in live bacteria. Here we report the visualization and characterization of the Salmonella type III secretion machine in live bacteria by 2D and 3D single-molecule switching superresolution microscopy. This approach provided access to transient components of this machine, which previously could not be analyzed. We determined the subcellular distribution of individual machines, the stoichiometry of the different components of this machine in situ, and the spatial distribution of the substrates of this machine before secretion. Furthermore, by visualizing this machine in Salmonella mutants we obtained major insights into the machine’s assembly. This study bridges a major resolution gap in the visualization of this nanomachine and may serve as a paradigm for the examination of other bacterially encoded molecular machines. PMID:28533372

Optical toolkits for in vivo deep tissue laser scanning microscopy: a primer

NASA Astrophysics Data System (ADS)

Lee, Woei Ming; McMenamin, Thomas; Li, Yongxiao

2018-06-01

Life at the microscale is animated and multifaceted. The impact of dynamic in vivo microscopy in small animals has opened up opportunities to peer into a multitude of biological processes at the cellular scale in their native microenvironments. Laser scanning microscopy (LSM) coupled with targeted fluorescent proteins has become an indispensable tool to enable dynamic imaging in vivo at high temporal and spatial resolutions. In the last few decades, the technique has been translated from imaging cells in thin samples to mapping cells in the thick biological tissue of living organisms. Here, we sought to provide a concise overview of the design considerations of a LSM that enables cellular and subcellular imaging in deep tissue. Individual components under review include: long working distance microscope objectives, laser scanning technologies, adaptive optics devices, beam shaping technologies and photon detectors, with an emphasis on more recent advances. The review will conclude with the latest innovations in automated optical microscopy, which would impact tracking and quantification of heterogeneous populations of cells in vivo.

Beyond Fluorescent Proteins: Hybrid and Bioluminescent Indicators for Imaging Neural Activities.

PubMed

Wang, Anqi; Feng, Jiesi; Li, Yulong; Zou, Peng

2018-04-18

Optical biosensors have been invaluable tools in neuroscience research, as they provide the ability to directly visualize neural activity in real time, with high specificity, and with exceptional spatial and temporal resolution. Notably, a majority of these sensors are based on fluorescent protein scaffolds, which offer the ability to target specific cell types or even subcellular compartments. However, fluorescent proteins are intrinsically bulky tags, often insensitive to the environment, and always require excitation light illumination. To address these limitations, there has been a proliferation of alternative sensor scaffolds developed in recent years, including hybrid sensors that combine the advantages of synthetic fluorophores and genetically encoded protein tags, as well as bioluminescent probes. While still in their early stage of development as compared with fluorescent protein-based sensors, these novel probes have offered complementary solutions to interrogate various aspects of neuronal communication, including transmitter release, changes in membrane potential, and the production of second messengers. In this Review, we discuss these important new developments with a particular focus on design strategies.

3D nanoscale imaging of the yeast, Schizosaccharomyces pombe, by full-field transmission x-ray microscopy at 5.4 keV

PubMed Central

Chen, Jie; Yang, Yunhao; Zhang, Xiaobo; Andrews, Joy C.; Pianetta, Piero; Guan, Yong; Liu, Gang; Xiong, Ying; Wu, Ziyu; Tian, Yangchao

2010-01-01

Three-dimensional (3D) nanoscale structures of the fission yeast, Schizosaccharomyces pombe, can be obtained by full-field transmission hard x-ray microscopy with 30 nm resolution using synchrotron radiation sources. Sample preparation is relatively simple and the samples are portable across various imaging environments, allowing for high throughput sample screening. The yeast cells were fixed and double stained with Reynold’s lead citrate and uranyl acetate. We performed both absorption contrast and Zernike phase contrast imaging on these cells in order to test this method. The membranes, nucleus and subcellular organelles of the cells were clearly visualized using absorption contrast mode. The x-ray images of the cells could be used to study the spatial distributions of the organelles in the cells. These results show unique structural information, demonstrating that hard x-ray microscopy is a complementary method for imaging and analyzing biological samples. PMID:20349228

Compartmental genomics in living cells revealed by single-cell nanobiopsy.

PubMed

Actis, Paolo; Maalouf, Michelle M; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R Adam; Pourmand, Nader

2014-01-28

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.

Single-cell analysis of pyroptosis dynamics reveals conserved GSDMD-mediated subcellular events that precede plasma membrane rupture.

PubMed

de Vasconcelos, Nathalia M; Van Opdenbosch, Nina; Van Gorp, Hanne; Parthoens, Eef; Lamkanfi, Mohamed

2018-04-17

Pyroptosis is rapidly emerging as a mechanism of anti-microbial host defense, and of extracellular release of the inflammasome-dependent cytokines interleukin (IL)-1β and IL-18, which contributes to autoinflammatory pathology. Caspases 1, 4, 5 and 11 trigger this regulated form of necrosis by cleaving the pyroptosis effector gasdermin D (GSDMD), causing its pore-forming amino-terminal domain to oligomerize and perforate the plasma membrane. However, the subcellular events that precede pyroptotic cell lysis are ill defined. In this study, we triggered primary macrophages to undergo pyroptosis from three inflammasome types and recorded their dynamics and morphology using high-resolution live-cell spinning disk confocal laser microscopy. Based on quantitative analysis of single-cell subcellular events, we propose a model of pyroptotic cell disintegration that is initiated by opening of GSDMD-dependent ion channels or pores that are more restrictive than recently proposed GSDMD pores, followed by osmotic cell swelling, commitment of mitochondria and other membrane-bound organelles prior to sudden rupture of the plasma membrane and full permeability to intracellular proteins. This study provides a dynamic framework for understanding cellular changes that occur during pyroptosis, and charts a chronological sequence of GSDMD-mediated subcellular events that define pyroptotic cell death at the single-cell level.

Univariate and multivariate methods for chemical mapping of cervical cancer cells

NASA Astrophysics Data System (ADS)

Duraipandian, Shiyamala; Zheng, Wei; Huang, Zhiwei

2012-01-01

Visualization of cells and subcellular organelles are currently carried out using available microscopy methods such as cryoelectron microscopy, and fluorescence microscopy. These methods require external labeling using fluorescent dyes and extensive sample preparations to access the subcellular structures. However, Raman micro-spectroscopy provides a non-invasive, label-free method for imaging the cells with chemical specificity at sub-micrometer spatial resolutions. The scope of this paper is to image the biochemical/molecular distributions in cells associated with cancerous changes. Raman map data sets were acquired from the human cervical carcinoma cell lines (HeLa) after fixation under 785 nm excitation wavelength. The individual spectrum was recorded by raster-scanning the laser beam over the sample with 1μm step size and 10s exposure time. Images revealing nucleic acids, lipids and proteins (phenylalanine, amide I) were reconstructed using univariate methods. In near future, the small pixel to pixel variations will also be imaged using different multivariate methods (PCA, clustering (HCA, K-means, FCM)) to determine the main cellular constitutions. The hyper-spectral image of cell was reconstructed utilizing the spectral contrast at different pixels of the cell (due to the variation in the biochemical distribution) without using fluorescent dyes. Normal cervical squamous cells will also be imaged in order to differentiate normal and cancer cells of cervix using the biochemical changes in different grades of cancer. Based on the information obtained from the pseudo-color maps, constructed from the hyper-spectral cubes, the primary cellular constituents of normal and cervical cancer cells were identified.

Quantification of asymmetric microtubule nucleation at sub-cellular structures

PubMed Central

Zhu, Xiaodong; Kaverina, Irina

2012-01-01

Cell polarization is important for multiple physiological processes. In polarized cells, microtubules (MTs) are organized into a spatially polarized array. Generally, in non-differentiated cells, it is assumed that MTs are symmetrically nucleated exclusively from centrosome (microtubule organizing center, MTOC) and then reorganized into the asymmetric array. We have recently identified the Golgi complex as an additional MTOC that asymmetrically nucleates MTs toward one side of the cell. Methods used for alternative MTOC identification include microtubule re-growth after complete drug-induced depolymerization and tracking of growing microtubules using fluorescence labeled MT +TIP binding proteins in living cells. These approaches can be used for quantification of MT nucleation sites at diverse sub-cellular structures. PMID:21773933

RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells.

PubMed

Roussis, Ioannis M; Guille, Matthew; Myers, Fiona A; Scarlett, Garry P

2016-01-01

Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein complexes as a consequence of the complexities associated with working with RNA. Here we present a method for the modification of the existing In Situ Hybridisation-Proximity Ligation Assay (ISH-PLA) protocol to adapt it to the study of RNA regulation (rISH-PLA). As proof of principle we used the well-characterised interaction of the Xenopus laevis Staufen RNA binding protein with Vg1 mRNA, the complex of which co-localises to the vegetal pole of Xenopus oocytes. The applicability of both the Stau1 antibody and the Locked Nucleic Acid probe (LNA) recognising Vg1 mRNA were independently validated by whole-mount Immunohistochemistry and whole-mount in situ hybridisation assays respectively prior to combining them in the rISH-PLA assay. The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogenous cell populations from which conventional RNA-protein interaction detection techniques suffer. This technique will be particularly usefully for studying the activity of RNA binding proteins (RBPs) in complex mixtures of cells, for example tissue sections or whole embryos.

Tracking protein dynamics with photoconvertible Dendra2 on spinning disk confocal systems.

PubMed

Woods, Elena; Courtney, Jane; Scholz, Dimitri; Hall, William W; Gautier, Virginie W

2014-12-01

Understanding the dynamic properties of cellular proteins in live cells and in real time is essential to delineate their function. In this context, we introduce the Fluorescence Recovery After Photobleaching-Photoactivation unit (Andor) combined with the Nikon Eclipse Ti E Spinning Disk (Andor) confocal microscope as an advantageous and robust platform to exploit the properties of the Dendra2 photoconvertible fluorescent protein (Evrogen) and analyse protein subcellular trafficking in living cells. A major advantage of the spinning disk confocal is the rapid acquisition speed, enabling high temporal resolution of cellular processes. Furthermore, photoconversion and imaging are less invasive on the spinning disk confocal as the cell exposition to illumination power is reduced, thereby minimizing photobleaching and increasing cell viability. We have tested this commercially available platform using experimental settings adapted to track the migration of fast trafficking proteins such as UBC9, Fibrillarin and have successfully characterized their differential motion between subnuclear structures. We describe here step-by-step procedures, with emphasis on cellular imaging parameters, to successfully perform the dynamic imaging and photoconversion of Dendra2-fused proteins at high spatial and temporal resolutions necessary to characterize the trafficking pathways of proteins. © 2014 The Authors. Journal of Microscopy published by John Wiley & Sons, Ltd on behalf of Royal Microscopical Society.

Time-lapse electrical impedance spectroscopy for monitoring the cell cycle of single immobilized S. pombe cells.

PubMed

Zhu, Zhen; Frey, Olivier; Haandbaek, Niels; Franke, Felix; Rudolf, Fabian; Hierlemann, Andreas

2015-11-26

As a complement and alternative to optical methods, wide-band electrical impedance spectroscopy (EIS) enables multi-parameter, label-free and real-time detection of cellular and subcellular features. We report on a microfluidics-based system designed to reliably capture single rod-shaped Schizosaccharomyces pombe cells by applying suction through orifices in a channel wall. The system enables subsequent culturing of immobilized cells in an upright position, while dynamic changes in cell-cycle state and morphology were continuously monitored through EIS over a broad frequency range. Besides measuring cell growth, clear impedance signals for nuclear division have been obtained. The EIS system has been characterized with respect to sensitivity and detection limits. The spatial resolution in measuring cell length was 0.25 μm, which corresponds to approximately a 5-min interval of cell growth under standard conditions. The comprehensive impedance data sets were also used to determine the occurrence of nuclear division and cytokinesis. The obtained results have been validated through concurrent confocal imaging and plausibilized through comparison with finite-element modeling data. The possibility to monitor cellular and intracellular features of single S. pombe cells during the cell cycle at high spatiotemporal resolution renders the presented microfluidics-based EIS system a suitable tool for dynamic single-cell investigations.

Time-lapse electrical impedance spectroscopy for monitoring the cell cycle of single immobilized S. pombe cells

PubMed Central

Zhu, Zhen; Frey, Olivier; Haandbaek, Niels; Franke, Felix; Rudolf, Fabian; Hierlemann, Andreas

2015-01-01

As a complement and alternative to optical methods, wide-band electrical impedance spectroscopy (EIS) enables multi-parameter, label-free and real-time detection of cellular and subcellular features. We report on a microfluidics-based system designed to reliably capture single rod-shaped Schizosaccharomyces pombe cells by applying suction through orifices in a channel wall. The system enables subsequent culturing of immobilized cells in an upright position, while dynamic changes in cell-cycle state and morphology were continuously monitored through EIS over a broad frequency range. Besides measuring cell growth, clear impedance signals for nuclear division have been obtained. The EIS system has been characterized with respect to sensitivity and detection limits. The spatial resolution in measuring cell length was 0.25 μm, which corresponds to approximately a 5-min interval of cell growth under standard conditions. The comprehensive impedance data sets were also used to determine the occurrence of nuclear division and cytokinesis. The obtained results have been validated through concurrent confocal imaging and plausibilized through comparison with finite-element modeling data. The possibility to monitor cellular and intracellular features of single S. pombe cells during the cell cycle at high spatiotemporal resolution renders the presented microfluidics-based EIS system a suitable tool for dynamic single-cell investigations. PMID:26608589

Generation of dense statistical connectomes from sparse morphological data

PubMed Central

Egger, Robert; Dercksen, Vincent J.; Udvary, Daniel; Hege, Hans-Christian; Oberlaender, Marcel

2014-01-01

Sensory-evoked signal flow, at cellular and network levels, is primarily determined by the synaptic wiring of the underlying neuronal circuitry. Measurements of synaptic innervation, connection probabilities and subcellular organization of synaptic inputs are thus among the most active fields of research in contemporary neuroscience. Methods to measure these quantities range from electrophysiological recordings over reconstructions of dendrite-axon overlap at light-microscopic levels to dense circuit reconstructions of small volumes at electron-microscopic resolution. However, quantitative and complete measurements at subcellular resolution and mesoscopic scales to obtain all local and long-range synaptic in/outputs for any neuron within an entire brain region are beyond present methodological limits. Here, we present a novel concept, implemented within an interactive software environment called NeuroNet, which allows (i) integration of sparsely sampled (sub)cellular morphological data into an accurate anatomical reference frame of the brain region(s) of interest, (ii) up-scaling to generate an average dense model of the neuronal circuitry within the respective brain region(s) and (iii) statistical measurements of synaptic innervation between all neurons within the model. We illustrate our approach by generating a dense average model of the entire rat vibrissal cortex, providing the required anatomical data, and illustrate how to measure synaptic innervation statistically. Comparing our results with data from paired recordings in vitro and in vivo, as well as with reconstructions of synaptic contact sites at light- and electron-microscopic levels, we find that our in silico measurements are in line with previous results. PMID:25426033

Pump-probe imaging of pigmented cutaneous melanoma primary lesions gives insight into metastatic potential

PubMed Central

Robles, Francisco E.; Deb, Sanghamitra; Wilson, Jesse W.; Gainey, Christina S.; Selim, M. Angelica; Mosca, Paul J.; Tyler, Douglas S.; Fischer, Martin C.; Warren, Warren S.

2015-01-01

Metastatic melanoma is associated with a poor prognosis, but no method reliably predicts which melanomas of a given stage will ultimately metastasize and which will not. While sentinel lymph node biopsy (SLNB) has emerged as the most powerful predictor of metastatic disease, the majority of people dying from metastatic melanoma still have a negative SLNB. Here we analyze pump-probe microscopy images of thin biopsy slides of primary melanomas to assess their metastatic potential. Pump-probe microscopy reveals detailed chemical information of melanin with subcellular spatial resolution. Quantification of the molecular signatures without reference standards is achieved using a geometrical representation of principal component analysis. Melanin structure is analyzed in unison with the chemical information by applying principles of mathematical morphology. Results show that melanin in metastatic primary lesions has lower chemical diversity than non-metastatic primary lesions, and contains two distinct phenotypes that are indicative of aggressive disease. Further, the mathematical morphology analysis reveals melanin in metastatic primary lesions has a distinct “dusty” quality. Finally, a statistical analysis shows that the combination of the chemical information with spatial structures predicts metastatic potential with much better sensitivity than SLNB and high specificity, suggesting pump-probe microscopy can be an important tool to help predict the metastatic potential of melanomas. PMID:26417529

In vivo metabolic fingerprinting of neutral lipids with hyperspectral stimulated Raman scattering microscopy.

PubMed

Fu, Dan; Yu, Yong; Folick, Andrew; Currie, Erin; Farese, Robert V; Tsai, Tsung-Huang; Xie, Xiaoliang Sunney; Wang, Meng C

2014-06-18

Metabolic fingerprinting provides valuable information on the physiopathological states of cells and tissues. Traditional imaging mass spectrometry and magnetic resonance imaging are unable to probe the spatial-temporal dynamics of metabolites at the subcellular level due to either lack of spatial resolution or inability to perform live cell imaging. Here we report a complementary metabolic imaging technique that is based on hyperspectral stimulated Raman scattering (hsSRS). We demonstrated the use of hsSRS imaging in quantifying two major neutral lipids: cholesteryl ester and triacylglycerol in cells and tissues. Our imaging results revealed previously unknown changes of lipid composition associated with obesity and steatohepatitis. We further used stable-isotope labeling to trace the metabolic dynamics of fatty acids in live cells and live Caenorhabditis elegans with hsSRS imaging. We found that unsaturated fatty acid has preferential uptake into lipid storage while saturated fatty acid exhibits toxicity in hepatic cells. Simultaneous metabolic fingerprinting of deuterium-labeled saturated and unsaturated fatty acids in living C. elegans revealed that there is a lack of interaction between the two, unlike previously hypothesized. Our findings provide new approaches for metabolic tracing of neutral lipids and their precursors in living cells and organisms, and could potentially serve as a general approach for metabolic fingerprinting of other metabolites.

Subcellular Localized Chemical Imaging of Benthic Algal Nutritional Content via HgCdTe Array FT-IR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wetzel, D.; Murdock, J; Dodds, W

2008-01-01

Algae respond rapidly and uniquely to changes in nutrient availability by adjusting pigment, storage product, and organelle content and quality. Cellular and subcellular variability of the relative abundance of macromolecular pools (e.g. protein, lipid, carbohydrate, and phosphodiesters) within the benthic (bottom dwelling) alga Cladophora glomerata (a common nuisance species in fresh and saline waters) was revealed by FT-IR microspectroscopic imaging. Nutrient heterogeneity was compared at the filament, cellular, and subcellular level, and localized nutrient uptake kinetics were studied by detecting the gradual incorporation of isotopically labeled nitrogen (N) (as K15NO3) from surrounding water into cellular proteins. Nutritional content differed substantiallymore » among filament cells, with differences driven by protein and lipid abundance. Whole cell imaging showed high subcellular macromolecular variability in all cells, including adjacent cells on a filament that developed clonally. N uptake was also very heterogeneous, both within and among cells, and did not appear to coincide with subcellular protein distribution. Despite high intercellular variability, some patterns emerged. Cells acquired more 15N the further they were away from the filament attachment point, and 15N incorporation was more closely correlated with phosphodiester content than protein, lipid, or carbohydrate content. Benthic algae are subject to substantial environmental heterogeneity induced by microscale hydrodynamic factors and spatial variability in nutrient availability. Species specific responses to nutrient heterogeneity are central to understanding this key component of aquatic ecosystems. FT-IR microspectroscopy, modified for benthic algae, allows determination of algal physiological responses at scales not available using current techniques.« less

Compartmental Genomics in Living Cells Revealed by Single-Cell Nanobiopsy

PubMed Central

Actis, Paolo; Maalouf, Michelle; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R. Adam; Pourmand, Nader

2014-01-01

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis. PMID:24279711

Understanding subcellular function on the nanometer scale in real time: Single-molecule imaging in living bacteria

NASA Astrophysics Data System (ADS)

Biteen, Julie

It has long been recognized that microorganisms play a central role in our lives. By beating the diffraction limit that restricts traditional light microscopy, single-molecule fluorescence imaging is a precise, noninvasive way to sensitively probe position and dynamics, even in living cells. We are pioneering this super-resolution imaging method for unraveling important biological processes in live bacteria, and I will discuss how we infer function from subcellular dynamics (Tuson and Biteen, Analytical Chemistry 2015). In particular, we have understood the mechanism of membrane-bound transcription regulation in the pathogenic Vibrio cholerae, revealed an intimate and dynamic coupling between DNA mismatch recognition and DNA replication, and measured starch utilization in an important member of the human gut microbiome.

The Secret Life of RNA: Lessons from Emerging Methodologies.

PubMed

Medioni, Caroline; Besse, Florence

2018-01-01

The last past decade has witnessed a revolution in our appreciation of transcriptome complexity and regulation. This remarkable expansion in our knowledge largely originates from the advent of high-throughput methodologies, and the consecutive discovery that up to 90% of eukaryotic genomes are transcribed, thus generating an unanticipated large range of noncoding RNAs (Hangauer et al., 15(4):112, 2014). Besides leading to the identification of new noncoding RNA species, transcriptome-wide studies have uncovered novel layers of posttranscriptional regulatory mechanisms controlling RNA processing, maturation or translation, and each contributing to the precise and dynamic regulation of gene expression. Remarkably, the development of systems-level studies has been accompanied by tremendous progress in the visualization of individual RNA molecules in single cells, such that it is now possible to image RNA species with a single-molecule resolution from birth to translation or decay. Monitoring quantitatively, with unprecedented spatiotemporal resolution, the fate of individual molecules has been key to understanding the molecular mechanisms underlying the different steps of RNA regulation. This has also revealed biologically relevant, intracellular and intercellular heterogeneities in RNA distribution or regulation. More recently, the convergence of imaging and high-throughput technologies has led to the emergence of spatially resolved transcriptomic techniques that provide a means to perform large-scale analyses while preserving spatial information. By generating transcriptome-wide data on single-cell RNA content, or even subcellular RNA distribution, these methodologies are opening avenues to a wide range of network-level studies at the cell and organ-level, and promise to strongly improve disease diagnostic and treatment.In this introductory chapter, we highlight how recently developed technologies aiming at detecting and visualizing RNA molecules have contributed to the emergence of entirely new research fields, and to dramatic progress in our understanding of gene expression regulation.

Spatial downscaling of soil prediction models based on weighted generalized additive models in smallholder farm settings.

PubMed

Xu, Yiming; Smith, Scot E; Grunwald, Sabine; Abd-Elrahman, Amr; Wani, Suhas P; Nair, Vimala D

2017-09-11

Digital soil mapping (DSM) is gaining momentum as a technique to help smallholder farmers secure soil security and food security in developing regions. However, communications of the digital soil mapping information between diverse audiences become problematic due to the inconsistent scale of DSM information. Spatial downscaling can make use of accessible soil information at relatively coarse spatial resolution to provide valuable soil information at relatively fine spatial resolution. The objective of this research was to disaggregate the coarse spatial resolution soil exchangeable potassium (K ex ) and soil total nitrogen (TN) base map into fine spatial resolution soil downscaled map using weighted generalized additive models (GAMs) in two smallholder villages in South India. By incorporating fine spatial resolution spectral indices in the downscaling process, the soil downscaled maps not only conserve the spatial information of coarse spatial resolution soil maps but also depict the spatial details of soil properties at fine spatial resolution. The results of this study demonstrated difference between the fine spatial resolution downscaled maps and fine spatial resolution base maps is smaller than the difference between coarse spatial resolution base maps and fine spatial resolution base maps. The appropriate and economical strategy to promote the DSM technique in smallholder farms is to develop the relatively coarse spatial resolution soil prediction maps or utilize available coarse spatial resolution soil maps at the regional scale and to disaggregate these maps to the fine spatial resolution downscaled soil maps at farm scale.

Investigation of subcellular localization and dynamics of membrane proteins in living bacteria by combining optical micromanipulation and high-resolution microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Barroso Peña, Álvaro; Nieves, Marcos; Teper, Konrad; Wedlich-Soldner, Roland; Denz, Cornelia

2016-09-01

The plasma membrane serves as protective interface between cells and their environment. It also constitutes a hub for selective nutrient uptake and signal transduction. Increasing evidence over the last years indicates that, similar to eukaryotic cells, lateral membrane organization plays an important role in the regulation of prokaryotic signaling pathways. However, the mechanisms underlying this phenomenon are still poorly understood. Spatiotemporal characterization of bacterial signal transduction demands very sensitive high-resolution microscopy techniques due to the low expression levels of most signaling proteins and the small size of bacterial cells. In addition, direct study of subcellular confinement and dynamics of bacterial signaling proteins during the different stages of the signal transduction also requires immobilization in order to avoid cell displacement caused by Brownian motion, local fluid flows and bacterial self-propulsion. In this work we present a novel approach based on the combination of high resolution imaging and optical manipulation that enables the investigation of the distribution and dynamics of proteins at the bacterial plasma membrane. For this purpose, we combine the versatility of holographic optical tweezers (HOT) with the sensitivity and resolution of total internal reflection fluorescence (TIRF) microscopy. Furthermore, we discuss the implementation of microfluidic devices in our integrated HOT+TIRF system for the control of growth conditions of bacterial cells. The capabilities of our workstation provides thus new valuable insights into the fundamental cellular and physical mechanisms underlying the regulation of bacterial signal transduction.

High-Resolution Intravital Microscopy

PubMed Central

Andresen, Volker; Pollok, Karolin; Rinnenthal, Jan-Leo; Oehme, Laura; Günther, Robert; Spiecker, Heinrich; Radbruch, Helena; Gerhard, Jenny; Sporbert, Anje; Cseresnyes, Zoltan; Hauser, Anja E.; Niesner, Raluca

2012-01-01

Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy - the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped-illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector. PMID:23251402

Micromanipulation by laser microbeam and optical tweezers: from plant cells to single molecules.

PubMed

Greulich, K O; Pilarczyk, G; Hoffmann, A; Meyer Zu Hörste, G; Schäfer, B; Uhl, V; Monajembashi, S

2000-06-01

Complete manipulation by laser light allows precise and gentle treatment of plant cells, subcellular structures, and even individual DNA molecules. Recently, affordable lasers have become available for the construction of microbeams as well as for optical tweezers. This may generate new interest in these tools for plant biologists. Early experiments, reviewed in this journal, showed that laser supported microinjection of material into plant cells or tissues circumvents mechanical problems encountered in microinjection by fragile glass capillaries. Plant protoplasts could be fused with each other when under microscopical observation, and it was no major problem to generate a triple or quadruple fusion product. In the present paper we review experiments where membrane material was prepared from root hair tips and microgravity was simulated in algae. As many plant cells are transparent, it is possible to work inside living, intact cells. New experiments show that it is possible to release by optical micromanipulation, with high spatial resolutions, intracellular calcium from caged compounds and to study calcium oscillations. An example for avian cardiac tissue is given, but the technique is also suitable for plant cell research. As a more technical tool, optical tweezers can be used to spatially fix subcellular structures otherwise moving inside a cell and thus make them available for investigation with a confocal microscope even when the time for image formation is extended (for example at low fluorescence emission). A molecular biological example is the handling of chromosomes and isolated individual DNA molecules by laser microtools. For example, chromosomes can be cut along complex trajectories, not only perpendicular to their long axis. Single DNA molecules are cut by the laser microbeam and, after coupling such a molecule to a polystrene microbead, are handled in complex geometries. Here, the individual DNA molecules are made visible with a conventional fluorescence microscope by fluorescent dyes such as SYBRGreen. The cutting of a single DNA molecule by molecules of the restriction endonuclease EcoRI can be observed directly, i.e. a type of single molecule restriction analysis is possible. Finally, mechanical properties of individual DNA molecules can be observed directly.

Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.

PubMed

Wegel, Eva; Göhler, Antonia; Lagerholm, B Christoffer; Wainman, Alan; Uphoff, Stephan; Kaufmann, Rainer; Dobbie, Ian M

2016-06-06

Many biological questions require fluorescence microscopy with a resolution beyond the diffraction limit of light. Super-resolution methods such as Structured Illumination Microscopy (SIM), STimulated Emission Depletion (STED) microscopy and Single Molecule Localisation Microscopy (SMLM) enable an increase in image resolution beyond the classical diffraction-limit. Here, we compare the individual strengths and weaknesses of each technique by imaging a variety of different subcellular structures in fixed cells. We chose examples ranging from well separated vesicles to densely packed three dimensional filaments. We used quantitative and correlative analyses to assess the performance of SIM, STED and SMLM with the aim of establishing a rough guideline regarding the suitability for typical applications and to highlight pitfalls associated with the different techniques.

In vivo observation of transient photoreceptor movement correlated with oblique light stimulation

NASA Astrophysics Data System (ADS)

Lu, Yiming; Liu, Changgeng; Yao, Xincheng

2018-02-01

Rod-dominated transient retinal phototropism (TRP) has been observed in freshly isolated retinas, promising a noninvasive biomarker for high resolution assessment of retinal physiology. However, in vivo mapping of TRP is challenging due to its fast time course and sub-cellular signal magnitude. By developing a line-scanning and virtually structured detection based super-resolution ophthalmoscope, we report here in vivo observation of TRP in frog retina. In vivo characterization of TRP time course and magnitude were implemented by using variable light stimulus intensities.

Molecular signaling in live cells studied by FRET

NASA Astrophysics Data System (ADS)

Chien, Shu; Wang, Yingxiao

2011-11-01

Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) enables visualization of signaling events in live cells with high spatiotemporal resolution. We have used FRET to assess temporal and spatial characteristics for signaling molecules, including tyrosine kinases Src and FAK, small GTPase Rac, calcium, and a membrane-bound matrix metalloproteinase MT1-MMP. Activations of Src and Rac by platelet derived growth factor (PDGF) led to distinct subcellular patterns during cell migration on micropatterned surface, and these two enzymes interact with each other to form a feedback loop with differential regulations at different subcellular locations. We have developed FRET biosensors to monitor FAK activities at rafts vs. non-raft regions of plasma membrane in live cells. In response to cell adhesion on matrix proteins or stimulation by PDGF, the raft-targeting FAK biosensor showed a stronger FRET response than that at non-rafts. The FAK activation at rafts induced by PDGF is mediated by Src. In contrast, the FAK activation at rafts induced by adhesion is independent of Src activity, but rather is essential for Src activation. Thus, Src is upstream to FAK in response to chemical stimulation (PDGF), but FAK is upstream to Src in response to mechanical stimulation (adhesion). A novel biosensor has been developed to dynamically visualize the activity of membrane type-1-matrix metalloproteinase (MT1-MMP), which proteolytically remodels the extracellular matrix. Epidermal growth factor (EGF) directed active MT1-MMP to the leading edge of migrating live cancer cells with local accumulation of EGF receptor via a process dependent on an intact cytoskeletal network. In summary, FRET-based biosensors enable the elucidation of molecular processes and hierarchies underlying spatiotemporal regulation of biological and pathological processes, thus advancing our knowledge on how cells perceive mechanical/chemical cues in space and time to coordinate molecular/cellular functions.

Molecular signaling in live cells studied by FRET

NASA Astrophysics Data System (ADS)

Chien, Shu; Wang, Yingxiao

2012-03-01

Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) enables visualization of signaling events in live cells with high spatiotemporal resolution. We have used FRET to assess temporal and spatial characteristics for signaling molecules, including tyrosine kinases Src and FAK, small GTPase Rac, calcium, and a membrane-bound matrix metalloproteinase MT1-MMP. Activations of Src and Rac by platelet derived growth factor (PDGF) led to distinct subcellular patterns during cell migration on micropatterned surface, and these two enzymes interact with each other to form a feedback loop with differential regulations at different subcellular locations. We have developed FRET biosensors to monitor FAK activities at rafts vs. non-raft regions of plasma membrane in live cells. In response to cell adhesion on matrix proteins or stimulation by PDGF, the raft-targeting FAK biosensor showed a stronger FRET response than that at non-rafts. The FAK activation at rafts induced by PDGF is mediated by Src. In contrast, the FAK activation at rafts induced by adhesion is independent of Src activity, but rather is essential for Src activation. Thus, Src is upstream to FAK in response to chemical stimulation (PDGF), but FAK is upstream to Src in response to mechanical stimulation (adhesion). A novel biosensor has been developed to dynamically visualize the activity of membrane type-1-matrix metalloproteinase (MT1-MMP), which proteolytically remodels the extracellular matrix. Epidermal growth factor (EGF) directed active MT1-MMP to the leading edge of migrating live cancer cells with local accumulation of EGF receptor via a process dependent on an intact cytoskeletal network. In summary, FRET-based biosensors enable the elucidation of molecular processes and hierarchies underlying spatiotemporal regulation of biological and pathological processes, thus advancing our knowledge on how cells perceive mechanical/chemical cues in space and time to coordinate molecular/cellular functions.

Visualizing Single Cell Biology: Nanosims Studies of Carbon and Nitrogen Metabolism in Diazotrophic Cyanobacteria

NASA Astrophysics Data System (ADS)

Pett-Ridge, J.; Finzi, J. A.; Capone, D. G.; Popa, R.; Nealson, K. H.; Ng, W.; Spormann, A. M.; Hutcheon, I. D.; Weber, P. K.

2007-12-01

Filamentous nitrogen fixing (diazotrophic) cyanobacteria are key players in global nutrient cycling, but the relationship between CO2- and N2-fixation and intercellular exchange of these elements remains poorly understood in many genera. These bacteria are faced with the challenge of isolating regions of N-fixation (O2 inhibited) and photosynthetic (O2 producing) activity. We used isotope labeling in conjunction with a high-resolution isotope and elemental mapping technique (NanoSIMS) to quantitatively describe 13C and 15N uptake and transport in two aquatic cyanobacteria grown on NaH13CO3 and 15N2. The technical challenges of tracing isotopes within individual bacteria can be overcome with high resolution Secondary Ion Mass Spectrometry (NanoSIMS). In NanoSIMS analysis, samples are sputtered with an energetic primary beam (Cs+, O-) liberating secondary ions that are separated by the mass spectrometer and detected in a suite of electron multipliers. Five isotopic species may be analyzed concurrently with spatial resolution as fine as 50nm. A high sensitivity isotope ratio 'map' can then be generated for the analyzed area. Using sequentially harvested cyanobacteria in conjunction with enriched H13CO3 and 15N2 incubations, we measured temporal enrichment patterns that evolve over the course of a day's growth and suggest tightly regulated changes in fixation kinetics. With a combination of TEM, SEM and NanoSIMS analyses, we also mapped the distribution of C, N and Mo (a critical nitrogenase co-factor) isotopes in intact cells. Our results suggest that NanoSIMS mapping of metal enzyme co-factors may be a powerful method of identifying physiological and morphological characteristics within individual bacterial cells, and could be used to provide a 3-dimensional context for more traditional analyses such as immunogold labeling. Finally, we resolved patterns of isotope enrichment at multiple spatial scales: sub-cellular variation, cell-cell differences along filaments, inter-species transfers (with Rhizobium epibionts), and within-cell depth profiles. Spatial enrichment patterns were correlated with morphological features evidenced in TEM images of microtomed filaments. These features indicate how 15N and 13C "hotspots" are dispersed throughout individual cells in different species, and may indicate isolated locations of increased N2 fixation, sites of amino acid/protein synthesis, or cyanophycin storage granules. This combination of Nano-Secondary Ion Mass Spectrometry (NanoSIMS) analysis and high resolution microscopy allows isotopic analysis to be linked to morphological features and holds great promise for fine-scale studies of bacteria metabolism.

Enhancing Spatial Resolution of Remotely Sensed Imagery Using Deep Learning

NASA Astrophysics Data System (ADS)

Beck, J. M.; Bridges, S.; Collins, C.; Rushing, J.; Graves, S. J.

2017-12-01

Researchers at the Information Technology and Systems Center at the University of Alabama in Huntsville are using Deep Learning with Convolutional Neural Networks (CNNs) to develop a method for enhancing the spatial resolutions of moderate resolution (10-60m) multispectral satellite imagery. This enhancement will effectively match the resolutions of imagery from multiple sensors to provide increased global temporal-spatial coverage for a variety of Earth science products. Our research is centered on using Deep Learning for automatically generating transformations for increasing the spatial resolution of remotely sensed images with different spatial, spectral, and temporal resolutions. One of the most important steps in using images from multiple sensors is to transform the different image layers into the same spatial resolution, preferably the highest spatial resolution, without compromising the spectral information. Recent advances in Deep Learning have shown that CNNs can be used to effectively and efficiently upscale or enhance the spatial resolution of multispectral images with the use of an auxiliary data source such as a high spatial resolution panchromatic image. In contrast, we are using both the spatial and spectral details inherent in low spatial resolution multispectral images for image enhancement without the use of a panchromatic image. This presentation will discuss how this technology will benefit many Earth Science applications that use remotely sensed images with moderate spatial resolutions.

Mass Measurements of Focal Adhesions in Single Cells Using High Resolution Surface Plasmon Resonance Microscopy.

PubMed

Peterson, Alexander W; Halter, Michael; Tona, Alessandro; Plant, Anne L; Elliott, John T

2018-01-01

Surface plasmon resonance microscopy (SPRM) is a powerful label-free imaging technique with spatial resolution approaching the optical diffraction limit. The high sensitivity of SPRM to small changes in index of refraction at an interface allows imaging of dynamic protein structures within a cell. Visualization of subcellular features, such as focal adhesions (FAs), can be performed on live cells using a high numerical aperture objective lens with a digital light projector to precisely position the incident angle of the excitation light. Within the cell-substrate region of the SPRM image, punctate regions of high contrast are putatively identified as the cellular FAs. Optical parameter analysis is achieved by application of the Fresnel model to the SPRM data and resulting refractive index measurements are used to calculate protein density and mass. FAs are known to be regions of high protein density that reside at the cell-substratum interface. Comparing SPRM with fluorescence images of antibody stained for vinculin, a component in FAs, reveals similar measurements of FA size. In addition, a positive correlation between FA size and protein density is revealed by SPRM. Comparing SPRM images for two cell types reveals a distinct difference in the protein density and mass of their respective FAs. Application of SPRM to quantify mass can greatly aid monitoring basic processes that control FA mass and growth and contribute to accurate models that describe cell-extracellular interactions.

Cell counting in whole mount tissue volumes using expansion OCT (Conference Presentation)

NASA Astrophysics Data System (ADS)

Liu, Yehe; Gu, Shi; Watanabe, Michiko; Rollins, Andrew M.; Jenkins, Michael W.

2017-02-01

Abnormal cell proliferation and migration during heart development can lead to severe congenital heart defects (CHDs). Studying the spatial distribution of cells during embryonic development helps our understanding of how the heart develops and the etiology of certain CHDs. However, imaging large groups of single cells in intact tissue volumes is challenging. No current technique can accomplish this task in both a time-efficient and cost-effective manner. OCT has potential with its large field of view and micron-scale resolution, but even the highest resolution OCT systems have poor contrast for counting cells and have a small field of view compared to conventional OCT. We propose using a conventional OCT system and processing the sample to enhance cellular contrast. Inspired by the recently developed Expansion Microscopy, we permeated whole-mount embryonic tissue with a superabsorbent monomer solution and polymerized into a hydrogel. When hydrated in DI water, the tissue-hydrogel complex was uniformly enlarged ( 5X in all dimensions) without distorting the microscopic structure. This had a twofold effect: it increased the resolution by a factor of 5 and decreased scattering, which allowed us to resolve cellular level features deep in the tissue with high contrast using conventional OCT. We noted that cell nuclei caused significantly more backscattering than the other subcellular structures after expansion. Based on this property, we were able to distinguish individual cell nuclei, and thus count cells, in expanded OCT images with simple intensity thresholding. We demonstrate the technique with embryonic quail hearts at various developmental stages.

Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina.

PubMed

Zawadzki, Robert J; Zhang, Pengfei; Zam, Azhar; Miller, Eric B; Goswami, Mayank; Wang, Xinlei; Jonnal, Ravi S; Lee, Sang-Hyuck; Kim, Dae Yu; Flannery, John G; Werner, John S; Burns, Marie E; Pugh, Edward N

2015-06-01

Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a large-scale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFP-expressing microglia, the resident macrophages of the retina, and GFP-expressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed.

Exploring photoinactivation of microbial biofilms using laser scanning microscopy and confined two-photon excitation.

PubMed

Thomsen, Hanna; Graf, Fabrice E; Farewell, Anne; Ericson, Marica B

2018-05-21

One pertinent complication in bacterial infection is the growth of biofilms, i.e., communities of surface-adhered bacteria resilient to antibiotics. Photodynamic inactivation has been proposed as an alternative to antibiotic treatment; however, novel techniques complementing standard efficacy measures are required. Herein, we present an approach employing multiphoton microscopy complemented with Airyscan super-resolution microscopy, to visualize the distribution of curcumin in Staphylococcus epidermidis biofilms. The effects of complexation of curcumin with hydroxypropyl-γ-cyclodextrin (HPγCD) were studied. It was shown that HPγCD-curcumin demonstrated higher bioavailability in the biofilms compared to curcumin, without affecting the subcellular uptake. Spectral quantification following photodynamic inactivation demonstrates a method for monitoring elimination of biofilms in real time using noninvasive 3D imaging. Additionally, spatially confined two-photon inactivation was demonstrated for the first time in biofilms. These results support the feasibility of advanced optical microscopy as a sensitive tool for evaluating treatment efficacy in biofilms towards improved mechanistic studies of photodynamic inactivation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

High-resolution microendoscope for the detection of cervical neoplasia.

PubMed

Grant, Benjamin D; Schwarz, Richard A; Quang, Timothy; Schmeler, Kathleen M; Richards-Kortum, Rebecca

2015-01-01

Cervical cancer causes 275,000 deaths each year with 85 % of these deaths occurring in the developing world. One of the primary reasons for the concentration of deaths in developing countries is a lack of effective screening methods suited for the infrastructure of these countries. In order to address this need, we have developed a high-resolution microendoscope (HRME). The HRME is a fiber-based fluorescence microscope with subcellular resolution. Using the vital stain proflavine, we are able to image cell nuclei in vivo and evaluate metrics such as nuclear-to-cytoplasmic ratio, critical to identifying precancerous epithelial regions. In this chapter, we detail the materials and methods necessary to build this system from commercially available parts.

Motion compensation for in vivo subcellular optical microscopy.

PubMed

Lucotte, B; Balaban, R S

2014-04-01

In this review, we focus on the impact of tissue motion on attempting to conduct subcellular resolution optical microscopy, in vivo. Our position is that tissue motion is one of the major barriers in conducting these studies along with light induced damage, optical probe loading as well as absorbing and scattering effects on the excitation point spread function and collection of emitted light. Recent developments in the speed of image acquisition have reached the limit, in most cases, where the signal from a subcellular voxel limits the speed and not the scanning rate of the microscope. Different schemes for compensating for tissue displacements due to rigid body and deformation are presented from tissue restriction, gating, adaptive gating and active tissue tracking. We argue that methods that minimally impact the natural physiological motion of the tissue are desirable because the major reason to perform in vivo studies is to evaluate normal physiological functions. Towards this goal, active tracking using the optical imaging data itself to monitor tissue displacement and either prospectively or retrospectively correct for the motion without affecting physiological processes is desirable. Critical for this development was the implementation of near real time image processing in conjunction with the control of the microscope imaging parameters. Clearly, the continuing development of methods of motion compensation as well as significant technological solutions to the other barriers to tissue subcellular optical imaging in vivo, including optical aberrations and overall signal-to-noise ratio, will make major contributions to the understanding of cell biology within the body.

Effects of spatial resolution ratio in image fusion

USGS Publications Warehouse

Ling, Y.; Ehlers, M.; Usery, E.L.; Madden, M.

2008-01-01

In image fusion, the spatial resolution ratio can be defined as the ratio between the spatial resolution of the high-resolution panchromatic image and that of the low-resolution multispectral image. This paper attempts to assess the effects of the spatial resolution ratio of the input images on the quality of the fused image. Experimental results indicate that a spatial resolution ratio of 1:10 or higher is desired for optimal multisensor image fusion provided the input panchromatic image is not downsampled to a coarser resolution. Due to the synthetic pixels generated from resampling, the quality of the fused image decreases as the spatial resolution ratio decreases (e.g. from 1:10 to 1:30). However, even with a spatial resolution ratio as small as 1:30, the quality of the fused image is still better than the original multispectral image alone for feature interpretation. In cases where the spatial resolution ratio is too small (e.g. 1:30), to obtain better spectral integrity of the fused image, one may downsample the input high-resolution panchromatic image to a slightly lower resolution before fusing it with the multispectral image.

High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels†

PubMed Central

Müller, Jan; Ballini, Marco; Livi, Paolo; Chen, Yihui; Radivojevic, Milos; Shadmani, Amir; Viswam, Vijay; Jones, Ian L.; Fiscella, Michele; Diggelmann, Roland; Stettler, Alexander; Frey, Urs; Bakkum, Douglas J.; Hierlemann, Andreas

2017-01-01

Studies on information processing and learning properties of neuronal networks would benefit from simultaneous and parallel access to the activity of a large fraction of all neurons in such networks. Here, we present a CMOS-based device, capable of simultaneously recording the electrical activity of over a thousand cells in in vitro neuronal networks. The device provides sufficiently high spatiotemporal resolution to enable, at the same time, access to neuronal preparations on subcellular, cellular, and network level. The key feature is a rapidly reconfigurable array of 26 400 microelectrodes arranged at low pitch (17.5 μm) within a large overall sensing area (3.85 × 2.10 mm2). An arbitrary subset of the electrodes can be simultaneously connected to 1024 low-noise readout channels as well as 32 stimulation units. Each electrode or electrode subset can be used to electrically stimulate or record the signals of virtually any neuron on the array. We demonstrate the applicability and potential of this device for various different experimental paradigms: large-scale recordings from whole networks of neurons as well as investigations of axonal properties of individual neurons. PMID:25973786

Emerging optical nanoscopy techniques

PubMed Central

Montgomery, Paul C; Leong-Hoi, Audrey

2015-01-01

To face the challenges of modern health care, new imaging techniques with subcellular resolution or detection over wide fields are required. Far field optical nanoscopy presents many new solutions, providing high resolution or detection at high speed. We present a new classification scheme to help appreciate the growing number of optical nanoscopy techniques. We underline an important distinction between superresolution techniques that provide improved resolving power and nanodetection techniques for characterizing unresolved nanostructures. Some of the emerging techniques within these two categories are highlighted with applications in biophysics and medicine. Recent techniques employing wider angle imaging by digital holography and scattering lens microscopy allow superresolution to be achieved for subcellular and even in vivo, imaging without labeling. Nanodetection techniques are divided into four subcategories using contrast, phase, deconvolution, and nanomarkers. Contrast enhancement is illustrated by means of a polarized light-based technique and with strobed phase-contrast microscopy to reveal nanostructures. Very high sensitivity phase measurement using interference microscopy is shown to provide nanometric surface roughness measurement or to reveal internal nanometric structures. Finally, the use of nanomarkers is illustrated with stochastic fluorescence microscopy for mapping intracellular structures. We also present some of the future perspectives of optical nanoscopy. PMID:26491270

Two-photon excited autofluorescence imaging of freshly isolated frog retinas.

PubMed

Lu, Rong-Wen; Li, Yi-Chao; Ye, Tong; Strang, Christianne; Keyser, Kent; Curcio, Christine A; Yao, Xin-Cheng

2011-06-01

The purpose of this study was to investigate cellular sources of autofluorescence signals in freshly isolated frog (Rana pipiens) retinas. Equipped with an ultrafast laser, a laser scanning two-photon excitation fluorescence microscope was employed for sub-cellular resolution examination of both sliced and flat-mounted retinas. Two-photon imaging of retinal slices revealed autofluorescence signals over multiple functional layers, including the photoreceptor layer (PRL), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Using flat-mounted retinas, depth-resolved imaging of individual retinal layers further confirmed multiple sources of autofluorescence signals. Cellular structures were clearly observed at the PRL, ONL, INL, and GCL. At the PRL, the autofluorescence was dominantly recorded from the intracellular compartment of the photoreceptors; while mixed intracellular and extracellular autofluorescence signals were observed at the ONL, INL, and GCL. High resolution autofluorescence imaging clearly revealed mosaic organization of rod and cone photoreceptors; and sub-cellular bright autofluorescence spots, which might relate to connecting cilium, was observed in the cone photoreceptors only. Moreover, single-cone and double-cone outer segments could be directly differentiated.

High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels.

PubMed

Müller, Jan; Ballini, Marco; Livi, Paolo; Chen, Yihui; Radivojevic, Milos; Shadmani, Amir; Viswam, Vijay; Jones, Ian L; Fiscella, Michele; Diggelmann, Roland; Stettler, Alexander; Frey, Urs; Bakkum, Douglas J; Hierlemann, Andreas

2015-07-07

Studies on information processing and learning properties of neuronal networks would benefit from simultaneous and parallel access to the activity of a large fraction of all neurons in such networks. Here, we present a CMOS-based device, capable of simultaneously recording the electrical activity of over a thousand cells in in vitro neuronal networks. The device provides sufficiently high spatiotemporal resolution to enable, at the same time, access to neuronal preparations on subcellular, cellular, and network level. The key feature is a rapidly reconfigurable array of 26 400 microelectrodes arranged at low pitch (17.5 μm) within a large overall sensing area (3.85 × 2.10 mm(2)). An arbitrary subset of the electrodes can be simultaneously connected to 1024 low-noise readout channels as well as 32 stimulation units. Each electrode or electrode subset can be used to electrically stimulate or record the signals of virtually any neuron on the array. We demonstrate the applicability and potential of this device for various different experimental paradigms: large-scale recordings from whole networks of neurons as well as investigations of axonal properties of individual neurons.

Quantitative evaluation of boron neutron capture therapy (BNCT) drugs for boron delivery and retention at subcellular scale resolution in human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS)

PubMed Central

Chandra, S.; Ahmad, T.; Barth, R. F.; Kabalka, G. W.

2014-01-01

Boron neutron capture therapy (BNCT) of cancer depends on the selective delivery of a sufficient number of boron-10 (10B) atoms to individual tumor cells. Cell killing results from the 10B (n, α)7Li neutron capture and fission reactions that occur if a sufficient number of 10B atoms are localized in the tumor cells. Intranuclear 10B localization enhances the efficiency of cell killing via damage to the DNA. The net cellular content of 10B atoms reflects both bound and free pools of boron in individual tumor cells. The assessment of these pools, delivered by a boron delivery agent, currently cannot be made at subcellular scale resolution by clinically applicable techniques such as PET and MRI. In this study, secondary ion mass spectrometry (SIMS) based imaging instrument, a CAMECA IMS 3f ion microscope, capable of 500 nm spatial resolution was employed. Cryogenically prepared cultured human T98G glioblastoma cells were evaluated for boron uptake and retention of two delivery agents. The first, L-p-boronophenylalanine (BPA), has been used clinically for BNCT of high grade gliomas, recurrent tumors of the head and neck region and melanomas. The second, a boron analogue of an unnatural amino acid, 1-amino-3-borono-cyclopentanecarboxylic acid (cis-ABCPC), has been studied in rodent glioma and melanoma models by quantification of boron in the nucleus and cytoplasm of individual tumor cells. The bound and free pools of boron were assessed by exposure of cells to boron-free nutrient medium. Both BPA and cis-ABCPC delivered almost 70% of the pool of boron in the free or loosely bound form to the nucleus and cytoplasm of human glioblastoma cells. This free pool of boron could be easily mobilized out of the cell and was in some sort of equilibrium with extracellular boron. In the case of BPA, the intracellular free pool of boron also was affected by the presence of phenylalanine in the nutrient medium. This suggests that it might be advantageous if patients were placed on a low phenylalanine diet prior to the initiation of BNCT. Since BPA currently is used clinically for BNCT, our observations may have direct relevance to future clinical studies utilizing this agent and provides support for individualized treatment planning regimens rather than the use of fixed BPA infusion protocols. PMID:24684609

Quantitative evaluation of boron neutron capture therapy (BNCT) drugs for boron delivery and retention at subcellular-scale resolution in human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS).

PubMed

Chandra, S; Ahmad, T; Barth, R F; Kabalka, G W

2014-06-01

Boron neutron capture therapy (BNCT) of cancer depends on the selective delivery of a sufficient number of boron-10 ((10)B) atoms to individual tumour cells. Cell killing results from the (10)B (n, α)(7) Li neutron capture and fission reactions that occur if a sufficient number of (10)B atoms are localized in the tumour cells. Intranuclear (10)B localization enhances the efficiency of cell killing via damage to the DNA. The net cellular content of (10)B atoms reflects both bound and free pools of boron in individual tumour cells. The assessment of these pools, delivered by a boron delivery agent, currently cannot be made at subcellular-scale resolution by clinically applicable techniques such as positron emission tomography and magnetic resonance imaging. In this study, a secondary ion mass spectrometry based imaging instrument, a CAMECA IMS 3f ion microscope, capable of 500 nm spatial resolution was employed. Cryogenically prepared cultured human T98G glioblastoma cells were evaluated for boron uptake and retention of two delivery agents. The first, L-p-boronophenylalanine (BPA), has been used clinically for BNCT of high-grade gliomas, recurrent tumours of the head and neck region and melanomas. The second, a boron analogue of an unnatural amino acid, 1-amino-3-borono-cyclopentanecarboxylic acid (cis-ABCPC), has been studied in rodent glioma and melanoma models by quantification of boron in the nucleus and cytoplasm of individual tumour cells. The bound and free pools of boron were assessed by exposure of cells to boron-free nutrient medium. Both BPA and cis-ABCPC delivered almost 70% of the pool of boron in the free or loosely bound form to the nucleus and cytoplasm of human glioblastoma cells. This free pool of boron could be easily mobilized out of the cell and was in some sort of equilibrium with extracellular boron. In the case of BPA, the intracellular free pool of boron also was affected by the presence of phenylalanine in the nutrient medium. This suggests that it might be advantageous if patients were placed on a low phenylalanine diet prior to the initiation of BNCT. Since BPA currently is used clinically for BNCT, our observations may have direct relevance to future clinical studies utilizing this agent and provides support for individualized treatment planning regimens rather than the use of fixed BPA infusion protocols. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

An Automatic Segmentation Method Combining an Active Contour Model and a Classification Technique for Detecting Polycomb-group Proteinsin High-Throughput Microscopy Images.

PubMed

Gregoretti, Francesco; Cesarini, Elisa; Lanzuolo, Chiara; Oliva, Gennaro; Antonelli, Laura

2016-01-01

The large amount of data generated in biological experiments that rely on advanced microscopy can be handled only with automated image analysis. Most analyses require a reliable cell image segmentation eventually capable of detecting subcellular structures.We present an automatic segmentation method to detect Polycomb group (PcG) proteins areas isolated from nuclei regions in high-resolution fluorescent cell image stacks. It combines two segmentation algorithms that use an active contour model and a classification technique serving as a tool to better understand the subcellular three-dimensional distribution of PcG proteins in live cell image sequences. We obtained accurate results throughout several cell image datasets, coming from different cell types and corresponding to different fluorescent labels, without requiring elaborate adjustments to each dataset.

IMART software for correction of motion artifacts in images collected in intravital microscopy

PubMed Central

Dunn, Kenneth W; Lorenz, Kevin S; Salama, Paul; Delp, Edward J

2014-01-01

Intravital microscopy is a uniquely powerful tool, providing the ability to characterize cell and organ physiology in the natural context of the intact, living animal. With the recent development of high-resolution microscopy techniques such as confocal and multiphoton microscopy, intravital microscopy can now characterize structures at subcellular resolution and capture events at sub-second temporal resolution. However, realizing the potential for high resolution requires remarkable stability in the tissue. Whereas the rigid structure of the skull facilitates high-resolution imaging of the brain, organs of the viscera are free to move with respiration and heartbeat, requiring additional apparatus for immobilization. In our experience, these methods are variably effective, so that many studies are compromised by residual motion artifacts. Here we demonstrate the use of IMART, a software tool for removing motion artifacts from intravital microscopy images collected in time series or in three dimensions. PMID:26090271

Compensation of missing wedge effects with sequential statistical reconstruction in electron tomography.

PubMed

Paavolainen, Lassi; Acar, Erman; Tuna, Uygar; Peltonen, Sari; Moriya, Toshio; Soonsawad, Pan; Marjomäki, Varpu; Cheng, R Holland; Ruotsalainen, Ulla

2014-01-01

Electron tomography (ET) of biological samples is used to study the organization and the structure of the whole cell and subcellular complexes in great detail. However, projections cannot be acquired over full tilt angle range with biological samples in electron microscopy. ET image reconstruction can be considered an ill-posed problem because of this missing information. This results in artifacts, seen as the loss of three-dimensional (3D) resolution in the reconstructed images. The goal of this study was to achieve isotropic resolution with a statistical reconstruction method, sequential maximum a posteriori expectation maximization (sMAP-EM), using no prior morphological knowledge about the specimen. The missing wedge effects on sMAP-EM were examined with a synthetic cell phantom to assess the effects of noise. An experimental dataset of a multivesicular body was evaluated with a number of gold particles. An ellipsoid fitting based method was developed to realize the quantitative measures elongation and contrast in an automated, objective, and reliable way. The method statistically evaluates the sub-volumes containing gold particles randomly located in various parts of the whole volume, thus giving information about the robustness of the volume reconstruction. The quantitative results were also compared with reconstructions made with widely-used weighted backprojection and simultaneous iterative reconstruction technique methods. The results showed that the proposed sMAP-EM method significantly suppresses the effects of the missing information producing isotropic resolution. Furthermore, this method improves the contrast ratio, enhancing the applicability of further automatic and semi-automatic analysis. These improvements in ET reconstruction by sMAP-EM enable analysis of subcellular structures with higher three-dimensional resolution and contrast than conventional methods.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duenas, Maria Emilia; Klein, Adam T.; Alexander, Liza E.

Metabolism in plants is compartmentalized among different tissues, cells and subcellular organelles. Mass spectrometry imaging (MSI) with matrix-assisted laser desorption ionization (MALDI) has recently advanced to allow for the visualization of metabolites at single-cell resolution. Here we applied 5- and 10 μm high spatial resolution MALDI-MSI to the asymmetric Kranz anatomy of Zea mays (maize) leaves to study the differential localization of two major anionic lipids in thylakoid membranes, sulfoquinovosyldiacylglycerols (SQDG) and phosphatidylglycerols (PG). The quantification and localization of SQDG and PG molecular species, among mesophyll (M) and bundle sheath (BS) cells, are compared across the leaf developmental gradient frommore » four maize genotypes (the inbreds B73 and Mo17, and the reciprocal hybrids B73 × Mo17 and Mo17 × B73). SQDG species are uniformly distributed in both photosynthetic cell types, regardless of leaf development or genotype; however, PG shows photosynthetic cell-specific differential localization depending on the genotype and the fatty acyl chain constituent. Overall, 16:1-containing PGs primarily contribute to the thylakoid membranes of M cells, whereas BS chloroplasts are mostly composed of 16:0-containing PGs. Furthermore, PG 32:0 shows genotype-specific differences in cellular distribution, with preferential localization in BS cells for B73, but more uniform distribution between BS and M cells in Mo17. Maternal inheritance is exhibited within the hybrids, such that the localization of PG 32:0 in B73 × Mo17 is similar to the distribution in the B73 parental inbred, whereas that of Mo17 × B73 resembles the Mo17 parent. As a result, this study demonstrates the power of MALDI-MSI to reveal unprecedented insights on metabolic outcomes in multicellular organisms at single-cell resolution.« less

Stimulated Emission Depletion Live-Cell Super-Resolution Imaging Shows Proliferative Remodeling of T-Tubule Membrane Structures After Myocardial Infarction

PubMed Central

Wagner, Eva; Lauterbach, Marcel A.; Kohl, Tobias; Westphal, Volker; Williams, George S.B.; Steinbrecher, Julia H.; Streich, Jan-Hendrik; Korff, Brigitte; Tuan, Hoang-Trong M.; Hagen, Brian; Luther, Stefan; Hasenfuss, Gerd; Parlitz, Ulrich; Jafri, M. Saleet; Hell, Stefan W.; Lederer, W. Jonathan; Lehnart, Stephan E.

2014-01-01

Rationale Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca2+ release and action potential prolongation. Conclusions This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing. PMID:22723297

Towards native-state imaging in biological context in the electron microscope

PubMed Central

Weston, Anne E.; Armer, Hannah E. J.

2009-01-01

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context. PMID:19916039

Discerning the Chemistry in Individual Organelles with Small-Molecule Fluorescent Probes.

PubMed

Xu, Wang; Zeng, Zebing; Jiang, Jian-Hui; Chang, Young-Tae; Yuan, Lin

2016-10-24

Principle has it that even the most advanced super-resolution microscope would be futile in providing biological insight into subcellular matrices without well-designed fluorescent tags/probes. Developments in biology have increasingly been boosted by advances of chemistry, with one prominent example being small-molecule fluorescent probes that not only allow cellular-level imaging, but also subcellular imaging. A majority, if not all, of the chemical/biological events take place inside cellular organelles, and researchers have been shifting their attention towards these substructures with the help of fluorescence techniques. This Review summarizes the existing fluorescent probes that target chemical/biological events within a single organelle. More importantly, organelle-anchoring strategies are described and emphasized to inspire the design of new generations of fluorescent probes, before concluding with future prospects on the possible further development of chemical biology. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Correlated optical and isotopic nanoscopy

NASA Astrophysics Data System (ADS)

Saka, Sinem K.; Vogts, Angela; Kröhnert, Katharina; Hillion, François; Rizzoli, Silvio O.; Wessels, Johannes T.

2014-04-01

The isotopic composition of different materials can be imaged by secondary ion mass spectrometry. In biology, this method is mainly used to study cellular metabolism and turnover, by pulsing the cells with marker molecules such as amino acids labelled with stable isotopes (15N, 13C). The incorporation of the markers is then imaged with a lateral resolution that can surpass 100 nm. However, secondary ion mass spectrometry cannot identify specific subcellular structures like organelles, and needs to be correlated with a second technique, such as fluorescence imaging. Here, we present a method based on stimulated emission depletion microscopy that provides correlated optical and isotopic nanoscopy (COIN) images. We use this approach to study the protein turnover in different organelles from cultured hippocampal neurons. Correlated optical and isotopic nanoscopy can be applied to a variety of biological samples, and should therefore enable the investigation of the isotopic composition of many organelles and subcellular structures.

Photothermally triggered actuation of hybrid materials as a new platform for in vitro cell manipulation

NASA Astrophysics Data System (ADS)

Sutton, Amy; Shirman, Tanya; Timonen, Jaakko V. I.; England, Grant T.; Kim, Philseok; Kolle, Mathias; Ferrante, Thomas; Zarzar, Lauren D.; Strong, Elizabeth; Aizenberg, Joanna

2017-03-01

Mechanical forces in the cell's natural environment have a crucial impact on growth, differentiation and behaviour. Few areas of biology can be understood without taking into account how both individual cells and cell networks sense and transduce physical stresses. However, the field is currently held back by the limitations of the available methods to apply physiologically relevant stress profiles on cells, particularly with sub-cellular resolution, in controlled in vitro experiments. Here we report a new type of active cell culture material that allows highly localized, directional and reversible deformation of the cell growth substrate, with control at scales ranging from the entire surface to the subcellular, and response times on the order of seconds. These capabilities are not matched by any other method, and this versatile material has the potential to bridge the performance gap between the existing single cell micro-manipulation and 2D cell sheet mechanical stimulation techniques.

A 1024-Channel CMOS Microelectrode Array With 26,400 Electrodes for Recording and Stimulation of Electrogenic Cells In Vitro

PubMed Central

Ballini, Marco; Müller, Jan; Livi, Paolo; Chen, Yihui; Frey, Urs; Stettler, Alexander; Shadmani, Amir; Viswam, Vijay; Jones, Ian Lloyd; Jäckel, David; Radivojevic, Milos; Lewandowska, Marta K.; Gong, Wei; Fiscella, Michele; Bakkum, Douglas J.; Heer, Flavio; Hierlemann, Andreas

2017-01-01

To advance our understanding of the functioning of neuronal ensembles, systems are needed to enable simultaneous recording from a large number of individual neurons at high spatiotemporal resolution and good signal-to-noise ratio. Moreover, stimulation capability is highly desirable for investigating, for example, plasticity and learning processes. Here, we present a microelectrode array (MEA) system on a single CMOS die for in vitro recording and stimulation. The system incorporates 26,400 platinum electrodes, fabricated by in-house post-processing, over a large sensing area (3.85 × 2.10 mm2) with sub-cellular spatial resolution (pitch of 17.5 μm). Owing to an area and power efficient implementation, we were able to integrate 1024 readout channels on chip to record extracellular signals from a user-specified selection of electrodes. These channels feature noise values of 2.4 μVrms in the action-potential band (300 Hz–10 kHz) and 5.4 μVrms in the local-field-potential band (1 Hz–300 Hz), and provide programmable gain (up to 78 dB) to accommodate various biological preparations. Amplified and filtered signals are digitized by 10 bit parallel single-slope ADCs at 20 kSamples/s. The system also includes 32 stimulation units, which can elicit neural spikes through either current or voltage pulses. The chip consumes only 75 mW in total, which obviates the need of active cooling even for sensitive cell cultures. PMID:28502989

Automated Interpretation of Subcellular Patterns in Fluorescence Microscope Images for Location Proteomics

PubMed Central

Chen, Xiang; Velliste, Meel; Murphy, Robert F.

2010-01-01

Proteomics, the large scale identification and characterization of many or all proteins expressed in a given cell type, has become a major area of biological research. In addition to information on protein sequence, structure and expression levels, knowledge of a protein’s subcellular location is essential to a complete understanding of its functions. Currently subcellular location patterns are routinely determined by visual inspection of fluorescence microscope images. We review here research aimed at creating systems for automated, systematic determination of location. These employ numerical feature extraction from images, feature reduction to identify the most useful features, and various supervised learning (classification) and unsupervised learning (clustering) methods. These methods have been shown to perform significantly better than human interpretation of the same images. When coupled with technologies for tagging large numbers of proteins and high-throughput microscope systems, the computational methods reviewed here enable the new subfield of location proteomics. This subfield will make critical contributions in two related areas. First, it will provide structured, high-resolution information on location to enable Systems Biology efforts to simulate cell behavior from the gene level on up. Second, it will provide tools for Cytomics projects aimed at characterizing the behaviors of all cell types before, during and after the onset of various diseases. PMID:16752421

Quantitative Proteomic Profiling of Low-Dose Ionizing Radiation Effects in a Human Skin Model

PubMed Central

Hengel, Shawna M.; Aldrich, Joshua T.; Waters, Katrina M.; Pasa-Tolic, Ljiljana; Stenoien, David L.

2014-01-01

To assess responses to low-dose ionizing radiation (LD-IR) exposures potentially encountered during medical diagnostic procedures, nuclear accidents or terrorist acts, a quantitative proteomic approach was used to identify changes in protein abundance in a reconstituted human skin tissue model treated with 0.1 Gy of ionizing radiation. To improve the dynamic range of the assay, subcellular fractionation was employed to remove highly abundant structural proteins and to provide insight into radiation-induced alterations in protein localization. Relative peptide quantification across cellular fractions, control and irradiated samples was performing using 8-plex iTRAQ labeling followed by online two-dimensional nano-scale liquid chromatography and high resolution MS/MS analysis. A total of 107 proteins were detected with statistically significant radiation-induced change in abundance (>1.5 fold) and/or subcellular localization compared to controls. The top biological pathways identified using bioinformatics include organ development, anatomical structure formation and the regulation of actin cytoskeleton. From the proteomic data, a change in proteolytic processing and subcellular localization of the skin barrier protein, filaggrin, was identified, and the results were confirmed by western blotting. This data indicate post-transcriptional regulation of protein abundance, localization and proteolytic processing playing an important role in regulating radiation response in human tissues. PMID:28250387

Host–virus dynamics and subcellular controls of cell fate in a natural coccolithophore population

PubMed Central

Vardi, Assaf; Haramaty, Liti; Van Mooy, Benjamin A. S.; Fredricks, Helen F.; Kimmance, Susan A.; Larsen, Aud; Bidle, Kay D.

2012-01-01

Marine viruses are major evolutionary and biogeochemical drivers in marine microbial foodwebs. However, an in-depth understanding of the cellular mechanisms and the signal transduction pathways mediating host–virus interactions during natural bloom dynamics has remained elusive. We used field-based mesocosms to examine the “arms race” between natural populations of the coccolithophore Emiliania huxleyi and its double-stranded DNA-containing coccolithoviruses (EhVs). Specifically, we examined the dynamics of EhV infection and its regulation of cell fate over the course of bloom development and demise using a diverse suite of molecular tools and in situ fluorescent staining to target different levels of subcellular resolution. We demonstrate the concomitant induction of reactive oxygen species, caspase-specific activity, metacaspase expression, and programmed cell death in response to the accumulation of virus-derived glycosphingolipids upon infection of natural E. huxleyi populations. These subcellular responses to viral infection simultaneously resulted in the enhanced production of transparent exopolymer particles, which can facilitate aggregation and stimulate carbon flux. Our results not only corroborate the critical role for glycosphingolipids and programmed cell death in regulating E. huxleyi–EhV interactions, but also elucidate promising molecular biomarkers and lipid-based proxies for phytoplankton host–virus interactions in natural systems. PMID:23134731

Analyzing the spatial positioning of nuclei in polynuclear giant cells

NASA Astrophysics Data System (ADS)

Stange, Maike; Hintsche, Marius; Sachse, Kirsten; Gerhardt, Matthias; Valleriani, Angelo; Beta, Carsten

2017-11-01

How cells establish and maintain a well-defined size is a fundamental question of cell biology. Here we investigated to what extent the microtubule cytoskeleton can set a predefined cell size, independent of an enclosing cell membrane. We used electropulse-induced cell fusion to form giant multinuclear cells of the social amoeba Dictyostelium discoideum. Based on dual-color confocal imaging of cells that expressed fluorescent markers for the cell nucleus and the microtubules, we determined the subcellular distributions of nuclei and centrosomes in the giant cells. Our two- and three-dimensional imaging results showed that the positions of nuclei in giant cells do not fall onto a regular lattice. However, a comparison with model predictions for random positioning showed that the subcellular arrangement of nuclei maintains a low but still detectable degree of ordering. This can be explained by the steric requirements of the microtubule cytoskeleton, as confirmed by the effect of a microtubule degrading drug.

Singlet oxygen and ROS in a new light: low-dose subcellular photodynamic treatment enhances proliferation at the single cell level.

PubMed

Blázquez-Castro, Alfonso; Breitenbach, Thomas; Ogilby, Peter R

2014-09-01

Two-photon excitation of a sensitizer with a focused laser beam was used to create a spatially-localized subcellular population of reactive oxygen species, ROS, in single HeLa cells. The sensitizer used was protoporphyrin IX, PpIX, endogenously derived from 5-aminolevulinic acid delivered to the cells. Although we infer that singlet oxygen, O2(a(1)Δg), is one ROS produced upon irradiation of PpIX under these conditions, it is possible that the superoxide ion, O2(-˙), may also play a role in this system. With a "high" dose of PpIX-sensitized ROS, the expected death of the cell was observed. However, under "low dose" conditions, clear signs of cell proliferation were observed. The present results facilitate studies of ROS-mediated signalling in imaging-based single cell experiments.

Global, quantitative and dynamic mapping of protein subcellular localization.

PubMed

Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg Hh

2016-06-09

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology.

EnLightenment: High resolution smartphone microscopy as an educational and public engagement platform.

PubMed

Wicks, Laura C; Cairns, Gemma S; Melnyk, Jacob; Bryce, Scott; Duncan, Rory R; Dalgarno, Paul A

2017-01-01

We developed a simple, cost-effective smartphone microscopy platform for use in educational and public engagement programs. We demonstrated its effectiveness, and potential for citizen science through a national imaging initiative, EnLightenment . The cost effectiveness of the instrument allowed for the program to deliver over 500 microscopes to more than 100 secondary schools throughout Scotland, targeting 1000's of 12-14 year olds. Through careful, quantified, selection of a high power, low-cost objective lens, our smartphone microscope has an imaging resolution of microns, with a working distance of 3 mm. It is therefore capable of imaging single cells and sub-cellular features, and retains usability for young children. The microscopes were designed in kit form and provided an interdisciplinary educational tool. By providing full lesson plans and support material, we developed a framework to explore optical design, microscope performance, engineering challenges on construction and real-world applications in life sciences, biological imaging, marine biology, art, and technology. A national online imaging competition framed EnLightenment ; with over 500 high quality images submitted of diverse content, spanning multiple disciplines. With examples of cellular and sub-cellular features clearly identifiable in some submissions, we show how young public can use these instruments for research-level imaging applications, and the potential of the instrument for citizen science programs.

The ultimate picture-the combination of live cell superresolution microscopy and single molecule tracking yields highest spatio-temporal resolution.

PubMed

Dersch, Simon; Graumann, Peter L

2018-06-01

We are witnessing a breathtaking development in light (fluorescence) microscopy, where structures can be resolved down to the size of a ribosome within cells. This has already yielded surprising insight into the subcellular structure of cells, including the smallest cells, bacteria. Moreover, it has become possible to visualize and track single fluorescent protein fusions in real time, and quantify molecule numbers within individual cells. Combined, super resolution and single molecule tracking are pushing the limits of our understanding of the spatio-temporal organization even of the smallest cells to an unprecedented depth. Copyright © 2017 Elsevier Ltd. All rights reserved.

Prospects of third-generation femtosecond laser technology in biological spectromicroscopy

NASA Astrophysics Data System (ADS)

Fattahi, Hanieh; Fattahi, Zohreh; Ghorbani, Asghar

2018-05-01

The next generation of biological imaging modalities will be a movement towards super-resolution, label-free approaches to realize subcellular images in a nonperturbative, non-invasive manner and towards new detection metrologies to reach a higher sensitivity and dynamic range. In this paper, we discuss how the third generation femtosecond laser technology in combination with the already existing concepts in time-resolved spectroscopy could fulfill the requirements of these exciting prospects. The expected enhanced specificity and sensitivity of the envisioned super-resolution microscope could lead us to a better understanding of the inter- and intra-cellular molecular transport and DNA-protein interaction.

High-resolution simultaneous voltage and Ca2+ imaging

PubMed Central

Vogt, Kaspar E; Gerharz, Stephan; Graham, Jeremy; Canepari, Marco

2011-01-01

Combining voltage and Ca2+ imaging allows the correlation of electrical and chemical activity at sub-cellular level. Here we describe a novel apparatus designed to obtain simultaneous voltage and Ca2+ measurements with single-trial resolution from sites as small as a few microns. These measurements can be obtained with negligible optical cross-talk between the two signals and negligible photo-damage of the preparation. The capability of the technique was assessed recording either from individual neurons in brain slices or from networks of cultured neurons. The present achievements open the gate to many novel physiological investigations requiring simultaneous measurement of voltage and Ca2+ signals. PMID:21115640

Monitoring wound healing by multiphoton tomography/endoscopy

NASA Astrophysics Data System (ADS)

König, Karsten; Weinigel, Martin; Bückle, Rainer; Kaatz, Martin; Hipler, Christina; Zens, Katharina; Schneider, Stefan W.; Huck, Volker

2015-02-01

Certified clinical multiphoton tomographs are employed to perform rapid label-free high-resolution in vivo histology. Novel tomographs include a flexible 360° scan head attached to a mechano-optical arm for autofluorescence and SHG imaging as well as rigid two-photon GRIN microendoscope. Mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen can be imaged with submicron resolution in human skin. The system was employed to study the healing of chronic wounds (venous leg ulcer) and acute wounds (curettage of actinic or seborrheic keratosis) on a subcellular level. Furthermore, a flexible sterile foil as interface between wound and focusing optic was tested.

Restoring the spatial resolution of refocus images on 4D light field

NASA Astrophysics Data System (ADS)

Lim, JaeGuyn; Park, ByungKwan; Kang, JooYoung; Lee, SeongDeok

2010-01-01

This paper presents the method for generating a refocus image with restored spatial resolution on a plenoptic camera, which functions controlling the depth of field after capturing one image unlike a traditional camera. It is generally known that the camera captures 4D light field (angular and spatial information of light) within a limited 2D sensor and results in reducing 2D spatial resolution due to inevitable 2D angular data. That's the reason why a refocus image is composed of a low spatial resolution compared with 2D sensor. However, it has recently been known that angular data contain sub-pixel spatial information such that the spatial resolution of 4D light field can be increased. We exploit the fact for improving the spatial resolution of a refocus image. We have experimentally scrutinized that the spatial information is different according to the depth of objects from a camera. So, from the selection of refocused regions (corresponding depth), we use corresponding pre-estimated sub-pixel spatial information for reconstructing spatial resolution of the regions. Meanwhile other regions maintain out-of-focus. Our experimental results show the effect of this proposed method compared to existing method.

Biosensors to measure inositol 1,4,5-trisphosphate concentration in living cells with spatiotemporal resolution.

PubMed

Remus, Timothy P; Zima, Aleksey V; Bossuyt, Julie; Bare, Dan J; Martin, Jody L; Blatter, Lothar A; Bers, Donald M; Mignery, Gregory A

2006-01-06

Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Destructive chemical approaches required to measure [InsP3] limit spatiotemporal understanding of subcellular InsP3 signaling. We constructed novel fluorescence resonance energy transfer-based InsP3 biosensors called FIRE (fluorescent InsP3-responsive element) by fusing plasmids encoding the InsP3-binding domain of InsP3 receptors (types 1-3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an approximately 11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent K(d) = 31.3 +/- 6.7 nm InsP3). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP3] using confocal microscopy (with free [InsP3] rising to approximately 30 nm). Local entry of intracellular InsP3 via membrane rupture by a patch pipette (containing InsP3)in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP3 diffusion. Both endothelin-1-induced and direct InsP3 application (via pipette rupture) revealed that InsP3 diffusion into the nucleus occurs with a delay and blunted rise of [InsP3] versus cytosolic [InsP3]. These new biosensors allow studying InsP3 dynamics at high temporal and spatial resolution that will be powerful in under-standing InsP3 signaling in intact cells.

High Spatial Resolution Commercial Satellite Imaging Product Characterization

NASA Technical Reports Server (NTRS)

Ryan, Robert E.; Pagnutti, Mary; Blonski, Slawomir; Ross, Kenton W.; Stnaley, Thomas

2005-01-01

NASA Stennis Space Center's Remote Sensing group has been characterizing privately owned high spatial resolution multispectral imaging systems, such as IKONOS, QuickBird, and OrbView-3. Natural and man made targets were used for spatial resolution, radiometric, and geopositional characterizations. Higher spatial resolution also presents significant adjacency effects for accurate reliable radiometry.

Automated Verification of Spatial Resolution in Remotely Sensed Imagery

NASA Technical Reports Server (NTRS)

Davis, Bruce; Ryan, Robert; Holekamp, Kara; Vaughn, Ronald

2011-01-01

Image spatial resolution characteristics can vary widely among sources. In the case of aerial-based imaging systems, the image spatial resolution characteristics can even vary between acquisitions. In these systems, aircraft altitude, speed, and sensor look angle all affect image spatial resolution. Image spatial resolution needs to be verified with estimators that include the ground sample distance (GSD), the modulation transfer function (MTF), and the relative edge response (RER), all of which are key components of image quality, along with signal-to-noise ratio (SNR) and dynamic range. Knowledge of spatial resolution parameters is important to determine if features of interest are distinguishable in imagery or associated products, and to develop image restoration algorithms. An automated Spatial Resolution Verification Tool (SRVT) was developed to rapidly determine the spatial resolution characteristics of remotely sensed aerial and satellite imagery. Most current methods for assessing spatial resolution characteristics of imagery rely on pre-deployed engineered targets and are performed only at selected times within preselected scenes. The SRVT addresses these insufficiencies by finding uniform, high-contrast edges from urban scenes and then using these edges to determine standard estimators of spatial resolution, such as the MTF and the RER. The SRVT was developed using the MATLAB programming language and environment. This automated software algorithm assesses every image in an acquired data set, using edges found within each image, and in many cases eliminating the need for dedicated edge targets. The SRVT automatically identifies high-contrast, uniform edges and calculates the MTF and RER of each image, and when possible, within sections of an image, so that the variation of spatial resolution characteristics across the image can be analyzed. The automated algorithm is capable of quickly verifying the spatial resolution quality of all images within a data set, enabling the appropriate use of those images in a number of applications.

Resolution Enhancement of Hyperion Hyperspectral Data using Ikonos Multispectral Data

DTIC Science & Technology

2007-09-01

spatial - resolution hyperspectral image to produce a sharpened product. The result is a product that has the spectral properties of the ...multispectral sensors. In this work, we examine the benefits of combining data from high- spatial - resolution , low- spectral - resolution spectral imaging...sensors with data obtained from high- spectral - resolution , low- spatial - resolution spectral imaging sensors.

Thematic and spatial resolutions affect model-based predictions of tree species distribution.

PubMed

Liang, Yu; He, Hong S; Fraser, Jacob S; Wu, ZhiWei

2013-01-01

Subjective decisions of thematic and spatial resolutions in characterizing environmental heterogeneity may affect the characterizations of spatial pattern and the simulation of occurrence and rate of ecological processes, and in turn, model-based tree species distribution. Thus, this study quantified the importance of thematic and spatial resolutions, and their interaction in predictions of tree species distribution (quantified by species abundance). We investigated how model-predicted species abundances changed and whether tree species with different ecological traits (e.g., seed dispersal distance, competitive capacity) had different responses to varying thematic and spatial resolutions. We used the LANDIS forest landscape model to predict tree species distribution at the landscape scale and designed a series of scenarios with different thematic (different numbers of land types) and spatial resolutions combinations, and then statistically examined the differences of species abundance among these scenarios. Results showed that both thematic and spatial resolutions affected model-based predictions of species distribution, but thematic resolution had a greater effect. Species ecological traits affected the predictions. For species with moderate dispersal distance and relatively abundant seed sources, predicted abundance increased as thematic resolution increased. However, for species with long seeding distance or high shade tolerance, thematic resolution had an inverse effect on predicted abundance. When seed sources and dispersal distance were not limiting, the predicted species abundance increased with spatial resolution and vice versa. Results from this study may provide insights into the choice of thematic and spatial resolutions for model-based predictions of tree species distribution.

Thematic and Spatial Resolutions Affect Model-Based Predictions of Tree Species Distribution

PubMed Central

Liang, Yu; He, Hong S.; Fraser, Jacob S.; Wu, ZhiWei

2013-01-01

Subjective decisions of thematic and spatial resolutions in characterizing environmental heterogeneity may affect the characterizations of spatial pattern and the simulation of occurrence and rate of ecological processes, and in turn, model-based tree species distribution. Thus, this study quantified the importance of thematic and spatial resolutions, and their interaction in predictions of tree species distribution (quantified by species abundance). We investigated how model-predicted species abundances changed and whether tree species with different ecological traits (e.g., seed dispersal distance, competitive capacity) had different responses to varying thematic and spatial resolutions. We used the LANDIS forest landscape model to predict tree species distribution at the landscape scale and designed a series of scenarios with different thematic (different numbers of land types) and spatial resolutions combinations, and then statistically examined the differences of species abundance among these scenarios. Results showed that both thematic and spatial resolutions affected model-based predictions of species distribution, but thematic resolution had a greater effect. Species ecological traits affected the predictions. For species with moderate dispersal distance and relatively abundant seed sources, predicted abundance increased as thematic resolution increased. However, for species with long seeding distance or high shade tolerance, thematic resolution had an inverse effect on predicted abundance. When seed sources and dispersal distance were not limiting, the predicted species abundance increased with spatial resolution and vice versa. Results from this study may provide insights into the choice of thematic and spatial resolutions for model-based predictions of tree species distribution. PMID:23861828

Trespassing cancer cells: ‘fingerprinting’ invasive protrusions reveals metastatic culprits

PubMed Central

Klemke, Richard L.

2012-01-01

Metastatic cancer cells produce invasive membrane protrusions called invadopodia and pseudopodia, which play a central role in driving cancer cell dissemination in the body. Malignant cells use these structures to attach to and degrade extracellular matrix proteins, generate force for cell locomotion, and to penetrate the vasculature. Recent work using unique subcellular fractionation methodologies combined with spatial genomic, proteomic, and phosphoproteomic profiling has provided insight into the invadopodiome and pseudopodiome signaling networks that control the protrusion of invasive membranes. Here I highlight how these powerful spatial “omics” approaches reveal important signatures of metastatic cancer cells and possible new therapeutic targets aimed at treating metastatic disease. PMID:22980730

Retrieved Products from Simulated Hyperspectral Observations of a Hurricane

NASA Technical Reports Server (NTRS)

Susskind, Joel; Kouvaris, Louis; Iredell, Lena; Blaisdell, John

2015-01-01

Demonstrate via Observing System Simulation Experiments (OSSEs) the potential utility of flying high spatial resolution AIRS class IR sounders on future LEO and GEO missions.The study simulates and analyzes radiances for 3 sounders with AIRS spectral and radiometric properties on different orbits with different spatial resolutions: 1) Control run 13 kilometers AIRS spatial resolution at nadir on LEO in Aqua orbit; 2) 2 kilometer spatial resolution LEO sounder at nadir ARIES; 3) 5 kilometers spatial resolution sounder on a GEO orbit, radiances simulated every 72 minutes.

Ultrahigh resolution optical coherence elastography using a Bessel beam for extended depth of field

NASA Astrophysics Data System (ADS)

Curatolo, Andrea; Villiger, Martin; Lorenser, Dirk; Wijesinghe, Philip; Fritz, Alexander; Kennedy, Brendan F.; Sampson, David D.

2016-03-01

Visualizing stiffness within the local tissue environment at the cellular and sub-cellular level promises to provide insight into the genesis and progression of disease. In this paper, we propose ultrahigh-resolution optical coherence elastography, and demonstrate three-dimensional imaging of local axial strain of tissues undergoing compressive loading. The technique employs a dual-arm extended focus optical coherence microscope to measure tissue displacement under compression. The system uses a broad bandwidth supercontinuum source for ultrahigh axial resolution, Bessel beam illumination and Gaussian beam detection, maintaining sub-2 μm transverse resolution over nearly 100 μm depth of field, and spectral-domain detection allowing high displacement sensitivity. The system produces strain elastograms with a record resolution (x,y,z) of 2×2×15 μm. We benchmark the advances in terms of resolution and strain sensitivity by imaging a suitable inclusion phantom. We also demonstrate this performance on freshly excised mouse aorta and reveal the mechanical heterogeneity of vascular smooth muscle cells and elastin sheets, otherwise unresolved in a typical, lower resolution optical coherence elastography system.

Live imaging of companion cells and sieve elements in Arabidopsis leaves.

PubMed

Cayla, Thibaud; Batailler, Brigitte; Le Hir, Rozenn; Revers, Frédéric; Anstead, James A; Thompson, Gary A; Grandjean, Olivier; Dinant, Sylvie

2015-01-01

The phloem is a complex tissue composed of highly specialized cells with unique subcellular structures and a compact organization that is challenging to study in vivo at cellular resolution. We used confocal scanning laser microscopy and subcellular fluorescent markers in companion cells and sieve elements, for live imaging of the phloem in Arabidopsis leaves. This approach provided a simple framework for identifying phloem cell types unambiguously. It highlighted the compactness of the meshed network of organelles within companion cells. By contrast, within the sieve elements, unknown bodies were observed in association with the PP2-A1:GFP, GFP:RTM1 and RTM2:GFP markers at the cell periphery. The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix. Its location differed from that of the P-protein filaments, which were visualized with SEOR1:GFP and SEOR2:GFP. PP2-A1:GFP surrounded two types of bodies, one of which was identified as mitochondria. This location suggested that it was embedded within the sieve element clamps, specific structures that may fix the organelles to each another or to the plasma membrane in the sieve tubes. GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids. PP2-A1:GFP was soluble in the cytosol of immature sieve elements. The changes in its subcellular localization during differentiation provide an in vivo blueprint for monitoring this process. The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo.

Live Imaging of Companion Cells and Sieve Elements in Arabidopsis Leaves

PubMed Central

Cayla, Thibaud; Batailler, Brigitte; Le Hir, Rozenn; Revers, Frédéric; Anstead, James A.; Thompson, Gary A.; Grandjean, Olivier; Dinant, Sylvie

2015-01-01

The phloem is a complex tissue composed of highly specialized cells with unique subcellular structures and a compact organization that is challenging to study in vivo at cellular resolution. We used confocal scanning laser microscopy and subcellular fluorescent markers in companion cells and sieve elements, for live imaging of the phloem in Arabidopsis leaves. This approach provided a simple framework for identifying phloem cell types unambiguously. It highlighted the compactness of the meshed network of organelles within companion cells. By contrast, within the sieve elements, unknown bodies were observed in association with the PP2-A1:GFP, GFP:RTM1 and RTM2:GFP markers at the cell periphery. The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix. Its location differed from that of the P-protein filaments, which were visualized with SEOR1:GFP and SEOR2:GFP. PP2-A1:GFP surrounded two types of bodies, one of which was identified as mitochondria. This location suggested that it was embedded within the sieve element clamps, specific structures that may fix the organelles to each another or to the plasma membrane in the sieve tubes. GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids. PP2-A1:GFP was soluble in the cytosol of immature sieve elements. The changes in its subcellular localization during differentiation provide an in vivo blueprint for monitoring this process. The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo. PMID:25714357

Detector motion method to increase spatial resolution in photon-counting detectors

NASA Astrophysics Data System (ADS)

Lee, Daehee; Park, Kyeongjin; Lim, Kyung Taek; Cho, Gyuseong

2017-03-01

Medical imaging requires high spatial resolution of an image to identify fine lesions. Photon-counting detectors in medical imaging have recently been rapidly replacing energy-integrating detectors due to the former`s high spatial resolution, high efficiency and low noise. Spatial resolution in a photon counting image is determined by the pixel size. Therefore, the smaller the pixel size, the higher the spatial resolution that can be obtained in an image. However, detector redesigning is required to reduce pixel size, and an expensive fine process is required to integrate a signal processing unit with reduced pixel size. Furthermore, as the pixel size decreases, charge sharing severely deteriorates spatial resolution. To increase spatial resolution, we propose a detector motion method using a large pixel detector that is less affected by charge sharing. To verify the proposed method, we utilized a UNO-XRI photon-counting detector (1-mm CdTe, Timepix chip) at the maximum X-ray tube voltage of 80 kVp. A similar spatial resolution of a 55- μm-pixel image was achieved by application of the proposed method to a 110- μm-pixel detector with a higher signal-to-noise ratio. The proposed method could be a way to increase spatial resolution without a pixel redesign when pixels severely suffer from charge sharing as pixel size is reduced.

SPED light sheet microscopy: fast mapping of biological system structure and function

PubMed Central

Tomer, Raju; Lovett-Barron, Matthew; Kauvar, Isaac; Andalman, Aaron; Burns, Vanessa M.; Sankaran, Sethuraman; Grosenick, Logan; Broxton, Michael; Yang, Samuel; Deisseroth, Karl

2016-01-01

The goal of understanding living nervous systems has driven interest in high-speed and large field-of-view volumetric imaging at cellular resolution. Light-sheet microscopy approaches have emerged for cellular-resolution functional brain imaging in small organisms such as larval zebrafish, but remain fundamentally limited in speed. Here we have developed SPED light sheet microscopy, which combines large volumetric field-of-view via an extended depth of field with the optical sectioning of light sheet microscopy, thereby eliminating the need to physically scan detection objectives for volumetric imaging. SPED enables scanning of thousands of volumes-per-second, limited only by camera acquisition rate, through the harnessing of optical mechanisms that normally result in unwanted spherical aberrations. We demonstrate capabilities of SPED microscopy by performing fast sub-cellular resolution imaging of CLARITY mouse brains and cellular-resolution volumetric Ca2+ imaging of entire zebrafish nervous systems. Together, SPED light sheet methods enable high-speed cellular-resolution volumetric mapping of biological system structure and function. PMID:26687363

The Effect of Remote Sensor Spatial Resolution in Monitoring U.S. Army Training Maneuver Sites

DTIC Science & Technology

1990-12-01

THE EFFECT OF REMOTE SENSOR SPATIAL RESOLUTION IN MONITORING U.S. ARMY...Multispectral Scanner with 6.5 meter spatial resolution provided the most effective digital data set for enhancing tank trails. However, this Airborne Scanner...primary objective of this research was to determine the capabilities and limitations of remote sensor systems having different spatial resolutions to

The effects of transient attention on spatial resolution and the size of the attentional cue.

PubMed

Yeshurun, Yaffa; Carrasco, Marisa

2008-01-01

It has been shown that transient attention enhances spatial resolution, but is the effect of transient attention on spatial resolution modulated by the size of the attentional cue? Would a gradual increase in the size of the cue lead to a gradual decrement in spatial resolution? To test these hypotheses, we used a texture segmentation task in which performance depends on spatial resolution, and systematically manipulated the size of the attentional cue: A bar of different lengths (Experiment 1) or a frame of different sizes (Experiments 2-3) indicated the target region in a texture segmentation display. Observers indicated whether a target patch region (oriented line elements in a background of an orthogonal orientation), appearing at a range of eccentricities, was present in the first or the second interval. We replicated the attentional enhancement of spatial resolution found with small cues; attention improved performance at peripheral locations but impaired performance at central locations. However, there was no evidence of gradual resolution decrement with large cues. Transient attention enhanced spatial resolution at the attended location when it was attracted to that location by a small cue but did not affect resolution when it was attracted by a large cue. These results indicate that transient attention cannot adapt its operation on spatial resolution on the basis of the size of the attentional cue.

Selecting a spatial resolution for estimation of per-field green leaf area index

NASA Technical Reports Server (NTRS)

Curran, Paul J.; Williamson, H. Dawn

1988-01-01

For any application of multispectral scanner (MSS) data, a user is faced with a number of choices concerning the characteristics of the data; one of these is their spatial resolution. A pilot study was undertaken to determine the spatial resolution that would be optimal for the per-field estimation of green leaf area index (GLAI) in grassland. By reference to empirically-derived data from three areas of grassland, the suitable spatial resolution was hypothesized to lie in the lower portion of a 2-18 m range. To estimate per-field GLAI, airborne MSS data were collected at spatial resolutions of 2 m, 5 m and 10 m. The highest accuracies of per-field GLAI estimation were achieved using MSS data with spatial resolutions of 2 m and 5 m.

Forest Classification Accuracy as Influenced by Multispectral Scanner Spatial Resolution. [Sam Houston National Forest, Texas

NASA Technical Reports Server (NTRS)

Nalepka, R. F. (Principal Investigator); Sadowski, F. E.; Sarno, J. E.

1976-01-01

The author has identified the following significant results. A supervised classification within two separate ground areas of the Sam Houston National Forest was carried out for two sq meters spatial resolution MSS data. Data were progressively coarsened to simulate five additional cases of spatial resolution ranging up to 64 sq meters. Similar processing and analysis of all spatial resolutions enabled evaluations of the effect of spatial resolution on classification accuracy for various levels of detail and the effects on area proportion estimation for very general forest features. For very coarse resolutions, a subset of spectral channels which simulated the proposed thematic mapper channels was used to study classification accuracy.

High Resolution Mesoscale Weather Data Improvement to Spatial Effects for Dose-Rate Contour Plot Predictions

DTIC Science & Technology

2007-03-01

time. This is a very powerful tool in determining fine spatial resolution , as boundary conditions are not only updated at every timestep, but the ...HIGH RESOLUTION MESOSCALE WEATHER DATA IMPROVEMENT TO SPATIAL EFFECTS FOR DOSE-RATE CONTOUR PLOT PREDICTIONS THESIS Christopher P...11 1 HIGH RESOLUTION MESOSCALE WEATHER DATA IMPROVEMENT TO SPATIAL EFFECTS FOR DOSE-RATE CONTOUR PLOT

Impact of the spatial resolution of satellite remote sensing sensors in the quantification of total suspended sediment concentration: A case study in turbid waters of Northern Western Australia.

PubMed

Dorji, Passang; Fearns, Peter

2017-01-01

The impact of anthropogenic activities on coastal waters is a cause of concern because such activities add to the total suspended sediment (TSS) budget of the coastal waters, which have negative impacts on the coastal ecosystem. Satellite remote sensing provides a powerful tool in monitoring TSS concentration at high spatiotemporal resolution, but coastal managers should be mindful that the satellite-derived TSS concentrations are dependent on the satellite sensor's radiometric properties, atmospheric correction approaches, the spatial resolution and the limitations of specific TSS algorithms. In this study, we investigated the impact of different spatial resolutions of satellite sensor on the quantification of TSS concentration in coastal waters of northern Western Australia. We quantified the TSS product derived from MODerate resolution Imaging Spectroradiometer (MODIS)-Aqua, Landsat-8 Operational Land Image (OLI), and WorldView-2 (WV2) at native spatial resolutions of 250 m, 30 m and 2 m respectively and coarser spatial resolution (resampled up to 5 km) to quantify the impact of spatial resolution on the derived TSS product in different turbidity conditions. The results from the study show that in the waters of high turbidity and high spatial variability, the high spatial resolution WV2 sensor reported TSS concentration as high as 160 mg L-1 while the low spatial resolution MODIS-Aqua reported a maximum TSS concentration of 23.6 mg L-1. Degrading the spatial resolution of each satellite sensor for highly spatially variable turbid waters led to variability in the TSS concentrations of 114.46%, 304.68% and 38.2% for WV2, Landsat-8 OLI and MODIS-Aqua respectively. The implications of this work are particularly relevant in the situation of compliance monitoring where operations may be required to restrict TSS concentrations to a pre-defined limit.

Impact of the spatial resolution of satellite remote sensing sensors in the quantification of total suspended sediment concentration: A case study in turbid waters of Northern Western Australia

PubMed Central

Fearns, Peter

2017-01-01

The impact of anthropogenic activities on coastal waters is a cause of concern because such activities add to the total suspended sediment (TSS) budget of the coastal waters, which have negative impacts on the coastal ecosystem. Satellite remote sensing provides a powerful tool in monitoring TSS concentration at high spatiotemporal resolution, but coastal managers should be mindful that the satellite-derived TSS concentrations are dependent on the satellite sensor’s radiometric properties, atmospheric correction approaches, the spatial resolution and the limitations of specific TSS algorithms. In this study, we investigated the impact of different spatial resolutions of satellite sensor on the quantification of TSS concentration in coastal waters of northern Western Australia. We quantified the TSS product derived from MODerate resolution Imaging Spectroradiometer (MODIS)-Aqua, Landsat-8 Operational Land Image (OLI), and WorldView-2 (WV2) at native spatial resolutions of 250 m, 30 m and 2 m respectively and coarser spatial resolution (resampled up to 5 km) to quantify the impact of spatial resolution on the derived TSS product in different turbidity conditions. The results from the study show that in the waters of high turbidity and high spatial variability, the high spatial resolution WV2 sensor reported TSS concentration as high as 160 mg L-1 while the low spatial resolution MODIS-Aqua reported a maximum TSS concentration of 23.6 mg L-1. Degrading the spatial resolution of each satellite sensor for highly spatially variable turbid waters led to variability in the TSS concentrations of 114.46%, 304.68% and 38.2% for WV2, Landsat-8 OLI and MODIS-Aqua respectively. The implications of this work are particularly relevant in the situation of compliance monitoring where operations may be required to restrict TSS concentrations to a pre-defined limit. PMID:28380059

Attention Modifies Spatial Resolution According to Task Demands.

PubMed

Barbot, Antoine; Carrasco, Marisa

2017-03-01

How does visual attention affect spatial resolution? In texture-segmentation tasks, exogenous (involuntary) attention automatically increases resolution at the attended location, which improves performance where resolution is too low (at the periphery) but impairs performance where resolution is already too high (at central locations). Conversely, endogenous (voluntary) attention improves performance at all eccentricities, which suggests a more flexible mechanism. Here, using selective adaptation to spatial frequency, we investigated the mechanism by which endogenous attention benefits performance in resolution tasks. Participants detected a texture target that could appear at several eccentricities. Adapting to high or low spatial frequencies selectively affected performance in a manner consistent with changes in resolution. Moreover, adapting to high, but not low, frequencies mitigated the attentional benefit at central locations where resolution was too high; this shows that attention can improve performance by decreasing resolution. Altogether, our results indicate that endogenous attention benefits performance by modulating the contribution of high-frequency information in order to flexibly adjust spatial resolution according to task demands.

Attention Modifies Spatial Resolution According to Task Demands

PubMed Central

Barbot, Antoine; Carrasco, Marisa

2017-01-01

How does visual attention affect spatial resolution? In texture-segmentation tasks, exogenous (involuntary) attention automatically increases resolution at the attended location, which improves performance where resolution is too low (at the periphery) but impairs performance where resolution is already too high (at central locations). Conversely, endogenous (voluntary) attention improves performance at all eccentricities, which suggests a more flexible mechanism. Here, using selective adaptation to spatial frequency, we investigated the mechanism by which endogenous attention benefits performance in resolution tasks. Participants detected a texture target that could appear at several eccentricities. Adapting to high or low spatial frequencies selectively affected performance in a manner consistent with changes in resolution. Moreover, adapting to high, but not low, frequencies mitigated the attentional benefit at central locations where resolution was too high; this shows that attention can improve performance by decreasing resolution. Altogether, our results indicate that endogenous attention benefits performance by modulating the contribution of high-frequency information in order to flexibly adjust spatial resolution according to task demands. PMID:28118103

A photocleavable rapamycin conjugate for spatiotemporal control of small GTPase activity.

PubMed

Umeda, Nobuhiro; Ueno, Tasuku; Pohlmeyer, Christopher; Nagano, Tetsuo; Inoue, Takanari

2011-01-12

We developed a novel method to spatiotemporally control the activity of signaling molecules. A newly synthesized photocaged rapamycin derivative induced rapid dimerization of FKBP (FK-506 binding protein) and FRB (FKBP-rapamycin binding protein) upon UV irradiation. With this system and the spatially confined UV irradiation, we achieved subcellularly localized activation of Rac, a member of small GTPases. Our technique offers a powerful approach to studies of dynamic intracellular signaling events.

Effects of spatial resolution

NASA Technical Reports Server (NTRS)

Abrams, M.

1982-01-01

Studies of the effects of spatial resolution on extraction of geologic information are woefully lacking but spatial resolution effects can be examined as they influence two general categories: detection of spatial features per se; and the effects of IFOV on the definition of spectral signatures and on general mapping abilities.

Spatial, Temporal and Spectral Satellite Image Fusion via Sparse Representation

NASA Astrophysics Data System (ADS)

Song, Huihui

Remote sensing provides good measurements for monitoring and further analyzing the climate change, dynamics of ecosystem, and human activities in global or regional scales. Over the past two decades, the number of launched satellite sensors has been increasing with the development of aerospace technologies and the growing requirements on remote sensing data in a vast amount of application fields. However, a key technological challenge confronting these sensors is that they tradeoff between spatial resolution and other properties, including temporal resolution, spectral resolution, swath width, etc., due to the limitations of hardware technology and budget constraints. To increase the spatial resolution of data with other good properties, one possible cost-effective solution is to explore data integration methods that can fuse multi-resolution data from multiple sensors, thereby enhancing the application capabilities of available remote sensing data. In this thesis, we propose to fuse the spatial resolution with temporal resolution and spectral resolution, respectively, based on sparse representation theory. Taking the study case of Landsat ETM+ (with spatial resolution of 30m and temporal resolution of 16 days) and MODIS (with spatial resolution of 250m ~ 1km and daily temporal resolution) reflectance, we propose two spatial-temporal fusion methods to combine the fine spatial information of Landsat image and the daily temporal resolution of MODIS image. Motivated by that the images from these two sensors are comparable on corresponding bands, we propose to link their spatial information on available Landsat- MODIS image pair (captured on prior date) and then predict the Landsat image from the MODIS counterpart on prediction date. To well-learn the spatial details from the prior images, we use a redundant dictionary to extract the basic representation atoms for both Landsat and MODIS images based on sparse representation. Under the scenario of two prior Landsat-MODIS image pairs, we build the corresponding relationship between the difference images of MODIS and ETM+ by training a low- and high-resolution dictionary pair from the given prior image pairs. In the second scenario, i.e., only one Landsat- MODIS image pair being available, we directly correlate MODIS and ETM+ data through an image degradation model. Then, the fusion stage is achieved by super-resolving the MODIS image combining the high-pass modulation in a two-layer fusion framework. Remarkably, the proposed spatial-temporal fusion methods form a unified framework for blending remote sensing images with phenology change or land-cover-type change. Based on the proposed spatial-temporal fusion models, we propose to monitor the land use/land cover changes in Shenzhen, China. As a fast-growing city, Shenzhen faces the problem of detecting the rapid changes for both rational city planning and sustainable development. However, the cloudy and rainy weather in region Shenzhen located makes the capturing circle of high-quality satellite images longer than their normal revisit periods. Spatial-temporal fusion methods are capable to tackle this problem by improving the spatial resolution of images with coarse spatial resolution but frequent temporal coverage, thereby making the detection of rapid changes possible. On two Landsat-MODIS datasets with annual and monthly changes, respectively, we apply the proposed spatial-temporal fusion methods to the task of multiple change detection. Afterward, we propose a novel spatial and spectral fusion method for satellite multispectral and hyperspectral (or high-spectral) images based on dictionary-pair learning and sparse non-negative matrix factorization. By combining the spectral information from hyperspectral image, which is characterized by low spatial resolution but high spectral resolution and abbreviated as LSHS, and the spatial information from multispectral image, which is featured by high spatial resolution but low spectral resolution and abbreviated as HSLS, this method aims to generate the fused data with both high spatial and high spectral resolutions. Motivated by the observation that each hyperspectral pixel can be represented by a linear combination of a few endmembers, this method first extracts the spectral bases of LSHS and HSLS images by making full use of the rich spectral information in LSHS data. The spectral bases of these two categories data then formulate a dictionary-pair due to their correspondence in representing each pixel spectra of LSHS data and HSLS data, respectively. Subsequently, the LSHS image is spatially unmixed by representing the HSLS image with respect to the corresponding learned dictionary to derive its representation coefficients. Combining the spectral bases of LSHS data and the representation coefficients of HSLS data, we finally derive the fused data characterized by the spectral resolution of LSHS data and the spatial resolution of HSLS data.

Results of the spatial resolution simulation for multispectral data (resolution brochures)

NASA Technical Reports Server (NTRS)

1982-01-01

The variable information content of Earth Resource products at different levels of spatial resolution and in different spectral bands is addressed. A low-cost brochure that scientists and laymen could use to visualize the effects of increasing the spatial resolution of multispectral scanner images was produced.

Two-colour live-cell nanoscale imaging of intracellular targets

NASA Astrophysics Data System (ADS)

Bottanelli, Francesca; Kromann, Emil B.; Allgeyer, Edward S.; Erdmann, Roman S.; Wood Baguley, Stephanie; Sirinakis, George; Schepartz, Alanna; Baddeley, David; Toomre, Derek K.; Rothman, James E.; Bewersdorf, Joerg

2016-03-01

Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile STED-compatible two-colour labelling strategy for intracellular targets in living cells. Here we demonstrate a universal labelling method based on the organic, membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-colour STED imaging in living cells below 50 nm resolution. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resolution.

Optical Coherence Microscopy

NASA Astrophysics Data System (ADS)

Aguirre, Aaron D.; Zhou, Chao; Lee, Hsiang-Chieh; Ahsen, Osman O.; Fujimoto, James G.

Cellular imaging of human tissues remains an important advance for many clinical applications of optical coherence tomography (OCT). Imaging cells with traditional OCT systems has not been possible due to the limited transverse resolution of such techniques. Optical coherence microscopy (OCM) refers to OCT methods that achieve high transverse resolution to visualize cells and subcellular features. This chapter provides a comprehensive discussion of the rationale for cellular imaging in human tissues as well as a review of the key technological advances required to achieve it. Time domain and Fourier domain OCM approaches are described with an emphasis on state of the art system designs, including miniaturized endoscopic imaging probes. Clinical applications are discussed and multiple examples of cellular imaging in human tissues are provided.

Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope

NASA Astrophysics Data System (ADS)

Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Morgado, António Miguel; König, Karsten

2016-03-01

Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.

Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope.

PubMed

Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Morgado, António Miguel; König, Karsten

2016-03-01

Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.

Phase-contrast tomography of neuronal tissues: from laboratory- to high resolution synchrotron CT

NASA Astrophysics Data System (ADS)

Töpperwien, Mareike; Krenkel, Martin; Müller, Kristin; Salditt, Tim

2016-10-01

Assessing the three-dimensional architecture of neuronal tissues with sub-cellular resolution presents a significant analytical challenge. Overcoming the limitations associated with serial slicing, phase-contrast x-ray tomography has the potential to contribute to this goal. Even compact laboratory CT at an optimized liquid-metal jet micro- focus source combined with suitable phase-retrieval algorithms and preparation protocols can yield renderings with single cell sensitivity in millimeter sized brain areas of mouse. Here, we show the capabilities of the setup by imaging a Golgi-Cox impregnated mouse brain. Towards higher resolution we extend these studies at our recently upgraded waveguide-based cone-beam holo-tomography instrument GINIX at DESY. This setup allows high resolution recordings with adjustable field of view and resolution, down to the voxel sizes in the range of a few ten nanometers. The recent results make us confident that important issues of neuronal connectivity can be addressed by these methods, and that 3D (virtual) histology with nanoscale resolution will become an attractive modality for neuroscience research.

Image sharpening for mixed spatial and spectral resolution satellite systems

NASA Technical Reports Server (NTRS)

Hallada, W. A.; Cox, S.

1983-01-01

Two methods of image sharpening (reconstruction) are compared. The first, a spatial filtering technique, extrapolates edge information from a high spatial resolution panchromatic band at 10 meters and adds it to the low spatial resolution narrow spectral bands. The second method, a color normalizing technique, is based on the ability to separate image hue and brightness components in spectral data. Using both techniques, multispectral images are sharpened from 30, 50, 70, and 90 meter resolutions. Error rates are calculated for the two methods and all sharpened resolutions. The results indicate that the color normalizing method is superior to the spatial filtering technique.

Spatial Resolution Requirements for Accurate Identification of Drivers of Atrial Fibrillation

PubMed Central

Roney, Caroline H.; Cantwell, Chris D.; Bayer, Jason D.; Qureshi, Norman A.; Lim, Phang Boon; Tweedy, Jennifer H.; Kanagaratnam, Prapa; Vigmond, Edward J.; Ng, Fu Siong

2017-01-01

Background— Recent studies have demonstrated conflicting mechanisms underlying atrial fibrillation (AF), with the spatial resolution of data often cited as a potential reason for the disagreement. The purpose of this study was to investigate whether the variation in spatial resolution of mapping may lead to misinterpretation of the underlying mechanism in persistent AF. Methods and Results— Simulations of rotors and focal sources were performed to estimate the minimum number of recording points required to correctly identify the underlying AF mechanism. The effects of different data types (action potentials and unipolar or bipolar electrograms) and rotor stability on resolution requirements were investigated. We also determined the ability of clinically used endocardial catheters to identify AF mechanisms using clinically recorded and simulated data. The spatial resolution required for correct identification of rotors and focal sources is a linear function of spatial wavelength (the distance between wavefronts) of the arrhythmia. Rotor localization errors are larger for electrogram data than for action potential data. Stationary rotors are more reliably identified compared with meandering trajectories, for any given spatial resolution. All clinical high-resolution multipolar catheters are of sufficient resolution to accurately detect and track rotors when placed over the rotor core although the low-resolution basket catheter is prone to false detections and may incorrectly identify rotors that are not present. Conclusions— The spatial resolution of AF data can significantly affect the interpretation of the underlying AF mechanism. Therefore, the interpretation of human AF data must be taken in the context of the spatial resolution of the recordings. PMID:28500175

Methods for Detection of Mitochondrial and Cellular Reactive Oxygen Species

PubMed Central

Harrison, David G.

2014-01-01

Abstract Significance: Mitochondrial and cellular reactive oxygen species (ROS) play important roles in both physiological and pathological processes. Different ROS, such as superoxide (O2•−), hydrogen peroxide, and peroxynitrite (ONOO•−), stimulate distinct cell-signaling pathways and lead to diverse outcomes depending on their amount and subcellular localization. A variety of methods have been developed for ROS detection; however, many of these methods are not specific, do not allow subcellular localization, and can produce artifacts. In this review, we will critically analyze ROS detection and present advantages and the shortcomings of several available methods. Recent Advances: In the past decade, a number of new fluorescent probes, electron-spin resonance approaches, and immunoassays have been developed. These new state-of-the-art methods provide improved selectivity and subcellular resolution for ROS detection. Critical Issues: Although new methods for HPLC superoxide detection, application of fluorescent boronate-containing probes, use of cell-targeted hydroxylamine spin probes, and immunospin trapping have been available for several years, there has been lack of translation of these into biomedical research, limiting their widespread use. Future Directions: Additional studies to translate these new technologies from the test tube to physiological applications are needed and could lead to a wider application of these approaches to study mitochondrial and cellular ROS. Antioxid. Redox Signal. 20, 372–382. PMID:22978713

Real-time quantification of subcellular H2O2 and glutathione redox potential in living cardiovascular tissues.

PubMed

Panieri, Emiliano; Millia, Carlo; Santoro, Massimo M

2017-08-01

Detecting and measuring the dynamic redox events that occur in vivo is a prerequisite for understanding the impact of oxidants and redox events in normal and pathological conditions. These aspects are particularly relevant in cardiovascular tissues wherein alterations of the redox balance are associated with stroke, aging, and pharmacological intervention. An ambiguous aspect of redox biology is how redox events occur in subcellular organelles including mitochondria, and nuclei. Genetically-encoded Rogfp2 fluorescent probes have become powerful tools for real-time detection of redox events. These probes detect hydrogen peroxide (H 2 O 2 ) levels and glutathione redox potential (E GSH ), both with high spatiotemporal resolution. By generating novel transgenic (Tg) zebrafish lines that express compartment-specific Rogfp2-Orp1 and Grx1-Rogfp2 sensors we analyzed cytosolic, mitochondrial, and the nuclear redox state of endothelial cells and cardiomyocytes of living zebrafish embryos. We provide evidence for the usefulness of these Tg lines for pharmacological compounds screening by addressing the blocking of pentose phosphate pathways (PPP) and glutathione synthesis, thus altering subcellular redox state in vivo. Rogfp2-based transgenic zebrafish lines represent valuable tools to characterize the impact of redox changes in living tissues and offer new opportunities for studying metabolic driven antioxidant response in biomedical research. Copyright © 2017 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirtley, John R., E-mail: jkirtley@stanford.edu; Rosenberg, Aaron J.; Palmstrom, Johanna C.

Superconducting QUantum Interference Device (SQUID) microscopy has excellent magnetic field sensitivity, but suffers from modest spatial resolution when compared with other scanning probes. This spatial resolution is determined by both the size of the field sensitive area and the spacing between this area and the sample surface. In this paper we describe scanning SQUID susceptometers that achieve sub-micron spatial resolution while retaining a white noise floor flux sensitivity of ≈2μΦ{sub 0}/Hz{sup 1/2}. This high spatial resolution is accomplished by deep sub-micron feature sizes, well shielded pickup loops fabricated using a planarized process, and a deep etch step that minimizes themore » spacing between the sample surface and the SQUID pickup loop. We describe the design, modeling, fabrication, and testing of these sensors. Although sub-micron spatial resolution has been achieved previously in scanning SQUID sensors, our sensors not only achieve high spatial resolution but also have integrated modulation coils for flux feedback, integrated field coils for susceptibility measurements, and batch processing. They are therefore a generally applicable tool for imaging sample magnetization, currents, and susceptibilities with higher spatial resolution than previous susceptometers.« less

Chromatic and Achromatic Spatial Resolution of Local Field Potentials in Awake Cortex

PubMed Central

Jansen, Michael; Li, Xiaobing; Lashgari, Reza; Kremkow, Jens; Bereshpolova, Yulia; Swadlow, Harvey A.; Zaidi, Qasim; Alonso, Jose-Manuel

2015-01-01

Local field potentials (LFPs) have become an important measure of neuronal population activity in the brain and could provide robust signals to guide the implant of visual cortical prosthesis in the future. However, it remains unclear whether LFPs can detect weak cortical responses (e.g., cortical responses to equiluminant color) and whether they have enough visual spatial resolution to distinguish different chromatic and achromatic stimulus patterns. By recording from awake behaving macaques in primary visual cortex, here we demonstrate that LFPs respond robustly to pure chromatic stimuli and exhibit ∼2.5 times lower spatial resolution for chromatic than achromatic stimulus patterns, a value that resembles the ratio of achromatic/chromatic resolution measured with psychophysical experiments in humans. We also show that, although the spatial resolution of LFP decays with visual eccentricity as is also the case for single neurons, LFPs have higher spatial resolution and show weaker response suppression to low spatial frequencies than spiking multiunit activity. These results indicate that LFP recordings are an excellent approach to measure spatial resolution from local populations of neurons in visual cortex including those responsive to color. PMID:25416722

Calculation of the spatial resolution in two-photon absorption spectroscopy applied to plasma diagnosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcia-Lechuga, M.; Laser Processing Group, Instituto de Óptica “Daza de Valdés,” CSIC, 28006-Madrid; Fuentes, L. M.

2014-10-07

We report a detailed characterization of the spatial resolution provided by two-photon absorption spectroscopy suited for plasma diagnosis via the 1S-2S transition of atomic hydrogen for optogalvanic detection and laser induced fluorescence (LIF). A precise knowledge of the spatial resolution is crucial for a correct interpretation of measurements, if the plasma parameters to be analysed undergo strong spatial variations. The present study is based on a novel approach which provides a reliable and realistic determination of the spatial resolution. Measured irradiance distribution of laser beam waists in the overlap volume, provided by a high resolution UV camera, are employed tomore » resolve coupled rate equations accounting for two-photon excitation, fluorescence decay and ionization. The resulting three-dimensional yield distributions reveal in detail the spatial resolution for optogalvanic and LIF detection and related saturation due to depletion. Two-photon absorption profiles broader than the Fourier transform-limited laser bandwidth are also incorporated in the calculations. The approach allows an accurate analysis of the spatial resolution present in recent and future measurements.« less

[An effective method for improving the imaging spatial resolution of terahertz time domain spectroscopy system].

PubMed

Zhang, Zeng-yan; Ji, Te; Zhu, Zhi-yong; Zhao, Hong-wei; Chen, Min; Xiao, Ti-qiao; Guo, Zhi

2015-01-01

Terahertz radiation is an electromagnetic radiation in the range between millimeter waves and far infrared. Due to its low energy and non-ionizing characters, THz pulse imaging emerges as a novel tool in many fields, such as material, chemical, biological medicine, and food safety. Limited spatial resolution is a significant restricting factor of terahertz imaging technology. Near field imaging method was proposed to improve the spatial resolution of terahertz system. Submillimeter scale's spauial resolution can be achieved if the income source size is smaller than the wawelength of the incoming source and the source is very close to the sample. But many changes were needed to the traditional terahertz time domain spectroscopy system, and it's very complex to analyze sample's physical parameters through the terahertz signal. A method of inserting a pinhole upstream to the sample was first proposed in this article to improve the spatial resolution of traditional terahertz time domain spectroscopy system. The measured spatial resolution of terahertz time domain spectroscopy system by knife edge method can achieve spatial resolution curves. The moving stage distance between 10 % and 90 Yo of the maximum signals respectively was defined as the, spatial resolution of the system. Imaging spatial resolution of traditional terahertz time domain spectroscopy system was improved dramatically after inserted a pinhole with diameter 0. 5 mm, 2 mm upstream to the sample. Experimental results show that the spatial resolution has been improved from 1. 276 mm to 0. 774 mm, with the increment about 39 %. Though this simple method, the spatial resolution of traditional terahertz time domain spectroscopy system was increased from millimeter scale to submillimeter scale. A pinhole with diameter 1 mm on a polyethylene plate was taken as sample, to terahertz imaging study. The traditional terahertz time domain spectroscopy system and pinhole inserted terahertz time domain spectroscopy system were applied in the imaging experiment respectively. The relative THz-power loss imaging of samples were use in this article. This method generally delivers the best signal to noise ratio in loss images, dispersion effects are cancelled. Terahertz imaging results show that the sample's boundary was more distinct after inserting the pinhole in front of, sample. The results also conform that inserting pinhole in front of sample can improve the imaging spatial resolution effectively. The theoretical analyses of the method which improve the spatial resolution by inserting a pinhole in front of sample were given in this article. The analyses also indicate that the smaller the pinhole size, the longer spatial coherence length of the system, the better spatial resolution of the system. At the same time the terahertz signal will be reduced accordingly. All the experimental results and theoretical analyses indicate that the method of inserting a pinhole in front of sample can improve the spatial resolution of traditional terahertz time domain spectroscopy system effectively, and it will further expand the application of terahertz imaging technology.

EnLightenment: High resolution smartphone microscopy as an educational and public engagement platform

PubMed Central

Wicks, Laura C.; Cairns, Gemma S.; Melnyk, Jacob; Bryce, Scott; Duncan, Rory R.; Dalgarno, Paul A.

2018-01-01

We developed a simple, cost-effective smartphone microscopy platform for use in educational and public engagement programs. We demonstrated its effectiveness, and potential for citizen science through a national imaging initiative, EnLightenment. The cost effectiveness of the instrument allowed for the program to deliver over 500 microscopes to more than 100 secondary schools throughout Scotland, targeting 1000’s of 12-14 year olds. Through careful, quantified, selection of a high power, low-cost objective lens, our smartphone microscope has an imaging resolution of microns, with a working distance of 3 mm. It is therefore capable of imaging single cells and sub-cellular features, and retains usability for young children. The microscopes were designed in kit form and provided an interdisciplinary educational tool. By providing full lesson plans and support material, we developed a framework to explore optical design, microscope performance, engineering challenges on construction and real-world applications in life sciences, biological imaging, marine biology, art, and technology. A national online imaging competition framed EnLightenment ; with over 500 high quality images submitted of diverse content, spanning multiple disciplines. With examples of cellular and sub-cellular features clearly identifiable in some submissions, we show how young public can use these instruments for research-level imaging applications, and the potential of the instrument for citizen science programs. PMID:29623296

Trespassing cancer cells: 'fingerprinting' invasive protrusions reveals metastatic culprits.

PubMed

Klemke, Richard L

2012-10-01

Metastatic cancer cells produce invasive membrane protrusions called invadopodia and pseudopodia, which play a central role in driving cancer cell dissemination in the body. Malignant cells use these structures to attach to and degrade extracellular matrix proteins, generate force for cell locomotion, and to penetrate the vasculature. Recent work using unique subcellular fractionation methodologies combined with spatial genomic, proteomic, and phosphoproteomic profiling has provided insight into the invadopodiome and pseudopodiome signaling networks that control the protrusion of invasive membranes. Here I highlight how these powerful spatial 'omics' approaches reveal important signatures of metastatic cancer cells and possible new therapeutic targets aimed at treating metastatic disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

Additional studies of forest classification accuracy as influenced by multispectral scanner spatial resolution

NASA Technical Reports Server (NTRS)

Sadowski, F. E.; Sarno, J. E.

1976-01-01

First, an analysis of forest feature signatures was used to help explain the large variation in classification accuracy that can occur among individual forest features for any one case of spatial resolution and the inconsistent changes in classification accuracy that were demonstrated among features as spatial resolution was degraded. Second, the classification rejection threshold was varied in an effort to reduce the large proportion of unclassified resolution elements that previously appeared in the processing of coarse resolution data when a constant rejection threshold was used for all cases of spatial resolution. For the signature analysis, two-channel ellipse plots showing the feature signature distributions for several cases of spatial resolution indicated that the capability of signatures to correctly identify their respective features is dependent on the amount of statistical overlap among signatures. Reductions in signature variance that occur in data of degraded spatial resolution may not necessarily decrease the amount of statistical overlap among signatures having large variance and small mean separations. Features classified by such signatures may thus continue to have similar amounts of misclassified elements in coarser resolution data, and thus, not necessarily improve in classification accuracy.

Handling Different Spatial Resolutions in Image Fusion by Multivariate Curve Resolution-Alternating Least Squares for Incomplete Image Multisets.

PubMed

Piqueras, Sara; Bedia, Carmen; Beleites, Claudia; Krafft, Christoph; Popp, Jürgen; Maeder, Marcel; Tauler, Romà; de Juan, Anna

2018-06-05

Data fusion of different imaging techniques allows a comprehensive description of chemical and biological systems. Yet, joining images acquired with different spectroscopic platforms is complex because of the different sample orientation and image spatial resolution. Whereas matching sample orientation is often solved by performing suitable affine transformations of rotation, translation, and scaling among images, the main difficulty in image fusion is preserving the spatial detail of the highest spatial resolution image during multitechnique image analysis. In this work, a special variant of the unmixing algorithm Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) for incomplete multisets is proposed to provide a solution for this kind of problem. This algorithm allows analyzing simultaneously images collected with different spectroscopic platforms without losing spatial resolution and ensuring spatial coherence among the images treated. The incomplete multiset structure concatenates images of the two platforms at the lowest spatial resolution with the image acquired with the highest spatial resolution. As a result, the constituents of the sample analyzed are defined by a single set of distribution maps, common to all platforms used and with the highest spatial resolution, and their related extended spectral signatures, covering the signals provided by each of the fused techniques. We demonstrate the potential of the new variant of MCR-ALS for multitechnique analysis on three case studies: (i) a model example of MIR and Raman images of pharmaceutical mixture, (ii) FT-IR and Raman images of palatine tonsil tissue, and (iii) mass spectrometry and Raman images of bean tissue.

Photothermally triggered actuation of hybrid materials as a new platform for in vitro cell manipulation

DOE PAGES

Sutton, Amy; Shirman, Tanya; Timonen, Jaakko V. I.; ...

2017-03-13

Mechanical forces in the cell’s natural environment have a crucial impact on growth, differentiation and behaviour. Few areas of biology can be understood without taking into account how both individual cells and cell networks sense and transduce physical stresses. However, the field is currently held back by the limitations of the available methods to apply physiologically relevant stress profiles on cells, particularly with sub-cellular resolution, in controlled in vitro experiments. Here we report a new type of active cell culture material that allows highly localized, directional and reversible deformation of the cell growth substrate, with control at scales ranging frommore » the entire surface to the subcellular, and response times on the order of seconds. These capabilities are not matched by any other method, and this versatile material has the potential to bridge the performance gap between the existing single cell micro-manipulation and 2D cell sheet mechanical stimulation techniques.« less

Probing embryonic tissue mechanics with laser hole drilling

NASA Astrophysics Data System (ADS)

Ma, Xiaoyan; Lynch, Holley E.; Scully, Peter C.; Hutson, M. Shane

2009-09-01

We use laser hole drilling to assess the mechanics of an embryonic epithelium during development—in vivo and with subcellular resolution. We ablate a subcellular cylindrical hole clean through the epithelium and track the subsequent recoil of adjacent cells (on ms time scales). We investigate dorsal closure in the fruit fly with emphasis on apical constriction of amnioserosa cells. The mechanical behavior of this epithelium falls between that of a continuous sheet and a 2D cellular foam (a network of tensile interfaces). Tensile stress is carried both by cell-cell interfaces and by the cells' apical actin networks. Our results show that stress is slightly concentrated along interfaces (1.6-fold), but only in early closure. Furthermore, closure is marked by a decrease in the recoil power-law exponent, implying a transition to a more solid-like tissue. We use the site and stage dependence of the recoil kinetics to constrain how the cellular mechanics change during closure. We apply these results to test extant computational models.

Photothermally triggered actuation of hybrid materials as a new platform for in vitro cell manipulation

PubMed Central

Sutton, Amy; Shirman, Tanya; Timonen, Jaakko V. I.; England, Grant T; Kim, Philseok; Kolle, Mathias; Ferrante, Thomas; Zarzar, Lauren D; Strong, Elizabeth; Aizenberg, Joanna

2017-01-01

Mechanical forces in the cell’s natural environment have a crucial impact on growth, differentiation and behaviour. Few areas of biology can be understood without taking into account how both individual cells and cell networks sense and transduce physical stresses. However, the field is currently held back by the limitations of the available methods to apply physiologically relevant stress profiles on cells, particularly with sub-cellular resolution, in controlled in vitro experiments. Here we report a new type of active cell culture material that allows highly localized, directional and reversible deformation of the cell growth substrate, with control at scales ranging from the entire surface to the subcellular, and response times on the order of seconds. These capabilities are not matched by any other method, and this versatile material has the potential to bridge the performance gap between the existing single cell micro-manipulation and 2D cell sheet mechanical stimulation techniques. PMID:28287116

Subcellular analysis of interaction between breast cancer cells and drug by digital holography

NASA Astrophysics Data System (ADS)

Zhao, Jie; Lin, Qiaowen; Wang, Dayong; Wang, Yunxin; Ouyang, Liting; Guo, Sha; Yao, Qian

2017-10-01

Digital holographic microscopy is a promising quantitative phase-contrast imaging technique, which exhibits the advantages of non-destruction, full field of view, quasi-real time, and don't need dye and external marker to the living biological sample. In this paper, the inverted off-axis image-plane digital holography with pre-magnification is built up to study the living MDA-MB-231 breast cancer cells. The lateral resolution of the proposed experimental setup is 0.87μm, which is verified by the standard USAF test target. Then the system is used to visualize the interaction between living breast cancer cells and drug. The blebbing is observed after the cells are treated by paclitaxel drug, and the distribution of the paclitaxel inside the cells is detected, which is near the cytomembrane, or in other words the end of the microtubules. It will stop the mitosis and cause the death of the cells. It is helpful to reveal the anticancer mechanism of paclitaxel in the subcellular scale.

Photothermally triggered actuation of hybrid materials as a new platform for in vitro cell manipulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sutton, Amy; Shirman, Tanya; Timonen, Jaakko V. I.

Mechanical forces in the cell’s natural environment have a crucial impact on growth, differentiation and behaviour. Few areas of biology can be understood without taking into account how both individual cells and cell networks sense and transduce physical stresses. However, the field is currently held back by the limitations of the available methods to apply physiologically relevant stress profiles on cells, particularly with sub-cellular resolution, in controlled in vitro experiments. Here we report a new type of active cell culture material that allows highly localized, directional and reversible deformation of the cell growth substrate, with control at scales ranging frommore » the entire surface to the subcellular, and response times on the order of seconds. These capabilities are not matched by any other method, and this versatile material has the potential to bridge the performance gap between the existing single cell micro-manipulation and 2D cell sheet mechanical stimulation techniques.« less

Assessing the Resolution Adaptability of the Zhang-McFarlane Cumulus Parameterization With Spatial and Temporal Averaging: RESOLUTION ADAPTABILITY OF ZM SCHEME

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yun, Yuxing; Fan, Jiwen; Xiao, Heng

Realistic modeling of cumulus convection at fine model resolutions (a few to a few tens of km) is problematic since it requires the cumulus scheme to adapt to higher resolution than they were originally designed for (~100 km). To solve this problem, we implement the spatial averaging method proposed in Xiao et al. (2015) and also propose a temporal averaging method for the large-scale convective available potential energy (CAPE) tendency in the Zhang-McFarlane (ZM) cumulus parameterization. The resolution adaptability of the original ZM scheme, the scheme with spatial averaging, and the scheme with both spatial and temporal averaging at 4-32more » km resolution is assessed using the Weather Research and Forecasting (WRF) model, by comparing with Cloud Resolving Model (CRM) results. We find that the original ZM scheme has very poor resolution adaptability, with sub-grid convective transport and precipitation increasing significantly as the resolution increases. The spatial averaging method improves the resolution adaptability of the ZM scheme and better conserves the total transport of moist static energy and total precipitation. With the temporal averaging method, the resolution adaptability of the scheme is further improved, with sub-grid convective precipitation becoming smaller than resolved precipitation for resolution higher than 8 km, which is consistent with the results from the CRM simulation. Both the spatial distribution and time series of precipitation are improved with the spatial and temporal averaging methods. The results may be helpful for developing resolution adaptability for other cumulus parameterizations that are based on quasi-equilibrium assumption.« less

Near-Atomic Three-Dimensional Mapping for Site-Specific Chemistry of 'Superbugs'.

PubMed

Adineh, Vahid R; Marceau, Ross K W; Velkov, Tony; Li, Jian; Fu, Jing

2016-11-09

Emergence of multidrug resistant Gram-negative bacteria has caused a global health crisis and last-line class of antibiotics such as polymyxins are increasingly used. The chemical composition at the cell surface plays a key role in antibiotic resistance. Unlike imaging the cellular ultrastructure with well-developed electron microscopy, the acquisition of a high-resolution chemical map of the bacterial surface still remains a technological challenge. In this study, we developed an atom probe tomography (APT) analysis approach to acquire mass spectra in the pulsed-voltage mode and reconstructed the 3D chemical distribution of atoms and molecules in the subcellular domain at the near-atomic scale. Using focused ion beam (FIB) milling together with micromanipulation, site-specific samples were retrieved from a single cell of Acinetobacter baumannii prepared as needle-shaped tips with end radii less than 60 nm, followed by a nanoscale coating of silver in the order of 10 nm. The significantly elevated conductivity provided by the metallic coating enabled successful and routine field evaporation of the biological material, with all the benefits of pulsed-voltage APT. In parallel with conventional cryo-TEM imaging, our novel approach was applied to investigate polymyxin-susceptible and -resistant strains of A. baumannii after treatment of polymyxin B. Acquired atom probe mass spectra from the cell envelope revealed characteristic fragments of phosphocholine from the polymyxin-susceptible strain, but limited signals from this molecule were detected in the polymyxin-resistant strain. This study promises unprecedented capacity for 3D nanoscale imaging and chemical mapping of bacterial cells at the ultimate 3D spatial resolution using APT.

Multimodal nonlinear microscopy of biopsy specimen: towards intraoperative diagnostics (Conference Presentation)

NASA Astrophysics Data System (ADS)

Schmitt, Michael; Heuke, Sandro; Meyer, Tobias; Chernavskaia, Olga; Bocklitz, Thomas W.; Popp, Juergen

2016-03-01

The realization of label-free molecule specific imaging of morphology and chemical composition of tissue at subcellular spatial resolution in real time is crucial for many envisioned applications in medicine, e.g., precise surgical guidance and non-invasive histopathologic examination of tissue. Thus, new approaches for a fast and reliable in vivo and near in vivo (ex corpore in vivo) tissue characterization to supplement routine pathological diagnostics is needed. Spectroscopic imaging approaches are particularly important since they have the potential to provide a pathologist with adequate support in the form of clinically-relevant information under both ex vivo and in vivo conditions. In this contribution it is demonstrated, that multimodal nonlinear microscopy combining coherent anti-Stokes Raman scattering (CARS), two photon excited fluorescence (TPEF) and second harmonic generation (SHG) enables the detection of characteristic structures and the accompanying molecular changes of widespread diseases, particularly of cancer and atherosclerosis. The detailed images enable an objective evaluation of the tissue samples for an early diagnosis of the disease status. Increasing the spectral resolution and analyzing CARS images at multiple Raman resonances improves the chemical specificity. To facilitate handling and interpretation of the image data characteristic properties can be automatically extracted by advanced image processing algorithms, e.g., for tissue classification. Overall, the presented examples show the great potential of multimodal imaging to augment standard intraoperative clinical assessment with functional multimodal CARS/SHG/TPEF images to highlight functional activity and tumor boundaries. It ensures fast, label-free and non-invasive intraoperative tissue classification paving the way towards in vivo optical pathology.

High-resolution synchrotron radiation-based phase tomography of the healthy and epileptic brain

NASA Astrophysics Data System (ADS)

Bikis, Christos; Janz, Philipp; Schulz, Georg; Schweighauser, Gabriel; Hench, Jürgen; Thalmann, Peter; Deyhle, Hans; Chicherova, Natalia; Rack, Alexander; Khimchenko, Anna; Hieber, Simone E.; Mariani, Luigi; Haas, Carola A.; Müller, Bert

2016-10-01

Phase-contrast micro-tomography using synchrotron radiation has yielded superior soft tissue visualization down to the sub-cellular level. The isotropic spatial resolution down to about one micron is comparable to the one of histology. The methods, however, provide different physical quantities and are thus complementary, also allowing for the extension of histology into the third dimension. To prepare for cross-sectional animal studies on epilepsy, we have standardized the specimen's preparation and scanning procedure for mouse brains, so that subsequent histology remains entirely unaffected and scanning of all samples (n = 28) is possible in a realistic time frame. For that, we have scanned five healthy and epileptic mouse brains at the ID19 beamline, ESRF, Grenoble, France, using grating- and propagation-based phase contrast micro-tomography. The resulting datasets clearly show the cortex, ventricular system, thalamus, hypothalamus, and hippocampus. Our focus is on the latter, having planned kainate-induced epilepsy experiments. The cell density and organization in the dentate gyrus and Ammon's horn region were clearly visualized in control animals. This proof of principle was required to initiate experiment. The resulting three-dimensional data have been correlated to histology. The goal is a brain-wide quantification of cell death or structural reorganization associated with epilepsy as opposed to histology alone that represents small volumes of the total brain only. Thus, the proposed technique bears the potential to correlate the gold standard in analysis with independently obtained data sets. Such an achievement also fuels interest for other groups in neuroscience research to closely collaborate with experts in phase micro-tomography.

Three-dimensional virtual histology of human cerebellum by X-ray phase-contrast tomography.

PubMed

Töpperwien, Mareike; van der Meer, Franziska; Stadelmann, Christine; Salditt, Tim

2018-06-18

To quantitatively evaluate brain tissue and its corresponding function, knowledge of the 3D cellular distribution is essential. The gold standard to obtain this information is histology, a destructive and labor-intensive technique where the specimen is sliced and examined under a light microscope, providing 3D information at nonisotropic resolution. To overcome the limitations of conventional histology, we use phase-contrast X-ray tomography with optimized optics, reconstruction, and image analysis, both at a dedicated synchrotron radiation endstation, which we have equipped with X-ray waveguide optics for coherence and wavefront filtering, and at a compact laboratory source. As a proof-of-concept demonstration we probe the 3D cytoarchitecture in millimeter-sized punches of unstained human cerebellum embedded in paraffin and show that isotropic subcellular resolution can be reached at both setups throughout the specimen. To enable a quantitative analysis of the reconstructed data, we demonstrate automatic cell segmentation and localization of over 1 million neurons within the cerebellar cortex. This allows for the analysis of the spatial organization and correlation of cells in all dimensions by borrowing concepts from condensed-matter physics, indicating a strong short-range order and local clustering of the cells in the granular layer. By quantification of 3D neuronal "packing," we can hence shed light on how the human cerebellum accommodates 80% of the total neurons in the brain in only 10% of its volume. In addition, we show that the distribution of neighboring neurons in the granular layer is anisotropic with respect to the Purkinje cell dendrites. Copyright © 2018 the Author(s). Published by PNAS.

Correlative two-photon and serial block face scanning electron microscopy in neuronal tissue using 3D near-infrared branding maps.

PubMed

Lees, Robert M; Peddie, Christopher J; Collinson, Lucy M; Ashby, Michael C; Verkade, Paul

2017-01-01

Linking cellular structure and function has always been a key goal of microscopy, but obtaining high resolution spatial and temporal information from the same specimen is a fundamental challenge. Two-photon (2P) microscopy allows imaging deep inside intact tissue, bringing great insight into the structural and functional dynamics of cells in their physiological environment. At the nanoscale, the complex ultrastructure of a cell's environment in tissue can be reconstructed in three dimensions (3D) using serial block face scanning electron microscopy (SBF-SEM). This provides a snapshot of high resolution structural information pertaining to the shape, organization, and localization of multiple subcellular structures at the same time. The pairing of these two imaging modalities in the same specimen provides key information to relate cellular dynamics to the ultrastructural environment. Until recently, approaches to relocate a region of interest (ROI) in tissue from 2P microscopy for SBF-SEM have been inefficient or unreliable. However, near-infrared branding (NIRB) overcomes this by using the laser from a multiphoton microscope to create fiducial markers for accurate correlation of 2P and electron microscopy (EM) imaging volumes. The process is quick and can be user defined for each sample. Here, to increase the efficiency of ROI relocation, multiple NIRB marks are used in 3D to target ultramicrotomy. A workflow is described and discussed to obtain a data set for 3D correlated light and electron microscopy, using three different preparations of brain tissue as examples. Copyright © 2017 Elsevier Inc. All rights reserved.

Nonlinear adaptive optics: aberration correction in three photon fluorescence microscopy for mouse brain imaging

NASA Astrophysics Data System (ADS)

Sinefeld, David; Paudel, Hari P.; Wang, Tianyu; Wang, Mengran; Ouzounov, Dimitre G.; Bifano, Thomas G.; Xu, Chris

2017-02-01

Multiphoton fluorescence microscopy is a well-established technique for deep-tissue imaging with subcellular resolution. Three-photon microscopy (3PM) when combined with long wavelength excitation was shown to allow deeper imaging than two-photon microscopy (2PM) in biological tissues, such as mouse brain, because out-of-focus background light can be further reduced due to the higher order nonlinear excitation. As was demonstrated in 2PM systems, imaging depth and resolution can be improved by aberration correction using adaptive optics (AO) techniques which are based on shaping the scanning beam using a spatial light modulator (SLM). In this way, it is possible to compensate for tissue low order aberration and to some extent, to compensate for tissue scattering. Here, we present a 3PM AO microscopy system for brain imaging. Soliton self-frequency shift is used to create a femtosecond source at 1675 nm and a microelectromechanical (MEMS) SLM serves as the wavefront shaping device. We perturb the 1020 segment SLM using a modified nonlinear version of three-point phase shifting interferometry. The nonlinearity of the fluorescence signal used for feedback ensures that the signal is increasing when the spot size decreases, allowing compensation of phase errors in an iterative optimization process without direct phase measurement. We compare the performance for different orders of nonlinear feedback, showing an exponential growth in signal improvement as the nonlinear order increases. We demonstrate the impact of the method by applying the 3PM AO system for in-vivo mouse brain imaging, showing improvement in signal at 1-mm depth inside the brain.

Achieving high spatial resolution using a microchannel plate detector with an economic and scalable approach

NASA Astrophysics Data System (ADS)

Wiggins, B. B.; deSouza, Z. O.; Vadas, J.; Alexander, A.; Hudan, S.; deSouza, R. T.

2017-11-01

A second generation position-sensitive microchannel plate detector using the induced signal approach has been realized. This detector is presently capable of measuring the incident position of electrons, photons, or ions. To assess the spatial resolution, the masked detector was illuminated by electrons. The initial, measured spatial resolution of 276 μm FWHM was improved by requiring a minimum signal amplitude on the anode and by employing digital signal processing techniques. The resulting measured spatial resolution of 119 μm FWHM corresponds to an intrinsic resolution of 98 μm FWHM when the effect of the finite slit width is de-convoluted. This measurement is a substantial improvement from the last reported spatial resolution of 466 μm FWHM using the induced signal approach. To understand the factors that limit the measured resolution, the performance of the detector is simulated.

Statistical model based iterative reconstruction (MBIR) in clinical CT systems. Part II. Experimental assessment of spatial resolution performance.

PubMed

Li, Ke; Garrett, John; Ge, Yongshuai; Chen, Guang-Hong

2014-07-01

Statistical model based iterative reconstruction (MBIR) methods have been introduced to clinical CT systems and are being used in some clinical diagnostic applications. The purpose of this paper is to experimentally assess the unique spatial resolution characteristics of this nonlinear reconstruction method and identify its potential impact on the detectabilities and the associated radiation dose levels for specific imaging tasks. The thoracic section of a pediatric phantom was repeatedly scanned 50 or 100 times using a 64-slice clinical CT scanner at four different dose levels [CTDIvol =4, 8, 12, 16 (mGy)]. Both filtered backprojection (FBP) and MBIR (Veo(®), GE Healthcare, Waukesha, WI) were used for image reconstruction and results were compared with one another. Eight test objects in the phantom with contrast levels ranging from 13 to 1710 HU were used to assess spatial resolution. The axial spatial resolution was quantified with the point spread function (PSF), while the z resolution was quantified with the slice sensitivity profile. Both were measured locally on the test objects and in the image domain. The dependence of spatial resolution on contrast and dose levels was studied. The study also features a systematic investigation of the potential trade-off between spatial resolution and locally defined noise and their joint impact on the overall image quality, which was quantified by the image domain-based channelized Hotelling observer (CHO) detectability index d'. (1) The axial spatial resolution of MBIR depends on both radiation dose level and image contrast level, whereas it is supposedly independent of these two factors in FBP. The axial spatial resolution of MBIR always improved with an increasing radiation dose level and/or contrast level. (2) The axial spatial resolution of MBIR became equivalent to that of FBP at some transitional contrast level, above which MBIR demonstrated superior spatial resolution than FBP (and vice versa); the value of this transitional contrast highly depended on the dose level. (3) The PSFs of MBIR could be approximated as Gaussian functions with reasonably good accuracy. (4) Thez resolution of MBIR showed similar contrast and dose dependence. (5) Noise standard deviation assessed on the edges of objects demonstrated a trade-off with spatial resolution in MBIR. (5) When both spatial resolution and image noise were considered using the CHO analysis, MBIR led to significant improvement in the overall CT image quality for both high and low contrast detection tasks at both standard and low dose levels. Due to the intrinsic nonlinearity of the MBIR method, many well-known CT spatial resolution and noise properties have been modified. In particular, dose dependence and contrast dependence have been introduced to the spatial resolution of CT images by MBIR. The method has also introduced some novel noise-resolution trade-off not seen in traditional CT images. While the benefits of MBIR regarding the overall image quality, as demonstrated in this work, are significant, the optimal use of this method in clinical practice demands a thorough understanding of its unique physical characteristics.

Microscopy with spatial filtering for sorting particles and monitoring subcellular morphology

NASA Astrophysics Data System (ADS)

Zheng, Jing-Yi; Qian, Zhen; Pasternack, Robert M.; Boustany, Nada N.

2009-02-01

Optical scatter imaging (OSI) was developed to non-invasively track real-time changes in particle morphology with submicron sensitivity in situ without exogenous labeling, cell fixing, or organelle isolation. For spherical particles, the intensity ratio of wide-to-narrow angle scatter (OSIR, Optical Scatter Image Ratio) was shown to decrease monotonically with diameter and agree with Mie theory. In living cells, we recently reported this technique is able to detect mitochondrial morphological alterations, which were mediated by the Bcl-xL transmembrane domain, and could not be observed by fluorescence or differential interference contrast images. Here we further extend the ability of morphology assessment by adopting a digital micromirror device (DMD) for Fourier filtering. When placed in the Fourier plane the DMD can be used to select scattering intensities at desired combination of scattering angles. We designed an optical filter bank consisting of Gabor-like filters with various scales and rotations based on Gabor filters, which have been widely used for localization of spatial and frequency information in digital images and texture analysis. Using a model system consisting of mixtures of polystyrene spheres and bacteria, we show how this system can be used to sort particles on a microscopic slide based on their size, orientation and aspect ratio. We are currently applying this technique to characterize the morphology of subcellular organelles to help understand fundamental biological processes.

Global, quantitative and dynamic mapping of protein subcellular localization

PubMed Central

Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH

2016-01-01

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

In vivo imaging of microscopic structures in the rat retina

PubMed Central

Geng, Ying; Greenberg, Kenneth P.; Wolfe, Robert; Gray, Daniel C.; Hunter, Jennifer J.; Dubra, Alfredo; Flannery, John G.; Williams, David R.; Porter, Jason

2010-01-01

Purpose The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. We have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo. Methods Green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy. Results Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics (AO). AO correction reduced the total root mean square wavefront error, on average, from 0.30 μm to 0.05 μm (1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was ∼1.84 μm, approximately 82% greater than the FWHM of the diffraction-limited LSF. Conclusions With perfect aberration compensation, the in vivo resolution in the rat eye could be ∼2× greater than that in the human eye due to its large numerical aperture (∼0.43). While the fAOSLO corrects a substantial fraction of the rat eye's aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using AO to investigate the rodent eye in vivo with high resolution. PMID:19578019

a Spiral-Based Downscaling Method for Generating 30 M Time Series Image Data

NASA Astrophysics Data System (ADS)

Liu, B.; Chen, J.; Xing, H.; Wu, H.; Zhang, J.

2017-09-01

The spatial detail and updating frequency of land cover data are important factors influencing land surface dynamic monitoring applications in high spatial resolution scale. However, the fragmentized patches and seasonal variable of some land cover types (e. g. small crop field, wetland) make it labor-intensive and difficult in the generation of land cover data. Utilizing the high spatial resolution multi-temporal image data is a possible solution. Unfortunately, the spatial and temporal resolution of available remote sensing data like Landsat or MODIS datasets can hardly satisfy the minimum mapping unit and frequency of current land cover mapping / updating at the same time. The generation of high resolution time series may be a compromise to cover the shortage in land cover updating process. One of popular way is to downscale multi-temporal MODIS data with other high spatial resolution auxiliary data like Landsat. But the usual manner of downscaling pixel based on a window may lead to the underdetermined problem in heterogeneous area, result in the uncertainty of some high spatial resolution pixels. Therefore, the downscaled multi-temporal data can hardly reach high spatial resolution as Landsat data. A spiral based method was introduced to downscale low spatial and high temporal resolution image data to high spatial and high temporal resolution image data. By the way of searching the similar pixels around the adjacent region based on the spiral, the pixel set was made up in the adjacent region pixel by pixel. The underdetermined problem is prevented to a large extent from solving the linear system when adopting the pixel set constructed. With the help of ordinary least squares, the method inverted the endmember values of linear system. The high spatial resolution image was reconstructed on the basis of high spatial resolution class map and the endmember values band by band. Then, the high spatial resolution time series was formed with these high spatial resolution images image by image. Simulated experiment and remote sensing image downscaling experiment were conducted. In simulated experiment, the 30 meters class map dataset Globeland30 was adopted to investigate the effect on avoid the underdetermined problem in downscaling procedure and a comparison between spiral and window was conducted. Further, the MODIS NDVI and Landsat image data was adopted to generate the 30m time series NDVI in remote sensing image downscaling experiment. Simulated experiment results showed that the proposed method had a robust performance in downscaling pixel in heterogeneous region and indicated that it was superior to the traditional window-based methods. The high resolution time series generated may be a benefit to the mapping and updating of land cover data.

Chromatic and Achromatic Spatial Resolution of Local Field Potentials in Awake Cortex.

PubMed

Jansen, Michael; Li, Xiaobing; Lashgari, Reza; Kremkow, Jens; Bereshpolova, Yulia; Swadlow, Harvey A; Zaidi, Qasim; Alonso, Jose-Manuel

2015-10-01

Local field potentials (LFPs) have become an important measure of neuronal population activity in the brain and could provide robust signals to guide the implant of visual cortical prosthesis in the future. However, it remains unclear whether LFPs can detect weak cortical responses (e.g., cortical responses to equiluminant color) and whether they have enough visual spatial resolution to distinguish different chromatic and achromatic stimulus patterns. By recording from awake behaving macaques in primary visual cortex, here we demonstrate that LFPs respond robustly to pure chromatic stimuli and exhibit ∼2.5 times lower spatial resolution for chromatic than achromatic stimulus patterns, a value that resembles the ratio of achromatic/chromatic resolution measured with psychophysical experiments in humans. We also show that, although the spatial resolution of LFP decays with visual eccentricity as is also the case for single neurons, LFPs have higher spatial resolution and show weaker response suppression to low spatial frequencies than spiking multiunit activity. These results indicate that LFP recordings are an excellent approach to measure spatial resolution from local populations of neurons in visual cortex including those responsive to color. © The Author 2014. Published by Oxford University Press.

The spatial resolution of silicon-based electron detectors in beta-autoradiography.

PubMed

Cabello, Jorge; Wells, Kevin

2010-03-21

Thin tissue autoradiography is an imaging modality where ex-vivo tissue sections are placed in direct contact with autoradiographic film. These tissue sections contain a radiolabelled ligand bound to a specific biomolecule under study. This radioligand emits beta - or beta+ particles ionizing silver halide crystals in the film. High spatial resolution autoradiograms are obtained using low energy radioisotopes, such as (3)H where an intrinsic 0.1-1 microm spatial resolution can be achieved. Several digital alternatives have been presented over the past few years to replace conventional film but their spatial resolution has yet to equal film, although silicon-based imaging technologies have demonstrated higher sensitivity compared to conventional film. It will be shown in this work how pixel size is a critical parameter for achieving high spatial resolution for low energy uncollimated beta imaging. In this work we also examine the confounding factors impeding silicon-based technologies with respect to spatial resolution. The study considers charge diffusion in silicon and detector noise, and this is applied to a range of radioisotopes typically used in autoradiography. Finally an optimal detector geometry to obtain the best possible spatial resolution for a specific technology and a specific radioisotope is suggested.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scaduto, DA; Hu, Y-H; Zhao, W

Purpose: Spatial resolution in digital breast tomosynthesis (DBT) is affected by inherent/binned detector resolution, oblique entry of x-rays, and focal spot size/motion; the limited angular range further limits spatial resolution in the depth-direction. While DBT is being widely adopted clinically, imaging performance metrics and quality control protocols have not been standardized. AAPM Task Group 245 on Tomosynthesis Quality Control has been formed to address this deficiency. Methods: Methods of measuring spatial resolution are evaluated using two prototype quality control phantoms for DBT. Spatial resolution in the detector plane is measured in projection and reconstruction domains using edge-spread function (ESF), point-spreadmore » function (PSF) and modulation transfer function (MTF). Spatial resolution in the depth-direction and effective slice thickness are measured in the reconstruction domain using slice sensitivity profile (SSP) and artifact spread function (ASF). An oversampled PSF in the depth-direction is measured using a 50 µm angulated tungsten wire, from which the MTF is computed. Object-dependent PSF is derived and compared with ASF. Sensitivity of these measurements to phantom positioning, imaging conditions and reconstruction algorithms is evaluated. Results are compared from systems of varying acquisition geometry (9–25 projections over 15–60°). Dependence of measurements on feature size is investigated. Results: Measurements of spatial resolution using PSF and LSF are shown to depend on feature size; depth-direction spatial resolution measurements are shown to similarly depend on feature size for ASF, though deconvolution with an object function removes feature size-dependence. A slanted wire may be used to measure oversampled PSFs, from which MTFs may be computed for both in-plane and depth-direction resolution. Conclusion: Spatial resolution measured using PSF is object-independent with sufficiently small object; MTF is object-independent. Depth-direction spatial resolution may be measured directly using MTF or indirectly using ASF or SSP as surrogate measurements. While MTF is object-independent, it is invalid for nonlinear reconstructions.« less

Spatial and temporal remote sensing data fusion for vegetation monitoring

USDA-ARS?s Scientific Manuscript database

The suite of available remote sensing instruments varies widely in terms of sensor characteristics, spatial resolution and acquisition frequency. For example, the Moderate-resolution Imaging Spectroradiometer (MODIS) provides daily global observations at 250m to 1km spatial resolution. While imagery...

Development of a large-area Multigap RPC with adequate spatial resolution for muon tomography

NASA Astrophysics Data System (ADS)

Wang, J.; Wang, Y.; Wang, X.; Zeng, M.; Xie, B.; Han, D.; Lyu, P.; Wang, F.; Li, Y.

2016-11-01

We study the performance of a large-area 2-D Multigap Resistive Plate Chamber (MRPC) designed for muon tomography with high spatial resolution. An efficiency up to 98% and a spatial resolution of around 270 μ m are obtained in cosmic ray and X-ray tests. The performance of the MRPC is also investigated for two working gases: standard gas and pure Freon. The result shows that the MRPC working in pure Freon can provide higher efficiency and better spatial resolution.

Label-free photoacoustic nanoscopy

PubMed Central

Danielli, Amos; Maslov, Konstantin; Garcia-Uribe, Alejandro; Winkler, Amy M.; Li, Chiye; Wang, Lidai; Chen, Yun; Dorn, Gerald W.; Wang, Lihong V.

2014-01-01

Abstract. Super-resolution microscopy techniques—capable of overcoming the diffraction limit of light—have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules. PMID:25104412

Light Sheet Fluorescence Microscopy (LSFM)

PubMed Central

Adams, Michael W.; Loftus, Andrew F.; Dunn, Sarah E.; Joens, Matthew S.; Fitzpatrick, James A.J.

2015-01-01

The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light Sheet Fluorescent Microscopy (LSFM), a century old idea (Siedentopf and Zsigmondy, 1902) made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light sheet based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements. PMID:25559221

Identification and super-resolution imaging of ligand-activated receptor dimers in live cells

NASA Astrophysics Data System (ADS)

Winckler, Pascale; Lartigue, Lydia; Giannone, Gregory; de Giorgi, Francesca; Ichas, François; Sibarita, Jean-Baptiste; Lounis, Brahim; Cognet, Laurent

2013-08-01

Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Förster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.

Estimation of Orbital Neutron Detector Spatial Resolution by Systematic Shifting of Differential Topographic Masks

NASA Technical Reports Server (NTRS)

McClanahan, T. P.; Mitrofanov, I. G.; Boynton, W. V.; Chin, G.; Livengood, T.; Starr, R. D.; Evans, L. G.; Mazarico, E.; Smith, D. E.

2012-01-01

We present a method and preliminary results related to determining the spatial resolution of orbital neutron detectors using epithermal maps and differential topographic masks. Our technique is similar to coded aperture imaging methods for optimizing photonic signals in telescopes [I]. In that approach photon masks with known spatial patterns in a telescope aperature are used to systematically restrict incoming photons which minimizes interference and enhances photon signal to noise. Three orbital neutron detector systems with different stated spatial resolutions are evaluated. The differing spatial resolutions arise due different orbital altitudes and the use of neutron collimation techniques. 1) The uncollimated Lunar Prospector Neutron Spectrometer (LPNS) system has spatial resolution of 45km FWHM from approx. 30km altitude mission phase [2]. The Lunar Rennaissance Orbiter (LRO) Lunar Exploration Neutron Detector (LEND) with two detectors at 50km altitude evaluated here: 2) the collimated 10km FWHM spatial resolution detector CSETN and 3) LEND's collimated Sensor for Epithermal Neutrons (SETN). Thus providing two orbital altitudes to study factors of: uncollimated vs collimated and two average altitudes for their effect on fields-of-view.

Some effects of finite spatial resolution on skin friction measurements in turbulent boundary layers

NASA Technical Reports Server (NTRS)

Westphal, Russell V.

1988-01-01

The effects of finite spatial resolution often cause serious errors in measurements in turbulent boundary layers, with particularly large effects for measurements of fluctuating skin friction and velocities within the sublayer. However, classical analyses of finite spatial resolution effects have generally not accounted for the substantial inhomogeneity and anisotropy of near-wall turbulence. The present study has made use of results from recent computational simulations of wall-bounded turbulent flows to examine spatial resolution effects for measurements made at a wall using both single-sensor probes and those employing two sensing volumes in a V shape. Results are presented to show the effects of finite spatial resolution on a variety of quantitites deduced from the skin friction field.

Plant subcellular proteomics: Application for exploring optimal cell function in soybean.

PubMed

Wang, Xin; Komatsu, Setsuko

2016-06-30

Plants have evolved complicated responses to developmental changes and stressful environmental conditions. Subcellular proteomics has the potential to elucidate localized cellular responses and investigate communications among subcellular compartments during plant development and in response to biotic and abiotic stresses. Soybean, which is a valuable legume crop rich in protein and vegetable oil, can grow in several climatic zones; however, the growth and yield of soybean are markedly decreased under stresses. To date, numerous proteomic studies have been performed in soybean to examine the specific protein profiles of cell wall, plasma membrane, nucleus, mitochondrion, chloroplast, and endoplasmic reticulum. In this review, methods for the purification and purity assessment of subcellular organelles from soybean are summarized. In addition, the findings from subcellular proteomic analyses of soybean during development and under stresses, particularly flooding stress, are presented and the proteins regulated among subcellular compartments are discussed. Continued advances in subcellular proteomics are expected to greatly contribute to the understanding of the responses and interactions that occur within and among subcellular compartments during development and under stressful environmental conditions. Subcellular proteomics has the potential to investigate the cellular events and interactions among subcellular compartments in response to development and stresses in plants. Soybean could grow in several climatic zones; however, the growth and yield of soybean are markedly decreased under stresses. Numerous proteomics of cell wall, plasma membrane, nucleus, mitochondrion, chloroplast, and endoplasmic reticulum was carried out to investigate the respecting proteins and their functions in soybean during development or under stresses. In this review, methods of subcellular-organelle enrichment and purity assessment are summarized. In addition, previous findings of subcellular proteomics are presented, and functional proteins regulated among different subcellular are discussed. Subcellular proteomics contributes greatly to uncovering responses and interactions among subcellular compartments during development and under stressful environmental conditions in soybean. Copyright © 2016 Elsevier B.V. All rights reserved.

An approach to estimate spatial distribution of analyte within cells using spectrally-resolved fluorescence microscopy.

PubMed

Sharma, Dharmendar Kumar; Irfanullah, Mir; Basu, Santanu Kumar; Madhu, Sheri; De, Suman; Jadhav, Sameer; Ravikanth, Mangalampalli; Chowdhury, Arindam

2017-01-18

While fluorescence microscopy has become an essential tool amongst chemists and biologists for the detection of various analyte within cellular environments, non-uniform spatial distribution of sensors within cells often restricts extraction of reliable information on relative abundance of analytes in different subcellular regions. As an alternative to existing sensing methodologies such as ratiometric or FRET imaging, where relative proportion of analyte with respect to the sensor can be obtained within cells, we propose a methodology using spectrally-resolved fluorescence microscopy, via which both the relative abundance of sensor as well as their relative proportion with respect to the analyte can be simultaneously extracted for local subcellular regions. This method is exemplified using a BODIPY sensor, capable of detecting mercury ions within cellular environments, characterized by spectral blue-shift and concurrent enhancement of emission intensity. Spectral emission envelopes collected from sub-microscopic regions allowed us to compare the shift in transition energies as well as integrated emission intensities within various intracellular regions. Construction of a 2D scatter plot using spectral shifts and emission intensities, which depend on the relative amount of analyte with respect to sensor and the approximate local amounts of the probe, respectively, enabled qualitative extraction of relative abundance of analyte in various local regions within a single cell as well as amongst different cells. Although the comparisons remain semi-quantitative, this approach involving analysis of multiple spectral parameters opens up an alternative way to extract spatial distribution of analyte in heterogeneous systems. The proposed method would be especially relevant for fluorescent probes that undergo relatively nominal shift in transition energies compared to their emission bandwidths, which often restricts their usage for quantitative ratiometric imaging in cellular media due to strong cross-talk between energetically separated detection channels.

An approach to estimate spatial distribution of analyte within cells using spectrally-resolved fluorescence microscopy

NASA Astrophysics Data System (ADS)

Sharma, Dharmendar Kumar; Irfanullah, Mir; Basu, Santanu Kumar; Madhu, Sheri; De, Suman; Jadhav, Sameer; Ravikanth, Mangalampalli; Chowdhury, Arindam

2017-03-01

While fluorescence microscopy has become an essential tool amongst chemists and biologists for the detection of various analyte within cellular environments, non-uniform spatial distribution of sensors within cells often restricts extraction of reliable information on relative abundance of analytes in different subcellular regions. As an alternative to existing sensing methodologies such as ratiometric or FRET imaging, where relative proportion of analyte with respect to the sensor can be obtained within cells, we propose a methodology using spectrally-resolved fluorescence microscopy, via which both the relative abundance of sensor as well as their relative proportion with respect to the analyte can be simultaneously extracted for local subcellular regions. This method is exemplified using a BODIPY sensor, capable of detecting mercury ions within cellular environments, characterized by spectral blue-shift and concurrent enhancement of emission intensity. Spectral emission envelopes collected from sub-microscopic regions allowed us to compare the shift in transition energies as well as integrated emission intensities within various intracellular regions. Construction of a 2D scatter plot using spectral shifts and emission intensities, which depend on the relative amount of analyte with respect to sensor and the approximate local amounts of the probe, respectively, enabled qualitative extraction of relative abundance of analyte in various local regions within a single cell as well as amongst different cells. Although the comparisons remain semi-quantitative, this approach involving analysis of multiple spectral parameters opens up an alternative way to extract spatial distribution of analyte in heterogeneous systems. The proposed method would be especially relevant for fluorescent probes that undergo relatively nominal shift in transition energies compared to their emission bandwidths, which often restricts their usage for quantitative ratiometric imaging in cellular media due to strong cross-talk between energetically separated detection channels. Dedicated to Professor Kankan Bhattacharyya.

The role of spatial organization of Ca2+ release sites in the generation of arrhythmogenic diastolic Ca2+ release in myocytes from failing hearts

PubMed Central

Ho, Hsiang-Ting; Bonilla, Ingrid M.; Terentyeva, Radmila; Schober, Karsten E.; Terentyev, Dmitry; Carnes, Cynthia A.

2018-01-01

In heart failure (HF), dysregulated cardiac ryanodine receptors (RyR2) contribute to the generation of diastolic Ca2+ waves (DCWs), thereby predisposing adrenergically stressed failing hearts to life-threatening arrhythmias. However, the specific cellular, subcellular, and molecular defects that account for cardiac arrhythmia in HF remain to be elucidated. Patch-clamp techniques and confocal Ca2+ imaging were applied to study spatially defined Ca2+ handling in ventricular myocytes isolated from normal (control) and failing canine hearts. Based on their activation time upon electrical stimulation, Ca2+ release sites were categorized as coupled, located in close proximity to the sarcolemmal Ca2+ channels, and uncoupled, the Ca2+ channel-free non-junctional Ca2+ release units. In control myocytes, stimulation of β-adrenergic receptors with isoproterenol (Iso) resulted in a preferential increase in Ca2+ spark rate at uncoupled sites. This site-specific effect of Iso was eliminated by the phosphatase inhibitor okadaic acid, which caused similar facilitation of Ca2+ sparks at coupled and uncoupled sites. Iso-challenged HF myocytes exhibited increased predisposition to DCWs compared to control myocytes. In addition, the overall frequency of Ca2+ sparks was increased in HF cells due to preferential stimulation of coupled sites. Furthermore, coupled sites exhibited accelerated recovery from functional refractoriness in HF myocytes compared to control myocytes. Spatially resolved subcellular Ca2+ mapping revealed that DCWs predominantly originated from coupled sites. Inhibition of CaMK∏ suppressed DCWs and prevented preferential stimulation of coupled sites in Iso-challenged HF myocytes. These results suggest that CaMK∏-(and phosphatase)-dependent dysregulation of junctional Ca2+ release sites contributes to Ca2+-dependent arrhythmogenesis in HF. PMID:28612155

Characterization and modelling of the spatially- and spectrally-varying point-spread function in hyperspectral imaging systems for computational correction of axial optical aberrations

NASA Astrophysics Data System (ADS)

Špiclin, Žiga; Bürmen, Miran; Pernuš, Franjo; Likar, Boštjan

2012-03-01

Spatial resolution of hyperspectral imaging systems can vary significantly due to axial optical aberrations that originate from wavelength-induced index-of-refraction variations of the imaging optics. For systems that have a broad spectral range, the spatial resolution will vary significantly both with respect to the acquisition wavelength and with respect to the spatial position within each spectral image. Variations of the spatial resolution can be effectively characterized as part of the calibration procedure by a local image-based estimation of the pointspread function (PSF) of the hyperspectral imaging system. The estimated PSF can then be used in the image deconvolution methods to improve the spatial resolution of the spectral images. We estimated the PSFs from the spectral images of a line grid geometric caliber. From individual line segments of the line grid, the PSF was obtained by a non-parametric estimation procedure that used an orthogonal series representation of the PSF. By using the non-parametric estimation procedure, the PSFs were estimated at different spatial positions and at different wavelengths. The variations of the spatial resolution were characterized by the radius and the fullwidth half-maximum of each PSF and by the modulation transfer function, computed from images of USAF1951 resolution target. The estimation and characterization of the PSFs and the image deconvolution based spatial resolution enhancement were tested on images obtained by a hyperspectral imaging system with an acousto-optic tunable filter in the visible spectral range. The results demonstrate that the spatial resolution of the acquired spectral images can be significantly improved using the estimated PSFs and image deconvolution methods.

Evaluating the Value of High Spatial Resolution in National Capacity Expansion Models using ReEDS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krishnan, Venkat; Cole, Wesley

2016-11-14

Power sector capacity expansion models (CEMs) have a broad range of spatial resolutions. This paper uses the Regional Energy Deployment System (ReEDS) model, a long-term national scale electric sector CEM, to evaluate the value of high spatial resolution for CEMs. ReEDS models the United States with 134 load balancing areas (BAs) and captures the variability in existing generation parameters, future technology costs, performance, and resource availability using very high spatial resolution data, especially for wind and solar modeled at 356 resource regions. In this paper we perform planning studies at three different spatial resolutions--native resolution (134 BAs), state-level, and NERCmore » region level--and evaluate how results change under different levels of spatial aggregation in terms of renewable capacity deployment and location, associated transmission builds, and system costs. The results are used to ascertain the value of high geographically resolved models in terms of their impact on relative competitiveness among renewable energy resources.« less

The Analysis of Burrows Recognition Accuracy in XINJIANG'S Pasture Area Based on Uav Visible Images with Different Spatial Resolution

NASA Astrophysics Data System (ADS)

Sun, D.; Zheng, J. H.; Ma, T.; Chen, J. J.; Li, X.

2018-04-01

The rodent disaster is one of the main biological disasters in grassland in northern Xinjiang. The eating and digging behaviors will cause the destruction of ground vegetation, which seriously affected the development of animal husbandry and grassland ecological security. UAV low altitude remote sensing, as an emerging technique with high spatial resolution, can effectively recognize the burrows. However, how to select the appropriate spatial resolution to monitor the calamity of the rodent disaster is the first problem we need to pay attention to. The purpose of this study is to explore the optimal spatial scale on identification of the burrows by evaluating the impact of different spatial resolution for the burrows identification accuracy. In this study, we shoot burrows from different flight heights to obtain visible images of different spatial resolution. Then an object-oriented method is used to identify the caves, and we also evaluate the accuracy of the classification. We found that the highest classification accuracy of holes, the average has reached more than 80 %. At the altitude of 24 m and the spatial resolution of 1cm, the accuracy of the classification is the highest We have created a unique and effective way to identify burrows by using UAVs visible images. We draw the following conclusion: the best spatial resolution of burrows recognition is 1 cm using DJI PHANTOM-3 UAV, and the improvement of spatial resolution does not necessarily lead to the improvement of classification accuracy. This study lays the foundation for future research and can be extended to similar studies elsewhere.

Design and evaluation of an ultra-slim objective for in-vivo deep optical biopsy

PubMed Central

Landau, Sara M.; Liang, Chen; Kester, Robert T.; Tkaczyk, Tomasz S.; Descour, Michael R.

2010-01-01

An estimated 1.6 million breast biopsies are performed in the US each year. In order to provide real-time, in-vivo imaging with sub-cellular resolution for optical biopsies, we have designed an ultra-slim objective to fit inside the 1-mm-diameter hypodermic needles currently used for breast biopsies to image tissue stained by the fluorescent probe proflavine. To ensure high-quality imaging performance, experimental tests were performed to characterize fiber bundle’s light-coupling efficiency and simulations were performed to evaluate the impact of candidate lens materials’ autofluorescence. A prototype of NA = 0.4, 250-µm field of view, ultra-slim objective optics was built and tested, yielding diffraction-limited performance and estimated resolution of 0.9 µm. When used in conjunction with a commercial coherent fiber bundle to relay the image formed by the objective, the measured resolution was 2.5 µm. PMID:20389489

Spirally-patterned pinhole arrays for long-term fluorescence cell imaging.

PubMed

Koo, Bon Ung; Kang, YooNa; Moon, SangJun; Lee, Won Gu

2015-11-07

Fluorescence cell imaging using a fluorescence microscope is an extensively used technique to examine the cell nucleus, internal structures, and other cellular molecules with fluorescence response time and intensity. However, it is difficult to perform high resolution cell imaging for a long period of time with this technique due to necrosis and apoptosis depending on the type and subcellular location of the damage caused by phototoxicity. A large number of studies have been performed to resolve this problem, but researchers have struggled to meet the challenge between cellular viability and image resolution. In this study, we employ a specially designed disc to reduce cell damage by controlling total fluorescence exposure time without deterioration of the image resolution. This approach has many advantages such as, the apparatus is simple, cost-effective, and easily integrated into the optical pathway through a conventional fluorescence microscope.

A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing.

PubMed

Murakami, Tatsuya C; Mano, Tomoyuki; Saikawa, Shu; Horiguchi, Shuhei A; Shigeta, Daichi; Baba, Kousuke; Sekiya, Hiroshi; Shimizu, Yoshihiro; Tanaka, Kenji F; Kiyonari, Hiroshi; Iino, Masamitsu; Mochizuki, Hideki; Tainaka, Kazuki; Ueda, Hiroki R

2018-04-01

A three-dimensional single-cell-resolution mammalian brain atlas will accelerate systems-level identification and analysis of cellular circuits underlying various brain functions. However, its construction requires efficient subcellular-resolution imaging throughout the entire brain. To address this challenge, we developed a fluorescent-protein-compatible, whole-organ clearing and homogeneous expansion protocol based on an aqueous chemical solution (CUBIC-X). The expanded, well-cleared brain enabled us to construct a point-based mouse brain atlas with single-cell annotation (CUBIC-Atlas). CUBIC-Atlas reflects inhomogeneous whole-brain development, revealing a significant decrease in the cerebral visual and somatosensory cortical areas during postnatal development. Probabilistic activity mapping of pharmacologically stimulated Arc-dVenus reporter mouse brains onto CUBIC-Atlas revealed the existence of distinct functional structures in the hippocampal dentate gyrus. CUBIC-Atlas is shareable by an open-source web-based viewer, providing a new platform for whole-brain cell profiling.

Automated motion artifact removal for intravital microscopy, without a priori information.

PubMed

Lee, Sungon; Vinegoni, Claudio; Sebas, Matthew; Weissleder, Ralph

2014-03-28

Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber.

Automated motion artifact removal for intravital microscopy, without a priori information

PubMed Central

Lee, Sungon; Vinegoni, Claudio; Sebas, Matthew; Weissleder, Ralph

2014-01-01

Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber. PMID:24676021

Experimental evaluation and basis function optimization of the spatially variant image-space PSF on the Ingenuity PET/MR scanner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kotasidis, Fotis A., E-mail: Fotis.Kotasidis@unige.ch; Zaidi, Habib; Geneva Neuroscience Centre, Geneva University, CH-1205 Geneva

2014-06-15

Purpose: The Ingenuity time-of-flight (TF) PET/MR is a recently developed hybrid scanner combining the molecular imaging capabilities of PET with the excellent soft tissue contrast of MRI. It is becoming common practice to characterize the system's point spread function (PSF) and understand its variation under spatial transformations to guide clinical studies and potentially use it within resolution recovery image reconstruction algorithms. Furthermore, due to the system's utilization of overlapping and spherical symmetric Kaiser-Bessel basis functions during image reconstruction, its image space PSF and reconstructed spatial resolution could be affected by the selection of the basis function parameters. Hence, a detailedmore » investigation into the multidimensional basis function parameter space is needed to evaluate the impact of these parameters on spatial resolution. Methods: Using an array of 12 × 7 printed point sources, along with a custom made phantom, and with the MR magnet on, the system's spatially variant image-based PSF was characterized in detail. Moreover, basis function parameters were systematically varied during reconstruction (list-mode TF OSEM) to evaluate their impact on the reconstructed resolution and the image space PSF. Following the spatial resolution optimization, phantom, and clinical studies were subsequently reconstructed using representative basis function parameters. Results: Based on the analysis and under standard basis function parameters, the axial and tangential components of the PSF were found to be almost invariant under spatial transformations (∼4 mm) while the radial component varied modestly from 4 to 6.7 mm. Using a systematic investigation into the basis function parameter space, the spatial resolution was found to degrade for basis functions with a large radius and small shape parameter. However, it was found that optimizing the spatial resolution in the reconstructed PET images, while having a good basis function superposition and keeping the image representation error to a minimum, is feasible, with the parameter combination range depending upon the scanner's intrinsic resolution characteristics. Conclusions: Using the printed point source array as a MR compatible methodology for experimentally measuring the scanner's PSF, the system's spatially variant resolution properties were successfully evaluated in image space. Overall the PET subsystem exhibits excellent resolution characteristics mainly due to the fact that the raw data are not under-sampled/rebinned, enabling the spatial resolution to be dictated by the scanner's intrinsic resolution and the image reconstruction parameters. Due to the impact of these parameters on the resolution properties of the reconstructed images, the image space PSF varies both under spatial transformations and due to basis function parameter selection. Nonetheless, for a range of basis function parameters, the image space PSF remains unaffected, with the range depending on the scanner's intrinsic resolution properties.« less

An evaluation of spatial resolution of a prototype proton CT scanner.

PubMed

Plautz, Tia E; Bashkirov, V; Giacometti, V; Hurley, R F; Johnson, R P; Piersimoni, P; Sadrozinski, H F-W; Schulte, R W; Zatserklyaniy, A

2016-12-01

To evaluate the spatial resolution of proton CT using both a prototype proton CT scanner and Monte Carlo simulations. A custom cylindrical edge phantom containing twelve tissue-equivalent inserts with four different compositions at varying radial displacements from the axis of rotation was developed for measuring the modulation transfer function (MTF) of a prototype proton CT scanner. Two scans of the phantom, centered on the axis of rotation, were obtained with a 200 MeV, low-intensity proton beam: one scan with steps of 4°, and one scan with the phantom continuously rotating. In addition, Monte Carlo simulations of the phantom scan were performed using scanners idealized to various degrees. The data were reconstructed using an iterative projection method with added total variation superiorization based on individual proton histories. Edge spread functions in the radial and azimuthal directions were obtained using the oversampling technique. These were then used to obtain the modulation transfer functions. The spatial resolution was defined by the 10% value of the modulation transfer function (MTF 10% ) in units of line pairs per centimeter (lp/cm). Data from the simulations were used to better understand the contributions of multiple Coulomb scattering in the phantom and the scanner hardware, as well as the effect of discretization of proton location. The radial spatial resolution of the prototype proton CT scanner depends on the total path length, W, of the proton in the phantom, whereas the azimuthal spatial resolution depends both on W and the position, u - , at which the most-likely path uncertainty is evaluated along the path. For protons contributing to radial spatial resolution, W varies with the radial position of the edge, whereas for protons contributing to azimuthal spatial resolution, W is approximately constant. For a pixel size of 0.625 mm, the radial spatial resolution of the image reconstructed from the fully idealized simulation data ranged between 6.31 ± 0.36 lp/cm for W = 197 mm i.e., close to the center of the phantom, and 13.79 ± 0.36 lp/cm for W = 97 mm, near the periphery of the phantom. The azimuthal spatial resolution ranged from 6.99 ± 0.23 lp/cm at u - = 75 mm (near the center) to 11.20 ± 0.26 lp/cm at u - = 20 mm (near the periphery). Multiple Coulomb scattering limits the radial spatial resolution for path lengths greater than approximately 130 mm, and the azimuthal spatial resolution for positions of evaluation greater than approximately 40 mm for W = 199 mm. The radial spatial resolution of the image reconstructed from data from the 4° stepped experimental scan ranged from 5.11 ± 0.61 lp/cm for W = 197 mm to 8.58 ± 0.50 lp/cm for W = 97 mm. In the azimuthal direction, the spatial resolution ranged from 5.37 ± 0.40 lp/cm at u - = 75 mm to 7.27 ± 0.39 lp/cm at u - = 20 mm. The continuous scan achieved the same spatial resolution as that of the stepped scan. Multiple Coulomb scattering in the phantom is the limiting physical factor of the achievable spatial resolution of proton CT; additional loss of spatial resolution in the prototype system is associated with scattering in the proton tracking system and inadequacies of the proton path estimate used in the iterative reconstruction algorithm. Improvement in spatial resolution may be achievable by improving the most likely path estimate by incorporating information about high and low density materials, and by minimizing multiple Coulomb scattering in the proton tracking system.

An evaluation of spatial resolution of a prototype proton CT scanner

PubMed Central

Plautz, Tia E.; Bashkirov, V.; Giacometti, V.; Hurley, R. F.; Piersimoni, P.; Sadrozinski, H. F.-W.; Schulte, R. W.; Zatserklyaniy, A.

2016-01-01

Purpose: To evaluate the spatial resolution of proton CT using both a prototype proton CT scanner and Monte Carlo simulations. Methods: A custom cylindrical edge phantom containing twelve tissue-equivalent inserts with four different compositions at varying radial displacements from the axis of rotation was developed for measuring the modulation transfer function (MTF) of a prototype proton CT scanner. Two scans of the phantom, centered on the axis of rotation, were obtained with a 200 MeV, low-intensity proton beam: one scan with steps of 4°, and one scan with the phantom continuously rotating. In addition, Monte Carlo simulations of the phantom scan were performed using scanners idealized to various degrees. The data were reconstructed using an iterative projection method with added total variation superiorization based on individual proton histories. Edge spread functions in the radial and azimuthal directions were obtained using the oversampling technique. These were then used to obtain the modulation transfer functions. The spatial resolution was defined by the 10% value of the modulation transfer function (MTF10%) in units of line pairs per centimeter (lp/cm). Data from the simulations were used to better understand the contributions of multiple Coulomb scattering in the phantom and the scanner hardware, as well as the effect of discretization of proton location. Results: The radial spatial resolution of the prototype proton CT scanner depends on the total path length, W, of the proton in the phantom, whereas the azimuthal spatial resolution depends both on W and the position, u−, at which the most-likely path uncertainty is evaluated along the path. For protons contributing to radial spatial resolution, W varies with the radial position of the edge, whereas for protons contributing to azimuthal spatial resolution, W is approximately constant. For a pixel size of 0.625 mm, the radial spatial resolution of the image reconstructed from the fully idealized simulation data ranged between 6.31 ± 0.36 lp/cm for W = 197 mm i.e., close to the center of the phantom, and 13.79 ± 0.36 lp/cm for W = 97 mm, near the periphery of the phantom. The azimuthal spatial resolution ranged from 6.99 ± 0.23 lp/cm at u− = 75 mm (near the center) to 11.20 ± 0.26 lp/cm at u− = 20 mm (near the periphery). Multiple Coulomb scattering limits the radial spatial resolution for path lengths greater than approximately 130 mm, and the azimuthal spatial resolution for positions of evaluation greater than approximately 40 mm for W = 199 mm. The radial spatial resolution of the image reconstructed from data from the 4° stepped experimental scan ranged from 5.11 ± 0.61 lp/cm for W = 197 mm to 8.58 ± 0.50 lp/cm for W = 97 mm. In the azimuthal direction, the spatial resolution ranged from 5.37 ± 0.40 lp/cm at u− = 75 mm to 7.27 ± 0.39 lp/cm at u− = 20 mm. The continuous scan achieved the same spatial resolution as that of the stepped scan. Conclusions: Multiple Coulomb scattering in the phantom is the limiting physical factor of the achievable spatial resolution of proton CT; additional loss of spatial resolution in the prototype system is associated with scattering in the proton tracking system and inadequacies of the proton path estimate used in the iterative reconstruction algorithm. Improvement in spatial resolution may be achievable by improving the most likely path estimate by incorporating information about high and low density materials, and by minimizing multiple Coulomb scattering in the proton tracking system. PMID:27908179

A comparison of high-throughput techniques for assaying circadian rhythms in plants.

PubMed

Tindall, Andrew J; Waller, Jade; Greenwood, Mark; Gould, Peter D; Hartwell, James; Hall, Anthony

2015-01-01

Over the last two decades, the development of high-throughput techniques has enabled us to probe the plant circadian clock, a key coordinator of vital biological processes, in ways previously impossible. With the circadian clock increasingly implicated in key fitness and signalling pathways, this has opened up new avenues for understanding plant development and signalling. Our tool-kit has been constantly improving through continual development and novel techniques that increase throughput, reduce costs and allow higher resolution on the cellular and subcellular levels. With circadian assays becoming more accessible and relevant than ever to researchers, in this paper we offer a review of the techniques currently available before considering the horizons in circadian investigation at ever higher throughputs and resolutions.

Effects of vegetation heterogeneity and surface topography on spatial scaling of net primary productivity

NASA Astrophysics Data System (ADS)

Chen, J. M.; Chen, X.; Ju, W.

2013-03-01

Due to the heterogeneous nature of the land surface, spatial scaling is an inevitable issue in the development of land models coupled with low-resolution Earth system models (ESMs) for predicting land-atmosphere interactions and carbon-climate feedbacks. In this study, a simple spatial scaling algorithm is developed to correct errors in net primary productivity (NPP) estimates made at a coarse spatial resolution based on sub-pixel information of vegetation heterogeneity and surface topography. An eco-hydrological model BEPS-TerrainLab, which considers both vegetation and topographical effects on the vertical and lateral water flows and the carbon cycle, is used to simulate NPP at 30 m and 1 km resolutions for a 5700 km2 watershed with an elevation range from 518 m to 3767 m in the Qinling Mountain, Shaanxi Province, China. Assuming that the NPP simulated at 30 m resolution represents the reality and that at 1 km resolution is subject to errors due to sub-pixel heterogeneity, a spatial scaling index (SSI) is developed to correct the coarse resolution NPP values pixel by pixel. The agreement between the NPP values at these two resolutions is improved considerably from R2 = 0.782 to R2 = 0.884 after the correction. The mean bias error (MBE) in NPP modeled at the 1 km resolution is reduced from 14.8 g C m-2 yr-1 to 4.8 g C m-2 yr-1 in comparison with NPP modeled at 30 m resolution, where the mean NPP is 668 g C m-2 yr-1. The range of spatial variations of NPP at 30 m resolution is larger than that at 1 km resolution. Land cover fraction is the most important vegetation factor to be considered in NPP spatial scaling, and slope is the most important topographical factor for NPP spatial scaling especially in mountainous areas, because of its influence on the lateral water redistribution, affecting water table, soil moisture and plant growth. Other factors including leaf area index (LAI), elevation and aspect have small and additive effects on improving the spatial scaling between these two resolutions.

Effects of vegetation heterogeneity and surface topography on spatial scaling of net primary productivity

NASA Astrophysics Data System (ADS)

Chen, J. M.; Chen, X.; Ju, W.

2013-07-01

Due to the heterogeneous nature of the land surface, spatial scaling is an inevitable issue in the development of land models coupled with low-resolution Earth system models (ESMs) for predicting land-atmosphere interactions and carbon-climate feedbacks. In this study, a simple spatial scaling algorithm is developed to correct errors in net primary productivity (NPP) estimates made at a coarse spatial resolution based on sub-pixel information of vegetation heterogeneity and surface topography. An eco-hydrological model BEPS-TerrainLab, which considers both vegetation and topographical effects on the vertical and lateral water flows and the carbon cycle, is used to simulate NPP at 30 m and 1 km resolutions for a 5700 km2 watershed with an elevation range from 518 m to 3767 m in the Qinling Mountain, Shanxi Province, China. Assuming that the NPP simulated at 30 m resolution represents the reality and that at 1 km resolution is subject to errors due to sub-pixel heterogeneity, a spatial scaling index (SSI) is developed to correct the coarse resolution NPP values pixel by pixel. The agreement between the NPP values at these two resolutions is improved considerably from R2 = 0.782 to R2 = 0.884 after the correction. The mean bias error (MBE) in NPP modelled at the 1 km resolution is reduced from 14.8 g C m-2 yr-1 to 4.8 g C m-2 yr-1 in comparison with NPP modelled at 30 m resolution, where the mean NPP is 668 g C m-2 yr-1. The range of spatial variations of NPP at 30 m resolution is larger than that at 1 km resolution. Land cover fraction is the most important vegetation factor to be considered in NPP spatial scaling, and slope is the most important topographical factor for NPP spatial scaling especially in mountainous areas, because of its influence on the lateral water redistribution, affecting water table, soil moisture and plant growth. Other factors including leaf area index (LAI) and elevation have small and additive effects on improving the spatial scaling between these two resolutions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Ke; Chen, Guang-Hong, E-mail: gchen7@wisc.edu; Garrett, John

Purpose: Statistical model based iterative reconstruction (MBIR) methods have been introduced to clinical CT systems and are being used in some clinical diagnostic applications. The purpose of this paper is to experimentally assess the unique spatial resolution characteristics of this nonlinear reconstruction method and identify its potential impact on the detectabilities and the associated radiation dose levels for specific imaging tasks. Methods: The thoracic section of a pediatric phantom was repeatedly scanned 50 or 100 times using a 64-slice clinical CT scanner at four different dose levels [CTDI{sub vol} =4, 8, 12, 16 (mGy)]. Both filtered backprojection (FBP) and MBIRmore » (Veo{sup ®}, GE Healthcare, Waukesha, WI) were used for image reconstruction and results were compared with one another. Eight test objects in the phantom with contrast levels ranging from 13 to 1710 HU were used to assess spatial resolution. The axial spatial resolution was quantified with the point spread function (PSF), while the z resolution was quantified with the slice sensitivity profile. Both were measured locally on the test objects and in the image domain. The dependence of spatial resolution on contrast and dose levels was studied. The study also features a systematic investigation of the potential trade-off between spatial resolution and locally defined noise and their joint impact on the overall image quality, which was quantified by the image domain-based channelized Hotelling observer (CHO) detectability index d′. Results: (1) The axial spatial resolution of MBIR depends on both radiation dose level and image contrast level, whereas it is supposedly independent of these two factors in FBP. The axial spatial resolution of MBIR always improved with an increasing radiation dose level and/or contrast level. (2) The axial spatial resolution of MBIR became equivalent to that of FBP at some transitional contrast level, above which MBIR demonstrated superior spatial resolution than FBP (and vice versa); the value of this transitional contrast highly depended on the dose level. (3) The PSFs of MBIR could be approximated as Gaussian functions with reasonably good accuracy. (4) Thez resolution of MBIR showed similar contrast and dose dependence. (5) Noise standard deviation assessed on the edges of objects demonstrated a trade-off with spatial resolution in MBIR. (5) When both spatial resolution and image noise were considered using the CHO analysis, MBIR led to significant improvement in the overall CT image quality for both high and low contrast detection tasks at both standard and low dose levels. Conclusions: Due to the intrinsic nonlinearity of the MBIR method, many well-known CT spatial resolution and noise properties have been modified. In particular, dose dependence and contrast dependence have been introduced to the spatial resolution of CT images by MBIR. The method has also introduced some novel noise-resolution trade-off not seen in traditional CT images. While the benefits of MBIR regarding the overall image quality, as demonstrated in this work, are significant, the optimal use of this method in clinical practice demands a thorough understanding of its unique physical characteristics.« less

High-resolution scanning precession electron diffraction: Alignment and spatial resolution.

PubMed

Barnard, Jonathan S; Johnstone, Duncan N; Midgley, Paul A

2017-03-01

Methods are presented for aligning the pivot point of a precessing electron probe in the scanning transmission electron microscope (STEM) and for assessing the spatial resolution in scanning precession electron diffraction (SPED) experiments. The alignment procedure is performed entirely in diffraction mode, minimising probe wander within the bright-field (BF) convergent beam electron diffraction (CBED) disk and is used to obtain high spatial resolution SPED maps. Through analysis of the power spectra of virtual bright-field images extracted from the SPED data, the precession-induced blur was measured as a function of precession angle. At low precession angles, SPED spatial resolution was limited by electronic noise in the scan coils; whereas at high precession angles SPED spatial resolution was limited by tilt-induced two-fold astigmatism caused by the positive spherical aberration of the probe-forming lens. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of Electric Field Gradient on Sub-nanometer Spatial Resolution of Tip-enhanced Raman Spectroscopy

PubMed Central

Meng, Lingyan; Yang, Zhilin; Chen, Jianing; Sun, Mengtao

2015-01-01

Tip-enhanced Raman spectroscopy (TERS) with sub-nanometer spatial resolution has been recently demonstrated experimentally. However, the physical mechanism underlying is still under discussion. Here we theoretically investigate the electric field gradient of a coupled tip-substrate system. Our calculations suggest that the ultra-high spatial resolution of TERS can be partially attributed to the electric field gradient effect owning to its tighter spatial confinement and sensitivity to the infrared (IR)-active of molecules. Particularly, in the case of TERS of flat-lying H2TBPP molecules,we find the electric field gradient enhancement is the dominating factor for the high spatial resolution, which qualitatively coincides with previous experimental report. Our theoretical study offers a new paradigm for understanding the mechanisms of the ultra-high spatial resolution demonstrated in tip-enhanced spectroscopy which is of importance but neglected. PMID:25784161

Spatial resolution properties of motion-compensated tomographic image reconstruction methods.

PubMed

Chun, Se Young; Fessler, Jeffrey A

2012-07-01

Many motion-compensated image reconstruction (MCIR) methods have been proposed to correct for subject motion in medical imaging. MCIR methods incorporate motion models to improve image quality by reducing motion artifacts and noise. This paper analyzes the spatial resolution properties of MCIR methods and shows that nonrigid local motion can lead to nonuniform and anisotropic spatial resolution for conventional quadratic regularizers. This undesirable property is akin to the known effects of interactions between heteroscedastic log-likelihoods (e.g., Poisson likelihood) and quadratic regularizers. This effect may lead to quantification errors in small or narrow structures (such as small lesions or rings) of reconstructed images. This paper proposes novel spatial regularization design methods for three different MCIR methods that account for known nonrigid motion. We develop MCIR regularization designs that provide approximately uniform and isotropic spatial resolution and that match a user-specified target spatial resolution. Two-dimensional PET simulations demonstrate the performance and benefits of the proposed spatial regularization design methods.

Development of a spatio-temporal disaggregation method (DisNDVI) for generating a time series of fine resolution NDVI images

NASA Astrophysics Data System (ADS)

Bindhu, V. M.; Narasimhan, B.

2015-03-01

Normalized Difference Vegetation Index (NDVI), a key parameter in understanding the vegetation dynamics, has high spatial and temporal variability. However, continuous monitoring of NDVI is not feasible at fine spatial resolution (<60 m) owing to the long revisit time needed by the satellites to acquire the fine spatial resolution data. Further, the study attains significance in the case of humid tropical regions of the earth, where the prevailing atmospheric conditions restrict availability of fine resolution cloud free images at a high temporal frequency. As an alternative to the lack of high resolution images, the current study demonstrates a novel disaggregation method (DisNDVI) which integrates the spatial information from a single fine resolution image and temporal information in terms of crop phenology from time series of coarse resolution images to generate estimates of NDVI at fine spatial and temporal resolution. The phenological variation of the pixels captured at the coarser scale provides the basis for relating the temporal variability of the pixel with the NDVI available at fine resolution. The proposed methodology was tested over a 30 km × 25 km spatially heterogeneous study area located in the south of Tamil Nadu, India. The robustness of the algorithm was assessed by an independent comparison of the disaggregated NDVI and observed NDVI obtained from concurrent Landsat ETM+ imagery. The results showed good spatial agreement across the study area dominated with agriculture and forest pixels, with a root mean square error of 0.05. The validation done at the coarser scale showed that disaggregated NDVI spatially averaged to 240 m compared well with concurrent MODIS NDVI at 240 m (R2 > 0.8). The validation results demonstrate the effectiveness of DisNDVI in improving the spatial and temporal resolution of NDVI images for utility in fine scale hydrological applications such as crop growth monitoring and estimation of evapotranspiration.

How Attention Affects Spatial Resolution

PubMed Central

Carrasco, Marisa; Barbot, Antoine

2015-01-01

We summarize and discuss a series of psychophysical studies on the effects of spatial covert attention on spatial resolution, our ability to discriminate fine patterns. Heightened resolution is beneficial in most, but not all, visual tasks. We show how endogenous attention (voluntary, goal driven) and exogenous attention (involuntary, stimulus driven) affect performance on a variety of tasks mediated by spatial resolution, such as visual search, crowding, acuity, and texture segmentation. Exogenous attention is an automatic mechanism that increases resolution regardless of whether it helps or hinders performance. In contrast, endogenous attention flexibly adjusts resolution to optimize performance according to task demands. We illustrate how psychophysical studies can reveal the underlying mechanisms of these effects and allow us to draw linking hypotheses with known neurophysiological effects of attention. PMID:25948640

Quantitative Analysis of Cell Nucleus Organisation

PubMed Central

Shiels, Carol; Adams, Niall M; Islam, Suhail A; Stephens, David A; Freemont, Paul S

2007-01-01

There are almost 1,300 entries for higher eukaryotes in the Nuclear Protein Database. The proteins' subcellular distribution patterns within interphase nuclei can be complex, ranging from diffuse to punctate or microspeckled, yet they all work together in a coordinated and controlled manner within the three-dimensional confines of the nuclear volume. In this review we describe recent advances in the use of quantitative methods to understand nuclear spatial organisation and discuss some of the practical applications resulting from this work. PMID:17676980

Super-resolution optical microscopy for studying membrane structure and dynamics.

PubMed

Sezgin, Erdinc

2017-07-12

Investigation of cell membrane structure and dynamics requires high spatial and temporal resolution. The spatial resolution of conventional light microscopy is limited due to the diffraction of light. However, recent developments in microscopy enabled us to access the nano-scale regime spatially, thus to elucidate the nanoscopic structures in the cellular membranes. In this review, we will explain the resolution limit, address the working principles of the most commonly used super-resolution microscopy techniques and summarise their recent applications in the biomembrane field.

Dynamic organization of myristoylated Src in the live cell plasma membrane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Adam W.; Huang, Hector H.; Endres, Nicholas F.

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell–cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescencemore » cross-correlation spectroscopy (PIE–FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10–80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Altogether, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.« less

Dynamic organization of myristoylated Src in the live cell plasma membrane

DOE PAGES

Smith, Adam W.; Huang, Hector H.; Endres, Nicholas F.; ...

2016-01-15

The spatial organization of lipid-anchored proteins in the plasma membrane directly influences cell signaling, but measuring such organization in situ is experimentally challenging. The canonical oncogene, c-Src, is a lipid anchored protein that plays a key role in integrin-mediated signal transduction within focal adhesions and cell–cell junctions. Because of its activity in specific plasma membrane regions, structural motifs within the protein have been hypothesized to play an important role in its subcellular localization. This study used a combination of time-resolved fluorescence fluctuation spectroscopy and super-resolution microscopy to quantify the dynamic organization of c-Src in live cell membranes. Pulsed-interleaved excitation fluorescencemore » cross-correlation spectroscopy (PIE–FCCS) showed that a small fraction of c-Src transiently sorts into membrane clusters that are several times larger than the monomers. Photoactivated localization microscopy (PALM) confirmed that c-Src partitions into clusters with low probability and showed that the characteristic size of the clusters is 10–80 nm. Finally, time-resolved fluorescence anisotropy measurements were used to quantify the rotational mobility of c-Src to determine how it interacts with its local environment. Altogether, these results build a quantitative description of the mobility and clustering behavior of the c-Src nonreceptor tyrosine kinase in the live cell plasma membrane.« less

Correlation of Particle Traversals with Clonogenic Survival Using Cell-Fluorescent Ion Track Hybrid Detector.

PubMed

Dokic, Ivana; Niklas, Martin; Zimmermann, Ferdinand; Mairani, Andrea; Seidel, Philipp; Krunic, Damir; Jäkel, Oliver; Debus, Jürgen; Greilich, Steffen; Abdollahi, Amir

2015-01-01

Development of novel approaches linking the physical characteristics of particles with biological responses are of high relevance for the field of particle therapy. In radiobiology, the clonogenic survival of cells is considered the gold standard assay for the assessment of cellular sensitivity to ionizing radiation. Toward further development of next generation biodosimeters in particle therapy, cell-fluorescent ion track hybrid detector (Cell-FIT-HD) was recently engineered by our group and successfully employed to study physical particle track information in correlation with irradiation-induced DNA damage in cell nuclei. In this work, we investigated the feasibility of Cell-FIT-HD as a tool to study the effects of clinical beams on cellular clonogenic survival. Tumor cells were grown on the fluorescent nuclear track detector as cell culture, mimicking the standard procedures for clonogenic assay. Cell-FIT-HD was used to detect the spatial distribution of particle tracks within colony-initiating cells. The physical data were associated with radiation-induced foci as surrogates for DNA double-strand breaks, the hallmark of radiation-induced cell lethality. Long-term cell fate was monitored to determine the ability of cells to form colonies. We report the first successful detection of particle traversal within colony-initiating cells at subcellular resolution using Cell-FIT-HD.

Spatio-temporal imaging of EGF-induced activation of protein kinase A by FRET in living cells

NASA Astrophysics Data System (ADS)

Wang, Jin Jun; Chen, Xiao-Chuan; Xing, Da

2004-07-01

Intracellular molecular interaction is important for the study of cell physiology, yet current relevant methods require fixation or microinjection and lack temporal or spatial resolution. We introduced a new method -- fluorescence resonance energy transfer (FRET) to detect molecular interaction in living cells. On the basis of FRET principle, A-kinase activity reporter (AKAR) protein was designed to consist of the fusions of cyan fluorescent protein (CFP), a phosphoamino acid binding domain, a consensus substrate for protein kinase-A (PKA), and yellow fluorescent protein (YFP). In this study, the designed pAKAR plasmid was used to transfect a human lung cancer cell line (ASTC-a-1). When the AKAR-transfected cells were treated by forskolin (Fsk), we were able to observe the efficient transfer of energy from excited CFP to YFP within the AKAR molecule by fluorescence microcopy, whereas no FRET was detected in the transfected cells without the treatment of Fsk. When the cells were treated by Epidermal growth factor (EGF), the change of FRET was observed at different subcellular locations, reflecting PKA activation inside the cells upon EGF stimulation. The successful design of a fluorescence reporter of PKA activation and its application demonstrated the superiority of this technology in the research of intracellular protein-protein interaction.

The smartest materials: the future of nanoelectronics in medicine.

PubMed

Cohen-Karni, Tzahi; Langer, Robert; Kohane, Daniel S

2012-08-28

Electronics have become central to many aspects of biomedicine, ranging from fundamental biophysical studies of excitable tissues to medical monitoring and electronic implants to restore limb movement. The development of new materials and approaches is needed to enable enhanced tissue integration, interrogation, and stimulation and other functionalities. Nanoscale materials offer many avenues for progress in this respect. New classes of molecular-scale bioelectronic interfaces can be constructed using either one-dimensional nanostructures, such as nanowires and nanotubes, or two-dimensional nanostructures, such as graphene. Nanodevices can create ultrasensitive sensors and can be designed with spatial resolution as fine as the subcellular regime. Structures on the nanoscale can enable the development of engineered tissues within which sensing elements are integrated as closely as the nervous system within native tissues. In addition, the close integration of nanomaterials with cells and tissues will also allow the development of in vitro platforms for basic research or diagnostics. Such lab-on-a-chip systems could, for example, enable testing of the effects of candidate therapeutic molecules on intercellular, single-cell, and even intracellular physiology. Finally, advances in nanoelectronics can lead to extremely sophisticated smart materials with multifunctional capabilities, enabling the spectrum of biomedical possibilities from diagnostic studies to the creation of cyborgs.

Spatial distribution of filament elasticity determines the migratory behaviors of a cell

PubMed Central

Harn, Hans I-Chen; Hsu, Chao-Kai; Wang, Yang-Kao; Huang, Yi-Wei; Chiu, Wen-Tai; Lin, Hsi-Hui; Cheng, Chao-Min; Tang, Ming-Jer

2016-01-01

ABSTRACT Any cellular response leading to morphological changes is highly tuned to balance the force generated from structural reorganization, provided by actin cytoskeleton. Actin filaments serve as the backbone of intracellular force, and transduce external mechanical signal via focal adhesion complex into the cell. During migration, cells not only undergo molecular changes but also rapid mechanical modulation. Here we focus on determining, the role of spatial distribution of mechanical changes of actin filaments in epithelial, mesenchymal, fibrotic and cancer cells with non-migration, directional migration, and non-directional migration behaviors using the atomic force microscopy. We found 1) non-migratory cells only generated one type of filament elasticity, 2) cells generating spatially distributed two types of filament elasticity showed directional migration, and 3) pathologic cells that autonomously generated two types of filament elasticity without spatial distribution were actively migrating non-directionally. The demonstration of spatial regulation of filament elasticity of different cell types at the nano-scale highlights the coupling of cytoskeletal function with physical characters at the sub-cellular level, and provides new research directions for migration related disease. PMID:26919488

Comparison of alternative spatial resolutions in the application of a spatially distributed biogeochemical model over complex terrain

USGS Publications Warehouse

Turner, D.P.; Dodson, R.; Marks, D.

1996-01-01

Spatially distributed biogeochemical models may be applied over grids at a range of spatial resolutions, however, evaluation of potential errors and loss of information at relatively coarse resolutions is rare. In this study, a georeferenced database at the 1-km spatial resolution was developed to initialize and drive a process-based model (Forest-BGC) of water and carbon balance over a gridded 54976 km2 area covering two river basins in mountainous western Oregon. Corresponding data sets were also prepared at 10-km and 50-km spatial resolutions using commonly employed aggregation schemes. Estimates were made at each grid cell for climate variables including daily solar radiation, air temperature, humidity, and precipitation. The topographic structure, water holding capacity, vegetation type and leaf area index were likewise estimated for initial conditions. The daily time series for the climatic drivers was developed from interpolations of meteorological station data for the water year 1990 (1 October 1989-30 September 1990). Model outputs at the 1-km resolution showed good agreement with observed patterns in runoff and productivity. The ranges for model inputs at the 10-km and 50-km resolutions tended to contract because of the smoothed topography. Estimates for mean evapotranspiration and runoff were relatively insensitive to changing the spatial resolution of the grid whereas estimates of mean annual net primary production varied by 11%. The designation of a vegetation type and leaf area at the 50-km resolution often subsumed significant heterogeneity in vegetation, and this factor accounted for much of the difference in the mean values for the carbon flux variables. Although area wide means for model outputs were generally similar across resolutions, difference maps often revealed large areas of disagreement. Relatively high spatial resolution analyses of biogeochemical cycling are desirable from several perspectives and may be particularly important in the study of the potential impacts of climate change.

Optical imaging modalities: From design to diagnosis of skin cancer

NASA Astrophysics Data System (ADS)

Korde, Vrushali Raj

This study investigates three high resolution optical imaging modalities to better detect and diagnose skin cancer. The ideal high resolution optical imaging system can visualize pre-malignant tissue growth non-invasively with resolution comparable to histology. I examined 3 modalities which approached this goal. The first method examined was high magnification microscopy of thin stained tissue sections, together with a statistical analysis of nuclear chromatin patterns termed Karyometry. This method has subcellular resolution, but it necessitates taking a biopsy at the desired tissue site and imaging the tissue ex-vivo. My part of this study was to develop an automated nuclear segmentation algorithm to segment cell nuclei in skin histology images for karyometric analysis. The results of this algorithm were compared to hand segmented cell nuclei in the same images, and it was concluded that the automated segmentations can be used for karyometric analysis. The second optical imaging modality I investigated was Optical Coherence Tomography (OCT). OCT is analogous to ultrasound, in which sound waves are delivered into the body and the echo time and reflected signal magnitude are measured. Due to the fast speed of light and detector temporal integration times, low coherence interferometry is needed to gate the backscattered light. OCT acquires cross sectional images, and has an axial resolution of 1-15 mum (depending on the source bandwidth) and a lateral resolution of 10-20 mum (depending on the sample arm optics). While it is not capable of achieving subcellular resolution, it is a non-invasive imaging modality. OCT was used in this study to evaluate skin along a continuum from normal to sun damaged to precancer. I developed algorithms to detect statistically significant differences between images of sun protected and sun damaged skin, as well as between undiseased and precancerous skin. An Optical Coherence Microscopy (OCM) endoscope was developed in the third portion of this study. OCM is a high resolution en-face imaging modality. It is a hybrid system that combines the principles of confocal microscopy with coherence gating to provide an increased imaging depth. It can also be described as an OCT system with a high NA objective. Similar to OCT, the axial resolution is determined by the source center wavelength and bandwidth. The NA of the sample arm optics determines the lateral resolution, usually on the order of 1-5 mum. My effort on this system was to develop a handheld endoscope. To my knowledge, an OCM endoscope has not been developed prior to this work. An image of skin was taken as a proof of concept. This rigid handheld OCM endoscope will be useful for applications ranging from minimally invasive surgical imaging to non-invasively assessing dysplasia and sun damage in skin.

Subcellular localization of leptin and leptin receptor in breast cancer detected in an electron microscopic study.

PubMed

Al-Shibli, Saad M; Amjad, Nasser M; Al-Kubaisi, Muna K; Mizan, Shaikh

2017-01-22

Leptin (LEP) and leptin receptor (LEPR) have long been found associated with breast cancer. So far no high-resolution method such as electron microscopy has been used to investigate the subcellular localization of leptin and leptin receptor in breast cancer. We collected cancer and non-cancer breast tissues from 51 women with invasive ductal breast cancer. Leptin and leptin receptor in the tissues were estimated using immunohistochemistry (IHC). LEP and LEPR were localized at subcellular level by immunocytochemistry (ICC) using ultra-fine gold particle conjugated antibody, and visualized with transmission electron microscopy (TEM). IHC showed high presence of LEP and LEPR in 65% and 67% respectively of the breast cancer samples, 100% and 0% respectively of the adipose tissue samples, and no high presence in the non-cancer breast tissue samples. On TEM views both LEP and LEPR were found highly concentrated within the nucleus of the cancer cells, indicating that nucleus is the principal seat of action. However, presence of high concentration of LEP does not necessarily prove its over-expression, as often concluded, because LEP could be internalized from outside by LEPR in the cells. In contrast, LEPR is definitely over-expressed in the ductal breast cancer cells. Therefore, we hypothesize that over-expression of LEPR, rather than that of LEP has a fundamental role in breast carcinogenesis in particular, and probably for LEP-LEPR associated tumors in general. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential growth of pavement cells of Arabidopsis thaliana leaf epidermis as revealed by microbead labeling.

PubMed

Elsner, Joanna; Lipowczan, Marcin; Kwiatkowska, Dorota

2018-02-01

In numerous vascular plants, pavement cells of the leaf epidermis are shaped like a jigsaw-puzzle piece. Knowledge about the subcellular pattern of growth that accompanies morphogenesis of such a complex shape is crucial for studies of the role of the cytoskeleton, cell wall and phytohormones in plant cell development. Because the detailed growth pattern of the anticlinal and periclinal cell walls remains unknown, our aim was to measure pavement cell growth at a subcellular resolution. Using fluorescent microbeads applied to the surface of the adaxial leaf epidermis of Arabidopsis thaliana as landmarks for growth computation, we directly assessed the growth rates for the outer periclinal and anticlinal cell walls at a subcellular scale. We observed complementary tendencies in the growth pattern of the outer periclinal and anticlinal cell walls. Central portions of periclinal walls were characterized by relatively slow growth, while growth of the other wall portions was heterogeneous. Local growth of the periclinal walls accompanying lobe development after initiation was relatively fast and anisotropic, with maximal extension usually in the direction along the lobe axis. This growth pattern of the periclinal walls was complemented by the extension of the anticlinal walls, which was faster on the lobe sides than at the tips. Growth of the anticlinal and outer periclinal walls of leaf pavement cells is heterogeneous. The growth of the lobes resembles cell elongation via diffuse growth rather than tip growth. © 2018 Botanical Society of America.

The fusion of satellite and UAV data: simulation of high spatial resolution band

NASA Astrophysics Data System (ADS)

Jenerowicz, Agnieszka; Siok, Katarzyna; Woroszkiewicz, Malgorzata; Orych, Agata

2017-10-01

Remote sensing techniques used in the precision agriculture and farming that apply imagery data obtained with sensors mounted on UAV platforms became more popular in the last few years due to the availability of low- cost UAV platforms and low- cost sensors. Data obtained from low altitudes with low- cost sensors can be characterised by high spatial and radiometric resolution but quite low spectral resolution, therefore the application of imagery data obtained with such technology is quite limited and can be used only for the basic land cover classification. To enrich the spectral resolution of imagery data acquired with low- cost sensors from low altitudes, the authors proposed the fusion of RGB data obtained with UAV platform with multispectral satellite imagery. The fusion is based on the pansharpening process, that aims to integrate the spatial details of the high-resolution panchromatic image with the spectral information of lower resolution multispectral or hyperspectral imagery to obtain multispectral or hyperspectral images with high spatial resolution. The key of pansharpening is to properly estimate the missing spatial details of multispectral images while preserving their spectral properties. In the research, the authors presented the fusion of RGB images (with high spatial resolution) obtained with sensors mounted on low- cost UAV platforms and multispectral satellite imagery with satellite sensors, i.e. Landsat 8 OLI. To perform the fusion of UAV data with satellite imagery, the simulation of the panchromatic bands from RGB data based on the spectral channels linear combination, was conducted. Next, for simulated bands and multispectral satellite images, the Gram-Schmidt pansharpening method was applied. As a result of the fusion, the authors obtained several multispectral images with very high spatial resolution and then analysed the spatial and spectral accuracies of processed images.

Large-watershed flood simulation and forecasting based on different-resolution distributed hydrological model

NASA Astrophysics Data System (ADS)

Li, J.

2017-12-01

Large-watershed flood simulation and forecasting is very important for a distributed hydrological model in the application. There are some challenges including the model's spatial resolution effect, model performance and accuracy and so on. To cope with the challenge of the model's spatial resolution effect, different model resolution including 1000m*1000m, 600m*600m, 500m*500m, 400m*400m, 200m*200m were used to build the distributed hydrological model—Liuxihe model respectively. The purpose is to find which one is the best resolution for Liuxihe model in Large-watershed flood simulation and forecasting. This study sets up a physically based distributed hydrological model for flood forecasting of the Liujiang River basin in south China. Terrain data digital elevation model (DEM), soil type and land use type are downloaded from the website freely. The model parameters are optimized by using an improved Particle Swarm Optimization(PSO) algorithm; And parameter optimization could reduce the parameter uncertainty that exists for physically deriving model parameters. The different model resolution (200m*200m—1000m*1000m ) are proposed for modeling the Liujiang River basin flood with the Liuxihe model in this study. The best model's spatial resolution effect for flood simulation and forecasting is 200m*200m.And with the model's spatial resolution reduction, the model performance and accuracy also become worse and worse. When the model resolution is 1000m*1000m, the flood simulation and forecasting result is the worst, also the river channel divided based on this resolution is differs from the actual one. To keep the model with an acceptable performance, minimum model spatial resolution is needed. The suggested threshold model spatial resolution for modeling the Liujiang River basin flood is a 500m*500m grid cell, but the model spatial resolution with a 200m*200m grid cell is recommended in this study to keep the model at a best performance.

Analysis of the impact of spatial resolution on land/water classifications using high-resolution aerial imagery

USGS Publications Warehouse

Enwright, Nicholas M.; Jones, William R.; Garber, Adrienne L.; Keller, Matthew J.

2014-01-01

Long-term monitoring efforts often use remote sensing to track trends in habitat or landscape conditions over time. To most appropriately compare observations over time, long-term monitoring efforts strive for consistency in methods. Thus, advances and changes in technology over time can present a challenge. For instance, modern camera technology has led to an increasing availability of very high-resolution imagery (i.e. submetre and metre) and a shift from analogue to digital photography. While numerous studies have shown that image resolution can impact the accuracy of classifications, most of these studies have focused on the impacts of comparing spatial resolution changes greater than 2 m. Thus, a knowledge gap exists on the impacts of minor changes in spatial resolution (i.e. submetre to about 1.5 m) in very high-resolution aerial imagery (i.e. 2 m resolution or less). This study compared the impact of spatial resolution on land/water classifications of an area dominated by coastal marsh vegetation in Louisiana, USA, using 1:12,000 scale colour-infrared analogue aerial photography (AAP) scanned at four different dot-per-inch resolutions simulating ground sample distances (GSDs) of 0.33, 0.54, 1, and 2 m. Analysis of the impact of spatial resolution on land/water classifications was conducted by exploring various spatial aspects of the classifications including density of waterbodies and frequency distributions in waterbody sizes. This study found that a small-magnitude change (1–1.5 m) in spatial resolution had little to no impact on the amount of water classified (i.e. percentage mapped was less than 1.5%), but had a significant impact on the mapping of very small waterbodies (i.e. waterbodies ≤ 250 m2). These findings should interest those using temporal image classifications derived from very high-resolution aerial photography as a component of long-term monitoring programs.

Polymeric spatial resolution test patterns for mass spectrometry imaging using nano-thermal analysis with atomic force microscopy

DOE PAGES

Tai, Tamin; Kertesz, Vilmos; Lin, Ming -Wei; ...

2017-05-11

As the spatial resolution of mass spectrometry imaging technologies has begun to reach into the nanometer regime, finding readily available or easily made resolution reference materials has become particularly challenging for molecular imaging purposes. This study describes the fabrication, characterization and use of vertical line array polymeric spatial resolution test patterns for nano-thermal analysis/atomic force microscopy/mass spectrometry chemical imaging.

Polymeric spatial resolution test patterns for mass spectrometry imaging using nano-thermal analysis with atomic force microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tai, Tamin; Kertesz, Vilmos; Lin, Ming -Wei

As the spatial resolution of mass spectrometry imaging technologies has begun to reach into the nanometer regime, finding readily available or easily made resolution reference materials has become particularly challenging for molecular imaging purposes. This study describes the fabrication, characterization and use of vertical line array polymeric spatial resolution test patterns for nano-thermal analysis/atomic force microscopy/mass spectrometry chemical imaging.

High density event-related potential data acquisition in cognitive neuroscience.

PubMed

Slotnick, Scott D

2010-04-16

Functional magnetic resonance imaging (fMRI) is currently the standard method of evaluating brain function in the field of Cognitive Neuroscience, in part because fMRI data acquisition and analysis techniques are readily available. Because fMRI has excellent spatial resolution but poor temporal resolution, this method can only be used to identify the spatial location of brain activity associated with a given cognitive process (and reveals virtually nothing about the time course of brain activity). By contrast, event-related potential (ERP) recording, a method that is used much less frequently than fMRI, has excellent temporal resolution and thus can track rapid temporal modulations in neural activity. Unfortunately, ERPs are under utilized in Cognitive Neuroscience because data acquisition techniques are not readily available and low density ERP recording has poor spatial resolution. In an effort to foster the increased use of ERPs in Cognitive Neuroscience, the present article details key techniques involved in high density ERP data acquisition. Critically, high density ERPs offer the promise of excellent temporal resolution and good spatial resolution (or excellent spatial resolution if coupled with fMRI), which is necessary to capture the spatial-temporal dynamics of human brain function.

Hyperspectral imagery super-resolution by compressive sensing inspired dictionary learning and spatial-spectral regularization.

PubMed

Huang, Wei; Xiao, Liang; Liu, Hongyi; Wei, Zhihui

2015-01-19

Due to the instrumental and imaging optics limitations, it is difficult to acquire high spatial resolution hyperspectral imagery (HSI). Super-resolution (SR) imagery aims at inferring high quality images of a given scene from degraded versions of the same scene. This paper proposes a novel hyperspectral imagery super-resolution (HSI-SR) method via dictionary learning and spatial-spectral regularization. The main contributions of this paper are twofold. First, inspired by the compressive sensing (CS) framework, for learning the high resolution dictionary, we encourage stronger sparsity on image patches and promote smaller coherence between the learned dictionary and sensing matrix. Thus, a sparsity and incoherence restricted dictionary learning method is proposed to achieve higher efficiency sparse representation. Second, a variational regularization model combing a spatial sparsity regularization term and a new local spectral similarity preserving term is proposed to integrate the spectral and spatial-contextual information of the HSI. Experimental results show that the proposed method can effectively recover spatial information and better preserve spectral information. The high spatial resolution HSI reconstructed by the proposed method outperforms reconstructed results by other well-known methods in terms of both objective measurements and visual evaluation.

Enhancing resolution and contrast in second-harmonic generation microscopy using an advanced maximum likelihood estimation restoration method

NASA Astrophysics Data System (ADS)

Sivaguru, Mayandi; Kabir, Mohammad M.; Gartia, Manas Ranjan; Biggs, David S. C.; Sivaguru, Barghav S.; Sivaguru, Vignesh A.; Berent, Zachary T.; Wagoner Johnson, Amy J.; Fried, Glenn A.; Liu, Gang Logan; Sadayappan, Sakthivel; Toussaint, Kimani C.

2017-02-01

Second-harmonic generation (SHG) microscopy is a label-free imaging technique to study collagenous materials in extracellular matrix environment with high resolution and contrast. However, like many other microscopy techniques, the actual spatial resolution achievable by SHG microscopy is reduced by out-of-focus blur and optical aberrations that degrade particularly the amplitude of the detectable higher spatial frequencies. Being a two-photon scattering process, it is challenging to define a point spread function (PSF) for the SHG imaging modality. As a result, in comparison with other two-photon imaging systems like two-photon fluorescence, it is difficult to apply any PSF-engineering techniques to enhance the experimental spatial resolution closer to the diffraction limit. Here, we present a method to improve the spatial resolution in SHG microscopy using an advanced maximum likelihood estimation (AdvMLE) algorithm to recover the otherwise degraded higher spatial frequencies in an SHG image. Through adaptation and iteration, the AdvMLE algorithm calculates an improved PSF for an SHG image and enhances the spatial resolution by decreasing the full-width-at-halfmaximum (FWHM) by 20%. Similar results are consistently observed for biological tissues with varying SHG sources, such as gold nanoparticles and collagen in porcine feet tendons. By obtaining an experimental transverse spatial resolution of 400 nm, we show that the AdvMLE algorithm brings the practical spatial resolution closer to the theoretical diffraction limit. Our approach is suitable for adaptation in micro-nano CT and MRI imaging, which has the potential to impact diagnosis and treatment of human diseases.

Epidermal Homeostasis and Radiation Responses in a Multiscale Tissue Modeling Framework

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Cucinotta, Francis A.

2013-01-01

The surface of skin is lined with several thin layers of epithelial cells that are maintained throughout life time by a small population of stem cells. High dose radiation exposures could injure and deplete the underlying proliferative cells and induce cutaneous radiation syndrome. In this work we propose a multiscale computational model for skin epidermal dynamics that links phenomena occurring at the subcellular, cellular, and tissue levels of organization, to simulate the experimental data of the radiation response of swine epidermis, which is closely similar to human epidermis. Incorporating experimentally measured histological and cell kinetic parameters, we obtain results of population kinetics and proliferation indexes comparable to observations in unirradiated and acutely irradiated swine experiments. At the sub-cellular level, several recently published Wnt signaling controlled cell-cycle models are applied and the roles of key components and parameters are analyzed. Based on our simulation results, we demonstrate that a moderate increase of proliferation rate for the survival proliferative cells is sufficient to fully repopulate the area denuded by high dose radiation, as long as the integrity of underlying basement membrane is maintained. Our work highlights the importance of considering proliferation kinetics as well as the spatial organization of tissues when conducting in vivo investigations of radiation responses. This integrated model allow us to test the validity of several basic biological rules at the cellular level and sub-cellular mechanisms by qualitatively comparing simulation results with published research, and enhance our understanding of the pathophysiological effects of ionizing radiation on skin.

Demonstration of Airborne Wide Area Assessment Technologies at Pueblo Precision Bombing Ranges, Colorado. Hyperspectral Imaging, Version 2.0

DTIC Science & Technology

2007-09-27

the spatial and spectral resolution ...variety of geological and vegetation mapping efforts, the Hymap sensor offered the best available combination of spectral and spatial resolution , signal... The limitations of the technology currently relate to spatial and spectral resolution and geo- correction accuracy. Secondly, HSI datasets

The effect of spatial resolution upon cloud optical property retrievals. I - Optical thickness

NASA Technical Reports Server (NTRS)

Feind, Rand E.; Christopher, Sundar A.; Welch, Ronald M.

1992-01-01

High spectral and spatial resolution Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) imagery is used to study the effects of spatial resolution upon fair weather cumulus cloud optical thickness retrievals. As a preprocessing step, a variation of the Gao and Goetz three-band ratio technique is used to discriminate clouds from the background. The combination of the elimination of cloud shadow pixels and using the first derivative of the histogram allows for accurate cloud edge discrimination. The data are progressively degraded from 20 m to 960 m spatial resolution. The results show that retrieved cloud area increases with decreasing spatial resolution. The results also show that there is a monotonic decrease in retrieved cloud optical thickness with decreasing spatial resolution. It is also demonstrated that the use of a single, monospectral reflectance threshold is inadequate for identifying cloud pixels in fair weather cumulus scenes and presumably in any inhomogeneous cloud field. Cloud edges have a distribution of reflectance thresholds. The incorrect identification of cloud edges significantly impacts the accurate retrieval of cloud optical thickness values.

Spatial resolution of a spherical x-ray crystal spectrometer at various magnifications

DOE PAGES

Gao, Lan; Hill, K. W.; Bitter, M.; ...

2016-08-23

Here, a high spatial resolution of a few μm is often required for probing small-scale high-energy-density plasmas using high resolution x-ray imaging spectroscopy. This resolution can be achieved by adjusting system magnification to overcome the inherent limitation of the detector pixel size. Laboratory experiments on investigating the relation between spatial resolution and system magnification for a spherical crystal spectrometer are presented. Tungsten Lβ 2 rays from a tungsten-target micro-focus x-ray tube were diffracted by a Ge 440 crystal, which was spherically bent to a radius of 223 mm, and imaged onto an x-ray CCD with 13-μm pixel size. The source-to-crystalmore » (p) and crystal-to-detector (q) distances were varied to produce spatial magnifications ( M = q/p) ranging from 2 to 10. The inferred instrumental spatial width reduces with increasing system magnification M. However, the experimental measurement at each M is larger than the theoretical value of pixel size divided by M. Future work will focus on investigating possible broadening mechanisms that limit the spatial resolution.« less

Emotional cues enhance the attentional effects on spatial and temporal resolution.

PubMed

Bocanegra, Bruno R; Zeelenberg, René

2011-12-01

In the present study, we demonstrated that the emotional significance of a spatial cue enhances the effect of covert attention on spatial and temporal resolution (i.e., our ability to discriminate small spatial details and fast temporal flicker). Our results indicated that fearful face cues, as compared with neutral face cues, enhanced the attentional benefits in spatial resolution but also enhanced the attentional deficits in temporal resolution. Furthermore, we observed that the overall magnitudes of individuals' attentional effects correlated strongly with the magnitude of the emotion × attention interaction effect. Combined, these findings provide strong support for the idea that emotion enhances the strength of a cue's attentional response.

HESS Opinions: The need for process-based evaluation of large-domain hyper-resolution models

NASA Astrophysics Data System (ADS)

Melsen, Lieke A.; Teuling, Adriaan J.; Torfs, Paul J. J. F.; Uijlenhoet, Remko; Mizukami, Naoki; Clark, Martyn P.

2016-03-01

A meta-analysis on 192 peer-reviewed articles reporting on applications of the variable infiltration capacity (VIC) model in a distributed way reveals that the spatial resolution at which the model is applied has increased over the years, while the calibration and validation time interval has remained unchanged. We argue that the calibration and validation time interval should keep pace with the increase in spatial resolution in order to resolve the processes that are relevant at the applied spatial resolution. We identified six time concepts in hydrological models, which all impact the model results and conclusions. Process-based model evaluation is particularly relevant when models are applied at hyper-resolution, where stakeholders expect credible results both at a high spatial and temporal resolution.

HESS Opinions: The need for process-based evaluation of large-domain hyper-resolution models

NASA Astrophysics Data System (ADS)

Melsen, L. A.; Teuling, A. J.; Torfs, P. J. J. F.; Uijlenhoet, R.; Mizukami, N.; Clark, M. P.

2015-12-01

A meta-analysis on 192 peer-reviewed articles reporting applications of the Variable Infiltration Capacity (VIC) model in a distributed way reveals that the spatial resolution at which the model is applied has increased over the years, while the calibration and validation time interval has remained unchanged. We argue that the calibration and validation time interval should keep pace with the increase in spatial resolution in order to resolve the processes that are relevant at the applied spatial resolution. We identified six time concepts in hydrological models, which all impact the model results and conclusions. Process-based model evaluation is particularly relevant when models are applied at hyper-resolution, where stakeholders expect credible results both at a high spatial and temporal resolution.

Interaction between the moss Physcomitrella patens and Phytophthora: a novel pathosystem for live-cell imaging of subcellular defence.

PubMed

Overdijk, Elysa J R; DE Keijzer, Jeroen; DE Groot, Deborah; Schoina, Charikleia; Bouwmeester, Klaas; Ketelaar, Tijs; Govers, Francine

2016-08-01

Live-cell imaging of plant-pathogen interactions is often hampered by the tissue complexity and multicell layered nature of the host. Here, we established a novel pathosystem with the moss Physcomitrella patens as host for Phytophthora. The tip-growing protonema cells of this moss are ideal for visualizing interactions with the pathogen over time using high-resolution microscopy. We tested four Phytophthora species for their ability to infect P. patens and showed that P. sojae and P. palmivora were only rarely capable to infect P. patens. In contrast, P. infestans and P. capsici frequently and successfully penetrated moss protonemal cells, showed intracellular hyphal growth and formed sporangia. Next to these successful invasions, many penetration attempts failed. Here the pathogen was blocked by a barrier of cell wall material deposited in papilla-like structures, a defence response that is common in higher plants. Another common response is the upregulation of defence-related genes upon infection and also in moss we observed this upregulation in tissues infected with Phytophthora. For more advanced analyses of the novel pathosystem we developed a special set-up that allowed live-cell imaging of subcellular defence processes by high-resolution microscopy. With this set-up, we revealed that Phytophthora infection of moss induces repositioning of the nucleus, accumulation of cytoplasm and rearrangement of the actin cytoskeleton, but not of microtubules. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Internalization of aggregated photosensitizers by tumor cells: subcellular time-resolved fluorescence spectroscopy on derivatives of pyropheophorbide-a ethers and chlorin e6 under femtosecond one- and two-photon excitations.

PubMed

Kelbauskas, L; Dietel, W

2002-12-01

Amphiphilic sensitizers self-associate in aqueous environments and form aggregated species that exhibit no or only negligible photodynamic activity. However, amphiphilic photosensitizers number among the most potent agents of photodynamic therapy. The processes by which these sensitizers are internalized into tumor cells have yet to be fully elucidated and thus remain the subject of debate. In this study the uptake of photosensitizer aggregates into tumor cells was examined directly using subcellular time-resolved fluorescence spectroscopy with a high temporal resolution (20-30 ps) and high sensitivity (time-correlated single-photon counting). The investigations were performed on selected sensitizers that exhibit short fluorescence decay times (< 50 ps) in aggregated form. Derivatives of pyropheophorbide-a ether and chlorin e6 with varying lipophilicity were used for the study. The characteristic fluorescence decay times and spectroscopic features of the sensitizer aggregates measured in aqueous solution also could be observed in A431 human endothelial carcinoma cells administered with these photosensitizers. This shows that tumor cells can internalize sensitizers in aggregated form. Uptake of aggregates and their monomerization inside cells were demonstrated directly for the first time by means of fluorescence lifetime imaging with a high temporal resolution. Internalization of the aggregates seems to be endocytosis mediated. The degree of their monomerization in tumor cells is strongly influenced by the lipophilicity of the compounds.

Effect of spatial resolution on remote sensing estimation of total evaporation in the uMngeni catchment, South Africa

NASA Astrophysics Data System (ADS)

Shoko, Cletah; Clark, David; Mengistu, Michael; Dube, Timothy; Bulcock, Hartley

2015-01-01

This study evaluated the effect of two readily available multispectral sensors: the newly launched 30 m spatial resolution Landsat 8 and the long-serving 1000 m moderate resolution imaging spectroradiometer (MODIS) datasets in the spatial representation of total evaporation in the heterogeneous uMngeni catchment, South Africa, using the surface energy balance system model. The results showed that sensor spatial resolution plays a critical role in the accurate estimation of energy fluxes and total evaporation across a heterogeneous catchment. Landsat 8 estimates showed better spatial representation of the biophysical parameters and total evaporation for different land cover types, due to the relatively higher spatial resolution compared to the coarse spatial resolution MODIS sensor. Moreover, MODIS failed to capture the spatial variations of total evaporation estimates across the catchment. Analysis of variance (ANOVA) results showed that MODIS-based total evaporation estimates did not show any significant differences across different land cover types (one-way ANOVA; F1.924=1.412, p=0.186). However, Landsat 8 images yielded significantly different estimates between different land cover types (one-way ANOVA; F1.993=5.185, p<0.001). The validation results showed that Landsat 8 estimates were more comparable to eddy covariance (EC) measurements than the MODIS-based total evaporation estimates. EC measurement on May 23, 2013, was 3.8 mm/day, whereas the Landsat 8 estimate on the same day was 3.6 mm/day, with MODIS showing significantly lower estimates of 2.3 mm/day. The findings of this study underscore the importance of spatial resolution in estimating spatial variations of total evaporation at the catchment scale, thus, they provide critical information on the relevance of the readily available remote sensing products in water resources management in data-scarce environments.

Spatial resolution limits for the isotropic-3D PET detector X’tal cube

NASA Astrophysics Data System (ADS)

Yoshida, Eiji; Tashima, Hideaki; Hirano, Yoshiyuki; Inadama, Naoko; Nishikido, Fumihiko; Murayama, Hideo; Yamaya, Taiga

2013-11-01

Positron emission tomography (PET) has become a popular imaging method in metabolism, neuroscience, and molecular imaging. For dedicated human brain and small animal PET scanners, high spatial resolution is needed to visualize small objects. To improve the spatial resolution, we are developing the X’tal cube, which is our new PET detector to achieve isotropic 3D positioning detectability. We have shown that the X’tal cube can achieve 1 mm3 uniform crystal identification performance with the Anger-type calculation even at the block edges. We plan to develop the X’tal cube with even smaller 3D grids for sub-millimeter crystal identification. In this work, we investigate spatial resolution of a PET scanner based on the X’tal cube using Monte Carlo simulations for predicting resolution performance in smaller 3D grids. For spatial resolution evaluation, a point source emitting 511 keV photons was simulated by GATE for all physical processes involved in emission and interaction of positrons. We simulated two types of animal PET scanners. The first PET scanner had a detector ring 14.6 cm in diameter composed of 18 detectors. The second PET scanner had a detector ring 7.8 cm in diameter composed of 12 detectors. After the GATE simulations, we converted the interacting 3D position information to digitalized positions for realistic segmented crystals. We simulated several X’tal cubes with cubic crystals from (0.5 mm)3 to (2 mm)3 in size. Also, for evaluating the effect of DOI resolution, we simulated several X’tal cubes with crystal thickness from (0.5 mm)3 to (9 mm)3. We showed that sub-millimeter spatial resolution was possible using cubic crystals smaller than (1.0 mm)3 even with the assumed physical processes. Also, the weighted average spatial resolutions of both PET scanners with (0.5 mm)3 cubic crystals were 0.53 mm (14.6 cm ring diameter) and 0.48 mm (7.8 cm ring diameter). For the 7.8 cm ring diameter, spatial resolution with 0.5×0.5×1.0 mm3 crystals was improved 39% relative to the (1 mm)3 cubic crystals. On the other hand, spatial resolution with (0.5 mm)3 cubic crystals was improved 47% relative to the (1 mm)3 cubic crystals. The X’tal cube promises better spatial resolution for the 3D crystal block with isotropic resolution.

Evaluating the Impact of Spatial Resolution of Landsat Predictors on the Accuracy of Biomass Models for Large-area Estimation Across the Eastern USA

NASA Astrophysics Data System (ADS)

Deo, R. K.; Domke, G. M.; Russell, M.; Woodall, C. W.

2017-12-01

Landsat data have been widely used to support strategic forest inventory and management decisions despite the limited success of passive optical remote sensing for accurate estimation of aboveground biomass (AGB). The archive of publicly available Landsat data, available at 30-m spatial resolutions since 1984, has been a valuable resource for cost-effective large-area estimation of AGB to inform national requirements such as for the US national greenhouse gas inventory (NGHGI). In addition, other optical satellite data such as MODIS imagery of wider spatial coverage and higher temporal resolution are enriching the domain of spatial predictors for regional scale mapping of AGB. Because NGHGIs require national scale AGB information and there are tradeoffs in the prediction accuracy versus operational efficiency of Landsat, this study evaluated the impact of various resolutions of Landsat predictors on the accuracy of regional AGB models across three different sites in the eastern USA: Maine, Pennsylvania-New Jersey, and South Carolina. We used recent national forest inventory (NFI) data with numerous Landsat-derived predictors at ten different spatial resolutions ranging from 30 to 1000 m to understand the optimal spatial resolution of the optical data for enhanced spatial inventory of AGB for NGHGI reporting. Ten generic spatial models at different spatial resolutions were developed for all sites and large-area estimates were evaluated (i) at the county-level against the independent designed-based estimates via the US NFI Evalidator tool and (ii) within a large number of strips ( 1 km wide) predicted via LiDAR metrics at a high spatial resolution. The county-level estimates by the Evalidator and Landsat models were statistically equivalent and produced coefficients of determination (R2) above 0.85 that varied with sites and resolution of predictors. The mean and standard deviation of county-level estimates followed increasing and decreasing trends, respectively, with models of decreasing resolutions. The Landsat-based total AGB estimates within the strips against the total AGB obtained using LiDAR metrics did not differ significantly and were within ±15 Mg/ha for each of the sites. We conclude that the optical satellite data at resolutions up to 1000 m provide acceptable accuracy for the US' NGHGI.

Spatial resolution limitation of liquid crystal spatial light modulator

NASA Astrophysics Data System (ADS)

Wang, Xinghua; Wang, Bin; McManamon, Paul F., III; Pouch, John J.; Miranda, Felix A.; Anderson, James E.; Bos, Philip J.

2004-10-01

The effect of fringing electric fields in a liquid crystal (LC) Optical Phased Array (OPA), also referred to as a spatial light modulator (SLM), is a governing factor that determines the diffraction efficiency (DE) of the LC OPA for high resolution spatial phase modulation. In this article, the fringing field effect in a high resolution LC OPA is studied by accurate modeling the DE of the LC blazed gratings by LC director simulation and Finite Difference Time Domain (FDTD) simulation. Influence factors that contribute significantly to the DE are discussed. Such results provide fundamental understanding for high resolution LC devices.

Impacts of spatial resolution and representation of flow connectivity on large-scale simulation of floods

NASA Astrophysics Data System (ADS)

Mateo, Cherry May R.; Yamazaki, Dai; Kim, Hyungjun; Champathong, Adisorn; Vaze, Jai; Oki, Taikan

2017-10-01

Global-scale river models (GRMs) are core tools for providing consistent estimates of global flood hazard, especially in data-scarce regions. Due to former limitations in computational power and input datasets, most GRMs have been developed to use simplified representations of flow physics and run at coarse spatial resolutions. With increasing computational power and improved datasets, the application of GRMs to finer resolutions is becoming a reality. To support development in this direction, the suitability of GRMs for application to finer resolutions needs to be assessed. This study investigates the impacts of spatial resolution and flow connectivity representation on the predictive capability of a GRM, CaMa-Flood, in simulating the 2011 extreme flood in Thailand. Analyses show that when single downstream connectivity (SDC) is assumed, simulation results deteriorate with finer spatial resolution; Nash-Sutcliffe efficiency coefficients decreased by more than 50 % between simulation results at 10 km resolution and 1 km resolution. When multiple downstream connectivity (MDC) is represented, simulation results slightly improve with finer spatial resolution. The SDC simulations result in excessive backflows on very flat floodplains due to the restrictive flow directions at finer resolutions. MDC channels attenuated these effects by maintaining flow connectivity and flow capacity between floodplains in varying spatial resolutions. While a regional-scale flood was chosen as a test case, these findings should be universal and may have significant impacts on large- to global-scale simulations, especially in regions where mega deltas exist.These results demonstrate that a GRM can be used for higher resolution simulations of large-scale floods, provided that MDC in rivers and floodplains is adequately represented in the model structure.

A technique for enhancing and matching the resolution of microwave measurements from the SSM/I instrument

NASA Technical Reports Server (NTRS)

Robinson, Wayne D.; Kummerrow, Christian; Olson, William S.

1992-01-01

A correction technique is presented for matching the resolution of all the frequencies of the satelliteborne Special Sensor Microwave/Imager (SSM/I) to the about-25-km spatial resolution of the 37-GHz channel. This entails, on the one hand, the enhancement of the spatial resolution of the 19- and 22-GHz channels, and on the other, the degrading of that of the 85-GHz channel. The Backus and Gilbert (1970) approach is found to yield sufficient spatial resolution to render such a correction worthwhile.

High spatial resolution distributed optical fiber dynamic strain sensor with enhanced frequency and strain resolution.

PubMed

Masoudi, Ali; Newson, Trevor P

2017-01-15

A distributed optical fiber dynamic strain sensor with high spatial and frequency resolution is demonstrated. The sensor, which uses the ϕ-OTDR interrogation technique, exhibited a higher sensitivity thanks to an improved optical arrangement and a new signal processing procedure. The proposed sensing system is capable of fully quantifying multiple dynamic perturbations along a 5 km long sensing fiber with a frequency and spatial resolution of 5 Hz and 50 cm, respectively. The strain resolution of the sensor was measured to be 40 nε.

Study of satellite retrieved aerosol optical depth spatial resolution effect on particulate matter concentration prediction

NASA Astrophysics Data System (ADS)

Strandgren, J.; Mei, L.; Vountas, M.; Burrows, J. P.; Lyapustin, A.; Wang, Y.

2014-10-01

The Aerosol Optical Depth (AOD) spatial resolution effect is investigated for the linear correlation between satellite retrieved AOD and ground level particulate matter concentrations (PM2.5). The Multi-Angle Implementation of Atmospheric Correction (MAIAC) algorithm was developed for the Moderate Resolution Imaging Spectroradiometer (MODIS) for obtaining AOD with a high spatial resolution of 1 km and provides a good dataset for the study of the AOD spatial resolution effect on the particulate matter concentration prediction. 946 Environmental Protection Agency (EPA) ground monitoring stations across the contiguous US have been used to investigate the linear correlation between AOD and PM2.5 using AOD at different spatial resolutions (1, 3 and 10 km) and for different spatial scales (urban scale, meso-scale and continental scale). The main conclusions are: (1) for both urban, meso- and continental scale the correlation between PM2.5 and AOD increased significantly with increasing spatial resolution of the AOD, (2) the correlation between AOD and PM2.5 decreased significantly as the scale of study region increased for the eastern part of the US while vice versa for the western part of the US, (3) the correlation between PM2.5 and AOD is much more stable and better over the eastern part of the US compared to western part due to the surface characteristics and atmospheric conditions like the fine mode fraction.

Employing temporal self-similarity across the entire time domain in computed tomography reconstruction

PubMed Central

Kazantsev, D.; Van Eyndhoven, G.; Lionheart, W. R. B.; Withers, P. J.; Dobson, K. J.; McDonald, S. A.; Atwood, R.; Lee, P. D.

2015-01-01

There are many cases where one needs to limit the X-ray dose, or the number of projections, or both, for high frame rate (fast) imaging. Normally, it improves temporal resolution but reduces the spatial resolution of the reconstructed data. Fortunately, the redundancy of information in the temporal domain can be employed to improve spatial resolution. In this paper, we propose a novel regularizer for iterative reconstruction of time-lapse computed tomography. The non-local penalty term is driven by the available prior information and employs all available temporal data to improve the spatial resolution of each individual time frame. A high-resolution prior image from the same or a different imaging modality is used to enhance edges which remain stationary throughout the acquisition time while dynamic features tend to be regularized spatially. Effective computational performance together with robust improvement in spatial and temporal resolution makes the proposed method a competitive tool to state-of-the-art techniques. PMID:25939621

Change of spatial information under rescaling: A case study using multi-resolution image series

NASA Astrophysics Data System (ADS)

Chen, Weirong; Henebry, Geoffrey M.

Spatial structure in imagery depends on a complicated interaction between the observational regime and the types and arrangements of entities within the scene that the image portrays. Although block averaging of pixels has commonly been used to simulate coarser resolution imagery, relatively little attention has been focused on the effects of simple rescaling on spatial structure and the explanation and a possible solution to the problem. Yet, if there are significant differences in spatial variance between rescaled and observed images, it may affect the reliability of retrieved biogeophysical quantities. To investigate these issues, a nested series of high spatial resolution digital imagery was collected at a research site in eastern Nebraska in 2001. An airborne Kodak DCS420IR camera acquired imagery at three altitudes, yielding nominal spatial resolutions ranging from 0.187 m to 1 m. The red and near infrared (NIR) bands of the co-registered image series were normalized using pseudo-invariant features, and the normalized difference vegetation index (NDVI) was calculated. Plots of grain sorghum planted in orthogonal crop row orientations were extracted from the image series. The finest spatial resolution data were then rescaled by averaging blocks of pixels to produce a rescaled image series that closely matched the spatial resolution of the observed image series. Spatial structures of the observed and rescaled image series were characterized using semivariogram analysis. Results for NDVI and its component bands show, as expected, that decreasing spatial resolution leads to decreasing spatial variability and increasing spatial dependence. However, compared to the observed data, the rescaled images contain more persistent spatial structure that exhibits limited variation in both spatial dependence and spatial heterogeneity. Rescaling via simple block averaging fails to consider the effect of scene object shape and extent on spatial information. As the features portrayed by pixels are equally weighted regardless of the shape and extent of the underlying scene objects, the rescaled image retains more of the original spatial information than would occur through direct observation at a coarser sensor spatial resolution. In contrast, for the observed images, due to the effect of the modulation transfer function (MTF) of the imaging system, high frequency features like edges are blurred or lost as the pixel size increases, resulting in greater variation in spatial structure. Successive applications of a low-pass spatial convolution filter are shown to mimic a MTF. Accordingly, it is recommended that such a procedure be applied prior to rescaling by simple block averaging, if insufficient image metadata exist to replicate the net MTF of the imaging system, as might be expected in land cover change analysis studies using historical imagery.

Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells.

PubMed

Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

2013-01-01

Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50-60 nm on a time scale of 2.3 s. Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level.

Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells

PubMed Central

Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

2016-01-01

Background Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. Results We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50–60 nm on a time scale of 2.3 s. Conclusion Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level. PMID:27795878

Satellite image fusion based on principal component analysis and high-pass filtering.

PubMed

Metwalli, Mohamed R; Nasr, Ayman H; Allah, Osama S Farag; El-Rabaie, S; Abd El-Samie, Fathi E

2010-06-01

This paper presents an integrated method for the fusion of satellite images. Several commercial earth observation satellites carry dual-resolution sensors, which provide high spatial resolution or simply high-resolution (HR) panchromatic (pan) images and low-resolution (LR) multi-spectral (MS) images. Image fusion methods are therefore required to integrate a high-spectral-resolution MS image with a high-spatial-resolution pan image to produce a pan-sharpened image with high spectral and spatial resolutions. Some image fusion methods such as the intensity, hue, and saturation (IHS) method, the principal component analysis (PCA) method, and the Brovey transform (BT) method provide HR MS images, but with low spectral quality. Another family of image fusion methods, such as the high-pass-filtering (HPF) method, operates on the basis of the injection of high frequency components from the HR pan image into the MS image. This family of methods provides less spectral distortion. In this paper, we propose the integration of the PCA method and the HPF method to provide a pan-sharpened MS image with superior spatial resolution and less spectral distortion. The experimental results show that the proposed fusion method retains the spectral characteristics of the MS image and, at the same time, improves the spatial resolution of the pan-sharpened image.

High-Spatial and High-Mass Resolution Imaging of Surface Metabolites of Arabidopsis thaliana by Laser Desorption-Ionization Mass Spectrometry Using Colloidal Silver

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jun, Ji Hyun; Song, Zhihong; Liu, Zhenjiu

High-spatial resolution and high-mass resolution techniques are developed and adopted for the mass spectrometric imaging of epicuticular lipids on the surface of Arabidopsis thaliana. Single cell level spatial resolution of {approx}12 {micro}m was achieved by reducing the laser beam size by using an optical fiber with 25 {micro}m core diameter in a vacuum matrix-assisted laser desorption ionization-linear ion trap (vMALDI-LTQ) mass spectrometer and improved matrix application using an oscillating capillary nebulizer. Fine chemical images of a whole flower were visualized in this high spatial resolution showing substructure of an anther and single pollen grains at the stigma and anthers. Themore » LTQ-Orbitrap with a MALDI ion source was adopted to achieve MS imaging in high mass resolution. Specifically, isobaric silver ion adducts of C29 alkane (m/z 515.3741) and C28 aldehyde (m/z 515.3377), indistinguishable in low-resolution LTQ, can now be clearly distinguished and their chemical images could be separately constructed. In the application to roots, the high spatial resolution allowed molecular MS imaging of secondary roots and the high mass resolution allowed direct identification of lipid metabolites on root surfaces.« less

The spatial resolution of a rotating gamma camera tomographic facility.

PubMed

Webb, S; Flower, M A; Ott, R J; Leach, M O; Inamdar, R

1983-12-01

An important feature determining the spatial resolution in transverse sections reconstructed by convolution and back-projection is the frequency filter corresponding to the convolution kernel. Equations have been derived giving the theoretical spatial resolution, for a perfect detector and noise-free data, using four filter functions. Experiments have shown that physical constraints will always limit the resolution that can be achieved with a given system. The experiments indicate that the region of the frequency spectrum between KN/2 and KN where KN is the Nyquist frequency does not contribute significantly to resolution. In order to investigate the physical effect of these filter functions, the spatial resolution of reconstructed images obtained with a GE 400T rotating gamma camera has been measured. The results obtained serve as an aid to choosing appropriate reconstruction filters for use with a rotating gamma camera system.

A high time and spatial resolution MRPC designed for muon tomography

NASA Astrophysics Data System (ADS)

Shi, L.; Wang, Y.; Huang, X.; Wang, X.; Zhu, W.; Li, Y.; Cheng, J.

2014-12-01

A prototype of cosmic muon scattering tomography system has been set up in Tsinghua University in Beijing. Multi-gap Resistive Plate Chamber (MRPC) is used in the system to get the muon tracks. Compared with other detectors, MRPC can not only provide the track but also the Time of Flight (ToF) between two detectors which can estimate the energy of particles. To get a more accurate track and higher efficiency of the tomography system, a new type of high time and two-dimensional spatial resolution MRPC has been developed. A series of experiments have been done to measure the efficiency, time resolution and spatial resolution. The results show that the efficiency can reach 95% and its time resolution is around 65 ps. The cluster size is around 4 and the spatial resolution can reach 200 μ m.

150-μm Spatial Resolution Using Photon-Counting Detector Computed Tomography Technology: Technical Performance and First Patient Images.

PubMed

Leng, Shuai; Rajendran, Kishore; Gong, Hao; Zhou, Wei; Halaweish, Ahmed F; Henning, Andre; Kappler, Steffen; Baer, Matthias; Fletcher, Joel G; McCollough, Cynthia H

2018-05-28

The aims of this study were to quantitatively assess two new scan modes on a photon-counting detector computed tomography system, each designed to maximize spatial resolution, and to qualitatively demonstrate potential clinical impact using patient data. This Health Insurance Portability Act-compliant study was approved by our institutional review board. Two high-spatial-resolution scan modes (Sharp and UHR) were evaluated using phantoms to quantify spatial resolution and image noise, and results were compared with the standard mode (Macro). Patients were scanned using a conventional energy-integrating detector scanner and the photon-counting detector scanner using the same radiation dose. In first patient images, anatomic details were qualitatively evaluated to demonstrate potential clinical impact. Sharp and UHR modes had a 69% and 87% improvement in in-plane spatial resolution, respectively, compared with Macro mode (10% modulation-translation-function values of 16.05, 17.69, and 9.48 lp/cm, respectively). The cutoff spatial frequency of the UHR mode (32.4 lp/cm) corresponded to a limiting spatial resolution of 150 μm. The full-width-at-half-maximum values of the section sensitivity profiles were 0.41, 0.44, and 0.67 mm for the thinnest image thickness for each mode (0.25, 0.25, and 0.5 mm, respectively). At the same in-plane spatial resolution, Sharp and UHR images had up to 15% lower noise than Macro images. Patient images acquired in Sharp mode demonstrated better delineation of fine anatomic structures compared with Macro mode images. Phantom studies demonstrated superior resolution and noise properties for the Sharp and UHR modes relative to the standard Macro mode and patient images demonstrated the potential benefit of these scan modes for clinical practice.

The Analytical Limits of Modeling Short Diffusion Timescales

NASA Astrophysics Data System (ADS)

Bradshaw, R. W.; Kent, A. J.

2016-12-01

Chemical and isotopic zoning in minerals is widely used to constrain the timescales of magmatic processes such as magma mixing and crystal residence, etc. via diffusion modeling. Forward modeling of diffusion relies on fitting diffusion profiles to measured compositional gradients. However, an individual measurement is essentially an average composition for a segment of the gradient defined by the spatial resolution of the analysis. Thus there is the potential for the analytical spatial resolution to limit the timescales that can be determined for an element of given diffusivity, particularly where the scale of the gradient approaches that of the measurement. Here we use a probabilistic modeling approach to investigate the effect of analytical spatial resolution on estimated timescales from diffusion modeling. Our method investigates how accurately the age of a synthetic diffusion profile can be obtained by modeling an "unknown" profile derived from discrete sampling of the synthetic compositional gradient at a given spatial resolution. We also include the effects of analytical uncertainty and the position of measurements relative to the diffusion gradient. We apply this method to the spatial resolutions of common microanalytical techniques (LA-ICP-MS, SIMS, EMP, NanoSIMS). Our results confirm that for a given diffusivity, higher spatial resolution gives access to shorter timescales, and that each analytical spacing has a minimum timescale, below which it overestimates the timescale. For example, for Ba diffusion in plagioclase at 750 °C timescales are accurate (within 20%) above 10, 100, 2,600, and 71,000 years at 0.3, 1, 5, and 25 mm spatial resolution, respectively. For Sr diffusion in plagioclase at 750 °C, timescales are accurate above 0.02, 0.2, 4, and 120 years at the same spatial resolutions. Our results highlight the importance of selecting appropriate analytical techniques to estimate accurate diffusion-based timescales.

Spatial and Angular Resolution Enhancement of Light Fields Using Convolutional Neural Networks

NASA Astrophysics Data System (ADS)

Gul, M. Shahzeb Khan; Gunturk, Bahadir K.

2018-05-01

Light field imaging extends the traditional photography by capturing both spatial and angular distribution of light, which enables new capabilities, including post-capture refocusing, post-capture aperture control, and depth estimation from a single shot. Micro-lens array (MLA) based light field cameras offer a cost-effective approach to capture light field. A major drawback of MLA based light field cameras is low spatial resolution, which is due to the fact that a single image sensor is shared to capture both spatial and angular information. In this paper, we present a learning based light field enhancement approach. Both spatial and angular resolution of captured light field is enhanced using convolutional neural networks. The proposed method is tested with real light field data captured with a Lytro light field camera, clearly demonstrating spatial and angular resolution improvement.

Spatial and Angular Resolution Enhancement of Light Fields Using Convolutional Neural Networks.

PubMed

Gul, M Shahzeb Khan; Gunturk, Bahadir K

2018-05-01

Light field imaging extends the traditional photography by capturing both spatial and angular distribution of light, which enables new capabilities, including post-capture refocusing, post-capture aperture control, and depth estimation from a single shot. Micro-lens array (MLA) based light field cameras offer a cost-effective approach to capture light field. A major drawback of MLA based light field cameras is low spatial resolution, which is due to the fact that a single image sensor is shared to capture both spatial and angular information. In this paper, we present a learning based light field enhancement approach. Both spatial and angular resolution of captured light field is enhanced using convolutional neural networks. The proposed method is tested with real light field data captured with a Lytro light field camera, clearly demonstrating spatial and angular resolution improvement.

Multi-Resolution Analysis of MODIS and ASTER Satellite Data for Water Classification

DTIC Science & Technology

2006-09-01

spectral bands, but also with different pixel resolutions . The overall goal... the total water surface. Due to the constraint that high spatial resolution satellite images are low temporal resolution , one needs a reliable method...at 15 m resolution , were processed. We used MODIS reflectance data from MOD02 Level 1B data. Even the spatial resolution of the 1240 nm

Definition of the Spatial Resolution of X-Ray Microanalysis in Thin Foils

NASA Technical Reports Server (NTRS)

Williams, D. B.; Michael, J. R.; Goldstein, J. I.; Romig, A. D., Jr.

1992-01-01

The spatial resolution of X-ray microanalysis in thin foils is defined in terms of the incident electron beam diameter and the average beam broadening. The beam diameter is defined as the full width tenth maximum of a Gaussian intensity distribution. The spatial resolution is calculated by a convolution of the beam diameter and the average beam broadening. This definition of the spatial resolution can be related simply to experimental measurements of composition profiles across interphase interfaces. Monte Carlo calculations using a high-speed parallel supercomputer show good agreement with this definition of the spatial resolution and calculations based on this definition. The agreement is good over a range of specimen thicknesses and atomic number, but is poor when excessive beam tailing distorts the assumed Gaussian electron intensity distributions. Beam tailing occurs in low-Z materials because of fast secondary electrons and in high-Z materials because of plural scattering.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krishnan, Venkat; Cole, Wesley

Power sector capacity expansion models (CEMs) have a broad range of spatial resolutions. This paper uses the Regional Energy Deployment System (ReEDS) model, a long-term national scale electric sector CEM, to evaluate the value of high spatial resolution for CEMs. ReEDS models the United States with 134 load balancing areas (BAs) and captures the variability in existing generation parameters, future technology costs, performance, and resource availability using very high spatial resolution data, especially for wind and solar modeled at 356 resource regions. In this paper we perform planning studies at three different spatial resolutions--native resolution (134 BAs), state-level, and NERCmore » region level--and evaluate how results change under different levels of spatial aggregation in terms of renewable capacity deployment and location, associated transmission builds, and system costs. The results are used to ascertain the value of high geographically resolved models in terms of their impact on relative competitiveness among renewable energy resources.« less

Easy way to determine quantitative spatial resolution distribution for a general inverse problem

NASA Astrophysics Data System (ADS)

An, M.; Feng, M.

2013-12-01

The spatial resolution computation of a solution was nontrivial and more difficult than solving an inverse problem. Most geophysical studies, except for tomographic studies, almost uniformly neglect the calculation of a practical spatial resolution. In seismic tomography studies, a qualitative resolution length can be indicatively given via visual inspection of the restoration of a synthetic structure (e.g., checkerboard tests). An effective strategy for obtaining quantitative resolution length is to calculate Backus-Gilbert resolution kernels (also referred to as a resolution matrix) by matrix operation. However, not all resolution matrices can provide resolution length information, and the computation of resolution matrix is often a difficult problem for very large inverse problems. A new class of resolution matrices, called the statistical resolution matrices (An, 2012, GJI), can be directly determined via a simple one-parameter nonlinear inversion performed based on limited pairs of random synthetic models and their inverse solutions. The total procedure were restricted to forward/inversion processes used in the real inverse problem and were independent of the degree of inverse skill used in the solution inversion. Spatial resolution lengths can be directly given during the inversion. Tests on 1D/2D/3D model inversion demonstrated that this simple method can be at least valid for a general linear inverse problem.

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.

PubMed

Trivedi, Vikas; Choi, Harry M T; Fraser, Scott E; Pierce, Niles A

2018-01-08

For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting and/or homogenization. Here, we eliminate the trade-off between quantitation and anatomical context, using quantitative in situ hybridization chain reaction (qHCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable bi-directional queries that open a new era for in situ hybridization. © 2018. Published by The Company of Biologists Ltd.

Measuring changes in the mass of single subcellular organelles using x-ray microscopy

NASA Astrophysics Data System (ADS)

Goncz, Kaarin K.; Moronne, Mario M.; Lin, W.; Rothman, Stephen S.

1993-01-01

Using quantitative scanning transmission x-ray microscopy, zymogen granules isolated from pancreatic acinar cells were observed suspended in aqueous medium at 50 nm resolution. From 3.64 nm x-ray absorption data, the protein content and rate of protein efflux from individual granules were determined. This was accomplished with a specially designed silicon nitride based wet-cell that allowed continuous perfusion and monitoring of individual granules in a variety of different aqueous environments. Granules suspended in 300 mM sucrose, 5 mM phosphate buffer (pH 6.0) were observed to continuously decrease in size and protein content over a period of several hours. Sudden lysis of the granules was not observed. From the flux data, the apparent protein permeability coefficients for individual granules were determined to range from 1 - 10 X 10-10 cm/sec with an average of 4.78 +/- 3.0 X 10-10 cm/sec. We believe this is the first quantitative population profile determined for a subcellular organelle developed from measurements of individual members of the population.

Multiphoton Intravital Calcium Imaging.

PubMed

Cheetham, Claire E J

2018-06-26

Multiphoton intravital calcium imaging is a powerful technique that enables high-resolution longitudinal monitoring of cellular and subcellular activity hundreds of microns deep in the living organism. This unit addresses the application of 2-photon microscopy to imaging of genetically encoded calcium indicators (GECIs) in the mouse brain. The protocols in this unit enable real-time intravital imaging of intracellular calcium concentration simultaneously in hundreds of neurons, or at the resolution of single synapses, as mice respond to sensory stimuli or perform behavioral tasks. Protocols are presented for implantation of a cranial imaging window to provide optical access to the brain and for 2-photon image acquisition. Protocols for implantation of both open skull and thinned skull windows for single or multi-session imaging are described. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.

Towards breaking the spatial resolution barriers: An optical flow and super-resolution approach for sea ice motion estimation

NASA Astrophysics Data System (ADS)

Petrou, Zisis I.; Xian, Yang; Tian, YingLi

2018-04-01

Estimation of sea ice motion at fine scales is important for a number of regional and local level applications, including modeling of sea ice distribution, ocean-atmosphere and climate dynamics, as well as safe navigation and sea operations. In this study, we propose an optical flow and super-resolution approach to accurately estimate motion from remote sensing images at a higher spatial resolution than the original data. First, an external example learning-based super-resolution method is applied on the original images to generate higher resolution versions. Then, an optical flow approach is applied on the higher resolution images, identifying sparse correspondences and interpolating them to extract a dense motion vector field with continuous values and subpixel accuracies. Our proposed approach is successfully evaluated on passive microwave, optical, and Synthetic Aperture Radar data, proving appropriate for multi-sensor applications and different spatial resolutions. The approach estimates motion with similar or higher accuracy than the original data, while increasing the spatial resolution of up to eight times. In addition, the adopted optical flow component outperforms a state-of-the-art pattern matching method. Overall, the proposed approach results in accurate motion vectors with unprecedented spatial resolutions of up to 1.5 km for passive microwave data covering the entire Arctic and 20 m for radar data, and proves promising for numerous scientific and operational applications.

Use of UAS remote sensing data to estimate crop ET at high spatial resolution

USDA-ARS?s Scientific Manuscript database

Estimation of the spatial distribution of evapotranspiration (ET) based on remotely sensed imagery has become useful for managing water in irrigated agricultural at various spatial scales. However, data acquired by conventional satellites (Landsat, ASTER, etc.) lack the spatial resolution to capture...

Quantitative imaging of electron transfer flavoprotein autofluorescence reveals the dynamics of lipid partitioning in living pancreatic islets.

PubMed

Lam, Alan K; Silva, Pamuditha N; Altamentova, Svetlana M; Rocheleau, Jonathan V

2012-08-01

Pancreatic islet β-cells metabolically sense nutrients to maintain blood glucose homeostasis through the regulated secretion of insulin. Long-term exposure to a mixed supply of excess glucose and fatty acids induces β-cell dysfunction and type II diabetes in a process termed glucolipotoxicity. Despite a number of documented mechanisms for glucolipotoxicity, the interplay between glucose and fatty acid oxidation in islets remains debated. Here, we develop confocal imaging of electron transfer flavoprotein (ETF) autofluorescence to reveal the dynamics of fatty acid oxidation in living pancreatic islets. This method further integrates microfluidic devices to hold the islets stationary in flow, and thus achieve ETF imaging in the β-cells with high spatial and temporal resolution. Our data first confirm that ETF autofluorescence reflects electron transport chain (ETC) activity downstream of Complex I, consistent with a response directly related to fatty acid metabolism. Together with two-photon imaging of NAD(P)H and confocal imaging of lipoamide dehydrogenase (LipDH) autofluorescence, we show that the ETC predominantly draws electrons from LipDH/NADH-dependent Complex I rather than from ETF/FADH(2)-dependent ETF:CoQ oxidoreductase (ETF-QO). Islets stimulated with palmitate also show increased ETF redox state that is dose-dependently diminished by glucose (>10 mM). Furthermore, stimulation with a glucose bolus causes a two-tier drop in the ETF redox state at ∼5 and ∼20 min, suggesting glucose metabolism immediately increases ETC activity and later decreases fatty acid oxidation. Our results demonstrate the utility of ETF imaging in characterizing fatty acid-induced redox responses with high subcellular and temporal resolution. Our results further demonstrate a dominant role of glucose metabolism over fatty acid oxidation in β-cells even when presented with a mixed nutrient condition associated with glucolipotoxicity.

Synchrotron IR microspectroscopy for protein structure analysis: Potential and questions

DOE PAGES

Yu, Peiqiang

2006-01-01

Synchrotron radiation-based Fourier transform infrared microspectroscopy (S-FTIR) has been developed as a rapid, direct, non-destructive, bioanalytical technique. This technique takes advantage of synchrotron light brightness and small effective source size and is capable of exploring the molecular chemical make-up within microstructures of a biological tissue without destruction of inherent structures at ultra-spatial resolutions within cellular dimension. To date there has been very little application of this advanced technique to the study of pure protein inherent structure at a cellular level in biological tissues. In this review, a novel approach was introduced to show the potential of the newly developed, advancedmore » synchrotron-based analytical technology, which can be used to localize relatively “pure“ protein in the plant tissues and relatively reveal protein inherent structure and protein molecular chemical make-up within intact tissue at cellular and subcellular levels. Several complex protein IR spectra data analytical techniques (Gaussian and Lorentzian multi-component peak modeling, univariate and multivariate analysis, principal component analysis (PCA), and hierarchical cluster analysis (CLA) are employed to relatively reveal features of protein inherent structure and distinguish protein inherent structure differences between varieties/species and treatments in plant tissues. By using a multi-peak modeling procedure, RELATIVE estimates (but not EXACT determinations) for protein secondary structure analysis can be made for comparison purpose. The issues of pro- and anti-multi-peaking modeling/fitting procedure for relative estimation of protein structure were discussed. By using the PCA and CLA analyses, the plant molecular structure can be qualitatively separate one group from another, statistically, even though the spectral assignments are not known. The synchrotron-based technology provides a new approach for protein structure research in biological tissues at ultraspatial resolutions.« less

Sub-millimetre DOI detector based on monolithic LYSO and digital SiPM for a dedicated small-animal PET system.

PubMed

Marcinkowski, Radosław; Mollet, Pieter; Van Holen, Roel; Vandenberghe, Stefaan

2016-03-07

The mouse model is widely used in a vast range of biomedical and preclinical studies. Thanks to the ability to detect and quantify biological processes at the molecular level in vivo, PET has become a well-established tool in these investigations. However, the need to visualize and quantify radiopharmaceuticals in anatomic structures of millimetre or less requires good spatial resolution and sensitivity from small-animal PET imaging systems.In previous work we have presented a proof-of-concept of a dedicated high-resolution small-animal PET scanner based on thin monolithic scintillator crystals and Digital Photon Counter photosensor. The combination of thin monolithic crystals and MLE positioning algorithm resulted in an excellent spatial resolution of 0.7 mm uniform in the entire field of view (FOV). However, the limitation of the scanner was its low sensitivity due to small thickness of the lutetium-yttrium oxyorthosilicate (LYSO) crystals (2 mm).Here we present an improved detector design for a small-animal PET system that simultaneously achieves higher sensitivity and sustains a sub-millimetre spatial resolution. The proposed detector consists of a 5 mm thick monolithic LYSO crystal optically coupled to a Digital Photon Counter. Mean nearest neighbour (MNN) positioning combined with depth of interaction (DOI) decoding was employed to achieve sub-millimetre spatial resolution. To evaluate detector performance the intrinsic spatial resolution, energy resolution and coincidence resolving time (CRT) were measured. The average intrinsic spatial resolution of the detector was 0.60 mm full-width-at-half-maximum (FWHM). A DOI resolution of 1.66 mm was achieved. The energy resolution was 23% FWHM at 511 keV and CRT of 529 ps were measured. The improved detector design overcomes the sensitivity limitation of the previous design by increasing the nominal sensitivity of the detector block and retains an excellent intrinsic spatial resolution.

The robustness of T2 value as a trabecular structural index at multiple spatial resolutions of 7 Tesla MRI.

PubMed

Lee, D K; Song, Y K; Park, B W; Cho, H P; Yeom, J S; Cho, G; Cho, H

2018-04-15

To evaluate the robustness of MR transverse relaxation times of trabecular bone from spin-echo and gradient-echo acquisitions at multiple spatial resolutions of 7 T. The effects of MRI resolutions to T 2 and T2* of trabecular bone were numerically evaluated by Monte Carlo simulations. T 2 , T2*, and trabecular structural indices from multislice multi-echo and UTE acquisitions were measured in defatted human distal femoral condyles on a 7 T scanner. Reference structural indices were extracted from high-resolution microcomputed tomography images. For bovine knee trabecular samples with intact bone marrow, T 2 and T2* were measured by degrading spatial resolutions on a 7 T system. In the defatted trabecular experiment, both T 2 and T2* values showed strong ( |r| > 0.80) correlations with trabecular spacing and number, at a high spatial resolution of 125 µm 3 . The correlations for MR image-segmentation-derived structural indices were significantly degraded ( |r| < 0.50) at spatial resolutions of 250 and 500 µm 3 . The correlations for T2* rapidly dropped ( |r| < 0.50) at a spatial resolution of 500 µm 3 , whereas those for T 2 remained consistently high ( |r| > 0.85). In the bovine trabecular experiments with intact marrow, low-resolution (approximately 1 mm 3 , 2 minutes) T 2 values did not shorten ( |r| > 0.95 with respect to approximately 0.4 mm 3 , 11 minutes) and maintained consistent correlations ( |r| > 0.70) with respect to trabecular spacing (turbo spin echo, 22.5 minutes). T 2 measurements of trabeculae at 7 T are robust with degrading spatial resolution and may be preferable in assessing trabecular spacing index with reduced scan time, when high-resolution 3D micro-MRI is difficult to obtain. © 2018 International Society for Magnetic Resonance in Medicine.

On the assessment of spatial resolution of PET systems with iterative image reconstruction

NASA Astrophysics Data System (ADS)

Gong, Kuang; Cherry, Simon R.; Qi, Jinyi

2016-03-01

Spatial resolution is an important metric for performance characterization in PET systems. Measuring spatial resolution is straightforward with a linear reconstruction algorithm, such as filtered backprojection, and can be performed by reconstructing a point source scan and calculating the full-width-at-half-maximum (FWHM) along the principal directions. With the widespread adoption of iterative reconstruction methods, it is desirable to quantify the spatial resolution using an iterative reconstruction algorithm. However, the task can be difficult because the reconstruction algorithms are nonlinear and the non-negativity constraint can artificially enhance the apparent spatial resolution if a point source image is reconstructed without any background. Thus, it was recommended that a background should be added to the point source data before reconstruction for resolution measurement. However, there has been no detailed study on the effect of the point source contrast on the measured spatial resolution. Here we use point source scans from a preclinical PET scanner to investigate the relationship between measured spatial resolution and the point source contrast. We also evaluate whether the reconstruction of an isolated point source is predictive of the ability of the system to resolve two adjacent point sources. Our results indicate that when the point source contrast is below a certain threshold, the measured FWHM remains stable. Once the contrast is above the threshold, the measured FWHM monotonically decreases with increasing point source contrast. In addition, the measured FWHM also monotonically decreases with iteration number for maximum likelihood estimate. Therefore, when measuring system resolution with an iterative reconstruction algorithm, we recommend using a low-contrast point source and a fixed number of iterations.

Full Spatial Resolution Infrared Sounding Application in the Preconvection Environment

NASA Astrophysics Data System (ADS)

Liu, C.; Liu, G.; Lin, T.

2013-12-01

Advanced infrared (IR) sounders such as the Atmospheric Infrared Sounder (AIRS) and Infrared Atmospheric Sounding Interferometer (IASI) provide atmospheric temperature and moisture profiles with high vertical resolution and high accuracy in preconvection environments. The derived atmospheric stability indices such as convective available potential energy (CAPE) and lifted index (LI) from advanced IR soundings can provide critical information 1 ; 6 h before the development of severe convective storms. Three convective storms are selected for the evaluation of applying AIRS full spatial resolution soundings and the derived products on providing warning information in the preconvection environments. In the first case, the AIRS full spatial resolution soundings revealed local extremely high atmospheric instability 3 h ahead of the convection on the leading edge of a frontal system, while the second case demonstrates that the extremely high atmospheric instability is associated with the local development of severe thunderstorm in the following hours. The third case is a local severe storm that occurred on 7-8 August 2010 in Zhou Qu, China, which caused more than 1400 deaths and left another 300 or more people missing. The AIRS full spatial resolution LI product shows the atmospheric instability 3.5 h before the storm genesis. The CAPE and LI from AIRS full spatial resolution and operational AIRS/AMSU soundings along with Geostationary Operational Environmental Satellite (GOES) Sounder derived product image (DPI) products were analyzed and compared. Case studies show that full spatial resolution AIRS retrievals provide more useful warning information in the preconvection environments for determining favorable locations for convective initiation (CI) than do the coarser spatial resolution operational soundings and lower spectral resolution GOES Sounder retrievals. The retrieved soundings are also tested in a regional data assimilation WRF 3D-var system to evaluate the potential assist in the NWP model.

Ultra high spatial and temporal resolution breast imaging at 7T.

PubMed

van de Bank, B L; Voogt, I J; Italiaander, M; Stehouwer, B L; Boer, V O; Luijten, P R; Klomp, D W J

2013-04-01

There is a need to obtain higher specificity in the detection of breast lesions using MRI. To address this need, Dynamic Contrast-Enhanced (DCE) MRI has been combined with other structural and functional MRI techniques. Unfortunately, owing to time constraints structural images at ultra-high spatial resolution can generally not be obtained during contrast uptake, whereas the relatively low spatial resolution of functional imaging (e.g. diffusion and perfusion) limits the detection of small lesions. To be able to increase spatial as well as temporal resolution simultaneously, the sensitivity of MR detection needs to increase as well as the ability to effectively accelerate the acquisition. The required gain in signal-to-noise ratio (SNR) can be obtained at 7T, whereas acceleration can be obtained with high-density receiver coil arrays. In this case, morphological imaging can be merged with DCE-MRI, and other functional techniques can be obtained at higher spatial resolution, and with less distortion [e.g. Diffusion Weighted Imaging (DWI)]. To test the feasibility of this concept, we developed a unilateral breast coil for 7T. It comprises a volume optimized dual-channel transmit coil combined with a 30-channel receive array coil. The high density of small coil elements enabled efficient acceleration in any direction to acquire ultra high spatial resolution MRI of close to 0.6 mm isotropic detail within a temporal resolution of 69 s, high spatial resolution MRI of 1.5 mm isotropic within an ultra high temporal resolution of 6.7 s and low distortion DWI at 7T, all validated in phantoms, healthy volunteers and a patient with a lesion in the right breast classified as Breast Imaging Reporting and Data System (BI-RADS) IV. Copyright © 2012 John Wiley & Sons, Ltd.

High Efficiency Multi-shot Interleaved Spiral-In/Out Acquisition for High Resolution BOLD fMRI

PubMed Central

Jung, Youngkyoo; Samsonov, Alexey A.; Liu, Thomas T.; Buracas, Giedrius T.

2012-01-01

Growing demand for high spatial resolution BOLD functional MRI faces a challenge of the spatial resolution vs. coverage or temporal resolution tradeoff, which can be addressed by methods that afford increased acquisition efficiency. Spiral acquisition trajectories have been shown to be superior to currently prevalent echo-planar imaging in terms of acquisition efficiency, and high spatial resolution can be achieved by employing multiple-shot spiral acquisition. The interleaved spiral in-out trajectory is preferred over spiral-in due to increased BOLD signal CNR and higher acquisition efficiency than that of spiral-out or non-interleaved spiral in/out trajectories (1), but to date applicability of the multi-shot interleaved spiral in-out for high spatial resolution imaging has not been studied. Herein we propose multi-shot interleaved spiral in-out acquisition and investigate its applicability for high spatial resolution BOLD fMRI. Images reconstructed from interleaved spiral-in and -out trajectories possess artifacts caused by differences in T2* decay, off-resonance and k-space errors associated with the two trajectories. We analyze the associated errors and demonstrate that application of conjugate phase reconstruction and spectral filtering can substantially mitigate these image artifacts. After applying these processing steps, the multishot interleaved spiral in-out pulse sequence yields high BOLD CNR images at in-plane resolution below 1x1 mm while preserving acceptable temporal resolution (4 s) and brain coverage (15 slices of 2 mm thickness). Moreover, this method yields sufficient BOLD CNR at 1.5 mm isotropic resolution for detection of activation in hippocampus associated with cognitive tasks (Stern memory task). The multi-shot interleaved spiral in-out acquisition is a promising technique for high spatial resolution BOLD fMRI applications. PMID:23023395

The influence of spatial resolution and smoothing on the detectability of resting-state and task fMRI.

PubMed

Molloy, Erin K; Meyerand, Mary E; Birn, Rasmus M

2014-02-01

Functional MRI blood oxygen level-dependent (BOLD) signal changes can be subtle, motivating the use of imaging parameters and processing strategies that maximize the temporal signal-to-noise ratio (tSNR) and thus the detection power of neuronal activity-induced fluctuations. Previous studies have shown that acquiring data at higher spatial resolutions results in greater percent BOLD signal changes, and furthermore that spatially smoothing higher resolution fMRI data improves tSNR beyond that of data originally acquired at a lower resolution. However, higher resolution images come at the cost of increased acquisition time, and the number of image volumes also influences detectability. The goal of our study is to determine how the detection power of neuronally induced BOLD fluctuations acquired at higher spatial resolutions and then spatially smoothed compares to data acquired at the lower resolutions with the same imaging duration. The number of time points acquired during a given amount of imaging time is a practical consideration given the limited ability of certain populations to lie still in the MRI scanner. We compare acquisitions at three different in-plane spatial resolutions (3.50×3.50mm(2), 2.33×2.33mm(2), 1.75×1.75mm(2)) in terms of their tSNR, contrast-to-noise ratio, and the power to detect both task-related activation and resting-state functional connectivity. The impact of SENSE acceleration, which speeds up acquisition time increasing the number of images collected, is also evaluated. Our results show that after spatially smoothing the data to the same intrinsic resolution, lower resolution acquisitions have a slightly higher detection power of task-activation in some, but not all, brain areas. There were no significant differences in functional connectivity as a function of resolution after smoothing. Similarly, the reduced tSNR of fMRI data acquired with a SENSE factor of 2 is offset by the greater number of images acquired, resulting in few significant differences in detection power of either functional activation or connectivity after spatial smoothing. © 2013.

Mapping Chinese tallow with color-infrared photography

USGS Publications Warehouse

Ramsey, Elijah W.; Nelson, G.A.; Sapkota, S.K.; Seeger, E.B.; Martella, K.D.

2002-01-01

Airborne color-infrared photography (CIR) (1:12,000 scale) was used to map localized occurrences of the widespread and aggressive Chinese tallow (Sapium sebiferum), an invasive species. Photography was collected during senescence when Chinese tallow's bright red leaves presented a high spectral contrast within the native bottomland hardwood and upland forests and marsh land-cover types. Mapped occurrences were conservative because not all senescing tallow leaves are bright red simultaneously. To simulate low spectral but high spatial resolution satellite/airborne image and digital video data, the CIR photography was transformed into raster images at spatial resolutions approximating 0.5 in and 1.0 m. The image data were then spectrally classified for the occurrence of bright red leaves associated with senescing Chinese tallow. Classification accuracies were greater than 95 percent at both spatial resolutions. There was no significant difference in either forest in the detection of tallow or inclusion of non-tallow trees associated with the two spatial resolutions. In marshes, slightly more tallow occurrences were mapped with the lower spatial resolution, but there were also more misclassifications of native land covers as tallow. Combining all land covers, there was no difference at detecting tallow occurrences (equal omission errors) between the two resolutions, but the higher spatial resolution was associated with less inclusion of non-tallow land covers as tallow (lower commission error). Overall, these results confirm that high spatial (???1 m) but low spectral resolution remote sensing data can be used for mapping Chinese tallow trees in dominant environments found in coastal and adjacent upland landscapes.

A Comparison of Spatial and Spectral Image Resolution for Mapping Invasive Plants in Coastal California

NASA Astrophysics Data System (ADS)

Underwood, Emma C.; Ustin, Susan L.; Ramirez, Carlos M.

2007-01-01

We explored the potential of detecting three target invasive species: iceplant ( Carpobrotus edulis), jubata grass ( Cortaderia jubata), and blue gum ( Eucalyptus globulus) at Vandenberg Air Force Base, California. We compared the accuracy of mapping six communities (intact coastal scrub, iceplant invaded coastal scrub, iceplant invaded chaparral, jubata grass invaded chaparral, blue gum invaded chaparral, and intact chaparral) using four images with different combinations of spatial and spectral resolution: hyperspectral AVIRIS imagery (174 wavebands, 4 m spatial resolution), spatially degraded AVIRIS (174 bands, 30 m), spectrally degraded AVIRIS (6 bands, 4 m), and both spatially and spectrally degraded AVIRIS (6 bands, 30 m, i.e., simulated Landsat ETM data). Overall success rates for classifying the six classes was 75% (kappa 0.7) using full resolution AVIRIS, 58% (kappa 0.5) for the spatially degraded AVIRIS, 42% (kappa 0.3) for the spectrally degraded AVIRIS, and 37% (kappa 0.3) for the spatially and spectrally degraded AVIRIS. A true Landsat ETM image was also classified to illustrate that the results from the simulated ETM data were representative, which provided an accuracy of 50% (kappa 0.4). Mapping accuracies using different resolution images are evaluated in the context of community heterogeneity (species richness, diversity, and percent species cover). Findings illustrate that higher mapping accuracies are achieved with images possessing high spectral resolution, thus capturing information across the visible and reflected infrared solar spectrum. Understanding the tradeoffs in spectral and spatial resolution can assist land managers in deciding the most appropriate imagery with respect to target invasives and community characteristics.

Error Estimation in an Optimal Interpolation Scheme for High Spatial and Temporal Resolution SST Analyses

NASA Technical Reports Server (NTRS)

Rigney, Matt; Jedlovec, Gary; LaFontaine, Frank; Shafer, Jaclyn

2010-01-01

Heat and moisture exchange between ocean surface and atmosphere plays an integral role in short-term, regional NWP. Current SST products lack both spatial and temporal resolution to accurately capture small-scale features that affect heat and moisture flux. NASA satellite is used to produce high spatial and temporal resolution SST analysis using an OI technique.

Instrumentation in molecular imaging.

PubMed

Wells, R Glenn

2016-12-01

In vivo molecular imaging is a challenging task and no single type of imaging system provides an ideal solution. Nuclear medicine techniques like SPECT and PET provide excellent sensitivity but have poor spatial resolution. Optical imaging has excellent sensitivity and spatial resolution, but light photons interact strongly with tissues and so only small animals and targets near the surface can be accurately visualized. CT and MRI have exquisite spatial resolution, but greatly reduced sensitivity. To overcome the limitations of individual modalities, molecular imaging systems often combine individual cameras together, for example, merging nuclear medicine cameras with CT or MRI to allow the visualization of molecular processes with both high sensitivity and high spatial resolution.

Microdome-gooved Gd(2)O(2)S:Tb scintillator for flexible and high resolution digital radiography.

PubMed

Jung, Phill Gu; Lee, Chi Hoon; Bae, Kong Myeong; Lee, Jae Min; Lee, Sang Min; Lim, Chang Hwy; Yun, Seungman; Kim, Ho Kyung; Ko, Jong Soo

2010-07-05

A flexible microdome-grooved Gd(2)O(2)S:Tb scintillator is simulated, fabricated, and characterized for digital radiography applications. According to Monte Carlo simulation results, the dome-grooved structure has a high spatial resolution, which is verified by X-ray image performance of the scintillator. The proposed scintillator has lower X-ray sensitivity than a nonstructured scintillator but almost two times higher spatial resolution at high spatial frequency. Through evaluation of the X-ray performance of the fabricated scintillators, we confirm that the microdome-grooved scintillator can be applied to next-generation flexible digital radiography systems requiring high spatial resolution.

Raman spectroscopy-based detection of chemical contaminants in food powders

NASA Astrophysics Data System (ADS)

Chao, Kuanglin; Dhakal, Sagar; Qin, Jianwei; Kim, Moon; Bae, Abigail

2016-05-01

Raman spectroscopy technique has proven to be a reliable method for qualitative detection of chemical contaminants in food ingredients and products. For quantitative imaging-based detection, each contaminant particle in a food sample must be detected and it is important to determine the necessary spatial resolution needed to effectively detect the contaminant particles. This study examined the effective spatial resolution required for detection of maleic acid in tapioca starch and benzoyl peroxide in wheat flour. Each chemical contaminant was mixed into its corresponding food powder at a concentration of 1% (w/w). Raman spectral images were collected for each sample, leveled across a 45 mm x 45 mm area, using different spatial resolutions. Based on analysis of these images, a spatial resolution of 0.5mm was selected as effective spatial resolution for detection of maleic acid in starch and benzoyl peroxide in flour. An experiment was then conducted using the 0.5mm spatial resolution to demonstrate Raman imaging-based quantitative detection of these contaminants for samples prepared at 0.1%, 0.3%, and 0.5% (w/w) concentrations. The results showed a linear correlation between the detected numbers of contaminant pixels and the actual concentrations of contaminant.

Fusion and quality analysis for remote sensing images using contourlet transform

NASA Astrophysics Data System (ADS)

Choi, Yoonsuk; Sharifahmadian, Ershad; Latifi, Shahram

2013-05-01

Recent developments in remote sensing technologies have provided various images with high spatial and spectral resolutions. However, multispectral images have low spatial resolution and panchromatic images have low spectral resolution. Therefore, image fusion techniques are necessary to improve the spatial resolution of spectral images by injecting spatial details of high-resolution panchromatic images. The objective of image fusion is to provide useful information by improving the spatial resolution and the spectral information of the original images. The fusion results can be utilized in various applications, such as military, medical imaging, and remote sensing. This paper addresses two issues in image fusion: i) image fusion method and ii) quality analysis of fusion results. First, a new contourlet-based image fusion method is presented, which is an improvement over the wavelet-based fusion. This fusion method is then applied to a case study to demonstrate its fusion performance. Fusion framework and scheme used in the study are discussed in detail. Second, quality analysis for the fusion results is discussed. We employed various quality metrics in order to analyze the fusion results both spatially and spectrally. Our results indicate that the proposed contourlet-based fusion method performs better than the conventional wavelet-based fusion methods.

Impaired temporal, not just spatial, resolution in amblyopia.

PubMed

Spang, Karoline; Fahle, Manfred

2009-11-01

In amblyopia, neuronal deficits deteriorate spatial vision including visual acuity, possibly because of a lack of use-dependent fine-tuning of afferents to the visual cortex during infancy; but temporal processing may deteriorate as well. Temporal, rather than spatial, resolution was investigated in patients with amblyopia by means of a task based on time-defined figure-ground segregation. Patients had to indicate the quadrant of the visual field where a purely time-defined square appeared. The results showed a clear decrease in temporal resolution of patients' amblyopic eyes compared with the dominant eyes in this task. The extent of this decrease in figure-ground segregation based on time of motion onset only loosely correlated with the decrease in spatial resolution and spanned a smaller range than did the spatial loss. Control experiments with artificially induced blur in normal observers confirmed that the decrease in temporal resolution was not simply due to the acuity loss. Amblyopia not only decreases spatial resolution, but also temporal factors such as time-based figure-ground segregation, even at high stimulus contrasts. This finding suggests that the realm of neuronal processes that may be disturbed in amblyopia is larger than originally thought.

Spatial Resolution and Refractive Index Contrast of Resonant Photonic Crystal Surfaces for Biosensing

PubMed Central

Triggs, G. J.; Fischer, M.; Stellinga, D.; Scullion, M. G.; Evans, G. J. O.; Krauss, T. F.

2015-01-01

By depositing a resolution test pattern on top of a Si3N4 photonic crystal resonant surface, we have measured the dependence of spatial resolution on refractive index contrast Δn. Our experimental results and finite-difference time-domain (FDTD) simulations at different refractive index contrasts show that the spatial resolution of our device reduces with reduced contrast, which is an important consideration in biosensing, where the contrast may be of order 10−2. We also compare 1-D and 2-D gratings, taking into account different incidence polarizations, leading to a better understanding of the excitation and propagation of the resonant modes in these structures, as well as how this contributes to the spatial resolution. At Δn = 0.077, we observe resolutions of 2 and 6 μm parallel to and perpendicular to the grooves of a 1-D grating, respectively, and show that for polarized illumination of a 2-D grating, resolution remains asymmetrical. Illumination of a 2-D grating at 45° results in symmetric resolution. At very low index contrast, the resolution worsens dramatically, particularly for Δn < 0.01, where we observe a resolution exceeding 10 μm for our device. In addition, we measure a reduction in the resonance linewidth as the index contrast becomes lower, corresponding to a longer resonant mode propagation length in the structure and contributing to the change in spatial resolution. PMID:26356353

Three-Dimensional Unstained Live-Cell Imaging Using Stimulated Parametric Emission Microscopy

NASA Astrophysics Data System (ADS)

Dang, Hieu M.; Kawasumi, Takehito; Omura, Gen; Umano, Toshiyuki; Kajiyama, Shin'ichiro; Ozeki, Yasuyuki; Itoh, Kazuyoshi; Fukui, Kiichi

2009-09-01

The ability to perform high-resolution unstained live imaging is very important to in vivo study of cell structures and functions. Stimulated parametric emission (SPE) microscopy is a nonlinear-optical microscopy based on ultra-fast electronic nonlinear-optical responses. For the first time, we have successfully applied this technique to archive three-dimensional (3D) images of unstained sub-cellular structures, such as, microtubules, nuclei, nucleoli, etc. in live cells. Observation of a complete cell division confirms the ability of SPE microscopy for long time-scale imaging.

Reflectance confocal microscopy of oral epithelial tissue using an electrically tunable lens

NASA Astrophysics Data System (ADS)

Jabbour, Joey M.; Malik, Bilal H.; Cuenca, Rodrigo; Cheng, Shuna; Jo, Javier A.; Cheng, Yi-Shing L.; Wright, John M.; Maitland, Kristen C.

2014-02-01

We present the use of a commercially available electrically tunable lens to achieve axial scanning in a reflectance confocal microscope. Over a 255 μm axial scan range, the lateral and axial resolutions varied from 1-2 μm and 4-14 μm, respectively, dependent on the variable focal length of the tunable lens. Confocal imaging was performed on normal human biopsies from the oral cavity ex vivo. Sub-cellular morphologic features were seen throughout the depth of the epithelium while axially scanning using the focus tunable lens.

High spatial resolution compressed sensing (HSPARSE) functional MRI.

PubMed

Fang, Zhongnan; Van Le, Nguyen; Choy, ManKin; Lee, Jin Hyung

2016-08-01

To propose a novel compressed sensing (CS) high spatial resolution functional MRI (fMRI) method and demonstrate the advantages and limitations of using CS for high spatial resolution fMRI. A randomly undersampled variable density spiral trajectory enabling an acceleration factor of 5.3 was designed with a balanced steady state free precession sequence to achieve high spatial resolution data acquisition. A modified k-t SPARSE method was then implemented and applied with a strategy to optimize regularization parameters for consistent, high quality CS reconstruction. The proposed method improves spatial resolution by six-fold with 12 to 47% contrast-to-noise ratio (CNR), 33 to 117% F-value improvement and maintains the same temporal resolution. It also achieves high sensitivity of 69 to 99% compared the original ground-truth, small false positive rate of less than 0.05 and low hemodynamic response function distortion across a wide range of CNRs. The proposed method is robust to physiological noise and enables detection of layer-specific activities in vivo, which cannot be resolved using the highest spatial resolution Nyquist acquisition. The proposed method enables high spatial resolution fMRI that can resolve layer-specific brain activity and demonstrates the significant improvement that CS can bring to high spatial resolution fMRI. Magn Reson Med 76:440-455, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.

Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome.

PubMed

Markert, Sebastian Matthias; Britz, Sebastian; Proppert, Sven; Lang, Marietta; Witvliet, Daniel; Mulcahy, Ben; Sauer, Markus; Zhen, Mei; Bessereau, Jean-Louis; Stigloher, Christian

2016-10-01

Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.

Electric crosstalk impairs spatial resolution of multi-electrode arrays in retinal implants

NASA Astrophysics Data System (ADS)

Wilke, R. G. H.; Khalili Moghadam, G.; Lovell, N. H.; Suaning, G. J.; Dokos, S.

2011-08-01

Active multi-electrode arrays are used in vision prostheses, including optic nerve cuffs and cortical and retinal implants for stimulation of neural tissue. For retinal implants, arrays with up to 1500 electrodes are used in clinical trials. The ability to convey information with high spatial resolution is critical for these applications. To assess the extent to which spatial resolution is impaired by electric crosstalk, finite-element simulation of electric field distribution in a simplified passive tissue model of the retina is performed. The effects of electrode size, electrode spacing, distance to target cells, and electrode return configuration (monopolar, tripolar, hexagonal) on spatial resolution is investigated in the form of a mathematical model of electric field distribution. Results show that spatial resolution is impaired with increased distance from the electrode array to the target cells. This effect can be partly compensated by non-monopolar electrode configurations and larger electrode diameters, albeit at the expense of lower pixel densities due to larger covering areas by each stimulation electrode. In applications where multi-electrode arrays can be brought into close proximity to target cells, as presumably with epiretinal implants, smaller electrodes in monopolar configuration can provide the highest spatial resolution. However, if the implantation site is further from the target cells, as is the case in suprachoroidal approaches, hexagonally guarded electrode return configurations can convey higher spatial resolution. This paper was originally submitted for the special issue containing contributions from the Sixth Biennial Research Congress of The Eye and the Chip.

Cryptic sexual dimorphism in spatial memory and hippocampal oxytocin receptors in prairie voles (Microtus ochrogaster).

PubMed

Rice, Marissa A; Hobbs, Lauren E; Wallace, Kelly J; Ophir, Alexander G

2017-09-01

Sex differences are well documented and are conventionally associated with intense sex-specific selection. For example, spatial memory is frequently better in males, presumably due to males' tendency to navigate large spaces to find mates. Alternatively, monogamy (in which sex-specific selection is relatively relaxed) should diminish or eliminate differences in spatial ability and the mechanisms associated with this behavior. Nevertheless, phenotypic differences between monogamous males and females persist, sometimes cryptically. We hypothesize that sex-specific cognitive demands are present in monogamous species that will influence neural and behavioral phenotypes. The effects of these demands should be observable in spatial learning performance and neural structures associated with spatial learning and memory. We analyzed spatial memory performance, hippocampal volume and cell density, and hippocampal oxytocin receptor (OTR) expression in the socially monogamous prairie vole. Compared to females, males performed better in a spatial memory and spatial learning test. Although we found no sex difference in hippocampal volume or cell density, male OTR density was significantly lower than females, suggesting that performance may be regulated by sub-cellular mechanisms within the hippocampus that are less obvious than classic neuroanatomical features. Our results suggest an expanded role for oxytocin beyond facilitating social interactions, which may function in part to integrate social and spatial information. Copyright © 2017 Elsevier Inc. All rights reserved.

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly

PubMed Central

Becker, Jordan T.

2017-01-01

ABSTRACT Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans. In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5′ packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM. IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins (gag-pol mRNAs) to distinct non-PM subcellular locales, such as cytoplasmic vesicles or the actin cytoskeleton, markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1 gag-pol mRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization may represent a novel antiviral strategy. PMID:28053097

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly.

PubMed

Becker, Jordan T; Sherer, Nathan M

2017-03-15

Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis -acting RNA regulatory elements: the 5' packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM. IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins ( gag-pol mRNAs) to distinct non-PM subcellular locales, such as cytoplasmic vesicles or the actin cytoskeleton, markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1 gag-pol mRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization may represent a novel antiviral strategy. Copyright © 2017 American Society for Microbiology.

The influence of multispectral scanner spatial resolution on forest feature classification

NASA Technical Reports Server (NTRS)

Sadowski, F. G.; Malila, W. A.; Sarno, J. E.; Nalepka, R. F.

1977-01-01

Inappropriate spatial resolution and corresponding data processing techniques may be major causes for non-optimal forest classification results frequently achieved from multispectral scanner (MSS) data. Procedures and results of empirical investigations are studied to determine the influence of MSS spatial resolution on the classification of forest features into levels of detail or hierarchies of information that might be appropriate for nationwide forest surveys and detailed in-place inventories. Two somewhat different, but related studies are presented. The first consisted of establishing classification accuracies for several hierarchies of features as spatial resolution was progressively coarsened from (2 meters) squared to (64 meters) squared. The second investigated the capabilities for specialized processing techniques to improve upon the results of conventional processing procedures for both coarse and fine resolution data.

Definition of SMOS Level 3 Land Products for the Villafranca del Castillo Data Processing Centre (CP34)

NASA Astrophysics Data System (ADS)

Lopez-Baeza, E.; Monsoriu Torres, A.; Font, J.; Alonso, O.

2009-04-01

The ESA SMOS (Soil Moisture and Ocean Salinity) Mission is planned to be launched in July 2009. The satellite will measure soil moisture over the continents and surface salinity of the oceans at resolutions that are sufficient for climatological-type studies. This paper describes the procedure to be used at the Spanish SMOS Level 3 and 4 Data Processing Centre (CP34) to generate Soil Moisture and other Land Surface Product maps from SMOS Level 2 data. This procedure can be used to map Soil Moisture, Vegetation Water Content and Soil Dielectric Constant data into different pre-defined spatial grids with fixed temporal frequency. The L3 standard Land Surface Products to be generated at CP34 are: Soil Moisture products: maximum spatial resolution with no spatial averaging, temporal averaging of 3 days, daily generation maximum spatial resolution with no spatial averaging, temporal averaging of 10 days, generation frequency of once every 10 days. b': maximum spatial resolution with no spatial averaging, temporal averaging of monthly decades (1st to 10th of the month, 11th to 20th of the month, 21st to last day of the month), generation frequency of once every decade monthly average, temporal averaging from L3 decade averages, monthly generation Seasonal average, temporal averaging from L3 monthly averages, seasonally generation yearly average, temporal averaging from L3 monthly averages, yearly generation Vegetation Water Content products: maximum spatial resolution with no spatial averaging, temporal averaging of 10 days, generation frequency of once every 10 days. a': maximum spatial resolution with no spatial averaging, temporal averaging of monthly decades (1st to 10th of the month, 11th to 20th of the month, 21st to last day of the month) using simple averaging method over the L2 products in ISEA grid, generation frequency of once every decade monthly average, temporal averaging from L3 decade averages, monthly generation seasonal average, temporal averaging from L3 monthly averages, seasonally generation yearly average, temporal averaging from L3 monthly averages, yearly generation Dielectric Constant products: (the dielectric constant products are delivered together with soil moisture products, with the same averaging periods and generation frequency): maximum spatial resolution with no spatial averaging, temporal averaging of 3 days, daily generation maximum spatial resolution with no spatial averaging, temporal averaging of 10 days, generation frequency of once every 10 days. b': maximum spatial resolution with no spatial averaging, temporal averaging of monthly decades (1st to 10th of the month, 11th to 20th of the month, 21st to last day of the month), generation frequency of once every decade monthly average, temporal averaging from L3 decade averages, monthly generation seasonal average, temporal averaging from L3 monthly averages, seasonally generation yearly average, temporal averaging from L3 monthly averages, yearly generation.

Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy

PubMed Central

Wu, Yicong; Chandris, Panagiotis; Winter, Peter W.; Kim, Edward Y.; Jaumouillé, Valentin; Kumar, Abhishek; Guo, Min; Leung, Jacqueline M.; Smith, Corey; Rey-Suarez, Ivan; Liu, Huafeng; Waterman, Clare M.; Ramamurthi, Kumaran S.; La Riviere, Patrick J.; Shroff, Hari

2016-01-01

Most fluorescence microscopes are inefficient, collecting only a small fraction of the emitted light at any instant. Besides wasting valuable signal, this inefficiency also reduces spatial resolution and causes imaging volumes to exhibit significant resolution anisotropy. We describe microscopic and computational techniques that address these problems by simultaneously capturing and subsequently fusing and deconvolving multiple specimen views. Unlike previous methods that serially capture multiple views, our approach improves spatial resolution without introducing any additional illumination dose or compromising temporal resolution relative to conventional imaging. When applying our methods to single-view wide-field or dual-view light-sheet microscopy, we achieve a twofold improvement in volumetric resolution (~235 nm × 235 nm × 340 nm) as demonstrated on a variety of samples including microtubules in Toxoplasma gondii, SpoVM in sporulating Bacillus subtilis, and multiple protein distributions and organelles in eukaryotic cells. In every case, spatial resolution is improved with no drawback by harnessing previously unused fluorescence. PMID:27761486

High Resolution Live Cell Raman Imaging Using Subcellular Organelle-Targeting SERS-Sensitive Gold Nanoparticles with Highly Narrow Intra-Nanogap

PubMed Central

Kang, Jeon Woong; So, Peter T. C.; Dasari, Ramachandra R.; Lim, Dong-Kwon

2015-01-01

We report a method to achieve high speed and high resolution live cell Raman images using small spherical gold nanoparticles with highly narrow intra-nanogap structures responding to NIR excitation (785 nm) and high-speed confocal Raman microscopy. The three different Raman-active molecules placed in the narrow intra-nanogap showed a strong and uniform Raman intensity in solution even under transient exposure time (10 ms) and low input power of incident laser (200 μW), which lead to obtain high-resolution single cell image within 30 s without inducing significant cell damage. The high resolution Raman image showed the distributions of gold nanoparticles for their targeted sites such as cytoplasm, mitochondria, or nucleus. The high speed Raman-based live cell imaging allowed us to monitor rapidly changing cell morphologies during cell death induced by the addition of highly toxic KCN solution to cells. These results strongly suggest that the use of SERS-active nanoparticle can greatly improve the current temporal resolution and image quality of Raman-based cell images enough to obtain the detailed cell dynamics and/or the responses of cells to potential drug molecules. PMID:25646716

The Effect of Spatial and Temporal Resolution of Cine Phase Contrast MRI on Wall Shear Stress and Oscillatory Shear Index Assessment

PubMed Central

Gijsen, Frank J.; Marquering, Henk; van Ooij, Pim; vanBavel, Ed; Wentzel, Jolanda J.; Nederveen, Aart J.

2016-01-01

Introduction Wall shear stress (WSS) and oscillatory shear index (OSI) are associated with atherosclerotic disease. Both parameters are derived from blood velocities, which can be measured with phase-contrast MRI (PC-MRI). Limitations in spatiotemporal resolution of PC-MRI are known to affect these measurements. Our aim was to investigate the effect of spatiotemporal resolution using a carotid artery phantom. Methods A carotid artery phantom was connected to a flow set-up supplying pulsatile flow. MRI measurement planes were placed at the common carotid artery (CCA) and internal carotid artery (ICA). Two-dimensional PC-MRI measurements were performed with thirty different spatiotemporal resolution settings. The MRI flow measurement was validated with ultrasound probe measurements. Mean flow, peak flow, flow waveform, WSS and OSI were compared for these spatiotemporal resolutions using regression analysis. The slopes of the regression lines were reported in %/mm and %/100ms. The distribution of low and high WSS and OSI was compared between different spatiotemporal resolutions. Results The mean PC-MRI CCA flow (2.5±0.2mL/s) agreed with the ultrasound probe measurements (2.7±0.02mL/s). Mean flow (mL/s) depended only on spatial resolution (CCA:-13%/mm, ICA:-49%/mm). Peak flow (mL/s) depended on both spatial (CCA:-13%/mm, ICA:-17%/mm) and temporal resolution (CCA:-19%/100ms, ICA:-24%/100ms). Mean WSS (Pa) was in inverse relationship only with spatial resolution (CCA:-19%/mm, ICA:-33%/mm). OSI was dependent on spatial resolution for CCA (-26%/mm) and temporal resolution for ICA (-16%/100ms). The regions of low and high WSS and OSI matched for most of the spatiotemporal resolutions (CCA:30/30, ICA:28/30 cases for WSS; CCA:23/30, ICA:29/30 cases for OSI). Conclusion We show that both mean flow and mean WSS are independent of temporal resolution. Peak flow and OSI are dependent on both spatial and temporal resolution. However, the magnitude of mean and peak flow, WSS and OSI, and the spatial distribution of OSI and WSS did not exhibit a strong dependency on spatiotemporal resolution. PMID:27669568

Zonal wavefront sensing with enhanced spatial resolution.

PubMed

Pathak, Biswajit; Boruah, Bosanta R

2016-12-01

In this Letter, we introduce a scheme to enhance the spatial resolution of a zonal wavefront sensor. The zonal wavefront sensor comprises an array of binary gratings implemented by a ferroelectric spatial light modulator (FLCSLM) followed by a lens, in lieu of the array of lenses in the Shack-Hartmann wavefront sensor. We show that the fast response of the FLCSLM device facilitates quick display of several laterally shifted binary grating patterns, and the programmability of the device enables simultaneous capturing of each focal spot array. This eventually leads to a wavefront estimation with an enhanced spatial resolution without much sacrifice on the sensor frame rate, thus making the scheme suitable for high spatial resolution measurement of transient wavefronts. We present experimental and numerical simulation results to demonstrate the importance of the proposed wavefront sensing scheme.

Hyperspectral and multispectral data fusion based on linear-quadratic nonnegative matrix factorization

NASA Astrophysics Data System (ADS)

Benhalouche, Fatima Zohra; Karoui, Moussa Sofiane; Deville, Yannick; Ouamri, Abdelaziz

2017-04-01

This paper proposes three multisharpening approaches to enhance the spatial resolution of urban hyperspectral remote sensing images. These approaches, related to linear-quadratic spectral unmixing techniques, use a linear-quadratic nonnegative matrix factorization (NMF) multiplicative algorithm. These methods begin by unmixing the observable high-spectral/low-spatial resolution hyperspectral and high-spatial/low-spectral resolution multispectral images. The obtained high-spectral/high-spatial resolution features are then recombined, according to the linear-quadratic mixing model, to obtain an unobservable multisharpened high-spectral/high-spatial resolution hyperspectral image. In the first designed approach, hyperspectral and multispectral variables are independently optimized, once they have been coherently initialized. These variables are alternately updated in the second designed approach. In the third approach, the considered hyperspectral and multispectral variables are jointly updated. Experiments, using synthetic and real data, are conducted to assess the efficiency, in spatial and spectral domains, of the designed approaches and of linear NMF-based approaches from the literature. Experimental results show that the designed methods globally yield very satisfactory spectral and spatial fidelities for the multisharpened hyperspectral data. They also prove that these methods significantly outperform the used literature approaches.

Anthropogenic heat flux: advisable spatial resolutions when input data are scarce

NASA Astrophysics Data System (ADS)

Gabey, A. M.; Grimmond, C. S. B.; Capel-Timms, I.

2018-02-01

Anthropogenic heat flux (QF) may be significant in cities, especially under low solar irradiance and at night. It is of interest to many practitioners including meteorologists, city planners and climatologists. QF estimates at fine temporal and spatial resolution can be derived from models that use varying amounts of empirical data. This study compares simple and detailed models in a European megacity (London) at 500 m spatial resolution. The simple model (LQF) uses spatially resolved population data and national energy statistics. The detailed model (GQF) additionally uses local energy, road network and workday population data. The Fractions Skill Score (FSS) and bias are used to rate the skill with which the simple model reproduces the spatial patterns and magnitudes of QF, and its sub-components, from the detailed model. LQF skill was consistently good across 90% of the city, away from the centre and major roads. The remaining 10% contained elevated emissions and "hot spots" representing 30-40% of the total city-wide energy. This structure was lost because it requires workday population, spatially resolved building energy consumption and/or road network data. Daily total building and traffic energy consumption estimates from national data were within ± 40% of local values. Progressively coarser spatial resolutions to 5 km improved skill for total QF, but important features (hot spots, transport network) were lost at all resolutions when residential population controlled spatial variations. The results demonstrate that simple QF models should be applied with conservative spatial resolution in cities that, like London, exhibit time-varying energy use patterns.

Generating High-Temporal and Spatial Resolution TIR Image Data

NASA Astrophysics Data System (ADS)

Herrero-Huerta, M.; Lagüela, S.; Alfieri, S. M.; Menenti, M.

2017-09-01

Remote sensing imagery to monitor global biophysical dynamics requires the availability of thermal infrared data at high temporal and spatial resolution because of the rapid development of crops during the growing season and the fragmentation of most agricultural landscapes. Conversely, no single sensor meets these combined requirements. Data fusion approaches offer an alternative to exploit observations from multiple sensors, providing data sets with better properties. A novel spatio-temporal data fusion model based on constrained algorithms denoted as multisensor multiresolution technique (MMT) was developed and applied to generate TIR synthetic image data at both temporal and spatial high resolution. Firstly, an adaptive radiance model is applied based on spectral unmixing analysis of . TIR radiance data at TOA (top of atmosphere) collected by MODIS daily 1-km and Landsat - TIRS 16-day sampled at 30-m resolution are used to generate synthetic daily radiance images at TOA at 30-m spatial resolution. The next step consists of unmixing the 30 m (now lower resolution) images using the information about their pixel land-cover composition from co-registered images at higher spatial resolution. In our case study, TIR synthesized data were unmixed to the Sentinel 2 MSI with 10 m resolution. The constrained unmixing preserves all the available radiometric information of the 30 m images and involves the optimization of the number of land-cover classes and the size of the moving window for spatial unmixing. Results are still being evaluated, with particular attention for the quality of the data streams required to apply our approach.

Spatial Scale Gap Filling Using an Unmanned Aerial System: A Statistical Downscaling Method for Applications in Precision Agriculture.

PubMed

Hassan-Esfahani, Leila; Ebtehaj, Ardeshir M; Torres-Rua, Alfonso; McKee, Mac

2017-09-14

Applications of satellite-borne observations in precision agriculture (PA) are often limited due to the coarse spatial resolution of satellite imagery. This paper uses high-resolution airborne observations to increase the spatial resolution of satellite data for related applications in PA. A new variational downscaling scheme is presented that uses coincident aerial imagery products from "AggieAir", an unmanned aerial system, to increase the spatial resolution of Landsat satellite data. This approach is primarily tested for downscaling individual band Landsat images that can be used to derive normalized difference vegetation index (NDVI) and surface soil moisture (SSM). Quantitative and qualitative results demonstrate promising capabilities of the downscaling approach enabling effective increase of the spatial resolution of Landsat imageries by orders of 2 to 4. Specifically, the downscaling scheme retrieved the missing high-resolution feature of the imageries and reduced the root mean squared error by 15, 11, and 10 percent in visual, near infrared, and thermal infrared bands, respectively. This metric is reduced by 9% in the derived NDVI and remains negligibly for the soil moisture products.

Spatial Scale Gap Filling Using an Unmanned Aerial System: A Statistical Downscaling Method for Applications in Precision Agriculture

PubMed Central

Hassan-Esfahani, Leila; Ebtehaj, Ardeshir M.; McKee, Mac

2017-01-01

Applications of satellite-borne observations in precision agriculture (PA) are often limited due to the coarse spatial resolution of satellite imagery. This paper uses high-resolution airborne observations to increase the spatial resolution of satellite data for related applications in PA. A new variational downscaling scheme is presented that uses coincident aerial imagery products from “AggieAir”, an unmanned aerial system, to increase the spatial resolution of Landsat satellite data. This approach is primarily tested for downscaling individual band Landsat images that can be used to derive normalized difference vegetation index (NDVI) and surface soil moisture (SSM). Quantitative and qualitative results demonstrate promising capabilities of the downscaling approach enabling effective increase of the spatial resolution of Landsat imageries by orders of 2 to 4. Specifically, the downscaling scheme retrieved the missing high-resolution feature of the imageries and reduced the root mean squared error by 15, 11, and 10 percent in visual, near infrared, and thermal infrared bands, respectively. This metric is reduced by 9% in the derived NDVI and remains negligibly for the soil moisture products. PMID:28906428

Calibration of Fuji BAS-SR type imaging plate as high spatial resolution x-ray radiography recorder

NASA Astrophysics Data System (ADS)

Yan, Ji; Zheng, Jianhua; Zhang, Xing; Chen, Li; Wei, Minxi

2017-05-01

Image Plates as x-ray recorder have advantages including reusable, high dynamic range, large active area, and so on. In this work, Fuji BAS-SR type image plate combined with BAS-5000 scanner is calibrated. The fade rates of Image Plates has been measured using x-ray diffractometric in different room temperature; the spectral response of Image Plates has been measured using 241Am radioactive sealed source and fitting with linear model; the spatial resolution of Image Plates has been measured using micro-focus x-ray tube. The results show that Image Plates has an exponent decade curve and double absorption edge response curve. The spatial resolution of Image Plates with 25μ/50μ scanner resolution is 6.5lp/mm, 11.9lp/mm respectively and gold grid radiography is collected with 80lp/mm spatial resolution using SR-type Image Plates. BAS-SR type Image Plates can do high spatial resolution and quantitative radiographic works. It can be widely used in High energy density physics (HEDP), inertial confinement fusion (ICF) and laboratory astronomy physics.

Simulations of the temporal and spatial resolution for a compact time-resolved electron diffractometer

NASA Astrophysics Data System (ADS)

Robinson, Matthew S.; Lane, Paul D.; Wann, Derek A.

2016-02-01

A novel compact electron gun for use in time-resolved gas electron diffraction experiments has recently been designed and commissioned. In this paper we present and discuss the extensive simulations that were performed to underpin the design in terms of the spatial and temporal qualities of the pulsed electron beam created by the ionisation of a gold photocathode using a femtosecond laser. The response of the electron pulses to a solenoid lens used to focus the electron beam has also been studied. The simulated results show that focussing the electron beam affects the overall spatial and temporal resolution of the experiment in a variety of ways, and that factors that improve the resolution of one parameter can often have a negative effect on the other. A balance must, therefore, be achieved between spatial and temporal resolution. The optimal experimental time resolution for the apparatus is predicted to be 416 fs for studies of gas-phase species, while the predicted spatial resolution of better than 2 nm-1 compares well with traditional time-averaged electron diffraction set-ups.

Coherent optical adaptive technique improves the spatial resolution of STED microscopy in thick samples

PubMed Central

Yan, Wei; Yang, Yanlong; Tan, Yu; Chen, Xun; Li, Yang; Qu, Junle; Ye, Tong

2018-01-01

Stimulated emission depletion microscopy (STED) is one of far-field optical microscopy techniques that can provide sub-diffraction spatial resolution. The spatial resolution of the STED microscopy is determined by the specially engineered beam profile of the depletion beam and its power. However, the beam profile of the depletion beam may be distorted due to aberrations of optical systems and inhomogeneity of specimens’ optical properties, resulting in a compromised spatial resolution. The situation gets deteriorated when thick samples are imaged. In the worst case, the sever distortion of the depletion beam profile may cause complete loss of the super resolution effect no matter how much depletion power is applied to specimens. Previously several adaptive optics approaches have been explored to compensate aberrations of systems and specimens. However, it is hard to correct the complicated high-order optical aberrations of specimens. In this report, we demonstrate that the complicated distorted wavefront from a thick phantom sample can be measured by using the coherent optical adaptive technique (COAT). The full correction can effectively maintain and improve the spatial resolution in imaging thick samples. PMID:29400356

Estimating Gross Primary Production in Cropland with High Spatial and Temporal Scale Remote Sensing Data

NASA Astrophysics Data System (ADS)

Lin, S.; Li, J.; Liu, Q.

2018-04-01

Satellite remote sensing data provide spatially continuous and temporally repetitive observations of land surfaces, and they have become increasingly important for monitoring large region of vegetation photosynthetic dynamic. But remote sensing data have their limitation on spatial and temporal scale, for example, higher spatial resolution data as Landsat data have 30-m spatial resolution but 16 days revisit period, while high temporal scale data such as geostationary data have 30-minute imaging period, which has lower spatial resolution (> 1 km). The objective of this study is to investigate whether combining high spatial and temporal resolution remote sensing data can improve the gross primary production (GPP) estimation accuracy in cropland. For this analysis we used three years (from 2010 to 2012) Landsat based NDVI data, MOD13 vegetation index product and Geostationary Operational Environmental Satellite (GOES) geostationary data as input parameters to estimate GPP in a small region cropland of Nebraska, US. Then we validated the remote sensing based GPP with the in-situ measurement carbon flux data. Results showed that: 1) the overall correlation between GOES visible band and in-situ measurement photosynthesis active radiation (PAR) is about 50 % (R2 = 0.52) and the European Center for Medium-Range Weather Forecasts ERA-Interim reanalysis data can explain 64 % of PAR variance (R2 = 0.64); 2) estimating GPP with Landsat 30-m spatial resolution data and ERA daily meteorology data has the highest accuracy(R2 = 0.85, RMSE < 3 gC/m2/day), which has better performance than using MODIS 1-km NDVI/EVI product import; 3) using daily meteorology data as input for GPP estimation in high spatial resolution data would have higher relevance than 8-day and 16-day input. Generally speaking, using the high spatial resolution and high frequency satellite based remote sensing data can improve GPP estimation accuracy in cropland.

Evaluating the influence of spatial resolution of Landsat predictors on the accuracy of biomass models for large-area estimation across the eastern USA

NASA Astrophysics Data System (ADS)

Deo, Ram K.; Domke, Grant M.; Russell, Matthew B.; Woodall, Christopher W.; Andersen, Hans-Erik

2018-05-01

Aboveground biomass (AGB) estimates for regional-scale forest planning have become cost-effective with the free access to satellite data from sensors such as Landsat and MODIS. However, the accuracy of AGB predictions based on passive optical data depends on spatial resolution and spatial extent of target area as fine resolution (small pixels) data are associated with smaller coverage and longer repeat cycles compared to coarse resolution data. This study evaluated various spatial resolutions of Landsat-derived predictors on the accuracy of regional AGB models at three different sites in the eastern USA: Maine, Pennsylvania-New Jersey, and South Carolina. We combined national forest inventory data with Landsat-derived predictors at spatial resolutions ranging from 30–1000 m to understand the optimal spatial resolution of optical data for large-area (regional) AGB estimation. Ten generic models were developed using the data collected in 2014, 2015 and 2016, and the predictions were evaluated (i) at the county-level against the estimates of the USFS Forest Inventory and Analysis Program which relied on EVALIDator tool and national forest inventory data from the 2009–2013 cycle and (ii) within a large number of strips (~1 km wide) predicted via LiDAR metrics at 30 m spatial resolution. The county-level estimates by the EVALIDator and Landsat models were highly related (R 2 > 0.66), although the R 2 varied significantly across sites and resolution of predictors. The mean and standard deviation of county-level estimates followed increasing and decreasing trends, respectively, with models of coarser resolution. The Landsat-based total AGB estimates were larger than the LiDAR-based total estimates within the strips, however the mean of AGB predictions by LiDAR were mostly within one-standard deviations of the mean predictions obtained from the Landsat-based model at any of the resolutions. We conclude that satellite data at resolutions up to 1000 m provide acceptable accuracy for continental scale analysis of AGB.

Protein phosphorylation in human peripheral blood lymphocytes. Phosphorylation of endogenous plasma membrane and cytoplasmic proteins

PubMed Central

Chaplin, David D.; Wedner, H. James; Parker, Charles W.

1979-01-01

Phosphorylation of endogenous proteins in subcellular fractions of human peripheral-blood lymphocytes was studied by one- and two-dimensional polyacrylamide-gel electrophoresis. Studies using extensively purified subcellular fractions indicated that the endogenous phosphorylating activity in the particulate fractions was derived primarily from the plasma membrane. Electrophoresis of 32P-labelled subcellular fractions in two dimensions [O'Farrell (1975) J. Biol. Chem. 250, 4007–4021] provided much greater resolution of the endogenous phosphoproteins than electrophoresis in one dimension, facilitating their excision from gels for quantification of 32P content. More than 100 cytoplasmic and 20 plasma-membrane phosphorylated species were observed. Phosphorylation of more than 10 cytoplasmic proteins was absolutely dependent on cyclic AMP. In the plasma membrane, cyclic AMP-dependent phosphoproteins were observed with mol.wts. of 42000, 42000, 80000 and 90000 and pI values of 6.1, 6.3, 6.25 and 6.5 respectively. Phosphorylation of endogenous cytoplasmic and plasma-membrane proteins was rapid with t½=5–12s at 25°C. Between 40 and 70% of the 32P was recovered as phosphoserine and phosphothreonine when acid hydrolysates of isolated plasma-membrane phosphoproteins were analysed by high-voltage paper electrophoresis. The presence of cyclic AMP-dependent protein kinase and endogenous phosphate-acceptor proteins in the plasma membranes of lymphocytes provides a mechanism by which these cells might respond to plasma-membrane pools of cyclic AMP generated in response to stimulation by mitogens or physiological modulators of lymphocyte function. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4. PMID:228657

Quantitative Multispectral Analysis Of Discrete Subcellular Particles By Digital Imaging Fluorescence Microscopy (DIFM)

NASA Astrophysics Data System (ADS)

Dorey, C. K.; Ebenstein, David B.

1988-10-01

Subcellular localization of multiple biochemical markers is readily achieved through their characteristic autofluorescence or through use of appropriately labelled antibodies. Recent development of specific probes has permitted elegant studies in calcium and pH in living cells. However, each of these methods measured fluorescence at one wavelength; precise quantitation of multiple fluorophores at individual sites within a cell has not been possible. Using DIFM, we have achieved spectral analysis of discrete subcellular particles 1-2 gm in diameter. The fluorescence emission is broken into narrow bands by an interference monochromator and visualized through the combined use of a silicon intensified target (SIT) camera, a microcomputer based framegrabber with 8 bit resolution, and a color video monitor. Image acquisition, processing, analysis and display are under software control. The digitized image can be corrected for the spectral distortions induced by the wavelength dependent sensitivity of the camera, and the displayed image can be enhanced or presented in pseudocolor to facilitate discrimination of variation in pixel intensity of individual particles. For rapid comparison of the fluorophore composition of granules, a ratio image is produced by dividing the image captured at one wavelength by that captured at another. In the resultant ratio image, a granule which has a fluorophore composition different from the majority is selectively colored. This powerful system has been utilized to obtain spectra of endogenous autofluorescent compounds in discrete cellular organelles of human retinal pigment epithelium, and to measure immunohistochemically labelled components of the extracellular matrix associated with the human optic nerve.

Fine and distributed subcellular retinotopy of excitatory inputs to the dendritic tree of a collision-detecting neuron

PubMed Central

Zhu, Ying

2016-01-01

Individual neurons in several sensory systems receive synaptic inputs organized according to subcellular topographic maps, yet the fine structure of this topographic organization and its relation to dendritic morphology have not been studied in detail. Subcellular topography is expected to play a role in dendritic integration, particularly when dendrites are extended and active. The lobula giant movement detector (LGMD) neuron in the locust visual system is known to receive topographic excitatory inputs on part of its dendritic tree. The LGMD responds preferentially to objects approaching on a collision course and is thought to implement several interesting dendritic computations. To study the fine retinotopic mapping of visual inputs onto the excitatory dendrites of the LGMD, we designed a custom microscope allowing visual stimulation at the native sampling resolution of the locust compound eye while simultaneously performing two-photon calcium imaging on excitatory dendrites. We show that the LGMD receives a distributed, fine retinotopic projection from the eye facets and that adjacent facets activate overlapping portions of the same dendritic branches. We also demonstrate that adjacent retinal inputs most likely make independent synapses on the excitatory dendrites of the LGMD. Finally, we show that the fine topographic mapping can be studied using dynamic visual stimuli. Our results reveal the detailed structure of the dendritic input originating from individual facets on the eye and their relation to that of adjacent facets. The mapping of visual space onto the LGMD's dendrites is expected to have implications for dendritic computation. PMID:27009157

Fabrication and characterization of a 0.5-mm lutetium oxyorthosilicate detector array for high-resolution PET applications.

PubMed

Stickel, Jennifer R; Qi, Jinyi; Cherry, Simon R

2007-01-01

With the increasing use of in vivo imaging in mouse models of disease, there are many interesting applications that demand imaging of organs and tissues with submillimeter resolution. Though there are other contributing factors, the spatial resolution in small-animal PET is still largely determined by the detector pixel dimensions. In this work, a pair of lutetium oxyorthosilicate (LSO) arrays with 0.5-mm pixels was coupled to multichannel photomultiplier tubes and evaluated for use as high-resolution PET detectors. Flood histograms demonstrated that most crystals were clearly identifiable. Energy resolution varied from 22% to 38%. The coincidence timing resolution was 1.42-ns full width at half maximum (FWHM). The intrinsic spatial resolution was 0.68-mm FWHM as measured with a 30-gauge needle filled with (18)F. The improvement in spatial resolution in a tomographic setting is demonstrated using images of a line source phantom reconstructed with filtered backprojection and compared with images obtained from 2 dedicated small-animal PET scanners. Finally, a projection image of the mouse foot is shown to demonstrate the application of these 0.5-mm LSO detectors to a biologic task. A pair of highly pixelated LSO detections has been constructed and characterized for use as high-spatial-resolution PET detectors. It appears that small-animal PET systems capable of a FWHM spatial resolution of 600 microm or less are feasible and should be pursued.

Characterization of spatial and spectral resolution of a rotating prism chromotomographic hyperspectral imager

NASA Astrophysics Data System (ADS)

Bostick, Randall L.; Perram, Glen P.; Tuttle, Ronald

2009-05-01

The Air Force Institute of Technology (AFIT) has built a rotating prism chromotomographic hyperspectral imager (CTI) with the goal of extending the technology to exploit spatially extended sources with quickly varying (> 10 Hz) phenomenology, such as bomb detonations and muzzle flashes. This technology collects successive frames of 2-D data dispersed at different angles multiplexing spatial and spectral information which can then be used to reconstruct any arbitrary spectral plane(s). In this paper, the design of the AFIT instrument is described and then tested against a spectral target with near point source spatial characteristics to measure spectral and spatial resolution. It will be shown that, in theory, the spectral and spatial resolution in the 3-D spectral image cube is the nearly the same as a simple prism spectrograph with the same design. However, error in the knowledge of the prism linear dispersion at the detector array as a function of wavelength and projection angle will degrade resolution without further corrections. With minimal correction for error and use of a simple shift-and-add reconstruction algorithm, the CTI is able to produce a spatial resolution of about 2 mm in the object plane (234 μrad IFOV) and is limited by chromatic aberration. A spectral resolution of less than 1nm at shorter wavelengths is shown, limited primarily by prism dispersion.

Evaluating an image-fusion algorithm with synthetic-image-generation tools

NASA Astrophysics Data System (ADS)

Gross, Harry N.; Schott, John R.

1996-06-01

An algorithm that combines spectral mixing and nonlinear optimization is used to fuse multiresolution images. Image fusion merges images of different spatial and spectral resolutions to create a high spatial resolution multispectral combination. High spectral resolution allows identification of materials in the scene, while high spatial resolution locates those materials. In this algorithm, conventional spectral mixing estimates the percentage of each material (called endmembers) within each low resolution pixel. Three spectral mixing models are compared; unconstrained, partially constrained, and fully constrained. In the partially constrained application, the endmember fractions are required to sum to one. In the fully constrained application, all fractions are additionally required to lie between zero and one. While negative fractions seem inappropriate, they can arise from random spectral realizations of the materials. In the second part of the algorithm, the low resolution fractions are used as inputs to a constrained nonlinear optimization that calculates the endmember fractions for the high resolution pixels. The constraints mirror the low resolution constraints and maintain consistency with the low resolution fraction results. The algorithm can use one or more higher resolution sharpening images to locate the endmembers to high spatial accuracy. The algorithm was evaluated with synthetic image generation (SIG) tools. A SIG developed image can be used to control the various error sources that are likely to impair the algorithm performance. These error sources include atmospheric effects, mismodeled spectral endmembers, and variability in topography and illumination. By controlling the introduction of these errors, the robustness of the algorithm can be studied and improved upon. The motivation for this research is to take advantage of the next generation of multi/hyperspectral sensors. Although the hyperspectral images will be of modest to low resolution, fusing them with high resolution sharpening images will produce a higher spatial resolution land cover or material map.

High Spatial Resolution Thermal Satellite Technologies

NASA Technical Reports Server (NTRS)

Ryan, Robert

2003-01-01

This document in the form of viewslides, reviews various low-cost alternatives to high spatial resolution thermal satellite technologies. There exists no follow-on to Landsat 7 or ASTER high spatial resolution thermal systems. This document reviews the results of the investigation in to the use of new technologies to create a low-cost useful alternative. Three suggested technologies are examined. 1. Conventional microbolometer pushbroom modes offers potential for low cost Landsat Data Continuity Mission (LDCM) thermal or ASTER capability with at least 60-120 ground sampling distance (GSD). 2. Backscanning could produce MultiSpectral Thermal Imager performance without cooled detectors. 3. Cooled detector could produce hyperspectral thermal class system or extremely high spatial resolution class instrument.

Improved spatial resolution in PET scanners using sampling techniques

PubMed Central

Surti, Suleman; Scheuermann, Ryan; Werner, Matthew E.; Karp, Joel S.

2009-01-01

Increased focus towards improved detector spatial resolution in PET has led to the use of smaller crystals in some form of light sharing detector design. In this work we evaluate two sampling techniques that can be applied during calibrations for pixelated detector designs in order to improve the reconstructed spatial resolution. The inter-crystal positioning technique utilizes sub-sampling in the crystal flood map to better sample the Compton scatter events in the detector. The Compton scatter rejection technique, on the other hand, rejects those events that are located further from individual crystal centers in the flood map. We performed Monte Carlo simulations followed by measurements on two whole-body scanners for point source data. The simulations and measurements were performed for scanners using scintillators with Zeff ranging from 46.9 to 63 for LaBr3 and LYSO, respectively. Our results show that near the center of the scanner, inter-crystal positioning technique leads to a gain of about 0.5-mm in reconstructed spatial resolution (FWHM) for both scanner designs. In a small animal LYSO scanner the resolution improves from 1.9-mm to 1.6-mm with the inter-crystal technique. The Compton scatter rejection technique shows higher gains in spatial resolution but at the cost of reduction in scanner sensitivity. The inter-crystal positioning technique represents a modest acquisition software modification for an improvement in spatial resolution, but at a cost of potentially longer data correction and reconstruction times. The Compton scatter rejection technique, while also requiring a modest acquisition software change with no increased data correction and reconstruction times, will be useful in applications where the scanner sensitivity is very high and larger improvements in spatial resolution are desirable. PMID:19779586

Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer

NASA Astrophysics Data System (ADS)

Jabbour, Joey M.; Cheng, Shuna; Malik, Bilal H.; Cuenca, Rodrigo; Jo, Javier A.; Wright, John; Cheng, Yi-Shing Lisa; Maitland, Kristen C.

2013-04-01

Optical imaging techniques using a variety of contrast mechanisms are under evaluation for early detection of epithelial precancer; however, tradeoffs in field of view (FOV) and resolution may limit their application. Therefore, we present a multiscale multimodal optical imaging system combining macroscopic biochemical imaging of fluorescence lifetime imaging (FLIM) with subcellular morphologic imaging of reflectance confocal microscopy (RCM). The FLIM module images a 16×16 mm2 tissue area with 62.5 μm lateral and 320 ps temporal resolution to guide cellular imaging of suspicious regions. Subsequently, coregistered RCM images are acquired at 7 Hz with 400 μm diameter FOV, <1 μm lateral and 3.5 μm axial resolution. FLIM-RCM imaging was performed on a tissue phantom, normal porcine buccal mucosa, and a hamster cheek pouch model of oral carcinogenesis. While FLIM is sensitive to biochemical and macroscopic architectural changes in tissue, RCM provides images of cell nuclear morphology, all key indicators of precancer progression.

Combined FLIM and reflectance confocal microscopy for epithelial imaging

NASA Astrophysics Data System (ADS)

Jabbour, Joey M.; Cheng, Shuna; Shrestha, Sebina; Malik, Bilal; Jo, Javier A.; Applegate, Brian; Maitland, Kristen C.

2012-03-01

Current methods for detection of oral cancer lack the ability to delineate between normal and precancerous tissue with adequate sensitivity and specificity. The usual diagnostic mechanism involves visual inspection and palpation followed by tissue biopsy and histopathology, a process both invasive and time-intensive. A more sensitive and objective screening method can greatly facilitate the overall process of detection of early cancer. To this end, we present a multimodal imaging system with fluorescence lifetime imaging (FLIM) for wide field of view guidance and reflectance confocal microscopy for sub-cellular resolution imaging of epithelial tissue. Moving from a 12 x 12 mm2 field of view with 157 ìm lateral resolution using FLIM to 275 x 200 μm2 with lateral resolution of 2.2 μm using confocal microscopy, hamster cheek pouch model is imaged both in vivo and ex vivo. The results indicate that our dual modality imaging system can identify and distinguish between different tissue features, and, therefore, can potentially serve as a guide in early oral cancer detection..

X-ray phase contrast tomography from whole organ down to single cells

NASA Astrophysics Data System (ADS)

Krenkel, Martin; Töpperwien, Mareike; Bartels, Matthias; Lingor, Paul; Schild, Detlev; Salditt, Tim

2014-09-01

We use propagation based hard x-ray phase contrast tomography to explore the three dimensional structure of neuronal tissues from the organ down to sub-cellular level, based on combinations of synchrotron radiation and laboratory sources. To this end a laboratory based microfocus tomography setup has been built in which the geometry was optimized for phase contrast imaging and tomography. By utilizing phase retrieval algorithms, quantitative reconstructions can be obtained that enable automatic renderings without edge artifacts. A high brightness liquid metal microfocus x-ray source in combination with a high resolution detector yielding a resolution down to 1.5 μm. To extend the method to nanoscale resolution we use a divergent x-ray waveguide beam geometry at the synchrotron. Thus, the magnification can be easily tuned by placing the sample at different defocus distances. Due to the small Fresnel numbers in this geometry the measured images are of holographic nature which poses a challenge in phase retrieval.

Focal calcium monitoring with targeted nanosensors at the cytosolic side of endoplasmic reticulum

NASA Astrophysics Data System (ADS)

Hou, Yanyan; Arai, Satoshi; Takei, Yoshiaki; Murata, Atsushi; Takeoka, Shinji; Suzuki, Madoka

2016-01-01

Ca2+ distribution is spatially and temporally non-uniform inside cells due to cellular compartmentalization. However, Ca2+ sensing with small organic dyes, such as fura-2 and fluo-4, has been practically applied at a single cell level where the averaged signal from freely diffusing dye molecules is acquired. In this study, we aimed to target azide-functionalized fura-2 (N3-fura-2) to a specific site of subcellular compartments to realize focal Ca2+ sensing. Using scAVD (single-chain avidin)-biotin interaction and a copper-free click reaction system, we linked N3-fura-2 to specifically-targeted scAVD protein fused with a red fluorescent protein mCherry, so that Ca2+ sensors conjugated with four N3-fura-2 dyes with dibenzocyclooctyne (DBCO)-PEG4-biotin as a linker were generated at subcellular compartments in living cells. In cytoplasm, N3-fura-2 showed a prolonged retention period after binding to scAVD. Furthermore, the reacted N3-fura-2 was retained inside cells even after free dyes were washed out by methanol fixation. When scAVD was overexpressed on endoplasmic reticulum (ER) membranes, N3-fura-2 was accumulated on ER membranes. Upon histamine stimulation, which increases cytosolic Ca2+ concentration, ER-localized N3-fura-2 successfully sensed the Ca2+ level changes at the cytosolic side of ER membrane. Our study demonstrated specific targeting of N3-fura-2 to subcellular compartments and the ability of sensing focal Ca2+ level changes with the specifically targeted Ca2+ sensors.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Lan; Hill, K. W.; Bitter, M.

Here, a high spatial resolution of a few μm is often required for probing small-scale high-energy-density plasmas using high resolution x-ray imaging spectroscopy. This resolution can be achieved by adjusting system magnification to overcome the inherent limitation of the detector pixel size. Laboratory experiments on investigating the relation between spatial resolution and system magnification for a spherical crystal spectrometer are presented. Tungsten Lβ 2 rays from a tungsten-target micro-focus x-ray tube were diffracted by a Ge 440 crystal, which was spherically bent to a radius of 223 mm, and imaged onto an x-ray CCD with 13-μm pixel size. The source-to-crystalmore » (p) and crystal-to-detector (q) distances were varied to produce spatial magnifications ( M = q/p) ranging from 2 to 10. The inferred instrumental spatial width reduces with increasing system magnification M. However, the experimental measurement at each M is larger than the theoretical value of pixel size divided by M. Future work will focus on investigating possible broadening mechanisms that limit the spatial resolution.« less

Scaling field data to calibrate and validate moderate spatial resolution remote sensing models

USGS Publications Warehouse

Baccini, A.; Friedl, M.A.; Woodcock, C.E.; Zhu, Z.

2007-01-01

Validation and calibration are essential components of nearly all remote sensing-based studies. In both cases, ground measurements are collected and then related to the remote sensing observations or model results. In many situations, and particularly in studies that use moderate resolution remote sensing, a mismatch exists between the sensor's field of view and the scale at which in situ measurements are collected. The use of in situ measurements for model calibration and validation, therefore, requires a robust and defensible method to spatially aggregate ground measurements to the scale at which the remotely sensed data are acquired. This paper examines this challenge and specifically considers two different approaches for aggregating field measurements to match the spatial resolution of moderate spatial resolution remote sensing data: (a) landscape stratification; and (b) averaging of fine spatial resolution maps. The results show that an empirically estimated stratification based on a regression tree method provides a statistically defensible and operational basis for performing this type of procedure. 

Evaluating Hyperspectral Imaging of Wetland Vegetation as a Tool for Detecting Estuarine Nutrient Enrichment

DTIC Science & Technology

2008-05-01

the vegetation’s uptake of water column nutrients produces a spectral response; and 3) the spectral and spatial resolutions ...analysis. This allowed us to evaluate these assumptions at the landscape level, by using the high spectral and spatial resolution of the hyperspectral... spatial resolution (2.5 m pixels) HyMap hyperspectral imagery of the entire wetland. After using a hand-held spectrometer to characterize

Atmospheric Correction Prototype Algorithm for High Spatial Resolution Multispectral Earth Observing Imaging Systems

NASA Technical Reports Server (NTRS)

Pagnutti, Mary

2006-01-01

This viewgraph presentation reviews the creation of a prototype algorithm for atmospheric correction using high spatial resolution earth observing imaging systems. The objective of the work was to evaluate accuracy of a prototype algorithm that uses satellite-derived atmospheric products to generate scene reflectance maps for high spatial resolution (HSR) systems. This presentation focused on preliminary results of only the satellite-based atmospheric correction algorithm.

Preliminary frequency-domain analysis for the reconstructed spatial resolution of muon tomography

NASA Astrophysics Data System (ADS)

Yu, B.; Zhao, Z.; Wang, X.; Wang, Y.; Wu, D.; Zeng, Z.; Zeng, M.; Yi, H.; Luo, Z.; Yue, X.; Cheng, J.

2014-11-01

Muon tomography is an advanced technology to non-destructively detect high atomic number materials. It exploits the multiple Coulomb scattering information of muon to reconstruct the scattering density image of the traversed object. Because of the statistics of muon scattering, the measurement error of system and the data incompleteness, the reconstruction is always accompanied with a certain level of interference, which will influence the reconstructed spatial resolution. While statistical noises can be reduced by extending the measuring time, system parameters determine the ultimate spatial resolution that one system can reach. In this paper, an effective frequency-domain model is proposed to analyze the reconstructed spatial resolution of muon tomography. The proposed method modifies the resolution analysis in conventional computed tomography (CT) to fit the different imaging mechanism in muon scattering tomography. The measured scattering information is described in frequency domain, then a relationship between the measurements and the original image is proposed in Fourier domain, which is named as "Muon Central Slice Theorem". Furthermore, a preliminary analytical expression of the ultimate reconstructed spatial is derived, and the simulations are performed for validation. While the method is able to predict the ultimate spatial resolution of a given system, it can also be utilized for the optimization of system design and construction.

Improving spectral resolution in spatial encoding dimension of single-scan nuclear magnetic resonance 2D spin echo correlated spectroscopy

NASA Astrophysics Data System (ADS)

Lin, Liangjie; Wei, Zhiliang; Yang, Jian; Lin, Yanqin; Chen, Zhong

2014-11-01

The spatial encoding technique can be used to accelerate the acquisition of multi-dimensional nuclear magnetic resonance spectra. However, with this technique, we have to make trade-offs between the spectral width and the resolution in the spatial encoding dimension (F1 dimension), resulting in the difficulty of covering large spectral widths while preserving acceptable resolutions for spatial encoding spectra. In this study, a selective shifting method is proposed to overcome the aforementioned drawback. This method is capable of narrowing spectral widths and improving spectral resolutions in spatial encoding dimensions by selectively shifting certain peaks in spectra of the ultrafast version of spin echo correlated spectroscopy (UFSECSY). This method can also serve as a powerful tool to obtain high-resolution correlated spectra in inhomogeneous magnetic fields for its resistance to any inhomogeneity in the F1 dimension inherited from UFSECSY. Theoretical derivations and experiments have been carried out to demonstrate performances of the proposed method. Results show that the spectral width in spatial encoding dimension can be reduced by shortening distances between cross peaks and axial peaks with the proposed method and the expected resolution improvement can be achieved. Finally, the shifting-absent spectrum can be recovered readily by post-processing.

Sympathy for the Devil: Detailing the Effects of Planning-Unit Size, Thematic Resolution of Reef Classes, and Socioeconomic Costs on Spatial Priorities for Marine Conservation

PubMed Central

Pressey, Robert L.; Weeks, Rebecca; Andréfouët, Serge; Moloney, James

2016-01-01

Spatial data characteristics have the potential to influence various aspects of prioritising biodiversity areas for systematic conservation planning. There has been some exploration of the combined effects of size of planning units and level of classification of physical environments on the pattern and extent of priority areas. However, these data characteristics have yet to be explicitly investigated in terms of their interaction with different socioeconomic cost data during the spatial prioritisation process. We quantify the individual and interacting effects of three factors—planning-unit size, thematic resolution of reef classes, and spatial variability of socioeconomic costs—on spatial priorities for marine conservation, in typical marine planning exercises that use reef classification maps as a proxy for biodiversity. We assess these factors by creating 20 unique prioritisation scenarios involving combinations of different levels of each factor. Because output data from these scenarios are analogous to ecological data, we applied ecological statistics to determine spatial similarities between reserve designs. All three factors influenced prioritisations to different extents, with cost variability having the largest influence, followed by planning-unit size and thematic resolution of reef classes. The effect of thematic resolution on spatial design depended on the variability of cost data used. In terms of incidental representation of conservation objectives derived from finer-resolution data, scenarios prioritised with uniform cost outperformed those prioritised with variable cost. Following our analyses, we make recommendations to help maximise the spatial and cost efficiency and potential effectiveness of future marine conservation plans in similar planning scenarios. We recommend that planners: employ the smallest planning-unit size practical; invest in data at the highest possible resolution; and, when planning across regional extents with the intention of incidentally representing fine-resolution features, prioritise the whole region with uniform costs rather than using coarse-resolution data on variable costs. PMID:27829042

Sympathy for the Devil: Detailing the Effects of Planning-Unit Size, Thematic Resolution of Reef Classes, and Socioeconomic Costs on Spatial Priorities for Marine Conservation.

PubMed

Cheok, Jessica; Pressey, Robert L; Weeks, Rebecca; Andréfouët, Serge; Moloney, James

2016-01-01

Spatial data characteristics have the potential to influence various aspects of prioritising biodiversity areas for systematic conservation planning. There has been some exploration of the combined effects of size of planning units and level of classification of physical environments on the pattern and extent of priority areas. However, these data characteristics have yet to be explicitly investigated in terms of their interaction with different socioeconomic cost data during the spatial prioritisation process. We quantify the individual and interacting effects of three factors-planning-unit size, thematic resolution of reef classes, and spatial variability of socioeconomic costs-on spatial priorities for marine conservation, in typical marine planning exercises that use reef classification maps as a proxy for biodiversity. We assess these factors by creating 20 unique prioritisation scenarios involving combinations of different levels of each factor. Because output data from these scenarios are analogous to ecological data, we applied ecological statistics to determine spatial similarities between reserve designs. All three factors influenced prioritisations to different extents, with cost variability having the largest influence, followed by planning-unit size and thematic resolution of reef classes. The effect of thematic resolution on spatial design depended on the variability of cost data used. In terms of incidental representation of conservation objectives derived from finer-resolution data, scenarios prioritised with uniform cost outperformed those prioritised with variable cost. Following our analyses, we make recommendations to help maximise the spatial and cost efficiency and potential effectiveness of future marine conservation plans in similar planning scenarios. We recommend that planners: employ the smallest planning-unit size practical; invest in data at the highest possible resolution; and, when planning across regional extents with the intention of incidentally representing fine-resolution features, prioritise the whole region with uniform costs rather than using coarse-resolution data on variable costs.

Investigation of spatial resolution improvement by use of a mouth-insert detector in the helmet PET scanner.

PubMed

Ahmed, Abdella M; Tashima, Hideaki; Yamaya, Taiga

2018-03-01

The dominant factor limiting the intrinsic spatial resolution of a positron emission tomography (PET) system is the size of the crystal elements in the detector. To increase sensitivity and achieve high spatial resolution, it is essential to use advanced depth-of-interaction (DOI) detectors and arrange them close to the subject. The DOI detectors help maintain high spatial resolution by mitigating the parallax error caused by the thickness of the scintillator near the peripheral regions of the field-of-view. As an optimal geometry for a brain PET scanner, with high sensitivity and spatial resolution, we proposed and developed the helmet-chin PET scanner using 54 four-layered DOI detectors consisting of a 16 × 16 × 4 array of GSOZ scintillator crystals with dimensions of 2.8 × 2.8 × 7.5 mm 3 . All the detectors used in the helmet-chin PET scanner had the same spatial resolution. In this study, we conducted a feasibility study of a new add-on detector arrangement for the helmet PET scanner by replacing the chin detector with a segmented crystal cube, having high spatial resolution in all directions, which can be placed inside the mouth. The crystal cube (which we have named the mouth-insert detector) has an array of 20 × 20 × 20 LYSO crystal segments with dimensions of 1 × 1 × 1 mm 3 . Thus, the scanner is formed by the combination of the helmet and mouth-insert detectors, and is referred to as the helmet-mouth-insert PET scanner. The results show that the helmet-mouth-insert PET scanner has comparable sensitivity and improved spatial resolution near the center of the hemisphere, compared to the helmet-chin PET scanner.

Added-values of high spatiotemporal remote sensing data in crop yield estimation

NASA Astrophysics Data System (ADS)

Gao, F.; Anderson, M. C.

2017-12-01

Timely and accurate estimation of crop yield before harvest is critical for food market and administrative planning. Remote sensing derived parameters have been used for estimating crop yield by using either empirical or crop growth models. The uses of remote sensing vegetation index (VI) in crop yield modeling have been typically evaluated at regional and country scales using coarse spatial resolution (a few hundred to kilo-meters) data or assessed over a small region at field level using moderate resolution spatial resolution data (10-100m). Both data sources have shown great potential in capturing spatial and temporal variability in crop yield. However, the added value of data with both high spatial and temporal resolution data has not been evaluated due to the lack of such data source with routine, global coverage. In recent years, more moderate resolution data have become freely available and data fusion approaches that combine data acquired from different spatial and temporal resolutions have been developed. These make the monitoring crop condition and estimating crop yield at field scale become possible. Here we investigate the added value of the high spatial and temporal VI for describing variability of crop yield. The explanatory ability of crop yield based on high spatial and temporal resolution remote sensing data was evaluated in a rain-fed agricultural area in the U.S. Corn Belt. Results show that the fused Landsat-MODIS (high spatial and temporal) VI explains yield variability better than single data source (Landsat or MODIS alone), with EVI2 performing slightly better than NDVI. The maximum VI describes yield variability better than cumulative VI. Even though VI is effective in explaining yield variability within season, the inter-annual variability is more complex and need additional information (e.g. weather, water use and management). Our findings augment the importance of high spatiotemporal remote sensing data and supports new moderate resolution satellite missions for agricultural applications.

Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins

PubMed Central

Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

2013-01-01

Purpose To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features. Materials and Methods Tissue excised from a genetically engineered mouse model of sarcoma was imaged using a subcellular resolution microendoscope after topical application of a fluorescent anatomical contrast agent: acriflavine. An algorithm based on sparse component analysis (SCA) and the circle transform (CT) was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma. Results Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity). For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach. Conclusion The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue. PMID:23824589

Dielectric elastomer actuator for the measurement of cell traction forces with sub-cellular resolution

NASA Astrophysics Data System (ADS)

Rosset, Samuel; Poulin, Alexandre; Zollinger, Alicia; Smith, Michael; Shea, Herbert

2017-04-01

We report on the use of dielectric elastomer actuators (DEAs) to measure the traction force field of cells with subcellular resolution. The study of cellular electrochemical and mechanical response to deformation is an important area of research, as mechanotransduction has been shown to be linked with fundamental cell functions, or the progression of diseases such as cancer or atherosclerosis. Experimental cell mechanics is based on two fundamental concepts: the ability to measure cell stiffness, and to apply controlled strains to small clusters of cells. However, there is a lack of tools capable of applying precise deformation to a small cell population while being compatible with an inverted microscope (stable focal plane, transparency, compactness, etc.). Here, we use an anisotropically prestretched silicone-based DEA to deform a soft (7.6kPa) polyacrylamide gel on which the cells are cultured. An array of micro-dots of fluorescent fibronectin is transferred on the gel by micro-contact printing and serves as attachment points for the cells. In addition, the fluorescent dots (which have a diameter of 2 μm with a spacing of 6 μm) are used during the experiment to monitor the traction forces of a single cell (or small cluster of cells). The cell locally exerts traction on the gel, thus deforming the matrix of dots. The position of dots versus time is monitored live when the cells are submitted to a uniaxial strain step. Our deformable bioreactor enables the measurement of the local stiffness of cells submitted to mechanical strain, and is fully compatible with an inverted microscope set-up.

Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins.

PubMed

Mueller, Jenna L; Harmany, Zachary T; Mito, Jeffrey K; Kennedy, Stephanie A; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G; Willett, Rebecca M; Brown, J Quincy; Ramanujam, Nimmi

2013-01-01

To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features. TISSUE EXCISED FROM A GENETICALLY ENGINEERED MOUSE MODEL OF SARCOMA WAS IMAGED USING A SUBCELLULAR RESOLUTION MICROENDOSCOPE AFTER TOPICAL APPLICATION OF A FLUORESCENT ANATOMICAL CONTRAST AGENT: acriflavine. An algorithm based on sparse component analysis (SCA) and the circle transform (CT) was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma. Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity). For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach. The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue.

Developing a CCD camera with high spatial resolution for RIXS in the soft X-ray range

NASA Astrophysics Data System (ADS)

Soman, M. R.; Hall, D. J.; Tutt, J. H.; Murray, N. J.; Holland, A. D.; Schmitt, T.; Raabe, J.; Schmitt, B.

2013-12-01

The Super Advanced X-ray Emission Spectrometer (SAXES) at the Swiss Light Source contains a high resolution Charge-Coupled Device (CCD) camera used for Resonant Inelastic X-ray Scattering (RIXS). Using the current CCD-based camera system, the energy-dispersive spectrometer has an energy resolution (E/ΔE) of approximately 12,000 at 930 eV. A recent study predicted that through an upgrade to the grating and camera system, the energy resolution could be improved by a factor of 2. In order to achieve this goal in the spectral domain, the spatial resolution of the CCD must be improved to better than 5 μm from the current 24 μm spatial resolution (FWHM). The 400 eV-1600 eV energy X-rays detected by this spectrometer primarily interact within the field free region of the CCD, producing electron clouds which will diffuse isotropically until they reach the depleted region and buried channel. This diffusion of the charge leads to events which are split across several pixels. Through the analysis of the charge distribution across the pixels, various centroiding techniques can be used to pinpoint the spatial location of the X-ray interaction to the sub-pixel level, greatly improving the spatial resolution achieved. Using the PolLux soft X-ray microspectroscopy endstation at the Swiss Light Source, a beam of X-rays of energies from 200 eV to 1400 eV can be focused down to a spot size of approximately 20 nm. Scanning this spot across the 16 μm square pixels allows the sub-pixel response to be investigated. Previous work has demonstrated the potential improvement in spatial resolution achievable by centroiding events in a standard CCD. An Electron-Multiplying CCD (EM-CCD) has been used to improve the signal to effective readout noise ratio achieved resulting in a worst-case spatial resolution measurement of 4.5±0.2 μm and 3.9±0.1 μm at 530 eV and 680 eV respectively. A method is described that allows the contribution of the X-ray spot size to be deconvolved from these worst-case resolution measurements, estimating the spatial resolution to be approximately 3.5 μm and 3.0 μm at 530 eV and 680 eV, well below the resolution limit of 5 μm required to improve the spectral resolution by a factor of 2.

Subcellular Redox Signaling.

PubMed

Zhu, Liping; Lu, Yankai; Zhang, Jiwei; Hu, Qinghua

2017-01-01

Oxidative and antioxidative system of cells and tissues maintains a balanced state under physiological conditions. A disruption in this balance of redox status has been associated with numerous pathological processes. Reactive oxygen species (ROS) as a major redox signaling generates in a spatiotemporally dependent manner. Subcellular organelles such as mitochondria, endoplasmic reticulum, plasma membrane and nuclei contribute to the production of ROS. In addition to downstream effects of ROS signaling regulated by average ROS changes in cytoplasm, whether subcelluar ROS mediate biological effect(s) has drawn greater attentions. With the advance in redox-sensitive probes targeted to different subcellular compartments, the investigation of subcellular ROS signaling and its associated cellular function has become feasible. In this review, we discuss the subcellular ROS signaling, with particular focus on mechanisms of subcellular ROS production and its downstream effects.

Coarse climate change projections for species living in a fine-scaled world.

PubMed

Nadeau, Christopher P; Urban, Mark C; Bridle, Jon R

2017-01-01

Accurately predicting biological impacts of climate change is necessary to guide policy. However, the resolution of climate data could be affecting the accuracy of climate change impact assessments. Here, we review the spatial and temporal resolution of climate data used in impact assessments and demonstrate that these resolutions are often too coarse relative to biologically relevant scales. We then develop a framework that partitions climate into three important components: trend, variance, and autocorrelation. We apply this framework to map different global climate regimes and identify where coarse climate data is most and least likely to reduce the accuracy of impact assessments. We show that impact assessments for many large mammals and birds use climate data with a spatial resolution similar to the biologically relevant area encompassing population dynamics. Conversely, impact assessments for many small mammals, herpetofauna, and plants use climate data with a spatial resolution that is orders of magnitude larger than the area encompassing population dynamics. Most impact assessments also use climate data with a coarse temporal resolution. We suggest that climate data with a coarse spatial resolution is likely to reduce the accuracy of impact assessments the most in climates with high spatial trend and variance (e.g., much of western North and South America) and the least in climates with low spatial trend and variance (e.g., the Great Plains of the USA). Climate data with a coarse temporal resolution is likely to reduce the accuracy of impact assessments the most in the northern half of the northern hemisphere where temporal climatic variance is high. Our framework provides one way to identify where improving the resolution of climate data will have the largest impact on the accuracy of biological predictions under climate change. © 2016 John Wiley & Sons Ltd.

A Study on the Effects of Spatial Scale on Snow Process in Hyper-Resolution Hydrological Modelling over Mountainous Areas

NASA Astrophysics Data System (ADS)

Garousi Nejad, I.; He, S.; Tang, Q.; Ogden, F. L.; Steinke, R. C.; Frazier, N.; Tarboton, D. G.; Ohara, N.; Lin, H.

2017-12-01

Spatial scale is one of the main considerations in hydrological modeling of snowmelt in mountainous areas. The size of model elements controls the degree to which variability can be explicitly represented versus what needs to be parameterized using effective properties such as averages or other subgrid variability parameterizations that may degrade the quality of model simulations. For snowmelt modeling terrain parameters such as slope, aspect, vegetation and elevation play an important role in the timing and quantity of snowmelt that serves as an input to hydrologic runoff generation processes. In general, higher resolution enhances the accuracy of the simulation since fine meshes represent and preserve the spatial variability of atmospheric and surface characteristics better than coarse resolution. However, this increases computational cost and there may be a scale beyond which the model response does not improve due to diminishing sensitivity to variability and irreducible uncertainty associated with the spatial interpolation of inputs. This paper examines the influence of spatial resolution on the snowmelt process using simulations of and data from the Animas River watershed, an alpine mountainous area in Colorado, USA, using an unstructured distributed physically based hydrological model developed for a parallel computing environment, ADHydro. Five spatial resolutions (30 m, 100 m, 250 m, 500 m, and 1 km) were used to investigate the variations in hydrologic response. This study demonstrated the importance of choosing the appropriate spatial scale in the implementation of ADHydro to obtain a balance between representing spatial variability and the computational cost. According to the results, variation in the input variables and parameters due to using different spatial resolution resulted in changes in the obtained hydrological variables, especially snowmelt, both at the basin-scale and distributed across the model mesh.

High efficiency multishot interleaved spiral-in/out: acquisition for high-resolution BOLD fMRI.

PubMed

Jung, Youngkyoo; Samsonov, Alexey A; Liu, Thomas T; Buracas, Giedrius T

2013-08-01

Growing demand for high spatial resolution blood oxygenation level dependent (BOLD) functional magnetic resonance imaging faces a challenge of the spatial resolution versus coverage or temporal resolution tradeoff, which can be addressed by methods that afford increased acquisition efficiency. Spiral acquisition trajectories have been shown to be superior to currently prevalent echo-planar imaging in terms of acquisition efficiency, and high spatial resolution can be achieved by employing multiple-shot spiral acquisition. The interleaved spiral in/out trajectory is preferred over spiral-in due to increased BOLD signal contrast-to-noise ratio (CNR) and higher acquisition efficiency than that of spiral-out or noninterleaved spiral in/out trajectories (Law & Glover. Magn Reson Med 2009; 62:829-834.), but to date applicability of the multishot interleaved spiral in/out for high spatial resolution imaging has not been studied. Herein we propose multishot interleaved spiral in/out acquisition and investigate its applicability for high spatial resolution BOLD functional magnetic resonance imaging. Images reconstructed from interleaved spiral-in and -out trajectories possess artifacts caused by differences in T2 decay, off-resonance, and k-space errors associated with the two trajectories. We analyze the associated errors and demonstrate that application of conjugate phase reconstruction and spectral filtering can substantially mitigate these image artifacts. After applying these processing steps, the multishot interleaved spiral in/out pulse sequence yields high BOLD CNR images at in-plane resolution below 1 × 1 mm while preserving acceptable temporal resolution (4 s) and brain coverage (15 slices of 2 mm thickness). Moreover, this method yields sufficient BOLD CNR at 1.5 mm isotropic resolution for detection of activation in hippocampus associated with cognitive tasks (Stern memory task). The multishot interleaved spiral in/out acquisition is a promising technique for high spatial resolution BOLD functional magnetic resonance imaging applications. © 2012 Wiley Periodicals, Inc.

Scaling effects on spring phenology detections from MODIS data at multiple spatial resolutions over the contiguous United States

NASA Astrophysics Data System (ADS)

Peng, Dailiang; Zhang, Xiaoyang; Zhang, Bing; Liu, Liangyun; Liu, Xinjie; Huete, Alfredo R.; Huang, Wenjiang; Wang, Siyuan; Luo, Shezhou; Zhang, Xiao; Zhang, Helin

2017-10-01

Land surface phenology (LSP) has been widely retrieved from satellite data at multiple spatial resolutions, but the spatial scaling effects on LSP detection are poorly understood. In this study, we collected enhanced vegetation index (EVI, 250 m) from collection 6 MOD13Q1 product over the contiguous United States (CONUS) in 2007 and 2008, and generated a set of multiple spatial resolution EVI data by resampling 250 m to 2 × 250 m and 3 × 250 m, 4 × 250 m, …, 35 × 250 m. These EVI time series were then used to detect the start of spring season (SOS) at various spatial resolutions. Further the SOS variation across scales was examined at each coarse resolution grid (35 × 250 m ≈ 8 km, refer to as reference grid) and ecoregion. Finally, the SOS scaling effects were associated with landscape fragment, proportion of primary land cover type, and spatial variability of seasonal greenness variation within each reference grid. The results revealed the influences of satellite spatial resolutions on SOS retrievals and the related impact factors. Specifically, SOS significantly varied lineally or logarithmically across scales although the relationship could be either positive or negative. The overall SOS values averaged from spatial resolutions between 250 m and 35 × 250 m at large ecosystem regions were generally similar with a difference less than 5 days, while the SOS values within the reference grid could differ greatly in some local areas. Moreover, the standard deviation of SOS across scales in the reference grid was less than 5 days in more than 70% of area over the CONUS, which was smaller in northeastern than in southern and western regions. The SOS scaling effect was significantly associated with heterogeneity of vegetation properties characterized using land landscape fragment, proportion of primary land cover type, and spatial variability of seasonal greenness variation, but the latter was the most important impact factor.

Sheet-scanned dual-axis confocal microscopy using Richardson-Lucy deconvolution.

PubMed

Wang, D; Meza, D; Wang, Y; Gao, L; Liu, J T C

2014-09-15

We have previously developed a line-scanned dual-axis confocal (LS-DAC) microscope with subcellular resolution suitable for high-frame-rate diagnostic imaging at shallow depths. Due to the loss of confocality along one dimension, the contrast (signal-to-background ratio) of a LS-DAC microscope is deteriorated compared to a point-scanned DAC microscope. However, by using a sCMOS camera for detection, a short oblique light-sheet is imaged at each scanned position. Therefore, by scanning the light sheet in only one dimension, a thin 3D volume is imaged. Both sequential two-dimensional deconvolution and three-dimensional deconvolution are performed on the thin image volume to improve the resolution and contrast of one en face confocal image section at the center of the volume, a technique we call sheet-scanned dual-axis confocal (SS-DAC) microscopy.

Radially polarized tip-enhanced near-field coherent anti-Stokes Raman scattering microscopy for vibrational nano-imaging

NASA Astrophysics Data System (ADS)

Lin, Jian; Zi Jian Er, Kenneth; Zheng, Wei; Huang, Zhiwei

2013-08-01

We report a radially polarized tip-enhanced near-field coherent anti-Stokes Raman scattering (RP-TE-CARS) microscopy technique for high-contrast vibrational imaging of subcellular organelles at nano-scale resolutions. The radially polarized pump and Stokes laser beams are tightly focused onto the sample while a gold-coated metallic probe is placed at the upper surface of the sample to enhance the electric field and CARS signals. The back-scattered CARS signal is measured with the gold-coated nano-tip being stationary at the focal region of laser beams. The RP-TE-CARS signal is ˜6-fold higher than that using linearly polarized laser excitation. We demonstrate the good performance of the RP-TE-CARS technique developed by imaging sub-micron polystyrene beads and mitochondria at nano-scale resolutions.

Visual resolution and contrast sensitivity in two benthic sharks.

PubMed

Ryan, Laura A; Hart, Nathan S; Collin, Shaun P; Hemmi, Jan M

2016-12-15

Sharks have long been described as having 'poor' vision. They are cone monochromats and anatomical estimates suggest they have low spatial resolution. However, there are no direct behavioural measurements of spatial resolution or contrast sensitivity. This study estimates contrast sensitivity and spatial resolution of two species of benthic sharks, the Port Jackson shark, Heterodontus portusjacksoni, and the brown-banded bamboo shark, Chiloscyllium punctatum, by recording eye movements in response to optokinetic stimuli. Both species tracked moving low spatial frequency gratings with weak but consistent eye movements. Eye movements ceased at 0.38 cycles per degree, even for high contrasts, suggesting low spatial resolution. However, at lower spatial frequencies, eye movements were elicited by low contrast gratings, 1.3% and 2.9% contrast in H portusjacksoni and C. punctatum, respectively. Contrast sensitivity was higher than in other vertebrates with a similar spatial resolving power, which may reflect an adaptation to the relatively low contrast encountered in aquatic environments. Optokinetic gain was consistently low and neither species stabilised the gratings on their retina. To check whether restraining the animals affected their optokinetic responses, we also analysed eye movements in free-swimming C. punctatum We found no eye movements that could compensate for body rotations, suggesting that vision may pass through phases of stabilisation and blur during swimming. As C. punctatum is a sedentary benthic species, gaze stabilisation during swimming may not be essential. Our results suggest that vision in sharks is not 'poor' as previously suggested, but optimised for contrast detection rather than spatial resolution. © 2016. Published by The Company of Biologists Ltd.

Study of spatial resolution of coordinate detectors based on Gas Electron Multipliers

NASA Astrophysics Data System (ADS)

Kudryavtsev, V. N.; Maltsev, T. V.; Shekhtman, L. I.

2017-02-01

Spatial resolution of GEM-based tracking detectors is determined in the simulation and measured in the experiments. The simulation includes GEANT4 implemented transport of high energy electrons with careful accounting of atomic relaxation processes including emission of fluorescent photons and Auger electrons and custom post-processing with accounting of diffusion, gas amplification fluctuations, distribution of signals on readout electrodes, electronics noise and particular algorithm of final coordinate calculation (center of gravity). The simulation demonstrates that the minimum of spatial resolution of about 10 μm can be achieved with a gas mixture of Ar -CO2 (75-25 %) at a strips pitch from 250 μm to 300 μm. At a larger pitch the resolution quickly degrades reaching 80-100 μm at a pitch of 460-500 μm. Spatial resolution of low-material triple-GEM detectors for the DEUTERON facility at the VEPP-3 storage ring is measured at the extracted beam facility of the VEPP-4 M collider. One-coordinate resolution of the DEUTERON detector is measured with electron beam of 500 MeV, 1 GeV and 3.5 GeV energies. The determined value of spatial resolution varies in the range from approximately 35 μm to 50 μm for orthogonal tracks in the experiments.

Horizontal Residual Mean Circulation: Evaluation of Spatial Correlations in Coarse Resolution Ocean Models

NASA Astrophysics Data System (ADS)

Li, Y.; McDougall, T. J.

2016-02-01

Coarse resolution ocean models lack knowledge of spatial correlations between variables on scales smaller than the grid scale. Some researchers have shown that these spatial correlations play a role in the poleward heat flux. In order to evaluate the poleward transport induced by the spatial correlations at a fixed horizontal position, an equation is obtained to calculate the approximate transport from velocity gradients. The equation involves two terms that can be added to the quasi-Stokes streamfunction (based on temporal correlations) to incorporate the contribution of spatial correlations. Moreover, these new terms do not need to be parameterized and is ready to be evaluated by using model data directly. In this study, data from a high resolution ocean model have been used to estimate the accuracy of this HRM approach for improving the horizontal property fluxes in coarse-resolution ocean models. A coarse grid is formed by sub-sampling and box-car averaging the fine grid scale. The transport calculated on the coarse grid is then compared to the transport on original high resolution grid scale accumulated over a corresponding number of grid boxes. The preliminary results have shown that the estimate on coarse resolution grids roughly match the corresponding transports on high resolution grids.

Spatial modeling of cell signaling networks.

PubMed

Cowan, Ann E; Moraru, Ion I; Schaff, James C; Slepchenko, Boris M; Loew, Leslie M

2012-01-01

The shape of a cell, the sizes of subcellular compartments, and the spatial distribution of molecules within the cytoplasm can all control how molecules interact to produce a cellular behavior. This chapter describes how these spatial features can be included in mechanistic mathematical models of cell signaling. The Virtual Cell computational modeling and simulation software is used to illustrate the considerations required to build a spatial model. An explanation of how to appropriately choose between physical formulations that implicitly or explicitly account for cell geometry and between deterministic versus stochastic formulations for molecular dynamics is provided, along with a discussion of their respective strengths and weaknesses. As a first step toward constructing a spatial model, the geometry needs to be specified and associated with the molecules, reactions, and membrane flux processes of the network. Initial conditions, diffusion coefficients, velocities, and boundary conditions complete the specifications required to define the mathematics of the model. The numerical methods used to solve reaction-diffusion problems both deterministically and stochastically are then described and some guidance is provided in how to set up and run simulations. A study of cAMP signaling in neurons ends the chapter, providing an example of the insights that can be gained in interpreting experimental results through the application of spatial modeling. Copyright © 2012 Elsevier Inc. All rights reserved.

Effects of configural processing on the perceptual spatial resolution for face features.

PubMed

Namdar, Gal; Avidan, Galia; Ganel, Tzvi

2015-11-01

Configural processing governs human perception across various domains, including face perception. An established marker of configural face perception is the face inversion effect, in which performance is typically better for upright compared to inverted faces. In two experiments, we tested whether configural processing could influence basic visual abilities such as perceptual spatial resolution (i.e., the ability to detect spatial visual changes). Face-related perceptual spatial resolution was assessed by measuring the just noticeable difference (JND) to subtle positional changes between specific features in upright and inverted faces. The results revealed robust inversion effect for spatial sensitivity to configural-based changes, such as the distance between the mouth and the nose, or the distance between the eyes and the nose. Critically, spatial resolution for face features within the region of the eyes (e.g., the interocular distance between the eyes) was not affected by inversion, suggesting that the eye region operates as a separate 'gestalt' unit which is relatively immune to manipulations that would normally hamper configural processing. Together these findings suggest that face orientation modulates fundamental psychophysical abilities including spatial resolution. Furthermore, they indicate that classic psychophysical methods can be used as a valid measure of configural face processing. Copyright © 2015 Elsevier Ltd. All rights reserved.

Super resolution PLIF demonstrated in turbulent jet flows seeded with I2

NASA Astrophysics Data System (ADS)

Xu, Wenjiang; Liu, Ning; Ma, Lin

2018-05-01

Planar laser induced fluorescence (PLIF) represents an indispensable tool for flow and flame imaging. However, the PLIF technique suffers from limited spatial resolution or blurring in many situations, which restricts its applicability and capability. This work describes a new method, named SR-PLIF (super-resolution PLIF), to overcome these limitations and enhance the capability of PLIF. The method uses PLIF images captured simultaneously from two (or more) orientations to reconstruct a final PLIF image with resolution enhanced or blurring removed. This paper reports the development of the reconstruction algorithm, and the experimental demonstration of the SR-PLIF method both with controlled samples and with turbulent flows seeded with iodine vapor. Using controlled samples with two cameras, the spatial resolution in the best case was improved from 0.06 mm in the projections to 0.03 mm in the SR image, in terms of the spreading width of a sharp edge. With turbulent flows, an image sharpness measure was developed to quantify the spatial resolution, and SR reconstruction with two cameras can effectively improve the spatial resolution compared to the projections in terms of the sharpness measure.

Multimodality hard-x-ray imaging of a chromosome with nanoscale spatial resolution

DOE PAGES

Yan, Hanfei; Nazaretski, Evgeny; Lauer, Kenneth R.; ...

2016-02-05

Here, we developed a scanning hard x-ray microscope using a new class of x-ray nano-focusing optic called a multilayer Laue lens and imaged a chromosome with nanoscale spatial resolution. The combination of the hard x-ray's superior penetration power, high sensitivity to elemental composition, high spatial-resolution and quantitative analysis creates a unique tool with capabilities that other microscopy techniques cannot provide. Using this microscope, we simultaneously obtained absorption-, phase-, and fluorescence-contrast images of Pt-stained human chromosome samples. The high spatial-resolution of the microscope and its multi-modality imaging capabilities enabled us to observe the internal ultra-structures of a thick chromosome without sectioningmore » it.« less

Multimodality hard-x-ray imaging of a chromosome with nanoscale spatial resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Hanfei; Nazaretski, Evgeny; Lauer, Kenneth R.

Here, we developed a scanning hard x-ray microscope using a new class of x-ray nano-focusing optic called a multilayer Laue lens and imaged a chromosome with nanoscale spatial resolution. The combination of the hard x-ray's superior penetration power, high sensitivity to elemental composition, high spatial-resolution and quantitative analysis creates a unique tool with capabilities that other microscopy techniques cannot provide. Using this microscope, we simultaneously obtained absorption-, phase-, and fluorescence-contrast images of Pt-stained human chromosome samples. The high spatial-resolution of the microscope and its multi-modality imaging capabilities enabled us to observe the internal ultra-structures of a thick chromosome without sectioningmore » it.« less

Land cover mapping and change detection in urban watersheds using QuickBird high spatial resolution satellite imagery

NASA Astrophysics Data System (ADS)

Hester, David Barry

The objective of this research was to develop methods for urban land cover analysis using QuickBird high spatial resolution satellite imagery. Such imagery has emerged as a rich commercially available remote sensing data source and has enjoyed high-profile broadcast news media and Internet applications, but methods of quantitative analysis have not been thoroughly explored. The research described here consists of three studies focused on the use of pan-sharpened 61-cm spatial resolution QuickBird imagery, the spatial resolution of which is the highest of any commercial satellite. In the first study, a per-pixel land cover classification method is developed for use with this imagery. This method utilizes a per-pixel classification approach to generate an accurate six-category high spatial resolution land cover map of a developing suburban area. The primary objective of the second study was to develop an accurate land cover change detection method for use with QuickBird land cover products. This work presents an efficient fuzzy framework for transforming map uncertainty into accurate and meaningful high spatial resolution land cover change analysis. The third study described here is an urban planning application of the high spatial resolution QuickBird-based land cover product developed in the first study. This work both meaningfully connects this exciting new data source to urban watershed management and makes an important empirical contribution to the study of suburban watersheds. Its analysis of residential roads and driveways as well as retail parking lots sheds valuable light on the impact of transportation-related land use on the suburban landscape. Broadly, these studies provide new methods for using state-of-the-art remote sensing data to inform land cover analysis and urban planning. These methods are widely adaptable and produce land cover products that are both meaningful and accurate. As additional high spatial resolution satellites are launched and the cost of high resolution imagery continues to decline, this research makes an important contribution to this exciting era in the science of remote sensing.

Integrating Eddy Covariance, Penman-Monteith and METRIC based Evapotranspiration estimates to generate high resolution space-time ET over the Brazos River Basin

NASA Astrophysics Data System (ADS)

Mbabazi, D.; Mohanty, B.; Gaur, N.

2017-12-01

Evapotranspiration (ET) is an important component of the water and energy balance and accounts for 60 -70% of precipitation losses. However, accurate estimates of ET are difficult to quantify at varying spatial and temporal scales. Eddy covariance methods estimate ET at high temporal resolutions but without capturing the spatial variation in ET within its footprint. On the other hand, remote sensing methods using Landsat imagery provide ET with high spatial resolution but low temporal resolution (16 days). In this study, we used both eddy covariance and remote sensing methods to generate high space-time resolution ET. Daily, monthly and seasonal ET estimates were obtained using the eddy covariance (EC) method, Penman-Monteith (PM) and Mapping Evapotranspiration with Internalized Calibration (METRIC) models to determine cotton and native prairie ET dynamics in the Brazos river basin characterized by varying hydro-climatic and geological gradients. Daily estimates of spatially distributed ET (30 m resolution) were generated using spatial autocorrelation and temporal interpolations between the EC flux variable footprints and METRIC ET for the 2016 and 2017 growing seasons. A comparison of the 2016 and 2017 preliminary daily ET estimates showed similar ET dynamics/trends among the EC, PM and METRIC methods, and 5-20% differences in seasonal ET estimates. This study will improve the spatial estimates of EC ET and temporal resolution of satellite derived ET thus providing better ET data for water use management.

Effects of finite hot-wire spatial resolution on turbulence statistics and velocity spectra in a round turbulent free jet

NASA Astrophysics Data System (ADS)

Sadeghi, Hamed; Lavoie, Philippe; Pollard, Andrew

2018-03-01

The effect of finite hot-wire spatial resolution on turbulence statistics and velocity spectra in a round turbulent free jet is investigated. To quantify spatial resolution effects, measurements were taken using a nano-scale thermal anemometry probe (NSTAP) and compared to results from conventional hot-wires with sensing lengths of l=0.5 and 1 mm. The NSTAP has a sensing length significantly smaller than the Kolmogorov length scale η for the present experimental conditions, whereas the sensing lengths for the conventional probes are larger than η. The spatial resolution is found to have a significant impact on the dissipation both on and off the jet centreline with the NSTAP results exceeding those obtained from the conventional probes. The resolution effects along the jet centreline are adequately predicted using a Wyngaard-type spectral technique (Wyngaard in J Sci Instr 1(2):1105-1108,1968), but additional attenuation on the measured turbulence quantities are observed off the centreline. The magnitude of this attenuation is a function of both the ratio of wire length to Kolmogorov length scale and the magnitude of the shear. The effect of spatial resolution is noted to have an impact on the power-law decay parameters for the turbulent kinetic energy that is computed. The effect of spatial filtering on the streamwise dissipation energy spectra is also considered. Empirical functions are proposed to estimate the effect of finite resolution, which take into account the mean shear.

Spatial resolution versus contrast trade-off enhancement in high-resolution surface plasmon resonance imaging (SPRI) by metal surface nanostructure design.

PubMed

Banville, Frederic A; Moreau, Julien; Sarkar, Mitradeep; Besbes, Mondher; Canva, Michael; Charette, Paul G

2018-04-16

Surface plasmon resonance imaging (SPRI) is an optical near-field method used for mapping the spatial distribution of chemical/physical perturbations above a metal surface without exogenous labeling. Currently, the majority of SPRI systems are used in microarray biosensing, requiring only modest spatial resolution. There is increasing interest in applying SPRI for label-free near-field imaging of biological cells to study cell/surface interactions. However, the required resolution (sub-µm) greatly exceeds what current systems can deliver. Indeed, the attenuation length of surface plasmon polaritons (SPP) severely limits resolution along one axis, typically to tens of µm. Strategies to date for improving spatial resolution result in a commensurate deterioration in other imaging parameters. Unlike the smooth metal surfaces used in SPRI that support purely propagating surface modes, nanostructured metal surfaces support "hybrid" SPP modes that share attributes from both propagating and localized modes. We show that these hybrid modes are especially well-suited to high-resolution imaging and demonstrate how the nanostructure geometry can be designed to achieve sub-µm resolution while mitigating the imaging parameter trade-off according to an application-specific optimum.

Raman, AFM and SNOM high resolution imaging of carotene crystals in a model carrot cell system.

PubMed

Rygula, Anna; Oleszkiewicz, Tomasz; Grzebelus, Ewa; Pacia, Marta Z; Baranska, Malgorzata; Baranski, Rafal

2018-05-15

Three non-destructive and complementary techniques, Raman imaging, Atomic Force Microscopy and Scanning Near-field Optical Microscopy were used simultaneously to show for the first time chemical and structural differences of carotenoid crystals. Spectroscopic and microscopic scanning probe measurements were applied to the released crystals or to crystals accumulated in a unique, carotenoids rich callus tissue growing in vitro that is considered as a new model system for plant carotenoid research. Three distinct morphological crystal types of various carotenoid composition were identified, a needle-like, rhomboidal and helical. Raman imaging using 532 and 488 nm excitation lines provided evidence that the needle-like and rhomboidal crystals had similar carotenoid composition and that they were composed mainly of β-carotene accompanied by α-carotene. However, the presence of α-carotene was not identified in the helical crystals, which had the characteristic spatial structure. AFM measurements of crystals identified by Raman imaging revealed the crystal topography and showed the needle-like and rhomboidal crystals were planar but they differed in all three dimensions. Combining SNOM and Raman imaging enabled indication of carotenoid rich structures and visualised their distribution in the cell. The morphology of identified subcellular structures was characteristic for crystalline, membraneous and tubular chromoplasts that are plant organelles responsible for carotenoid accumulation in cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Toward unstained cytology and complete blood counts at the point of care (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zuluaga, Andres F.; Pierce, Mark C.; MacAulay, Calum E.

2017-02-01

Cytology tests, whether performed on body fluids, aspirates, or scrapings are commonly used to detect, diagnose, and monitor a wide variety of health conditions. Complete blood counts (CBCs) quantify the number of red and white blood cells in a blood volume, as well as the different types of white blood cells. There is a critical unmet need for an instrument that can perform CBCs at the point of care (POC), and there is currently no product in the US that can perform this test at the bedside. We have developed a system that is capable of tomographic images with sub-cellular resolution with consumer-grade broadband (LED) sources and CMOS detectors suitable for POC implementation of CBC tests. The systems consists of cascaded static Michelson and Sagnac interferometers that map phase (encoding depth) and a transverse spatial dimension onto a two-dimensional output plane. Our approach requires a 5 microliter sample, can be performed in 5 minutes or less, and does not require staining or other processing as it relies on intrinsic contrast. We will show results directly imaging and differentiating unstained blood cells using supercontinuum fiber lasers and LEDs as sources and CMOS cameras as sensors. We will also lay out the follow up steps needed, including image segmentation, analysis and classification, to verify performance and advance toward CBCs that can be performed bedside and do not require CLIA-certified laboratories.

Detection of tumorigenesis in urinary bladder with optical coherence tomography: optical characterization of morphological changes

NASA Astrophysics Data System (ADS)

Xie, T.-Q.; Zeidel, M. L.; Pan, Yingtian

2002-12-01

Most transitional cell tumorigenesis involves three stages of subcellular morphological changes: hyperplasia, dysplasia and neoplasia. Previous studies demonstrated that owing to its high spatial resolution and intermediate penetration depth, current OCT technology including endoscopic OCT could delineate the urothelium, submucosa and the upper muscular layers of the bladder wall. In this paper, we will discuss the sensitivity and limitations of OCT in diagnosing and staging bladder cancer. Based on histomorphometric evaluations of nuclear morphology, we modeled the resultant backscattering changes and the characteristic changes in OCT image contrast. In the theoretical modeling, we assumed that nuclei were the primary sources of scattering and were uniformly distributed in the uroepithelium, and compared with the results of the corresponding prior OCT measurements. According to our theoretical modeling, normal bladder shows a thin, uniform and low scattering urothelium, so does an inflammatory lesion except thickening in the submucosa. Compared with a normal bladder, a hyperplastic lesion exhibits a thickened, low scattering urothelium whereas a neoplastic lesion shows a thickened urothelium with increased backscattering. These results support our previous animal study that OCT has the potential to differentiate inflammation, hyperplasia, and neoplasia by quantifying the changes in urothelial thickening and backscattering. The results also suggest that OCT might not have the sensitivity to differentiate the subtle morphological changes between hyperplasia and dysplasia based on minor backscattering differences.

Inverse problems biomechanical imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Oberai, Assad A.

2016-03-01

It is now well recognized that a host of imaging modalities (a list that includes Ultrasound, MRI, Optical Coherence Tomography, and optical microscopy) can be used to "watch" tissue as it deforms in response to an internal or external excitation. The result is a detailed map of the deformation field in the interior of the tissue. This deformation field can be used in conjunction with a material mechanical response to determine the spatial distribution of material properties of the tissue by solving an inverse problem. Images of material properties thus obtained can be used to quantify the health of the tissue. Recently, they have been used to detect, diagnose and monitor cancerous lesions, detect vulnerable plaque in arteries, diagnose liver cirrhosis, and possibly detect the onset of Alzheimer's disease. In this talk I will describe the mathematical and computational aspects of solving this class of inverse problems, and their applications in biology and medicine. In particular, I will discuss the well-posedness of these problems and quantify the amount of displacement data necessary to obtain a unique property distribution. I will describe an efficient algorithm for solving the resulting inverse problem. I will also describe some recent developments based on Bayesian inference in estimating the variance in the estimates of material properties. I will conclude with the applications of these techniques in diagnosing breast cancer and in characterizing the mechanical properties of cells with sub-cellular resolution.

Detection of tumorigenesis in urinary bladder with optical coherence tomography: optical characterization of morphological changes.

PubMed

Xie, T; Zeidel, M; Pan, Yingtian

2002-12-02

Most transitional cell tumorigenesis involves three stages of subcellular morphological changes: hyperplasia, dysplasia and neoplasia. Previous studies demonstrated that owing to its high spatial resolution and intermediate penetration depth, current OCT technology including endoscopic OCT could delineate the urothelium, submucosa and the upper muscular layers of the bladder wall. In this paper, we will discuss the sensitivity and limitations of OCT in diagnosing and staging bladder cancer. Based on histomorphometric evaluations of nuclear morphology, we modeled the resultant backscattering changes and the characteristic changes in OCT image contrast. In the theoretical modeling, we assumed that nuclei were the primary sources of scattering and were uniformly distributed in the uroepithelium, and compared with the results of the corresponding prior OCT measurements. According to our theoretical modeling, normal bladder shows a thin, uniform and low scattering urothelium, so does an inflammatory lesion except thickening in the submucosa. Compared with a normal bladder, a hyperplastic lesion exhibits a thickened, low scattering urothelium whereas a neoplastic lesion shows a thickened urothelium with increased backscattering. These results support our previous animal study that OCT has the potential to differentiate inflammation, hyperplasia, and neoplasia by quantifying the changes in urothelial thickening and backscattering. The results also suggest that OCT might not have the sensitivity to differentiate the subtle morphological changes between hyperplasia and dysplasia based on minor backscattering differences.

Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution.

PubMed

Lillis, Kyle P; Eng, Alfred; White, John A; Mertz, Jerome

2008-07-30

We describe a simple two-photon fluorescence imaging strategy, called targeted path scanning (TPS), to monitor the dynamics of spatially extended neuronal networks with high spatiotemporal resolution. Our strategy combines the advantages of mirror-based scanning, minimized dead time, ease of implementation, and compatibility with high-resolution low-magnification objectives. To demonstrate the performance of TPS, we monitor the calcium dynamics distributed across an entire juvenile rat hippocampus (>1.5mm), at scan rates of 100 Hz, with single cell resolution and single action potential sensitivity. Our strategy for fast, efficient two-photon microscopy over spatially extended regions provides a particularly attractive solution for monitoring neuronal population activity in thick tissue, without sacrificing the signal-to-noise ratio or high spatial resolution associated with standard two-photon microscopy. Finally, we provide the code to make our technique generally available.

High Speed Computational Ghost Imaging via Spatial Sweeping

NASA Astrophysics Data System (ADS)

Wang, Yuwang; Liu, Yang; Suo, Jinli; Situ, Guohai; Qiao, Chang; Dai, Qionghai

2017-03-01

Computational ghost imaging (CGI) achieves single-pixel imaging by using a Spatial Light Modulator (SLM) to generate structured illuminations for spatially resolved information encoding. The imaging speed of CGI is limited by the modulation frequency of available SLMs, and sets back its practical applications. This paper proposes to bypass this limitation by trading off SLM’s redundant spatial resolution for multiplication of the modulation frequency. Specifically, a pair of galvanic mirrors sweeping across the high resolution SLM multiply the modulation frequency within the spatial resolution gap between SLM and the final reconstruction. A proof-of-principle setup with two middle end galvanic mirrors achieves ghost imaging as fast as 42 Hz at 80 × 80-pixel resolution, 5 times faster than state-of-the-arts, and holds potential for one magnitude further multiplication by hardware upgrading. Our approach brings a significant improvement in the imaging speed of ghost imaging and pushes ghost imaging towards practical applications.

Edge-Preserving Image Smoothing Constraint in Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) of Hyperspectral Data.

PubMed

Hugelier, Siewert; Vitale, Raffaele; Ruckebusch, Cyril

2018-03-01

This article explores smoothing with edge-preserving properties as a spatial constraint for the resolution of hyperspectral images with multivariate curve resolution-alternating least squares (MCR-ALS). For each constrained component image (distribution map), irrelevant spatial details and noise are smoothed applying an L 1 - or L 0 -norm penalized least squares regression, highlighting in this way big changes in intensity of adjacent pixels. The feasibility of the constraint is demonstrated on three different case studies, in which the objects under investigation are spatially clearly defined, but have significant spectral overlap. This spectral overlap is detrimental for obtaining a good resolution and additional spatial information should be provided. The final results show that the spatial constraint enables better image (map) abstraction, artifact removal, and better interpretation of the results obtained, compared to a classical MCR-ALS analysis of hyperspectral images.

Implications of sensor design for coral reef detection: Upscaling ground hyperspectral imagery in spatial and spectral scales

NASA Astrophysics Data System (ADS)

Caras, Tamir; Hedley, John; Karnieli, Arnon

2017-12-01

Remote sensing offers a potential tool for large scale environmental surveying and monitoring. However, remote observations of coral reefs are difficult especially due to the spatial and spectral complexity of the target compared to sensor specifications as well as the environmental implications of the water medium above. The development of sensors is driven by technological advances and the desired products. Currently, spaceborne systems are technologically limited to a choice between high spectral resolution and high spatial resolution, but not both. The current study explores the dilemma of whether future sensor design for marine monitoring should prioritise on improving their spatial or spectral resolution. To address this question, a spatially and spectrally resampled ground-level hyperspectral image was used to test two classification elements: (1) how the tradeoff between spatial and spectral resolutions affects classification; and (2) how a noise reduction by majority filter might improve classification accuracy. The studied reef, in the Gulf of Aqaba (Eilat), Israel, is heterogeneous and complex so the local substrate patches are generally finer than currently available imagery. Therefore, the tested spatial resolution was broadly divided into four scale categories from five millimeters to one meter. Spectral resolution resampling aimed to mimic currently available and forthcoming spaceborne sensors such as (1) Environmental Mapping and Analysis Program (EnMAP) that is characterized by 25 bands of 6.5 nm width; (2) VENμS with 12 narrow bands; and (3) the WorldView series with broadband multispectral resolution. Results suggest that spatial resolution should generally be prioritized for coral reef classification because the finer spatial scale tested (pixel size < 0.1 m) may compensate for some low spectral resolution drawbacks. In this regard, it is shown that the post-classification majority filtering substantially improves the accuracy of all pixel sizes up to the point where the kernel size reaches the average unit size (pixel < 0.25 m). However, careful investigation as to the effect of band distribution and choice could improve the sensor suitability for the marine environment task. This in mind, while the focus in this study was on the technologically limited spaceborne design, aerial sensors may presently provide an opportunity to implement the suggested setup.

Inter-kingdom prediction certainty evaluation of protein subcellular localization tools: microbial pathogenesis approach for deciphering host microbe interaction.

PubMed

Khan, Abdul Arif; Khan, Zakir; Kalam, Mohd Abul; Khan, Azmat Ali

2018-01-01

Microbial pathogenesis involves several aspects of host-pathogen interactions, including microbial proteins targeting host subcellular compartments and subsequent effects on host physiology. Such studies are supported by experimental data, but recent detection of bacterial proteins localization through computational eukaryotic subcellular protein targeting prediction tools has also come into practice. We evaluated inter-kingdom prediction certainty of these tools. The bacterial proteins experimentally known to target host subcellular compartments were predicted with eukaryotic subcellular targeting prediction tools, and prediction certainty was assessed. The results indicate that these tools alone are not sufficient for inter-kingdom protein targeting prediction. The correct prediction of pathogen's protein subcellular targeting depends on several factors, including presence of localization signal, transmembrane domain and molecular weight, etc., in addition to approach for subcellular targeting prediction. The detection of protein targeting in endomembrane system is comparatively difficult, as the proteins in this location are channelized to different compartments. In addition, the high specificity of training data set also creates low inter-kingdom prediction accuracy. Current data can help to suggest strategy for correct prediction of bacterial protein's subcellular localization in host cell. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Super-resolution reconstruction of diffusion parameters from diffusion-weighted images with different slice orientations.

PubMed

Van Steenkiste, Gwendolyn; Jeurissen, Ben; Veraart, Jelle; den Dekker, Arnold J; Parizel, Paul M; Poot, Dirk H J; Sijbers, Jan

2016-01-01

Diffusion MRI is hampered by long acquisition times, low spatial resolution, and a low signal-to-noise ratio. Recently, methods have been proposed to improve the trade-off between spatial resolution, signal-to-noise ratio, and acquisition time of diffusion-weighted images via super-resolution reconstruction (SRR) techniques. However, during the reconstruction, these SRR methods neglect the q-space relation between the different diffusion-weighted images. An SRR method that includes a diffusion model and directly reconstructs high resolution diffusion parameters from a set of low resolution diffusion-weighted images was proposed. Our method allows an arbitrary combination of diffusion gradient directions and slice orientations for the low resolution diffusion-weighted images, optimally samples the q- and k-space, and performs motion correction with b-matrix rotation. Experiments with synthetic data and in vivo human brain data show an increase of spatial resolution of the diffusion parameters, while preserving a high signal-to-noise ratio and low scan time. Moreover, the proposed SRR method outperforms the previous methods in terms of the root-mean-square error. The proposed SRR method substantially increases the spatial resolution of MRI that can be obtained in a clinically feasible scan time. © 2015 Wiley Periodicals, Inc.

Camera system resolution and its influence on digital image correlation

DOE PAGES

Reu, Phillip L.; Sweatt, William; Miller, Timothy; ...

2014-09-21

Digital image correlation (DIC) uses images from a camera and lens system to make quantitative measurements of the shape, displacement, and strain of test objects. This increasingly popular method has had little research on the influence of the imaging system resolution on the DIC results. This paper investigates the entire imaging system and studies how both the camera and lens resolution influence the DIC results as a function of the system Modulation Transfer Function (MTF). It will show that when making spatial resolution decisions (including speckle size) the resolution limiting component should be considered. A consequence of the loss ofmore » spatial resolution is that the DIC uncertainties will be increased. This is demonstrated using both synthetic and experimental images with varying resolution. The loss of image resolution and DIC accuracy can be compensated for by increasing the subset size, or better, by increasing the speckle size. The speckle-size and spatial resolution are now a function of the lens resolution rather than the more typical assumption of the pixel size. The study will demonstrate the tradeoffs associated with limited lens resolution.« less

Epidermal Growth Factor Receptor mediated cellular and subcellular targeted delivery of Iron oxide core-Titanium dioxide shell nanoparticles

NASA Astrophysics Data System (ADS)

Yuan, Ye

TiO2 nanomaterials can carry a multitude of therapeutic and diagnostic agents and the semiconductor properties of TiO2 allow for the production of cytotoxic reactive oxygen species following photoactivation. However, the delivery of these nanomaterials to specific cancer cells and specific subcellular organelles within these cells can have a substantial impact on the efficacy and safety of TiO2 nanoparticle therapeutics. Targeting cell surface receptors that are overexpressed by cancer cells is one strategy to improve the specificity of nanoparticle delivery. Therefore we decided to target the Epidermal Growth Factor Receptor (EGFR) because ligand- binding induces rapid receptor endocytosis and ligand-bound EGFR can translocate to the nucleus of cancer cells. To create NPs that can bind EGFR, we identified a peptide derived from the B-loop of Epidermal Growth Factor (EGF) that has been shown to bind and activate EGFR and conjugated it to the surface of Fe3O4 core-TiO2 shell NPs to produce B-loop NCs. We then devised a pulldown assay to show that B-loop NCs, but not bare NPs or NCs carrying a scrambled B-loop peptide, can bind and extract EGFR from HeLa cell protein extracts. Interestingly, B-loop NCs can also pulldown importin-beta, a protein that can transport EGFR to the nucleus. Furthermore, we used flow cytometry and fluorescently labeled NPs to show that B-loop peptides can significantly improve the internalization of NPs by EGFR-expressing HeLa cells. We determined that B-loop NCs can bind EGFR on the membrane of HeLa cells and that these NCs can be transported to the nucleus, by using a combination of confocal microscopy and X-ray Fluorescence Microscopy (XFM) to indirectly and directly track the subcellular distribution of NCs. Finally, we demonstrate how the Bionanoprobe, a novel high-resolution XFM apparatus that can scan whole-mounted, frozen-hydrated cells at multiple angles can be used to verify the subcellular distribution of B-loop NCs.

Attenuated total reflection-Fourier transform infrared imaging of large areas using inverted prism crystals and combining imaging and mapping.

PubMed

Chan, K L Andrew; Kazarian, Sergei G

2008-10-01

Attenuated total reflection-Fourier transform infrared (ATR-FT-IR) imaging is a very useful tool for capturing chemical images of various materials due to the simple sample preparation and the ability to measure wet samples or samples in an aqueous environment. However, the size of the array detector used for image acquisition is often limited and there is usually a trade off between spatial resolution and the field of view (FOV). The combination of mapping and imaging can be used to acquire images with a larger FOV without sacrificing spatial resolution. Previous attempts have demonstrated this using an infrared microscope and a Germanium hemispherical ATR crystal to achieve images of up to 2.5 mm x 2.5 mm but with varying spatial resolution and depth of penetration across the imaged area. In this paper, we demonstrate a combination of mapping and imaging with a different approach using an external optics housing for large ATR accessories and inverted ATR prisms to achieve ATR-FT-IR images with a large FOV and reasonable spatial resolution. The results have shown that a FOV of 10 mm x 14 mm can be obtained with a spatial resolution of approximately 40-60 microm when using an accessory that gives no magnification. A FOV of 1.3 mm x 1.3 mm can be obtained with spatial resolution of approximately 15-20 microm when using a diamond ATR imaging accessory with 4x magnification. No significant change in image quality such as spatial resolution or depth of penetration has been observed across the whole FOV with this method and the measurement time was approximately 15 minutes for an image consisting of 16 image tiles.

Evaluating the effect of remote sensing image spatial resolution on soil exchangeable potassium prediction models in smallholder farm settings.

PubMed

Xu, Yiming; Smith, Scot E; Grunwald, Sabine; Abd-Elrahman, Amr; Wani, Suhas P

2017-09-15

Major end users of Digital Soil Mapping (DSM) such as policy makers and agricultural extension workers are faced with choosing the appropriate remote sensing data. The objective of this research is to analyze the spatial resolution effects of different remote sensing images on soil prediction models in two smallholder farms in Southern India called Kothapally (Telangana State), and Masuti (Karnataka State), and provide empirical guidelines to choose the appropriate remote sensing images in DSM. Bayesian kriging (BK) was utilized to characterize the spatial pattern of exchangeable potassium (K ex ) in the topsoil (0-15 cm) at different spatial resolutions by incorporating spectral indices from Landsat 8 (30 m), RapidEye (5 m), and WorldView-2/GeoEye-1/Pleiades-1A images (2 m). Some spectral indices such as band reflectances, band ratios, Crust Index and Atmospherically Resistant Vegetation Index from multiple images showed relatively strong correlations with soil K ex in two study areas. The research also suggested that fine spatial resolution WorldView-2/GeoEye-1/Pleiades-1A-based and RapidEye-based soil prediction models would not necessarily have higher prediction performance than coarse spatial resolution Landsat 8-based soil prediction models. The end users of DSM in smallholder farm settings need select the appropriate spectral indices and consider different factors such as the spatial resolution, band width, spectral resolution, temporal frequency, cost, and processing time of different remote sensing images. Overall, remote sensing-based Digital Soil Mapping has potential to be promoted to smallholder farm settings all over the world and help smallholder farmers implement sustainable and field-specific soil nutrient management scheme. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Use of Coarse Resolution Satellite Imagery to Predict Human Puumala Virus Epidemics in Sweden.

DTIC Science & Technology

1992-09-11

the adverse effects on NDVI data quality can occur in both the spatial and temporal dimension. In other words, a specific pixel value recorded in...are compared to the land-oriented systems.22 On the other hand, the very course spatial resolution has the advantage of greatly reducing the volume...necessary on the scale of individual fields, in which case LANDSAT-TM has higher spatial resolution ; and secondly, when specific

Investigation of noise properties in grating-based x-ray phase tomography with reverse projection method

NASA Astrophysics Data System (ADS)

Bao, Yuan; Wang, Yan; Gao, Kun; Wang, Zhi-Li; Zhu, Pei-Ping; Wu, Zi-Yu

2015-10-01

The relationship between noise variance and spatial resolution in grating-based x-ray phase computed tomography (PCT) imaging is investigated with reverse projection extraction method, and the noise variances of the reconstructed absorption coefficient and refractive index decrement are compared. For the differential phase contrast method, the noise variance in the differential projection images follows the same inverse-square law with spatial resolution as in conventional absorption-based x-ray imaging projections. However, both theoretical analysis and simulations demonstrate that in PCT the noise variance of the reconstructed refractive index decrement scales with spatial resolution follows an inverse linear relationship at fixed slice thickness, while the noise variance of the reconstructed absorption coefficient conforms with the inverse cubic law. The results indicate that, for the same noise variance level, PCT imaging may enable higher spatial resolution than conventional absorption computed tomography (ACT), while ACT benefits more from degraded spatial resolution. This could be a useful guidance in imaging the inner structure of the sample in higher spatial resolution. Project supported by the National Basic Research Program of China (Grant No. 2012CB825800), the Science Fund for Creative Research Groups, the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant Nos. KJCX2-YW-N42 and Y4545320Y2), the National Natural Science Foundation of China (Grant Nos. 11475170, 11205157, 11305173, 11205189, 11375225, 11321503, 11179004, and U1332109).