Quantitative super-resolution single molecule microscopy dataset of YFP-tagged growth factor receptors.

PubMed

Lukeš, Tomáš; Pospíšil, Jakub; Fliegel, Karel; Lasser, Theo; Hagen, Guy M

2018-03-01

Super-resolution single molecule localization microscopy (SMLM) is a method for achieving resolution beyond the classical limit in optical microscopes (approx. 200 nm laterally). Yellow fluorescent protein (YFP) has been used for super-resolution single molecule localization microscopy, but less frequently than other fluorescent probes. Working with YFP in SMLM is a challenge because a lower number of photons are emitted per molecule compared with organic dyes, which are more commonly used. Publically available experimental data can facilitate development of new data analysis algorithms. Four complete, freely available single molecule super-resolution microscopy datasets on YFP-tagged growth factor receptors expressed in a human cell line are presented, including both raw and analyzed data. We report methods for sample preparation, for data acquisition, and for data analysis, as well as examples of the acquired images. We also analyzed the SMLM datasets using a different method: super-resolution optical fluctuation imaging (SOFI). The 2 modes of analysis offer complementary information about the sample. A fifth single molecule super-resolution microscopy dataset acquired with the dye Alexa 532 is included for comparison purposes. This dataset has potential for extensive reuse. Complete raw data from SMLM experiments have typically not been published. The YFP data exhibit low signal-to-noise ratios, making data analysis a challenge. These datasets will be useful to investigators developing their own algorithms for SMLM, SOFI, and related methods. The data will also be useful for researchers investigating growth factor receptors such as ErbB3.

Aberrations and adaptive optics in super-resolution microscopy.

PubMed

Booth, Martin; Andrade, Débora; Burke, Daniel; Patton, Brian; Zurauskas, Mantas

2015-08-01

As one of the most powerful tools in the biological investigation of cellular structures and dynamic processes, fluorescence microscopy has undergone extraordinary developments in the past decades. The advent of super-resolution techniques has enabled fluorescence microscopy - or rather nanoscopy - to achieve nanoscale resolution in living specimens and unravelled the interior of cells with unprecedented detail. The methods employed in this expanding field of microscopy, however, are especially prone to the detrimental effects of optical aberrations. In this review, we discuss how super-resolution microscopy techniques based upon single-molecule switching, stimulated emission depletion and structured illumination each suffer from aberrations in different ways that are dependent upon intrinsic technical aspects. We discuss the use of adaptive optics as an effective means to overcome this problem. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy.

Aberrations and adaptive optics in super-resolution microscopy

PubMed Central

Booth, Martin; Andrade, Débora; Burke, Daniel; Patton, Brian; Zurauskas, Mantas

2015-01-01

As one of the most powerful tools in the biological investigation of cellular structures and dynamic processes, fluorescence microscopy has undergone extraordinary developments in the past decades. The advent of super-resolution techniques has enabled fluorescence microscopy – or rather nanoscopy – to achieve nanoscale resolution in living specimens and unravelled the interior of cells with unprecedented detail. The methods employed in this expanding field of microscopy, however, are especially prone to the detrimental effects of optical aberrations. In this review, we discuss how super-resolution microscopy techniques based upon single-molecule switching, stimulated emission depletion and structured illumination each suffer from aberrations in different ways that are dependent upon intrinsic technical aspects. We discuss the use of adaptive optics as an effective means to overcome this problem. PMID:26124194

Super resolution imaging of HER2 gene amplification

NASA Astrophysics Data System (ADS)

Okada, Masaya; Kubo, Takuya; Masumoto, Kanako; Iwanaga, Shigeki

2016-02-01

HER2 positive breast cancer is currently examined by counting HER2 genes using fluorescence in situ hybridization (FISH)-stained breast carcinoma samples. In this research, two-dimensional super resolution fluorescence microscopy based on stochastic optical reconstruction microscopy (STORM), with a spatial resolution of approximately 20 nm in the lateral direction, was used to more precisely distinguish and count HER2 genes in a FISH-stained tissue section. Furthermore, by introducing double-helix point spread function (DH-PSF), an optical phase modulation technique, to super resolution microscopy, three-dimensional images were obtained of HER2 in a breast carcinoma sample approximately 4 μm thick.

A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

PubMed Central

Tam, Johnny; Cordier, Guillaume Alan; Bálint, Štefan; Sandoval Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike

2014-01-01

Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images. PMID:25545548

Deep learning massively accelerates super-resolution localization microscopy.

PubMed

Ouyang, Wei; Aristov, Andrey; Lelek, Mickaël; Hao, Xian; Zimmer, Christophe

2018-06-01

The speed of super-resolution microscopy methods based on single-molecule localization, for example, PALM and STORM, is limited by the need to record many thousands of frames with a small number of observed molecules in each. Here, we present ANNA-PALM, a computational strategy that uses artificial neural networks to reconstruct super-resolution views from sparse, rapidly acquired localization images and/or widefield images. Simulations and experimental imaging of microtubules, nuclear pores, and mitochondria show that high-quality, super-resolution images can be reconstructed from up to two orders of magnitude fewer frames than usually needed, without compromising spatial resolution. Super-resolution reconstructions are even possible from widefield images alone, though adding localization data improves image quality. We demonstrate super-resolution imaging of >1,000 fields of view containing >1,000 cells in ∼3 h, yielding an image spanning spatial scales from ∼20 nm to ∼2 mm. The drastic reduction in acquisition time and sample irradiation afforded by ANNA-PALM enables faster and gentler high-throughput and live-cell super-resolution imaging.

Nano-scale measurement of biomolecules by optical microscopy and semiconductor nanoparticles

PubMed Central

Ichimura, Taro; Jin, Takashi; Fujita, Hideaki; Higuchi, Hideo; Watanabe, Tomonobu M.

2014-01-01

Over the past decade, great developments in optical microscopy have made this technology increasingly compatible with biological studies. Fluorescence microscopy has especially contributed to investigating the dynamic behaviors of live specimens and can now resolve objects with nanometer precision and resolution due to super-resolution imaging. Additionally, single particle tracking provides information on the dynamics of individual proteins at the nanometer scale both in vitro and in cells. Complementing advances in microscopy technologies has been the development of fluorescent probes. The quantum dot, a semi-conductor fluorescent nanoparticle, is particularly suitable for single particle tracking and super-resolution imaging. This article overviews the principles of single particle tracking and super resolution along with describing their application to the nanometer measurement/observation of biological systems when combined with quantum dot technologies. PMID:25120488

Conical diffraction as a versatile building block to implement new imaging modalities for superresolution in fluorescence microscopy

NASA Astrophysics Data System (ADS)

Fallet, Clément; Caron, Julien; Oddos, Stephane; Tinevez, Jean-Yves; Moisan, Lionel; Sirat, Gabriel Y.; Braitbart, Philippe O.; Shorte, Spencer L.

2014-08-01

We present a new technology for super-resolution fluorescence imaging, based on conical diffraction. Conical diffraction is a linear, singular phenomenon taking place when a polarized beam is diffracted through a biaxial crystal. The illumination patterns generated by conical diffraction are more compact than the classical Gaussian beam; we use them to generate a super-resolution imaging modality. Conical Diffraction Microscopy (CODIM) resolution enhancement can be achieved with any type of objective on any kind of sample preparation and standard fluorophores. Conical diffraction can be used in multiple fashion to create new and disruptive technologies for super-resolution microscopy. This paper will focus on the first one that has been implemented and give a glimpse at what the future of microscopy using conical diffraction could be.

Nanoscopy for nanoscience: how super-resolution microscopy extends imaging for nanotechnology.

PubMed

Johnson, Sam A

2015-01-01

Imaging methods have presented scientists with powerful means of investigation for centuries. The ability to resolve structures using light microscopes is though limited to around 200 nm. Fluorescence-based super-resolution light microscopy techniques of several principles and methods have emerged in recent years and offer great potential to extend the capabilities of microscopy. This resolution improvement is especially promising for nanoscience where the imaging of nanoscale structures is inherently restricted by the resolution limit of standard forms of light microscopy. Resolution can be improved by several distinct approaches including structured illumination microscopy, stimulated emission depletion, and single-molecule positioning methods such as photoactivated localization microscopy and stochastic optical reconstruction microscopy and several derivative variations of each of these. These methods involve substantial differences in the resolutions achievable in the different axes, speed of acquisition, compatibility with different labels, ease of use, hardware complexity, and compatibility with live biological samples. The field of super-resolution imaging and its application to nanotechnology is relatively new and still rapidly developing. An overview of how these methods may be used with nanomaterials is presented with some examples of pioneering uses of these approaches. © 2014 Wiley Periodicals, Inc.

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.

Localization-based super-resolution imaging of cellular structures.

PubMed

Kanchanawong, Pakorn; Waterman, Clare M

2013-01-01

Fluorescence microscopy allows direct visualization of fluorescently tagged proteins within cells. However, the spatial resolution of conventional fluorescence microscopes is limited by diffraction to ~250 nm, prompting the development of super-resolution microscopy which offers resolution approaching the scale of single proteins, i.e., ~20 nm. Here, we describe protocols for single molecule localization-based super-resolution imaging, using focal adhesion proteins as an example and employing either photoswitchable fluorophores or photoactivatable fluorescent proteins. These protocols should also be easily adaptable to imaging a broad array of macromolecular assemblies in cells whose components can be fluorescently tagged and assemble into high density structures.

Dances with Membranes: Breakthroughs from Super-resolution Imaging

PubMed Central

Curthoys, Nikki M.; Parent, Matthew; Mlodzianoski, Michael; Nelson, Andrew J.; Lilieholm, Jennifer; Butler, Michael B.; Valles, Matthew; Hess, Samuel T.

2017-01-01

Biological membrane organization mediates numerous cellular functions and has also been connected with an immense number of human diseases. However, until recently, experimental methodologies have been unable to directly visualize the nanoscale details of biological membranes, particularly in intact living cells. Numerous models explaining membrane organization have been proposed, but testing those models has required indirect methods; the desire to directly image proteins and lipids in living cell membranes is a strong motivation for the advancement of technology. The development of super-resolution microscopy has provided powerful tools for quantification of membrane organization at the level of individual proteins and lipids, and many of these tools are compatible with living cells. Previously inaccessible questions are now being addressed, and the field of membrane biology is developing rapidly. This chapter discusses how the development of super-resolution microscopy has led to fundamental advances in the field of biological membrane organization. We summarize the history and some models explaining how proteins are organized in cell membranes, and give an overview of various super-resolution techniques and methods of quantifying super-resolution data. We discuss the application of super-resolution techniques to membrane biology in general, and also with specific reference to the fields of actin and actin-binding proteins, virus infection, mitochondria, immune cell biology, and phosphoinositide signaling. Finally, we present our hopes and expectations for the future of super-resolution microscopy in the field of membrane biology. PMID:26015281

Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy.

PubMed

Traenkle, Bjoern; Rothbauer, Ulrich

2017-01-01

Single-domain antibodies (sdAbs) have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies) have become versatile tools to study dynamics of endogenous proteins in living cells. Additionally, sdAbs conjugated to organic dyes were shown to label cellular structures with high density and minimal fluorophore displacement making them highly attractive probes for super-resolution microscopy. Here, we review recent advances of the chromobody technology to visualize localization and dynamics of cellular targets and the application of chromobody-based cell models for compound screening. Acknowledging the emerging importance of super-resolution microscopy in cell biology, we further discuss advantages and challenges of sdAbs for this technology.

Differential localization of SAP102 and PSD-95 is revealed in hippocampal spines using super-resolution light microscopy.

PubMed

Zheng, Chan-Ying; Wang, Ya-Xia; Kachar, Bechara; Petralia, Ronald S

2011-01-01

Synapse-associated protein 102 (SAP102) and postsynaptic density 95 (PSD-95) are two major cytoskeleton proteins in the postsynaptic density (PSD). Both of them belong to the membrane-associated guanylate kinase (MAGUK) family, which clusters and anchors glutamate receptors and other proteins at synapses. In our previous study, we found that SAP102 and PSD-95 have different distributions, using combined light/electron microscopy (LM/EM) methods.1 Here, we double labeled endogenous SAP102 and PSD-95 in mature hippocampal neurons, and then took images by two different kinds of super resolution microscopy-Stimulated Emission Depletion microscopy (STED) and DeltaVision OMX 3D super resolution microscopy. We found that our 2D and 3D super resolution data were consistent with our previous LM/EM data, showing significant differences in the localization of SAP102 and PSD-95 in spines: SAP102 is distributed in both the PSD and cytoplasm of spines, while PSD-95 is concentrated only in the PSD area. These results indicate functional differences between SAP102 and PSD-95 in synaptic organization and plasticity.

Super-Resolution Microscopy Techniques and Their Potential for Applications in Radiation Biophysics.

PubMed

Eberle, Jan Philipp; Rapp, Alexander; Krufczik, Matthias; Eryilmaz, Marion; Gunkel, Manuel; Erfle, Holger; Hausmann, Michael

2017-01-01

Fluorescence microscopy is an essential tool for imaging tagged biological structures. Due to the wave nature of light, the resolution of a conventional fluorescence microscope is limited laterally to about 200 nm and axially to about 600 nm, which is often referred to as the Abbe limit. This hampers the observation of important biological structures and dynamics in the nano-scaled range ~10 nm to ~100 nm. Consequentially, various methods have been developed circumventing this limit of resolution. Super-resolution microscopy comprises several of those methods employing physical and/or chemical properties, such as optical/instrumental modifications and specific labeling of samples. In this article, we will give a brief insight into a variety of selected optical microscopy methods reaching super-resolution beyond the Abbe limit. We will survey three different concepts in connection to biological applications in radiation research without making a claim to be complete.

DMD-based LED-illumination super-resolution and optical sectioning microscopy.

PubMed

Dan, Dan; Lei, Ming; Yao, Baoli; Wang, Wen; Winterhalder, Martin; Zumbusch, Andreas; Qi, Yujiao; Xia, Liang; Yan, Shaohui; Yang, Yanlong; Gao, Peng; Ye, Tong; Zhao, Wei

2013-01-01

Super-resolution three-dimensional (3D) optical microscopy has incomparable advantages over other high-resolution microscopic technologies, such as electron microscopy and atomic force microscopy, in the study of biological molecules, pathways and events in live cells and tissues. We present a novel approach of structured illumination microscopy (SIM) by using a digital micromirror device (DMD) for fringe projection and a low-coherence LED light for illumination. The lateral resolution of 90 nm and the optical sectioning depth of 120 μm were achieved. The maximum acquisition speed for 3D imaging in the optical sectioning mode was 1.6×10(7) pixels/second, which was mainly limited by the sensitivity and speed of the CCD camera. In contrast to other SIM techniques, the DMD-based LED-illumination SIM is cost-effective, ease of multi-wavelength switchable and speckle-noise-free. The 2D super-resolution and 3D optical sectioning modalities can be easily switched and applied to either fluorescent or non-fluorescent specimens.

DMD-based LED-illumination Super-resolution and optical sectioning microscopy

PubMed Central

Dan, Dan; Lei, Ming; Yao, Baoli; Wang, Wen; Winterhalder, Martin; Zumbusch, Andreas; Qi, Yujiao; Xia, Liang; Yan, Shaohui; Yang, Yanlong; Gao, Peng; Ye, Tong; Zhao, Wei

2013-01-01

Super-resolution three-dimensional (3D) optical microscopy has incomparable advantages over other high-resolution microscopic technologies, such as electron microscopy and atomic force microscopy, in the study of biological molecules, pathways and events in live cells and tissues. We present a novel approach of structured illumination microscopy (SIM) by using a digital micromirror device (DMD) for fringe projection and a low-coherence LED light for illumination. The lateral resolution of 90 nm and the optical sectioning depth of 120 μm were achieved. The maximum acquisition speed for 3D imaging in the optical sectioning mode was 1.6×107 pixels/second, which was mainly limited by the sensitivity and speed of the CCD camera. In contrast to other SIM techniques, the DMD-based LED-illumination SIM is cost-effective, ease of multi-wavelength switchable and speckle-noise-free. The 2D super-resolution and 3D optical sectioning modalities can be easily switched and applied to either fluorescent or non-fluorescent specimens. PMID:23346373

DURIP: Super-Resolution Module for Confocal Microscopy of Reconfigurable Matter

DTIC Science & Technology

2014-09-28

Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 superresolution microscopy, colloidal particles, self-assembly REPORT...previously have been resolved by optical microscopy. Results of Super Resolution Technique Evaluation Commercially available superresolution imaging...Weaknesses of the method are that is fundamentally a measurement that can only be deployed for fixed samples. Because superresolution is obtained by

Super-resolution and super-localization microscopy: A novel tool for imaging chemical and biological processes

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

Dong, Bin

2015-01-01

Optical microscopy imaging of single molecules and single particles is an essential method for studying fundamental biological and chemical processes at the molecular and nanometer scale. The best spatial resolution (~ λ/2) achievable in traditional optical microscopy is governed by the diffraction of light. However, single molecule-based super-localization and super-resolution microscopy imaging techniques have emerged in the past decade. Individual molecules can be localized with nanometer scale accuracy and precision for studying of biological and chemical processes.This work uncovered the heterogeneous properties of the pore structures. In this dissertation, the coupling of molecular transport and catalytic reaction at the singlemore » molecule and single particle level in multilayer mesoporous nanocatalysts was elucidated. Most previous studies dealt with these two important phenomena separately. A fluorogenic oxidation reaction of non-fluorescent amplex red to highly fluorescent resorufin was tested. The diffusion behavior of single resorufin molecules in aligned nanopores was studied using total internal reflection fluorescence microscopy (TIRFM).« less

Analyzing Protein Clusters on the Plasma Membrane: Application of Spatial Statistical Analysis Methods on Super-Resolution Microscopy Images.

PubMed

Paparelli, Laura; Corthout, Nikky; Pavie, Benjamin; Annaert, Wim; Munck, Sebastian

2016-01-01

The spatial distribution of proteins within the cell affects their capability to interact with other molecules and directly influences cellular processes and signaling. At the plasma membrane, multiple factors drive protein compartmentalization into specialized functional domains, leading to the formation of clusters in which intermolecule interactions are facilitated. Therefore, quantifying protein distributions is a necessity for understanding their regulation and function. The recent advent of super-resolution microscopy has opened up the possibility of imaging protein distributions at the nanometer scale. In parallel, new spatial analysis methods have been developed to quantify distribution patterns in super-resolution images. In this chapter, we provide an overview of super-resolution microscopy and summarize the factors influencing protein arrangements on the plasma membrane. Finally, we highlight methods for analyzing clusterization of plasma membrane proteins, including examples of their applications.

Super-resolved terahertz microscopy by knife-edge scan

NASA Astrophysics Data System (ADS)

Giliberti, V.; Flammini, M.; Ciano, C.; Pontecorvo, E.; Del Re, E.; Ortolani, M.

2017-08-01

We present a compact, all solid-state THz confocal microscope operating at 0.30 THz that achieves super-resolution by using the knife-edge scan approach. In the final reconstructed image, a lateral resolution of 60 μm ≍ λ/17 is demonstrated when the knife-edge is deep in the near-field of the sample surface. When the knife-edge is lifted up to λ/4 from the sample surface, a certain degree of super-resolution is maintained with a resolution of 0.4 mm, i.e. more than a factor 2 if compared to the diffraction-limited scheme. The present results open an interesting path towards super-resolved imaging with in-depth information that would be peculiar to THz microscopy systems.

FALCON: fast and unbiased reconstruction of high-density super-resolution microscopy data

NASA Astrophysics Data System (ADS)

Min, Junhong; Vonesch, Cédric; Kirshner, Hagai; Carlini, Lina; Olivier, Nicolas; Holden, Seamus; Manley, Suliana; Ye, Jong Chul; Unser, Michael

2014-04-01

Super resolution microscopy such as STORM and (F)PALM is now a well known method for biological studies at the nanometer scale. However, conventional imaging schemes based on sparse activation of photo-switchable fluorescent probes have inherently slow temporal resolution which is a serious limitation when investigating live-cell dynamics. Here, we present an algorithm for high-density super-resolution microscopy which combines a sparsity-promoting formulation with a Taylor series approximation of the PSF. Our algorithm is designed to provide unbiased localization on continuous space and high recall rates for high-density imaging, and to have orders-of-magnitude shorter run times compared to previous high-density algorithms. We validated our algorithm on both simulated and experimental data, and demonstrated live-cell imaging with temporal resolution of 2.5 seconds by recovering fast ER dynamics.

FALCON: fast and unbiased reconstruction of high-density super-resolution microscopy data

PubMed Central

Min, Junhong; Vonesch, Cédric; Kirshner, Hagai; Carlini, Lina; Olivier, Nicolas; Holden, Seamus; Manley, Suliana; Ye, Jong Chul; Unser, Michael

2014-01-01

Super resolution microscopy such as STORM and (F)PALM is now a well known method for biological studies at the nanometer scale. However, conventional imaging schemes based on sparse activation of photo-switchable fluorescent probes have inherently slow temporal resolution which is a serious limitation when investigating live-cell dynamics. Here, we present an algorithm for high-density super-resolution microscopy which combines a sparsity-promoting formulation with a Taylor series approximation of the PSF. Our algorithm is designed to provide unbiased localization on continuous space and high recall rates for high-density imaging, and to have orders-of-magnitude shorter run times compared to previous high-density algorithms. We validated our algorithm on both simulated and experimental data, and demonstrated live-cell imaging with temporal resolution of 2.5 seconds by recovering fast ER dynamics. PMID:24694686

An introduction to optical super-resolution microscopy for the adventurous biologist

NASA Astrophysics Data System (ADS)

Vangindertael, J.; Camacho, R.; Sempels, W.; Mizuno, H.; Dedecker, P.; Janssen, K. P. F.

2018-04-01

Ever since the inception of light microscopy, the laws of physics have seemingly thwarted every attempt to visualize the processes of life at its most fundamental, sub-cellular, level. The diffraction limit has restricted our view to length scales well above 250 nm and in doing so, severely compromised our ability to gain true insights into many biological systems. Fortunately, continuous advancements in optics, electronics and mathematics have since provided the means to once again make physics work to our advantage. Even though some of the fundamental concepts enabling super-resolution light microscopy have been known for quite some time, practically feasible implementations have long remained elusive. It should therefore not come as a surprise that the 2014 Nobel Prize in Chemistry was awarded to the scientists who, each in their own way, contributed to transforming super-resolution microscopy from a technological tour de force to a staple of the biologist’s toolkit. By overcoming the diffraction barrier, light microscopy could once again be established as an indispensable tool in an age where the importance of understanding life at the molecular level cannot be overstated. This review strives to provide the aspiring life science researcher with an introduction to optical microscopy, starting from the fundamental concepts governing compound and fluorescent confocal microscopy to the current state-of-the-art of super-resolution microscopy techniques and their applications.

Super-Resolution Imaging Strategies for Cell Biologists Using a Spinning Disk Microscope

PubMed Central

Hosny, Neveen A.; Song, Mingying; Connelly, John T.; Ameer-Beg, Simon; Knight, Martin M.; Wheeler, Ann P.

2013-01-01

In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging. PMID:24130668

Super Resolution Imaging of Genetically Labeled Synapses in Drosophila Brain Tissue

PubMed Central

Spühler, Isabelle A.; Conley, Gaurasundar M.; Scheffold, Frank; Sprecher, Simon G.

2016-01-01

Understanding synaptic connectivity and plasticity within brain circuits and their relationship to learning and behavior is a fundamental quest in neuroscience. Visualizing the fine details of synapses using optical microscopy remains however a major technical challenge. Super resolution microscopy opens the possibility to reveal molecular features of synapses beyond the diffraction limit. With direct stochastic optical reconstruction microscopy, dSTORM, we image synaptic proteins in the brain tissue of the fruit fly, Drosophila melanogaster. Super resolution imaging of brain tissue harbors difficulties due to light scattering and the density of signals. In order to reduce out of focus signal, we take advantage of the genetic tools available in the Drosophila and have fluorescently tagged synaptic proteins expressed in only a small number of neurons. These neurons form synapses within the calyx of the mushroom body, a distinct brain region involved in associative memory formation. Our results show that super resolution microscopy, in combination with genetically labeled synaptic proteins, is a powerful tool to investigate synapses in a quantitative fashion providing an entry point for studies on synaptic plasticity during learning and memory formation. PMID:27303270

Super Resolution Imaging of Genetically Labeled Synapses in Drosophila Brain Tissue.

PubMed

Spühler, Isabelle A; Conley, Gaurasundar M; Scheffold, Frank; Sprecher, Simon G

2016-01-01

Understanding synaptic connectivity and plasticity within brain circuits and their relationship to learning and behavior is a fundamental quest in neuroscience. Visualizing the fine details of synapses using optical microscopy remains however a major technical challenge. Super resolution microscopy opens the possibility to reveal molecular features of synapses beyond the diffraction limit. With direct stochastic optical reconstruction microscopy, dSTORM, we image synaptic proteins in the brain tissue of the fruit fly, Drosophila melanogaster. Super resolution imaging of brain tissue harbors difficulties due to light scattering and the density of signals. In order to reduce out of focus signal, we take advantage of the genetic tools available in the Drosophila and have fluorescently tagged synaptic proteins expressed in only a small number of neurons. These neurons form synapses within the calyx of the mushroom body, a distinct brain region involved in associative memory formation. Our results show that super resolution microscopy, in combination with genetically labeled synaptic proteins, is a powerful tool to investigate synapses in a quantitative fashion providing an entry point for studies on synaptic plasticity during learning and memory formation.

Techniques for super-resolution microscopy using NV-diamond

NASA Astrophysics Data System (ADS)

Trifonov, Alexei; Glenn, David; Bar-Gill, Nir; Le Sage, David; Walsworth, Ronald

2011-05-01

We discuss the development and application of techniques for super-resolution microscopy using NV centers in diamond: stimulated emission depletion (STED), metastable ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM). NV centers do not bleach under optical excitation, are not biotoxic, and have long-lived electronic spin coherence and spin-state-dependent fluorescence. Thus NV-diamond has great potential as a fluorescent biomarker and as a magnetic biosensor.

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.

Label-free super-resolution with coherent nonlinear structured-illumination microscopy

NASA Astrophysics Data System (ADS)

Huttunen, Mikko J.; Abbas, Aazad; Upham, Jeremy; Boyd, Robert W.

2017-08-01

Structured-illumination microscopy enables up to a two-fold lateral resolution improvement by spatially modulating the intensity profile of the illumination beam. We propose a novel way to generalize the concept of structured illumination to nonlinear widefield modalities by spatially modulating, instead of field intensities, the phase of the incident field while interferometrically measuring the complex-valued scattered field. We numerically demonstrate that for second-order and third-order processes an almost four- and six-fold increase in lateral resolution is achievable, respectively. This procedure overcomes the conventional Abbe diffraction limit and provides new possibilities for label-free super-resolution microscopy.

Enhancing multi-spot structured illumination microscopy with fluorescence difference

NASA Astrophysics Data System (ADS)

Ward, Edward N.; Torkelsen, Frida H.; Pal, Robert

2018-03-01

Structured illumination microscopy is a super-resolution technique used extensively in biological research. However, this technique is limited in the maximum possible resolution increase. Here we report the results of simulations of a novel enhanced multi-spot structured illumination technique. This method combines the super-resolution technique of difference microscopy with structured illumination deconvolution. Initial results give at minimum a 1.4-fold increase in resolution over conventional structured illumination in a low-noise environment. This new technique also has the potential to be expanded to further enhance axial resolution with three-dimensional difference microscopy. The requirement for precise pattern determination in this technique also led to the development of a new pattern estimation algorithm which proved more efficient and reliable than other methods tested.

Three-dimensional nanometre localization of nanoparticles to enhance super-resolution microscopy

NASA Astrophysics Data System (ADS)

Bon, Pierre; Bourg, Nicolas; Lécart, Sandrine; Monneret, Serge; Fort, Emmanuel; Wenger, Jérôme; Lévêque-Fort, Sandrine

2015-07-01

Meeting the nanometre resolution promised by super-resolution microscopy techniques (pointillist: PALM, STORM, scanning: STED) requires stabilizing the sample drifts in real time during the whole acquisition process. Metal nanoparticles are excellent probes to track the lateral drifts as they provide crisp and photostable information. However, achieving nanometre axial super-localization is still a major challenge, as diffraction imposes large depths-of-fields. Here we demonstrate fast full three-dimensional nanometre super-localization of gold nanoparticles through simultaneous intensity and phase imaging with a wavefront-sensing camera based on quadriwave lateral shearing interferometry. We show how to combine the intensity and phase information to provide the key to the third axial dimension. Presently, we demonstrate even in the occurrence of large three-dimensional fluctuations of several microns, unprecedented sub-nanometre localization accuracies down to 0.7 nm in lateral and 2.7 nm in axial directions at 50 frames per second. We demonstrate that nanoscale stabilization greatly enhances the image quality and resolution in direct stochastic optical reconstruction microscopy imaging.

Understanding Super-Resolution Nanoscopy and Its Biological Applications in Cell Imaging

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

Hu, Dehong; Zhao, Baoming; Xie, Yumei

2013-01-01

Optical microscopy has been an ideal tool to study phenomena in live cells because visible light at reasonable intensity does not perturb much of the normal biological functions. However, optical resolution using visible light is significantly limited by the wavelength. Overcoming this diffraction-limit barrier will reveal biological mechanisms, cellular structures, and physiological processes at nanometer scale, orders of magnitude lower than current optical microscopy. Although this appears to be a daunting task, recently developed photoswitchable probes enable reconstruction of individual images into a super-resolution image, thus the emergence of nanoscopy. Harnessing the resolution power of nanoscopy, we report here nano-resolutionmore » fluorescence imaging of microtubules and their network structures in biological cells. The super-resolution nanoscopy successfully resolved nanostructures of microtubule network—a daunting task that cannot be completed using conventional wide-field microscopy.« less

Interrogating Surface Functional Group Heterogeneity of Activated Thermoplastics Using Super-Resolution Fluorescence Microscopy.

PubMed

ONeil, Colleen E; Jackson, Joshua M; Shim, Sang-Hee; Soper, Steven A

2016-04-05

We present a novel approach for characterizing surfaces utilizing super-resolution fluorescence microscopy with subdiffraction limit spatial resolution. Thermoplastic surfaces were activated by UV/O3 or O2 plasma treatment under various conditions to generate pendant surface-confined carboxylic acids (-COOH). These surface functional groups were then labeled with a photoswitchable dye and interrogated using single-molecule, localization-based, super-resolution fluorescence microscopy to elucidate the surface heterogeneity of these functional groups across the activated surface. Data indicated nonuniform distributions of these functional groups for both COC and PMMA thermoplastics with the degree of heterogeneity being dose dependent. In addition, COC demonstrated relative higher surface density of functional groups compared to PMMA for both UV/O3 and O2 plasma treatment. The spatial distribution of -COOH groups secured from super-resolution imaging were used to simulate nonuniform patterns of electroosmotic flow in thermoplastic nanochannels. Simulations were compared to single-particle tracking of fluorescent nanoparticles within thermoplastic nanoslits to demonstrate the effects of surface functional group heterogeneity on the electrokinetic transport process.

Wavelength scanning achieves pixel super-resolution in holographic on-chip microscopy

NASA Astrophysics Data System (ADS)

Luo, Wei; Göröcs, Zoltan; Zhang, Yibo; Feizi, Alborz; Greenbaum, Alon; Ozcan, Aydogan

2016-03-01

Lensfree holographic on-chip imaging is a potent solution for high-resolution and field-portable bright-field imaging over a wide field-of-view. Previous lensfree imaging approaches utilize a pixel super-resolution technique, which relies on sub-pixel lateral displacements between the lensfree diffraction patterns and the image sensor's pixel-array, to achieve sub-micron resolution under unit magnification using state-of-the-art CMOS imager chips, commonly used in e.g., mobile-phones. Here we report, for the first time, a wavelength scanning based pixel super-resolution technique in lensfree holographic imaging. We developed an iterative super-resolution algorithm, which generates high-resolution reconstructions of the specimen from low-resolution (i.e., under-sampled) diffraction patterns recorded at multiple wavelengths within a narrow spectral range (e.g., 10-30 nm). Compared with lateral shift-based pixel super-resolution, this wavelength scanning approach does not require any physical shifts in the imaging setup, and the resolution improvement is uniform in all directions across the sensor-array. Our wavelength scanning super-resolution approach can also be integrated with multi-height and/or multi-angle on-chip imaging techniques to obtain even higher resolution reconstructions. For example, using wavelength scanning together with multi-angle illumination, we achieved a halfpitch resolution of 250 nm, corresponding to a numerical aperture of 1. In addition to pixel super-resolution, the small scanning steps in wavelength also enable us to robustly unwrap phase, revealing the specimen's optical path length in our reconstructed images. We believe that this new wavelength scanning based pixel super-resolution approach can provide competitive microscopy solutions for high-resolution and field-portable imaging needs, potentially impacting tele-pathology applications in resource-limited-settings.

Fast and efficient molecule detection in localization-based super-resolution microscopy by parallel adaptive histogram equalization.

PubMed

Li, Yiming; Ishitsuka, Yuji; Hedde, Per Niklas; Nienhaus, G Ulrich

2013-06-25

In localization-based super-resolution microscopy, individual fluorescent markers are stochastically photoactivated and subsequently localized within a series of camera frames, yielding a final image with a resolution far beyond the diffraction limit. Yet, before localization can be performed, the subregions within the frames where the individual molecules are present have to be identified-oftentimes in the presence of high background. In this work, we address the importance of reliable molecule identification for the quality of the final reconstructed super-resolution image. We present a fast and robust algorithm (a-livePALM) that vastly improves the molecule detection efficiency while minimizing false assignments that can lead to image artifacts.

Plasmonics and metamaterials based super-resolution imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Liu, Zhaowei

2017-05-01

In recent years, surface imaging of various biological dynamics and biomechanical phenomena has seen a surge of interest. Imaging of processes such as exocytosis and kinesin motion are most effective when depth is limited to a very thin region of interest at the edge of the cell or specimen. However, many objects and processes of interest are of size scales below the diffraction limit for safe, visible wavelength illumination. Super-resolution imaging methods such as structured illumination microscopy and others have offered various compromises between resolution, imaging speed, and bio-compatibility. In this talk, I will present our most recent progress in plasmonic structured illumination microscopy (PSIM) and localized plasmonic structured illumination microscopy (LPSIM), and their applications in bio-imaging. We have achieved wide-field surface imaging with resolution down to 75 nm while maintaining reasonable speed and compatibility with biological specimens. These plasmonic enhanced super resolution techniques offer unique solutions to obtain 50nm spatial resolution and 50 frames per second wide imaging speed at the same time.

Enhancing multi-spot structured illumination microscopy with fluorescence difference

PubMed Central

Torkelsen, Frida H.

2018-01-01

Structured illumination microscopy is a super-resolution technique used extensively in biological research. However, this technique is limited in the maximum possible resolution increase. Here we report the results of simulations of a novel enhanced multi-spot structured illumination technique. This method combines the super-resolution technique of difference microscopy with structured illumination deconvolution. Initial results give at minimum a 1.4-fold increase in resolution over conventional structured illumination in a low-noise environment. This new technique also has the potential to be expanded to further enhance axial resolution with three-dimensional difference microscopy. The requirement for precise pattern determination in this technique also led to the development of a new pattern estimation algorithm which proved more efficient and reliable than other methods tested. PMID:29657751

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.

Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples

PubMed Central

Kopek, Benjamin G.; Paez-Segala, Maria G.; Shtengel, Gleb; Sochacki, Kem A.; Sun, Mei G.; Wang, Yalin; Xu, C. Shan; van Engelenburg, Schuyler B.; Taraska, Justin W.; Looger, Loren L.; Hess, Harald F.

2017-01-01

Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM datasets on aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. Choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replica creates high-contrast, 3-dimensional images of the cytoplasmic surface of the plasma membrane, but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples, but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (~10–50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2–7 days, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology. PMID:28384138

Perspectives in Super-resolved Fluorescence Microscopy: What comes next?

NASA Astrophysics Data System (ADS)

Cremer, Christoph; Birk, Udo

2016-04-01

The Nobel Prize in Chemistry 2014 has been awarded to three scientists involved in the development of STED and PALM super-resolution fluorescence microscopy (SRM) methods. They have proven that it is possible to overcome the hundred year old theoretical limit for the resolution potential of light microscopy (of about 200 nm for visible light), which for decades has precluded a direct glimpse of the molecular machinery of life. None of the present-day super-resolution techniques have invalidated the Abbe limit for light optical detection; however, they have found clever ways around it. In this report, we discuss some of the challenges still to be resolved before arising SRM approaches will be fit to bring about the revolution in Biology and Medicine envisaged. Some of the challenges discussed are the applicability to image live and/or large samples, the further enhancement of resolution, future developments of labels, and multi-spectral approaches.

Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ

PubMed Central

Müller, Marcel; Mönkemöller, Viola; Hennig, Simon; Hübner, Wolfgang; Huser, Thomas

2016-01-01

Super-resolved structured illumination microscopy (SR-SIM) is an important tool for fluorescence microscopy. SR-SIM microscopes perform multiple image acquisitions with varying illumination patterns, and reconstruct them to a super-resolved image. In its most frequent, linear implementation, SR-SIM doubles the spatial resolution. The reconstruction is performed numerically on the acquired wide-field image data, and thus relies on a software implementation of specific SR-SIM image reconstruction algorithms. We present fairSIM, an easy-to-use plugin that provides SR-SIM reconstructions for a wide range of SR-SIM platforms directly within ImageJ. For research groups developing their own implementations of super-resolution structured illumination microscopy, fairSIM takes away the hurdle of generating yet another implementation of the reconstruction algorithm. For users of commercial microscopes, it offers an additional, in-depth analysis option for their data independent of specific operating systems. As a modular, open-source solution, fairSIM can easily be adapted, automated and extended as the field of SR-SIM progresses. PMID:26996201

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

3D single-molecule super-resolution microscopy with a tilted light sheet.

PubMed

Gustavsson, Anna-Karin; Petrov, Petar N; Lee, Maurice Y; Shechtman, Yoav; Moerner, W E

2018-01-09

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.

Controlled power delivery for super-resolution imaging of biological samples using digital micromirror device

NASA Astrophysics Data System (ADS)

Valiya Peedikakkal, Liyana; Cadby, Ashley

2017-02-01

Localization based super resolution images of a biological sample is generally achieved by using high power laser illumination with long exposure time which unfortunately increases photo-toxicity of a sample, making super resolution microscopy, in general, incompatible with live cell imaging. Furthermore, the limitation of photobleaching reduces the ability to acquire time lapse images of live biological cells using fluorescence microscopy. Digital Light Processing (DLP) technology can deliver light at grey scale levels by flickering digital micromirrors at around 290 Hz enabling highly controlled power delivery to samples. In this work, Digital Micromirror Device (DMD) is implemented in an inverse Schiefspiegler telescope setup to control the power and pattern of illumination for super resolution microscopy. We can achieve spatial and temporal patterning of illumination by controlling the DMD pixel by pixel. The DMD allows us to control the power and spatial extent of the laser illumination. We have used this to show that we can reduce the power delivered to the sample to allow for longer time imaging in one area while achieving sub-diffraction STORM imaging in another using higher power densities.

Correlative Super-Resolution Microscopy: New Dimensions and New Opportunities.

PubMed

Hauser, Meghan; Wojcik, Michal; Kim, Doory; Mahmoudi, Morteza; Li, Wan; Xu, Ke

2017-06-14

Correlative microscopy, the integration of two or more microscopy techniques performed on the same sample, produces results that emphasize the strengths of each technique while offsetting their individual weaknesses. Light microscopy has historically been a central method in correlative microscopy due to its widespread availability, compatibility with hydrated and live biological samples, and excellent molecular specificity through fluorescence labeling. However, conventional light microscopy can only achieve a resolution of ∼300 nm, undercutting its advantages in correlations with higher-resolution methods. The rise of super-resolution microscopy (SRM) over the past decade has drastically improved the resolution of light microscopy to ∼10 nm, thus creating exciting new opportunities and challenges for correlative microscopy. Here we review how these challenges are addressed to effectively correlate SRM with other microscopy techniques, including light microscopy, electron microscopy, cryomicroscopy, atomic force microscopy, and various forms of spectroscopy. Though we emphasize biological studies, we also discuss the application of correlative SRM to materials characterization and single-molecule reactions. Finally, we point out current limitations and discuss possible future improvements and advances. We thus demonstrate how a correlative approach adds new dimensions of information and provides new opportunities in the fast-growing field of SRM.

SNSMIL, a real-time single molecule identification and localization algorithm for super-resolution fluorescence microscopy

PubMed Central

Tang, Yunqing; Dai, Luru; Zhang, Xiaoming; Li, Junbai; Hendriks, Johnny; Fan, Xiaoming; Gruteser, Nadine; Meisenberg, Annika; Baumann, Arnd; Katranidis, Alexandros; Gensch, Thomas

2015-01-01

Single molecule localization based super-resolution fluorescence microscopy offers significantly higher spatial resolution than predicted by Abbe’s resolution limit for far field optical microscopy. Such super-resolution images are reconstructed from wide-field or total internal reflection single molecule fluorescence recordings. Discrimination between emission of single fluorescent molecules and background noise fluctuations remains a great challenge in current data analysis. Here we present a real-time, and robust single molecule identification and localization algorithm, SNSMIL (Shot Noise based Single Molecule Identification and Localization). This algorithm is based on the intrinsic nature of noise, i.e., its Poisson or shot noise characteristics and a new identification criterion, QSNSMIL, is defined. SNSMIL improves the identification accuracy of single fluorescent molecules in experimental or simulated datasets with high and inhomogeneous background. The implementation of SNSMIL relies on a graphics processing unit (GPU), making real-time analysis feasible as shown for real experimental and simulated datasets. PMID:26098742

Super-resolution optical telescopes with local light diffraction shrinkage

PubMed Central

Wang, Changtao; Tang, Dongliang; Wang, Yanqin; Zhao, Zeyu; Wang, Jiong; Pu, Mingbo; Zhang, Yudong; Yan, Wei; Gao, Ping; Luo, Xiangang

2015-01-01

Suffering from giant size of objective lenses and infeasible manipulations of distant targets, telescopes could not seek helps from present super-resolution imaging, such as scanning near-field optical microscopy, perfect lens and stimulated emission depletion microscopy. In this paper, local light diffraction shrinkage associated with optical super-oscillatory phenomenon is proposed for real-time and optically restoring super-resolution imaging information in a telescope system. It is found that fine target features concealed in diffraction-limited optical images of a telescope could be observed in a small local field of view, benefiting from a relayed metasurface-based super-oscillatory imaging optics in which some local Fourier components beyond the cut-off frequency of telescope could be restored. As experimental examples, a minimal resolution to 0.55 of Rayleigh criterion is obtained, and imaging complex targets and large targets by superimposing multiple local fields of views are demonstrated as well. This investigation provides an access for real-time, incoherent and super-resolution telescopes without the manipulation of distant targets. More importantly, it gives counterintuitive evidence to the common knowledge that relayed optics could not deliver more imaging details than objective systems. PMID:26677820

Multiple signal classification algorithm for super-resolution fluorescence microscopy

PubMed Central

Agarwal, Krishna; Macháň, Radek

2016-01-01

Single-molecule localization techniques are restricted by long acquisition and computational times, or the need of special fluorophores or biologically toxic photochemical environments. Here we propose a statistical super-resolution technique of wide-field fluorescence microscopy we call the multiple signal classification algorithm which has several advantages. It provides resolution down to at least 50 nm, requires fewer frames and lower excitation power and works even at high fluorophore concentrations. Further, it works with any fluorophore that exhibits blinking on the timescale of the recording. The multiple signal classification algorithm shows comparable or better performance in comparison with single-molecule localization techniques and four contemporary statistical super-resolution methods for experiments of in vitro actin filaments and other independently acquired experimental data sets. We also demonstrate super-resolution at timescales of 245 ms (using 49 frames acquired at 200 frames per second) in samples of live-cell microtubules and live-cell actin filaments imaged without imaging buffers. PMID:27934858

Three-Dimensional Orientation of Anisotropic Plasmonic Aggregates at Intracellular Nuclear Indentation Sites by Integrated Light Sheet Super-Resolution Microscopy.

PubMed

Chakkarapani, Suresh Kumar; Sun, Yucheng; Lee, Seungah; Fang, Ning; Kang, Seong Ho

2018-05-22

Three-dimensional (3D) orientations of individual anisotropic plasmonic nanoparticles in aggregates were observed in real time by integrated light sheet super-resolution microscopy ( iLSRM). Asymmetric light scattering of a gold nanorod (AuNR) was used to trigger signals based on the polarizer angle. Controlled photoswitching was achieved by turning the polarizer and obtaining a series of images at different polarization directions. 3D subdiffraction-limited super-resolution images were obtained by superlocalization of scattering signals as a function of the anisotropic optical properties of AuNRs. Varying the polarizer angle allowed resolution of the orientation of individual AuNRs. 3D images of individual nanoparticles were resolved in aggregated regions, resulting in as low as 64 nm axial resolution and 28 nm spatial resolution. The proposed imaging setup and localization approach demonstrates a convenient method for imaging under a noisy environment where the majority of scattering noise comes from cellular components. This integrated 3D iLSRM and localization technique was shown to be reliable and useful in the field of 3D nonfluorescence super-resolution imaging.

Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria.

PubMed

Wheeler, Richard; Mesnage, Stéphane; Boneca, Ivo G; Hobbs, Jamie K; Foster, Simon J

2011-12-01

Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super-resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms. © 2011 Blackwell Publishing Ltd.

Reconstitution radicicol containing apolipoprotein B lipoparticle and tracing its cell uptake process by super resolution fluorescent microscopy.

NASA Astrophysics Data System (ADS)

Lin, Chung Ching; Lin, Po-Yen; Chang, Chia-Ching

Apolipoprotein B (apoB) is the only protein of LDL. LDL delivers cholesterol, triacylglycerides and lipids to the target cells. Reconstitute apoB lipoparticle (rABL) will be an idea drug delivery vehicle for hydrophobic and amphiphilic materials delivery. It is challenged to renature ApoB into its functional state from denatured state. By using modified bile salt and radicicol (Rad) added over-critical refolding process, apoB can be restored into its native like state. The intrinsic fluorescence of apoB increased during the refolding process. Moreover, radicicol (Rad) molecules have been encapsulated into reconstitute rABL (Rad@rABL). To investigate the cell uptake mechanism of Rad@rABL, a super resolution ground state depletion (GSD) microscopy is used in this research. Fluorescence labeled Rad@rABL can be traced within the tumor cell. Key words: LDL, radicicol, protein refolding, super resolution microscopy.

Lensfree on-chip microscopy over a wide field-of-view using pixel super-resolution

PubMed Central

Bishara, Waheb; Su, Ting-Wei; Coskun, Ahmet F.; Ozcan, Aydogan

2010-01-01

We demonstrate lensfree holographic microscopy on a chip to achieve ~0.6 µm spatial resolution corresponding to a numerical aperture of ~0.5 over a large field-of-view of ~24 mm2. By using partially coherent illumination from a large aperture (~50 µm), we acquire lower resolution lensfree in-line holograms of the objects with unit fringe magnification. For each lensfree hologram, the pixel size at the sensor chip limits the spatial resolution of the reconstructed image. To circumvent this limitation, we implement a sub-pixel shifting based super-resolution algorithm to effectively recover much higher resolution digital holograms of the objects, permitting sub-micron spatial resolution to be achieved across the entire sensor chip active area, which is also equivalent to the imaging field-of-view (24 mm2) due to unit magnification. We demonstrate the success of this pixel super-resolution approach by imaging patterned transparent substrates, blood smear samples, as well as Caenoharbditis Elegans. PMID:20588977

Compact three-dimensional super-resolution system based on fluorescence emission difference microscopy

NASA Astrophysics Data System (ADS)

Zhu, Dazhao; Chen, Youhua; Fang, Yue; Hussain, Anwar; Kuang, Cuifang; Zhou, Xiaoxu; Xu, Yingke; Liu, Xu

2017-12-01

A compact microscope system for three-dimensional (3-D) super-resolution imaging is presented. The super-resolution capability of the system is based on a size-reduced effective 3-D point spread function generated through the fluorescence emission difference (FED) method. The appropriate polarization direction distribution and manipulation allows the panel active area of the spatial light modulator to be fully utilized. This allows simultaneous modulation of the incident light by two kinds of phase masks to be performed with a single spatial light modulator in order to generate a 3-D negative spot. The system is more compact than standard 3-D FED systems while maintaining all the advantages of 3-D FED microscopy. The experimental results demonstrated the improvement in 3-D resolution by nearly 1.7 times and 1.6 times compared to the classic confocal resolution in the lateral and axial directions, respectively.

Lateral resolution improvement in scanning nonlinear dielectric microscopy by measuring super-higher-order nonlinear dielectric constants

NASA Astrophysics Data System (ADS)

Chinone, N.; Yamasue, K.; Hiranaga, Y.; Honda, K.; Cho, Y.

2012-11-01

Scanning nonlinear dielectric microscopy (SNDM) can be used to visualize polarization distributions in ferroelectric materials and dopant profiles in semiconductor devices. Without using a special sharp tip, we achieved an improved lateral resolution in SNDM through the measurement of super-higher-order nonlinearity up to the fourth order. We observed a multidomain single crystal congruent LiTaO3 (CLT) sample, and a cross section of a metal-oxide-semiconductor (MOS) field-effect-transistor (FET). The imaged domain boundaries of the CLT were narrower in the super-higher-order images than in the conventional image. Compared to the conventional method, the super-higher-order method resolved the more detailed structure of the MOSFET.

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.

Interferometric temporal focusing microscopy using three-photon excitation fluorescence.

PubMed

Toda, Keisuke; Isobe, Keisuke; Namiki, Kana; Kawano, Hiroyuki; Miyawaki, Atsushi; Midorikawa, Katsumi

2018-04-01

Super-resolution microscopy has become a powerful tool for biological research. However, its spatial resolution and imaging depth are limited, largely due to background light. Interferometric temporal focusing (ITF) microscopy, which combines structured illumination microscopy and three-photon excitation fluorescence microscopy, can overcome these limitations. Here, we demonstrate ITF microscopy using three-photon excitation fluorescence, which has a spatial resolution of 106 nm at an imaging depth of 100 µm with an excitation wavelength of 1060 nm.

Recent advancements in structured-illumination microscopy toward live-cell imaging.

PubMed

Hirano, Yasuhiro; Matsuda, Atsushi; Hiraoka, Yasushi

2015-08-01

Fluorescence microscopy allows us to observe fluorescently labeled molecules in diverse biological processes and organelle structures within living cells. However, the diffraction limit restricts its spatial resolution to about half of its wavelength, limiting the capability of biological observation at the molecular level. Structured-illumination microscopy (SIM), a type of super-resolution microscopy, doubles the spatial resolution in all three dimensions by illuminating the sample with a patterned excitation light, followed by computer reconstruction. SIM uses a relatively low illumination power compared with other methods of super-resolution microscopy and is easily available for multicolor imaging. SIM has great potential for meeting the requirements of live-cell imaging. Recent developments in diverse types of SIM have achieved higher spatial (∼50 nm lateral) and temporal (∼100 Hz) resolutions. Here, we review recent advancements in SIM and discuss its application in noninvasive live-cell imaging. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

STED super-resolution microscopy reveals an array of MINOS clusters along human mitochondria

PubMed Central

Jans, Daniel C.; Wurm, Christian A.; Riedel, Dietmar; Wenzel, Dirk; Stagge, Franziska; Deckers, Markus; Rehling, Peter; Jakobs, Stefan

2013-01-01

The mitochondrial inner membrane organizing system (MINOS) is a conserved large hetero-oligomeric protein complex in the mitochondrial inner membrane, crucial for the maintenance of cristae morphology. MINOS has been suggested to represent the core of an extended protein network that controls mitochondrial function and structure, and has been linked to several human diseases. The spatial arrangement of MINOS within mitochondria is ill-defined, however. Using super-resolution stimulated emission depletion (STED) microscopy and immunogold electron microscopy, we determined the distribution of three known human MINOS subunits (mitofilin, MINOS1, and CHCHD3) in mammalian cells. Super-resolution microscopy revealed that all three subunits form similar clusters within mitochondria, and that MINOS is more abundant in mitochondria around the nucleus than in peripheral mitochondria. At the submitochondrial level, mitofilin, a core MINOS subunit, is preferentially localized at cristae junctions. In primary human fibroblasts, mitofilin labeling uncovered a regularly spaced pattern of clusters arranged in parallel to the cell growth surfaces. We suggest that this array of MINOS complexes might explain the observed phenomenon of largely horizontally arranged cristae junctions that connect the inner boundary membrane to lamellar cristae. The super-resolution images demonstrate an unexpectedly high level of regularity in the nanoscale distribution of the MINOS complex in human mitochondria, supporting an integrating role of MINOS in the structural organization of the organelle. PMID:23676277

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

PubMed Central

Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

2016-01-01

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min−1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics. PMID:27991512

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

NASA Astrophysics Data System (ADS)

Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

2016-12-01

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min-1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions.

PubMed

Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

2016-12-19

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min -1 . The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

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

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localizationmore » patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.« less

Live-cell super-resolution imaging of intrinsically fast moving flagellates

NASA Astrophysics Data System (ADS)

Glogger, M.; Stichler, S.; Subota, I.; Bertlein, S.; Spindler, M.-C.; Teßmar, J.; Groll, J.; Engstler, M.; Fenz, S. F.

2017-02-01

Recent developments in super-resolution microscopy make it possible to resolve structures in biological cells at a spatial resolution of a few nm and observe dynamical processes with a temporal resolution of ms to μs. However, the optimal structural resolution requires repeated illumination cycles and is thus limited to chemically fixed cells. For live cell applications substantial improvement over classical Abbe-limited imaging can already be obtained in adherent or slow moving cells. Nonetheless, a large group of cells are fast moving and thus could not yet be addressed with live cell super-resolution microscopy. These include flagellate pathogens like African trypanosomes, the causative agents of sleeping sickness in humans and nagana in livestock. Here, we present an embedding method based on a in situ forming cytocompatible UV-crosslinked hydrogel. The fast cross-linking hydrogel immobilizes trypanosomes efficiently to allow microscopy on the nanoscale. We characterized both the trypanosomes and the hydrogel with respect to their autofluorescence properties and found them suitable for single-molecule fluorescence microscopy (SMFM). As a proof of principle, SMFM was applied to super-resolve a structure inside the living trypanosome. We present an image of a flagellar axoneme component recorded by using the intrinsic blinking behavior of eYFP. , which features invited work from the best early-career researchers working within the scope of J Phys D. This project is part of the Journal of Physics series’ 50th anniversary celebrations in 2017. Susanne Fenz was selected by the Editorial Board of J Phys D as an Emerging Talent/Leader.

Super-resolution photoacoustic microscopy using a localized near-field of a plasmonic nanoaperture: a three-dimensional simulation study

NASA Astrophysics Data System (ADS)

Park, Byullee; Lee, Hongki; Upputuri, Paul Kumar; Pramanik, Manojit; Kim, Donghyun; Kim, Chulhong

2018-02-01

Super-resolution microscopy has been increasingly important to delineate nanoscale biological structures or nanoparticles. With these increasing demands, several imaging modalities, including super-resolution fluorescence microscope (SRFM) and electron microscope (EM), have been developed and commercialized. These modalities achieve nanoscale resolution, however, SRFM cannot image without fluorescence, and sample preparation of EM is not suitable for biological specimens. To overcome those disadvantages, we have numerically studied the possibility of superresolution photoacoustic microscopy (SR-PAM) based on near-field localization of light. Photoacoustic (PA) signal is generally acquired based on optical absorption contrast; thus it requires no agents or pre-processing for the samples. The lateral resolution of the conventional photoacoustic microscopy is limited to 200 nm by diffraction limit, therefore reducing the lateral resolution is a major research impetus. Our approach to breaking resolution limit is to use laser pulses of extremely small spot size as a light source. In this research, we simulated the PA signal by constructing the three dimensional SR-PAM system environment using the k-Wave toolbox. As the light source, we simulated ultrashort light pulses using geometrical nanoaperture with near-field localization of surface plasmons. Through the PA simulation, we have successfully distinguish cuboids spaced 3 nm apart. In the near future, we will develop the SR-PAM and it will contribute to biomedical and material sciences.

Two-photon speckle illumination for super-resolution microscopy.

PubMed

Negash, Awoke; Labouesse, Simon; Chaumet, Patrick C; Belkebir, Kamal; Giovannini, Hugues; Allain, Marc; Idier, Jérôme; Sentenac, Anne

2018-06-01

We present a numerical study of a microscopy setup in which the sample is illuminated with uncontrolled speckle patterns and the two-photon excitation fluorescence is collected on a camera. We show that, using a simple deconvolution algorithm for processing the speckle low-resolution images, this wide-field imaging technique exhibits resolution significantly better than that of two-photon excitation scanning microscopy or one-photon excitation bright-field microscopy.

Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy.

PubMed

Siegel, Nisan; Brooker, Gary

2014-09-22

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called "CINCH".

Two-color CW STED nanoscopy

NASA Astrophysics Data System (ADS)

Chen, Xuanze; Liu, Yujia; Yang, Xusan; Wang, Tingting; Alonas, Eric; Santangelo, Philip J.; Ren, Qiushi; Xi, Peng

2013-02-01

Fluorescent microscopy has become an essential tool to study biological molecules, pathways and events in living cells, tissues and animals. Meanwhile even the most advanced confocal microscopy can only yield optical resolution approaching Abbe diffraction limit of 200 nm. This is still larger than many subcellular structures, which are too small to be resolved in detail. These limitations have driven the development of super-resolution optical imaging methodologies over the past decade. In stimulated emission depletion (STED) microscopy, the excitation focus is overlapped by an intense doughnut-shaped spot to instantly de-excite markers from their fluorescent state to the ground state by stimulated emission. This effectively eliminates the periphery of the Point Spread Function (PSF), resulting in a narrower focal region, or super-resolution. Scanning a sharpened spot through the specimen renders images with sub-diffraction resolution. Multi-color STED imaging can present important structural and functional information for protein-protein interaction. In this work, we presented a two-color, synchronization-free STED microscopy with a Ti:Sapphire oscillator. The excitation wavelengths were 532nm and 635nm, respectively. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. Human respiratory syncytial virus (hRSV) proteins were imaged with our two-color CW STED for co-localization analysis.

Next-generation endomyocardial biopsy: the potential of confocal and super-resolution microscopy.

PubMed

Crossman, David J; Ruygrok, Peter N; Hou, Yu Feng; Soeller, Christian

2015-03-01

Confocal laser scanning microscopy and super-resolution microscopy provide high-contrast and high-resolution fluorescent imaging, which has great potential to increase the diagnostic yield of endomyocardial biopsy (EMB). EMB is currently the gold standard for identification of cardiac allograft rejection, myocarditis, and infiltrative and storage diseases. However, standard analysis is dominated by low-contrast bright-field light and electron microscopy (EM); this lack of contrast makes quantification of pathological features difficult. For example, assessment of cardiac allograft rejection relies on subjective grading of H&E histology, which may lead to diagnostic variability between pathologists. This issue could be solved by utilising the high contrast provided by fluorescence methods such as confocal to quantitatively assess the degree of lymphocytic infiltrate. For infiltrative diseases such as amyloidosis, the nanometre resolution provided by EM can be diagnostic in identifying disease-causing fibrils. The recent advent of super-resolution imaging, particularly direct stochastic optical reconstruction microscopy (dSTORM), provides high-contrast imaging at resolution approaching that of EM. Moreover, dSTORM utilises conventional fluorescence dyes allowing for the same structures to be routinely imaged at the cellular scale and then at the nanoscale. The key benefit of these technologies is that the high contrast facilitates quantitative digital analysis and thereby provides a means to robustly assess critical pathological features. Ultimately, this technology has the ability to provide greater accuracy and precision to EMB assessment, which could result in better outcomes for patients.

Structured Illumination Microscopy for the Investigation of Synaptic Structure and Function.

PubMed

Hong, Soyon; Wilton, Daniel K; Stevens, Beth; Richardson, Douglas S

2017-01-01

The neuronal synapse is a primary building block of the nervous system to which alterations in structure or function can result in numerous pathologies. Studying its formation and elimination is the key to understanding how brains are wired during development, maintained throughout adulthood plasticity, and disrupted during disease. However, due to its diffraction-limited size, investigations of the synaptic junction at the structural level have primarily relied on labor-intensive electron microscopy or ultra-thin section array tomography. Recent advances in the field of super-resolution light microscopy now allow researchers to image synapses and associated molecules with high-spatial resolution, while taking advantage of the key characteristics of light microscopy, such as easy sample preparation and the ability to detect multiple targets with molecular specificity. One such super-resolution technique, Structured Illumination Microscopy (SIM), has emerged as an attractive method to examine synapse structure and function. SIM requires little change in standard light microscopy sample preparation steps, but results in a twofold improvement in both lateral and axial resolutions compared to widefield microscopy. The following protocol outlines a method for imaging synaptic structures at resolutions capable of resolving the intricacies of these neuronal connections.

Polarization sensitive localization based super-resolution microscopy with a birefringent wedge

NASA Astrophysics Data System (ADS)

Sinkó, József; Gajdos, Tamás; Czvik, Elvira; Szabó, Gábor; Erdélyi, Miklós

2017-03-01

A practical method has been presented for polarization sensitive localization based super-resolution microscopy using a birefringent dual wedge. The measurement of the polarization degree at the single molecule level can reveal the chemical and physical properties of the local environment of the fluorescent dye molecule and can hence provide information about the sub-diffraction sized structure of biological samples. Polarization sensitive STORM imaging of the F-Actins proved correlation between the orientation of fluorescent dipoles and the axis of the fibril.

Multicolor Super-Resolution Fluorescence Imaging via Multi-Parameter Fluorophore Detection

PubMed Central

Bates, Mark; Dempsey, Graham T; Chen, Kok Hao; Zhuang, Xiaowei

2012-01-01

Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution. PMID:22213647

Localization-based super-resolution imaging meets high-content screening.

PubMed

Beghin, Anne; Kechkar, Adel; Butler, Corey; Levet, Florian; Cabillic, Marine; Rossier, Olivier; Giannone, Gregory; Galland, Rémi; Choquet, Daniel; Sibarita, Jean-Baptiste

2017-12-01

Single-molecule localization microscopy techniques have proven to be essential tools for quantitatively monitoring biological processes at unprecedented spatial resolution. However, these techniques are very low throughput and are not yet compatible with fully automated, multiparametric cellular assays. This shortcoming is primarily due to the huge amount of data generated during imaging and the lack of software for automation and dedicated data mining. We describe an automated quantitative single-molecule-based super-resolution methodology that operates in standard multiwell plates and uses analysis based on high-content screening and data-mining software. The workflow is compatible with fixed- and live-cell imaging and allows extraction of quantitative data like fluorophore photophysics, protein clustering or dynamic behavior of biomolecules. We demonstrate that the method is compatible with high-content screening using 3D dSTORM and DNA-PAINT based super-resolution microscopy as well as single-particle tracking.

Tilted Light Sheet Microscopy with 3D Point Spread Functions for Single-Molecule Super-Resolution Imaging in Mammalian Cells.

PubMed

Gustavsson, Anna-Karin; Petrov, Petar N; Lee, Maurice Y; Shechtman, Yoav; Moerner, W E

2018-02-01

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy

PubMed Central

Siegel, Nisan; Brooker, Gary

2014-01-01

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called “CINCH”. PMID:25321701

Re-scan confocal microscopy: scanning twice for better resolution.

PubMed

De Luca, Giulia M R; Breedijk, Ronald M P; Brandt, Rick A J; Zeelenberg, Christiaan H C; de Jong, Babette E; Timmermans, Wendy; Azar, Leila Nahidi; Hoebe, Ron A; Stallinga, Sjoerd; Manders, Erik M M

2013-01-01

We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required.

Fast myopic 2D-SIM super resolution microscopy with joint modulation pattern estimation

NASA Astrophysics Data System (ADS)

Orieux, François; Loriette, Vincent; Olivo-Marin, Jean-Christophe; Sepulveda, Eduardo; Fragola, Alexandra

2017-12-01

Super-resolution in structured illumination microscopy (SIM) is obtained through de-aliasing of modulated raw images, in which high frequencies are measured indirectly inside the optical transfer function. Usual approaches that use 9 or 15 images are often too slow for dynamic studies. Moreover, as experimental conditions change with time, modulation parameters must be estimated within the images. This paper tackles the problem of image reconstruction for fast super resolution in SIM, where the number of available raw images is reduced to four instead of nine or fifteen. Within an optimization framework, the solution is inferred via a joint myopic criterion for image and modulation (or acquisition) parameters, leading to what is frequently called a myopic or semi-blind inversion problem. The estimate is chosen as the minimizer of the nonlinear criterion, numerically calculated by means of a block coordinate optimization algorithm. The effectiveness of the proposed method is demonstrated for simulated and experimental examples. The results show precise estimation of the modulation parameters jointly with the reconstruction of the super resolution image. The method also shows its effectiveness for thick biological samples.

Improved localization accuracy in stochastic super-resolution fluorescence microscopy by K-factor image deshadowing

PubMed Central

Ilovitsh, Tali; Meiri, Amihai; Ebeling, Carl G.; Menon, Rajesh; Gerton, Jordan M.; Jorgensen, Erik M.; Zalevsky, Zeev

2013-01-01

Localization of a single fluorescent particle with sub-diffraction-limit accuracy is a key merit in localization microscopy. Existing methods such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) achieve localization accuracies of single emitters that can reach an order of magnitude lower than the conventional resolving capabilities of optical microscopy. However, these techniques require a sparse distribution of simultaneously activated fluorophores in the field of view, resulting in larger time needed for the construction of the full image. In this paper we present the use of a nonlinear image decomposition algorithm termed K-factor, which reduces an image into a nonlinear set of contrast-ordered decompositions whose joint product reassembles the original image. The K-factor technique, when implemented on raw data prior to localization, can improve the localization accuracy of standard existing methods, and also enable the localization of overlapping particles, allowing the use of increased fluorophore activation density, and thereby increased data collection speed. Numerical simulations of fluorescence data with random probe positions, and especially at high densities of activated fluorophores, demonstrate an improvement of up to 85% in the localization precision compared to single fitting techniques. Implementing the proposed concept on experimental data of cellular structures yielded a 37% improvement in resolution for the same super-resolution image acquisition time, and a decrease of 42% in the collection time of super-resolution data with the same resolution. PMID:24466491

Super-resolution atomic force photoactivated microscopy of biological samples (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lee, Seunghyun; Kim, Hyemin; Shin, Seungjun; Doh, Junsang; Kim, Chulhong

2017-03-01

Optical microscopy (OM) and photoacoustic microscopy (PAM) have previously been used to image the optical absorption of intercellular features of biological cells. However, the optical diffraction limit ( 200 nm) makes it difficult for these modalities to image nanoscale inner cell structures and the distribution of internal cell components. Although super-resolution fluorescence microscopy, such as stimulated emission depletion microscopy (STED) and stochastic optical reconstruction microscopy (STORM), has successfully performed nanoscale biological imaging, these modalities require the use of exogenous fluorescence agents, which are unfavorable for biological samples. Our newly developed atomic force photoactivated microscopy (AFPM) can provide optical absorption images with nanoscale lateral resolution without any exogenous contrast agents. AFPM combines conventional atomic force microscopy (AFM) and an optical excitation system, and simultaneously provides multiple contrasts, such as the topography and magnitude of optical absorption. AFPM can detect the intrinsic optical absorption of samples with 8 nm lateral resolution, easily overcoming the diffraction limit. Using the label-free AFPM system, we have successfully imaged the optical absorption properties of a single melanoma cell (B16F10) and a rosette leaf epidermal cell of Arabidopsis (ecotype Columbia (Col-0)) with nanoscale lateral resolution. The remarkable images show the melanosome distribution of a melanoma cell and the biological structures of a plant cell. AFPM provides superior imaging of optical absorption with a nanoscale lateral resolution, and it promises to become widely used in biological and chemical research.

Single cell systems biology by super-resolution imaging and combinatorial labeling

PubMed Central

Lubeck, Eric; Cai, Long

2012-01-01

Fluorescence microscopy is a powerful quantitative tool for exploring regulatory networks in single cells. However, the number of molecular species that can be measured simultaneously is limited by the spectral separability of fluorophores. Here we demonstrate a simple but general strategy to drastically increase the capacity for multiplex detection of molecules in single cells by using optical super-resolution microscopy (SRM) and combinatorial labeling. As a proof of principle, we labeled mRNAs with unique combinations of fluorophores using Fluorescence in situ Hybridization (FISH), and resolved the sequences and combinations of fluorophores with SRM. We measured the mRNA levels of 32 genes simultaneously in single S. cerevisiae cells. These experiments demonstrate that combinatorial labeling and super-resolution imaging of single cells provides a natural approach to bring systems biology into single cells. PMID:22660740

Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

PubMed Central

Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; Edwards, Thayne L.; James, Conrad D.; Lidke, Keith A.

2016-01-01

We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet. PMID:27375939

Re-scan confocal microscopy: scanning twice for better resolution

PubMed Central

De Luca, Giulia M.R.; Breedijk, Ronald M.P.; Brandt, Rick A.J.; Zeelenberg, Christiaan H.C.; de Jong, Babette E.; Timmermans, Wendy; Azar, Leila Nahidi; Hoebe, Ron A.; Stallinga, Sjoerd; Manders, Erik M.M.

2013-01-01

We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required. PMID:24298422

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.

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy†

PubMed Central

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-soo; Torelli, Marco D.; Hamers, Robert J.; Murhpy, Catherine J.; Orr, Galya

2015-01-01

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate eficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells. PMID:24816810

A user's guide to localization-based super-resolution fluorescence imaging.

PubMed

Dempsey, Graham T

2013-01-01

Advances in far-field fluorescence microscopy over the past decade have led to the development of super-resolution imaging techniques that provide more than an order of magnitude improvement in spatial resolution compared to conventional light microscopy. One such approach, called Stochastic Optical Reconstruction Microscopy (STORM) uses the sequential, nanometer-scale localization of individual fluorophores to reconstruct a high-resolution image of a structure of interest. This is an attractive method for biological investigation at the nanoscale due to its relative simplicity, both conceptually and practically in the laboratory. Like most research tools, however, the devil is in the details. The aim of this chapter is to serve as a guide for applying STORM to the study of biological samples. This chapter will discuss considerations for choosing a photoswitchable fluorescent probe, preparing a sample, selecting hardware for data acquisition, and collecting and analyzing data for image reconstruction. Copyright © 2013 Elsevier Inc. All rights reserved.

Tilted light sheet microscopy with 3D point spread functions for single-molecule super-resolution imaging in mammalian cells

NASA Astrophysics Data System (ADS)

Gustavsson, Anna-Karin; Petrov, Petar N.; Lee, Maurice Y.; Shechtman, Yoav; Moerner, W. E.

2018-02-01

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D superresolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography).

PubMed

Siegel, Nisan; Storrie, Brian; Bruce, Marc; Brooker, Gary

2015-02-07

FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called "CINCH". An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution.

Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

DOE PAGES

Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; ...

2016-01-01

Here, we have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single moleculemore » super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.« less

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

Stimulated emission depletion microscopy resolves individual nitrogen vacancy centers in diamond nanocrystals.

PubMed

Arroyo-Camejo, Silvia; Adam, Marie-Pierre; Besbes, Mondher; Hugonin, Jean-Paul; Jacques, Vincent; Greffet, Jean-Jacques; Roch, Jean-François; Hell, Stefan W; Treussart, François

2013-12-23

Nitrogen-vacancy (NV) color centers in nanodiamonds are highly promising for bioimaging and sensing. However, resolving individual NV centers within nanodiamond particles and the controlled addressing and readout of their spin state has remained a major challenge. Spatially stochastic super-resolution techniques cannot provide this capability in principle, whereas coordinate-controlled super-resolution imaging methods, like stimulated emission depletion (STED) microscopy, have been predicted to fail in nanodiamonds. Here we show that, contrary to these predictions, STED can resolve single NV centers in 40-250 nm sized nanodiamonds with a resolution of ≈10 nm. Even multiple adjacent NVs located in single nanodiamonds can be imaged individually down to relative distances of ≈15 nm. Far-field optical super-resolution of NVs inside nanodiamonds is highly relevant for bioimaging applications of these fluorescent nanolabels. The targeted addressing and readout of individual NV(-) spins inside nanodiamonds by STED should also be of high significance for quantum sensing and information applications.

Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping

NASA Astrophysics Data System (ADS)

Bongiovanni, Marie N.; Godet, Julien; Horrocks, Mathew H.; Tosatto, Laura; Carr, Alexander R.; Wirthensohn, David C.; Ranasinghe, Rohan T.; Lee, Ji-Eun; Ponjavic, Aleks; Fritz, Joelle V.; Dobson, Christopher M.; Klenerman, David; Lee, Steven F.

2016-12-01

Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.

Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping

PubMed Central

Bongiovanni, Marie N.; Godet, Julien; Horrocks, Mathew H.; Tosatto, Laura; Carr, Alexander R.; Wirthensohn, David C.; Ranasinghe, Rohan T.; Lee, Ji-Eun; Ponjavic, Aleks; Fritz, Joelle V.; Dobson, Christopher M.; Klenerman, David; Lee, Steven F.

2016-01-01

Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane. PMID:27929085

Superresolution microscopy for microbiology

PubMed Central

Coltharp, Carla; Xiao, Jie

2014-01-01

Summary This review provides a practical introduction to superresolution microscopy from the perspective of microbiological research. Because of the small sizes of bacterial cells, superresolution methods are particularly powerful and suitable for revealing details of cellular structures that are not resolvable under conventional fluorescence light microscopy. Here we describe the methodological concepts behind three major categories of super-resolution light microscopy: photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) and stimulated emission-depletion (STED) microscopy. We then present recent applications of each of these techniques to microbial systems, which have revealed novel conformations of cellular structures and described new properties of in vivo protein function and interactions. Finally, we discuss the unique issues related to implementing each of these superresolution techniques with bacterial specimens and suggest avenues for future development. The goal of this review is to provide the necessary technical background for interested microbiologists to choose the appropriate super-resolution method for their biological systems, and to introduce the practical considerations required for designing and analysing superresolution imaging experiments. PMID:22947061

Super-resolution differential interference contrast microscopy by structured illumination.

PubMed

Chen, Jianling; Xu, Yan; Lv, Xiaohua; Lai, Xiaomin; Zeng, Shaoqun

2013-01-14

We propose a structured illumination differential interference contrast (SI-DIC) microscopy, breaking the diffraction resolution limit of differential interference contrast (DIC) microscopy. SI-DIC extends the bandwidth of coherent transfer function of the DIC imaging system, thus the resolution is improved. With 0.8 numerical aperture condenser and objective, the reconstructed SI-DIC image of 53 nm polystyrene beads reveals lateral resolution of approximately 190 nm, doubling that of the conventional DIC image. We also demonstrate biological observations of label-free cells with improved spatial resolution. The SI-DIC microscopy can provide sub-diffraction resolution and high contrast images with marker-free specimens, and has the potential for achieving sub-diffraction resolution quantitative phase imaging.

Super-Resolution Imaging of Molecular Emission Spectra and Single Molecule Spectral Fluctuations

PubMed Central

Mlodzianoski, Michael J.; Curthoys, Nikki M.; Gunewardene, Mudalige S.; Carter, Sean; Hess, Samuel T.

2016-01-01

Localization microscopy can image nanoscale cellular details. To address biological questions, the ability to distinguish multiple molecular species simultaneously is invaluable. Here, we present a new version of fluorescence photoactivation localization microscopy (FPALM) which detects the emission spectrum of each localized molecule, and can quantify changes in emission spectrum of individual molecules over time. This information can allow for a dramatic increase in the number of different species simultaneously imaged in a sample, and can create super-resolution maps showing how single molecule emission spectra vary with position and time in a sample. PMID:27002724

CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography)

PubMed Central

Siegel, Nisan; Storrie, Brian; Bruce, Marc

2016-01-01

FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called “CINCH”. An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution. PMID:26839443

Fluorescence microscopy.

PubMed

Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

2014-10-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

Fluorescence Microscopy

PubMed Central

Sanderson, Michael J.; Smith, Ian; Parker, Ian; Bootman, Martin D.

2016-01-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. PMID:25275114

Sequential Super-Resolution Imaging of Bacterial Regulatory Proteins: The Nucleoid and the Cell Membrane in Single, Fixed E. coli Cells.

PubMed

Spahn, Christoph; Glaesmann, Mathilda; Gao, Yunfeng; Foo, Yong Hwee; Lampe, Marko; Kenney, Linda J; Heilemann, Mike

2017-01-01

Despite their small size and the lack of compartmentalization, bacteria exhibit a striking degree of cellular organization, both in time and space. During the last decade, a group of new microscopy techniques emerged, termed super-resolution microscopy or nanoscopy, which facilitate visualizing the organization of proteins in bacteria at the nanoscale. Single-molecule localization microscopy (SMLM) is especially well suited to reveal a wide range of new information regarding protein organization, interaction, and dynamics in single bacterial cells. Recent developments in click chemistry facilitate the visualization of bacterial chromatin with a resolution of ~20 nm, providing valuable information about the ultrastructure of bacterial nucleoids, especially at short generation times. In this chapter, we describe a simple-to-realize protocol that allows determining precise structural information of bacterial nucleoids in fixed cells, using direct stochastic optical reconstruction microscopy (dSTORM). In combination with quantitative photoactivated localization microscopy (PALM), the spatial relationship of proteins with the bacterial chromosome can be studied. The position of a protein of interest with respect to the nucleoids and the cell cylinder can be visualized by super-resolving the membrane using point accumulation for imaging in nanoscale topography (PAINT). The combination of the different SMLM techniques in a sequential workflow maximizes the information that can be extracted from single cells, while maintaining optimal imaging conditions for each technique.

Measurement of replication structures at the nanometer scale using super-resolution light microscopy

PubMed Central

Baddeley, D.; Chagin, V. O.; Schermelleh, L.; Martin, S.; Pombo, A.; Carlton, P. M.; Gahl, A.; Domaing, P.; Birk, U.; Leonhardt, H.; Cremer, C.; Cardoso, M. C.

2010-01-01

DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses. PMID:19864256

Multi-color localization microscopy of fixed cells as a promising tool to study organization of bacterial cytoskeleton

NASA Astrophysics Data System (ADS)

Vedyaykin, A. D.; Gorbunov, V. V.; Sabantsev, A. V.; Polinovskaya, V. S.; Vishnyakov, I. E.; Melnikov, A. S.; Serdobintsev, P. Yu; Khodorkovskii, M. A.

2015-11-01

Localization microscopy allows visualization of biological structures with resolution well below the diffraction limit. Localization microscopy was used to study FtsZ organization in Escherichia coli previously in combination with fluorescent protein labeling, but the fact that fluorescent chimeric protein was unable to rescue temperature-sensitive ftsZ mutants suggests that obtained images may not represent native FtsZ structures faithfully. Indirect immunolabeling of FtsZ not only overcomes this problem, but also allows the use of the powerful visualization methods arsenal available for different structures in fixed cells. In this work we simultaneously obtained super-resolution images of FtsZ structures and diffraction-limited or super-resolution images of DNA and cell surface in E. coli, which allows for the study of the spatial arrangement of FtsZ structures with respect to the nucleoid positions and septum formation.

Operating organic light-emitting diodes imaged by super-resolution spectroscopy

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

King, John T.; Granick, Steve

Super-resolution stimulated emission depletion (STED) microscopy is adapted here for materials characterization that would not otherwise be possible. With the example of organic light-emitting diodes (OLEDs), spectral imaging with pixel-by-pixel wavelength discrimination allows us to resolve local-chain environment encoded in the spectral response of the semi-conducting polymer, and correlate chain packing with local electroluminescence by using externally applied current as the excitation source. We observe nanoscopic defects that would be unresolvable by traditional microscopy. They are revealed in electroluminescence maps in operating OLEDs with 50 nm spatial resolution. We find that brightest emission comes from regions with more densely packedmore » chains. Conventional microscopy of an operating OLED would lack the resolution needed to discriminate these features, while traditional methods to resolve nanoscale features generally cannot be performed when the device is operating. As a result, this points the way towards real-time analysis of materials design principles in devices as they actually operate.« less

Fast, long-term, super-resolution imaging with Hessian structured illumination microscopy.

PubMed

Huang, Xiaoshuai; Fan, Junchao; Li, Liuju; Liu, Haosen; Wu, Runlong; Wu, Yi; Wei, Lisi; Mao, Heng; Lal, Amit; Xi, Peng; Tang, Liqiang; Zhang, Yunfeng; Liu, Yanmei; Tan, Shan; Chen, Liangyi

2018-06-01

To increase the temporal resolution and maximal imaging time of super-resolution (SR) microscopy, we have developed a deconvolution algorithm for structured illumination microscopy based on Hessian matrixes (Hessian-SIM). It uses the continuity of biological structures in multiple dimensions as a priori knowledge to guide image reconstruction and attains artifact-minimized SR images with less than 10% of the photon dose used by conventional SIM while substantially outperforming current algorithms at low signal intensities. Hessian-SIM enables rapid imaging of moving vesicles or loops in the endoplasmic reticulum without motion artifacts and with a spatiotemporal resolution of 88 nm and 188 Hz. Its high sensitivity allows the use of sub-millisecond excitation pulses followed by dark recovery times to reduce photobleaching of fluorescent proteins, enabling hour-long time-lapse SR imaging of actin filaments in live cells. Finally, we observed the structural dynamics of mitochondrial cristae and structures that, to our knowledge, have not been observed previously, such as enlarged fusion pores during vesicle exocytosis.

Operating organic light-emitting diodes imaged by super-resolution spectroscopy

DOE PAGES

King, John T.; Granick, Steve

2016-06-21

Super-resolution stimulated emission depletion (STED) microscopy is adapted here for materials characterization that would not otherwise be possible. With the example of organic light-emitting diodes (OLEDs), spectral imaging with pixel-by-pixel wavelength discrimination allows us to resolve local-chain environment encoded in the spectral response of the semi-conducting polymer, and correlate chain packing with local electroluminescence by using externally applied current as the excitation source. We observe nanoscopic defects that would be unresolvable by traditional microscopy. They are revealed in electroluminescence maps in operating OLEDs with 50 nm spatial resolution. We find that brightest emission comes from regions with more densely packedmore » chains. Conventional microscopy of an operating OLED would lack the resolution needed to discriminate these features, while traditional methods to resolve nanoscale features generally cannot be performed when the device is operating. As a result, this points the way towards real-time analysis of materials design principles in devices as they actually operate.« less

Particle tracking and extended object imaging by interferometric super resolution microscopy

NASA Astrophysics Data System (ADS)

Gdor, Itay; Yoo, Seunghwan; Wang, Xiaolei; Daddysman, Matthew; Wilton, Rosemarie; Ferrier, Nicola; Hereld, Mark; Cossairt, Oliver (Ollie); Katsaggelos, Aggelos; Scherer, Norbert F.

2018-02-01

An interferometric fluorescent microscope and a novel theoretic image reconstruction approach were developed and used to obtain super-resolution images of live biological samples and to enable dynamic real time tracking. The tracking utilizes the information stored in the interference pattern of both the illuminating incoherent light and the emitted light. By periodically shifting the interferometer phase and a phase retrieval algorithm we obtain information that allow localization with sub-2 nm axial resolution at 5 Hz.

Measuring true localization accuracy in super resolution microscopy with DNA-origami nanostructures

NASA Astrophysics Data System (ADS)

Reuss, Matthias; Fördős, Ferenc; Blom, Hans; Öktem, Ozan; Högberg, Björn; Brismar, Hjalmar

2017-02-01

A common method to assess the performance of (super resolution) microscopes is to use the localization precision of emitters as an estimate for the achieved resolution. Naturally, this is widely used in super resolution methods based on single molecule stochastic switching. This concept suffers from the fact that it is hard to calibrate measures against a real sample (a phantom), because true absolute positions of emitters are almost always unknown. For this reason, resolution estimates are potentially biased in an image since one is blind to true position accuracy, i.e. deviation in position measurement from true positions. We have solved this issue by imaging nanorods fabricated with DNA-origami. The nanorods used are designed to have emitters attached at each end in a well-defined and highly conserved distance. These structures are widely used to gauge localization precision. Here, we additionally determined the true achievable localization accuracy and compared this figure of merit to localization precision values for two common super resolution microscope methods STED and STORM.

Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy

NASA Astrophysics Data System (ADS)

Jünger, Felix; Olshausen, Philipp V.; Rohrbach, Alexander

2016-07-01

Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes.

Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy

PubMed Central

Jünger, Felix; Olshausen, Philipp v.; Rohrbach, Alexander

2016-01-01

Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes. PMID:27465033

Lensfree super-resolution holographic microscopy using wetting films on a chip

NASA Astrophysics Data System (ADS)

Mudanyali, Onur; Bishara, Waheb; Ozcan, Aydogan

2011-08-01

We investigate the use of wetting films to significantly improve the imaging performance of lensfree pixel super-resolution on-chip microscopy, achieving < 1 μm spatial resolution over a large imaging area of ~24 mm2. Formation of an ultra-thin wetting film over the specimen effectively creates a micro-lens effect over each object, which significantly improves the signal-to-noise-ratio and therefore the resolution of our lensfree images. We validate the performance of this approach through lensfree on-chip imaging of various objects having fine morphological features (with dimensions of e.g., ≤0.5 μm) such as Escherichia coli (E. coli), human sperm, Giardia lamblia trophozoites, polystyrene micro beads as well as red blood cells. These results are especially important for the development of highly sensitive field-portable microscopic analysis tools for resource limited settings.

Custom Super-Resolution Microscope for the Structural Analysis of Nanostructures

DTIC Science & Technology

2018-05-29

research community. As part of our validation of the new design approach, we performed two - color imaging of pairs of adjacent oligo probes hybridized...nanostructures and biological targets. Our microscope features a large field of view and custom optics that facilitate 3D imaging and enhanced contrast in...our imaging throughput by creating two microscopy platforms for high-throughput, super-resolution materials characterization, with the AO set-up being

Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins.

PubMed

Venkataramani, Varun; Kardorff, Markus; Herrmannsdörfer, Frank; Wieneke, Ralph; Klein, Alina; Tampé, Robert; Heilemann, Mike; Kuner, Thomas

2018-04-03

With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the 'labeling barrier' and to bypass photobleaching in multi-plane, whole-cell 3D experiments.

Assessing resolution in live cell structured illumination microscopy

NASA Astrophysics Data System (ADS)

Pospíšil, Jakub; Fliegel, Karel; Klíma, Miloš

2017-12-01

Structured Illumination Microscopy (SIM) is a powerful super-resolution technique, which is able to enhance the resolution of optical microscope beyond the Abbe diffraction limit. In the last decade, numerous SIM methods that achieve the resolution of 100 nm in the lateral dimension have been developed. The SIM setups with new high-speed cameras and illumination pattern generators allow rapid acquisition of the live specimen. Therefore, SIM is widely used for investigation of the live structures in molecular and live cell biology. Quantitative evaluation of resolution enhancement in a real sample is essential to describe the efficiency of super-resolution microscopy technique. However, measuring the resolution of a live cell sample is a challenging task. Based on our experimental findings, the widely used Fourier ring correlation (FRC) method does not seem to be well suited for measuring the resolution of SIM live cell video sequences. Therefore, the resolution assessing methods based on Fourier spectrum analysis are often used. We introduce a measure based on circular average power spectral density (PSDca) estimated from a single SIM image (one video frame). PSDca describes the distribution of the power of a signal with respect to its spatial frequency. Spatial resolution corresponds to the cut-off frequency in Fourier space. In order to estimate the cut-off frequency from a noisy signal, we use a spectral subtraction method for noise suppression. In the future, this resolution assessment approach might prove useful also for single-molecule localization microscopy (SMLM) live cell imaging.

Structured illumination microscopy for dual-modality 3D sub-diffraction resolution fluorescence and refractive-index reconstruction

PubMed Central

Chowdhury, Shwetadwip; Eldridge, Will J.; Wax, Adam; Izatt, Joseph A.

2017-01-01

Though structured illumination (SI) microscopy is a popular imaging technique conventionally associated with fluorescent super-resolution, recent works have suggested its applicability towards sub-diffraction resolution coherent imaging with quantitative endogenous biological contrast. Here, we demonstrate that SI can efficiently integrate together the principles of fluorescent super-resolution and coherent synthetic aperture to achieve 3D dual-modality sub-diffraction resolution, fluorescence and refractive-index (RI) visualizations of biological samples. We experimentally demonstrate this framework by introducing a SI microscope capable of 3D sub-diffraction resolution fluorescence and RI imaging, and verify its biological visualization capabilities by experimentally reconstructing 3D RI/fluorescence visualizations of fluorescent calibration microspheres as well as alveolar basal epithelial adenocarcinoma (A549) and human colorectal adenocarcinmoa (HT-29) cells, fluorescently stained for F-actin. This demonstration may suggest SI as an especially promising imaging technique to enable future biological studies that explore synergistically operating biophysical/biochemical and molecular mechanisms at sub-diffraction resolutions. PMID:29296504

Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots.

PubMed

Chizhik, Anna M; Stein, Simon; Dekaliuk, Mariia O; Battle, Christopher; Li, Weixing; Huss, Anja; Platen, Mitja; Schaap, Iwan A T; Gregor, Ingo; Demchenko, Alexander P; Schmidt, Christoph F; Enderlein, Jörg; Chizhik, Alexey I

2016-01-13

Success in super-resolution imaging relies on a proper choice of fluorescent probes. Here, we suggest novel easily produced and biocompatible nanoparticles-carbon nanodots-for super-resolution optical fluctuation bioimaging (SOFI). The particles revealed an intrinsic dual-color fluorescence, which corresponds to two subpopulations of particles of different electric charges. The neutral nanoparticles localize to cellular nuclei suggesting their potential use as an inexpensive, easily produced nucleus-specific label. The single particle study revealed that the carbon nanodots possess a unique hybrid combination of fluorescence properties exhibiting characteristics of both dye molecules and semiconductor nanocrystals. The results suggest that charge trapping and redistribution on the surface of the particles triggers their transitions between emissive and dark states. These findings open up new possibilities for the utilization of carbon nanodots in the various super-resolution microscopy methods based on stochastic optical switching.

Super-resolution optical microscopy study of telomere structure.

PubMed

Phipps, Mary Lisa; Goodwin, Peter M; Martinez, Jennifer S; Goodwin, Edwin H

2016-09-01

Chromosome ends are shielded from exonucleolytic attack and inappropriate end-joining by terminal structures called telomeres; these structures are potential targets for anticancer drugs. Telomeres are composed of a simple DNA sequence (5?-TTAGGG-3? in humans) repeated more than a thousand times, a short 3? single-stranded overhang, and numerous proteins. Electron microscopy has shown that the 3? overhang pairs with the complementary strand at an internal site creating a small displacement loop and a large double-stranded “t-loop.” Our goal is to determine whether all telomeres adopt the t-loop configuration, or whether there are two or more distinct configurations. Progress in optimizing super-resolution (SR) microscopy for this ongoing investigation is reported here. Results suggest that under certain conditions sample preparation procedures may disrupt chromatin by causing loss of nucleosomes. This finding may limit the use of SR microscopy in telomere studies.

Super-resolution optical microscopy study of telomere structure

NASA Astrophysics Data System (ADS)

Phipps, Mary Lisa; Goodwin, Peter M.; Martinez, Jennifer S.; Goodwin, Edwin H.

2016-09-01

Chromosome ends are shielded from exonucleolytic attack and inappropriate end-joining by terminal structures called telomeres; these structures are potential targets for anticancer drugs. Telomeres are composed of a simple DNA sequence (5‧-TTAGGG-3‧ in humans) repeated more than a thousand times, a short 3‧ single-stranded overhang, and numerous proteins. Electron microscopy has shown that the 3‧ overhang pairs with the complementary strand at an internal site creating a small displacement loop and a large double-stranded "t-loop." Our goal is to determine whether all telomeres adopt the t-loop configuration, or whether there are two or more distinct configurations. Progress in optimizing super-resolution (SR) microscopy for this ongoing investigation is reported here. Results suggest that under certain conditions sample preparation procedures may disrupt chromatin by causing loss of nucleosomes. This finding may limit the use of SR microscopy in telomere studies.

Fluorescent Nano-Probes to Image Plant Cell Walls by Super-Resolution STED Microscopy

PubMed Central

Paës, Gabriel; Habrant, Anouck; Terryn, Christine

2018-01-01

Lignocellulosic biomass is a complex network of polymers making up the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals, and materials. Because of its complex architecture, lignocellulose is a recalcitrant material that requires some pretreatments and several types of catalysts to be transformed efficiently. Gaining more knowledge in the architecture of plant cell walls is therefore important to understand and optimize transformation processes. For the first time, super-resolution imaging of poplar wood samples has been performed using the Stimulated Emission Depletion (STED) technique. In comparison to standard confocal images, STED reveals new details in cell wall structure, allowing the identification of secondary walls and middle lamella with fine details, while keeping open the possibility to perform topochemistry by the use of relevant fluorescent nano-probes. In particular, the deconvolution of STED images increases the signal-to-noise ratio so that images become very well defined. The obtained results show that the STED super-resolution technique can be easily implemented by using cheap commercial fluorescent rhodamine-PEG nano-probes which outline the architecture of plant cell walls due to their interaction with lignin. Moreover, the sample preparation only requires easily-prepared plant sections of a few tens of micrometers, in addition to an easily-implemented post-treatment of images. Overall, the STED super-resolution technique in combination with a variety of nano-probes can provide a new vision of plant cell wall imaging by filling in the gap between classical photon microscopy and electron microscopy. PMID:29415498

Fluorescent Nano-Probes to Image Plant Cell Walls by Super-Resolution STED Microscopy.

PubMed

Paës, Gabriel; Habrant, Anouck; Terryn, Christine

2018-02-06

Lignocellulosic biomass is a complex network of polymers making up the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals, and materials. Because of its complex architecture, lignocellulose is a recalcitrant material that requires some pretreatments and several types of catalysts to be transformed efficiently. Gaining more knowledge in the architecture of plant cell walls is therefore important to understand and optimize transformation processes. For the first time, super-resolution imaging of poplar wood samples has been performed using the Stimulated Emission Depletion (STED) technique. In comparison to standard confocal images, STED reveals new details in cell wall structure, allowing the identification of secondary walls and middle lamella with fine details, while keeping open the possibility to perform topochemistry by the use of relevant fluorescent nano-probes. In particular, the deconvolution of STED images increases the signal-to-noise ratio so that images become very well defined. The obtained results show that the STED super-resolution technique can be easily implemented by using cheap commercial fluorescent rhodamine-PEG nano-probes which outline the architecture of plant cell walls due to their interaction with lignin. Moreover, the sample preparation only requires easily-prepared plant sections of a few tens of micrometers, in addition to an easily-implemented post-treatment of images. Overall, the STED super-resolution technique in combination with a variety of nano-probes can provide a new vision of plant cell wall imaging by filling in the gap between classical photon microscopy and electron microscopy.

Super-resolution binding activated localization microscopy through reversible change of DNA conformation.

PubMed

Szczurek, Aleksander; Birk, Udo; Knecht, Hans; Dobrucki, Jurek; Mai, Sabine; Cremer, Christoph

2018-01-01

Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future applications in biology and medicine.

Super-resolution binding activated localization microscopy through reversible change of DNA conformation

PubMed Central

Knecht, Hans; Dobrucki, Jurek; Mai, Sabine

2018-01-01

ABSTRACT Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future applications in biology and medicine. PMID:29297245

Autofocusing Airy beam STED microscopy with long focal distance

NASA Astrophysics Data System (ADS)

Hu, Di; Liang, Yao; Chen, Yin; Chen, Zan Hui; Huang, Xu Guang

2017-12-01

Stimulated emission depletion (STED) is a very important technique in super-resolution microscopy. Until now, while autofocusing Airy beam (AAB) has been an attractive theme for both theoretical and applied researches, there are almost no report on AABs being used in STED microscopy. In this paper, we propose a novel STED microscopy based on AABs. A radially symmetric 3/2 phase plate is involved to simultaneously generate autofocusing excitation- and depletion-Airy beams. Remarkably, the AAB can auto-focus to a wavelength-scale spot with a long focal depth (several millimeters): on the contrary, the working distance of a conventional high numerical aperture (NA) objective is usually very short (about 200 μm). Our calculations indicate that the AAB based STED microscopy can achieve a super-resolution spot with FWHM of 58 nm while the focal length is 4.638 mm. Moreover, with properties of non-diffracting and self-healing, the Airy beam could enable a reduction of the scattering distortion induced by the specimens and has a great potential in imaging thick specimens.

Localization microscopy of DNA in situ using Vybrant{sup ®} DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution

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

Żurek-Biesiada, Dominika; Szczurek, Aleksander T.; Prakash, Kirti

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant{sup ®} DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei ofmore » fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10{sup 6} signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100 nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy. - Highlights: • Super-resolution imaging of nuclear DNA with Vybrant Violet and blue excitation. • 90nm resolution images of DNA structures in optically thick eukaryotic nuclei. • Enhanced resolution confirms the existence of DNA-free regions inside the nucleus. • Optimized imaging conditions enable multicolor super-resolution imaging.« less

Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states

NASA Astrophysics Data System (ADS)

Ehmann, Nadine; van de Linde, Sebastian; Alon, Amit; Ljaschenko, Dmitrij; Keung, Xi Zhen; Holm, Thorge; Rings, Annika; Diantonio, Aaron; Hallermann, Stefan; Ashery, Uri; Heckmann, Manfred; Sauer, Markus; Kittel, Robert J.

2014-08-01

The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.

Super-Chelators for Advanced Protein Labeling in Living Cells.

PubMed

Gatterdam, Karl; Joest, Eike F; Dietz, Marina S; Heilemann, Mike; Tampé, Robert

2018-05-14

Live-cell labeling, super-resolution microscopy, single-molecule applications, protein localization, or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Herein, we define a new class of hexavalent N-nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA-based small interaction pairs described so far. Coupled to bright organic fluorophores with fine-tuned photophysical properties, the super-chelator probes were delivered into human cells by chemically gated nanopores. These super-chelators permit kinetic profiling, multiplexed labeling of His 6 - and His 12 -tagged proteins as well as single-molecule-based super-resolution imaging. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Annular solid-immersion lenslet array super-resolution optical microscopy

NASA Astrophysics Data System (ADS)

Liau, Z. L.

2012-10-01

We describe a novel solid-immersion lenslet array, micro-fabricated in a chip form in the high-index (3.45) gallium phosphide. The innovatively designed lenslet features an annular aperture with appropriately patterned light absorbers and antireflection coatings. The array chip is easy to handle and enables the direct deposition of the specimen of interest onto its back-plane for tight adhesion and good optical coupling. The ensuing diffraction from the near field can yield supercritical rays inside the high-index lenslet and can, therefore, overcome the refraction and critical-angle limitations. This model showed agreement with the experimental observation of the solid-immersion fluorescence microscopy imaging, in which the refracted rays were completely blocked by the annular aperture. A large longitudinal (depth) magnification effect was also predicted and showed agreement with experiment. The annular lenslet's additional advantages of improved resolution and contrast were also discussed. Resolution of nested-L patterns with grating pitch as small as 100 nm was experimentally demonstrated. The demonstrated annular solid-immersion lenslet array concept is promising for a wider use in super-resolution optical microscopy.

Quantitative evaluation of software packages for single-molecule localization microscopy.

PubMed

Sage, Daniel; Kirshner, Hagai; Pengo, Thomas; Stuurman, Nico; Min, Junhong; Manley, Suliana; Unser, Michael

2015-08-01

The quality of super-resolution images obtained by single-molecule localization microscopy (SMLM) depends largely on the software used to detect and accurately localize point sources. In this work, we focus on the computational aspects of super-resolution microscopy and present a comprehensive evaluation of localization software packages. Our philosophy is to evaluate each package as a whole, thus maintaining the integrity of the software. We prepared synthetic data that represent three-dimensional structures modeled after biological components, taking excitation parameters, noise sources, point-spread functions and pixelation into account. We then asked developers to run their software on our data; most responded favorably, allowing us to present a broad picture of the methods available. We evaluated their results using quantitative and user-interpretable criteria: detection rate, accuracy, quality of image reconstruction, resolution, software usability and computational resources. These metrics reflect the various tradeoffs of SMLM software packages and help users to choose the software that fits their needs.

Single-Molecule Tracking Photoactivated Localization Microscopy to Map Nano-Scale Structure and Dynamics in Living Spines

PubMed Central

MacGillavry, Harold D.; Blanpied, Thomas A.

2013-01-01

Super-resolution microscopy has rapidly become an indispensable tool in cell biology and neuroscience by enabling measurement in live cells of structures smaller than the classical limit imposed by diffraction. The most widely applied super-resolution method currently is localization microscopy, which takes advantage of the ability to determine the position of individual fluorescent molecules with nanometer accuracy even in cells. By iteratively measuring sparse subsets of photoactivatable fluorescent proteins, protein distribution in macromolecular structures can be accurately reconstructed. Moreover, the motion trajectories of individual molecules within cells can be measured, providing unique ability to measure transport kinetics, exchange rates, and binding affinities of even small subsets of molecules with high temporal resolution and great spatial specificity. This unit describes protocols to measure and quantify the distribution of scaffold proteins within single synapses of cultured hippocampal neurons, and to track and measure the diffusion of intracellular constituents of the neuronal plasma membrane. PMID:25429311

Saturated virtual fluorescence emission difference microscopy based on detector array

NASA Astrophysics Data System (ADS)

Liu, Shaocong; Sun, Shiyi; Kuang, Cuifang; Ge, Baoliang; Wang, Wensheng; Liu, Xu

2017-07-01

Virtual fluorescence emission difference microscopy (vFED) has been proposed recently to enhance the lateral resolution of confocal microscopy with a detector array, implemented by scanning a doughnut-shaped pattern. Theoretically, the resolution can be enhanced by around 1.3-fold compared with that in confocal microscopy. For further improvement of the resolving ability of vFED, a novel method is presented utilizing fluorescence saturation for super-resolution imaging, which we called saturated virtual fluorescence emission difference microscopy (svFED). With a point detector array, matched solid and hollow point spread functions (PSF) can be obtained by photon reassignment, and the difference results between them can be used to boost the transverse resolution. Results show that the diffraction barrier can be surpassed by at least 34% compared with that in vFED and the resolution is around 2-fold higher than that in confocal microscopy.

Super-resolution study of polymer mobility fluctuations near c*.

PubMed

King, John T; Yu, Changqian; Wilson, William L; Granick, Steve

2014-09-23

Nanoscale dynamic heterogeneities in synthetic polymer solutions are detected using super-resolution optical microscopy. To this end, we map concentration fluctuations in polystyrene-toluene solutions with spatial resolution below the diffraction limit, focusing on critical fluctuations near the polymer overlap concentration, c*. Two-photon super-resolution microscopy was adapted to be applicable in an organic solvent, and a home-built STED-FCS system with stimulated emission depletion (STED) was used to perform fluorescence correlation spectroscopy (FCS). The polystyrene serving as the tracer probe (670 kg mol(-1), radius of gyration RG ≈ 35 nm, end-labeled with a bodipy derivative chromophore) was dissolved in toluene at room temperature (good solvent) and mixed with matrix polystyrene (3,840 kg mol(-1), RG ≈ 97 nm, Mw/Mn = 1.04) whose concentration was varied from dilute to more than 10c*. Whereas for dilute solutions the intensity-intensity correlation function follows a single diffusion process, it splits starting at c* to imply an additional relaxation process provided that the experimental focal area does not greatly exceed the polymer blob size. We identify the slower mode as self-diffusion and the increasingly rapid mode as correlated segment fluctuations that reflect the cooperative diffusion coefficient, Dcoop. These real-space measurements find quantitative agreement between correlation lengths inferred from dynamic measurements and those from determining the limit below which diffusion coefficients are independent of spot size. This study is considered to illustrate the potential of importing into polymer science the techniques of super-resolution imaging.

Advances in pancreatic islet monolayer culture on glass surfaces enable super-resolution microscopy and insights into beta cell ciliogenesis and proliferation

PubMed Central

Phelps, Edward A.; Cianciaruso, Chiara; Santo-Domingo, Jaime; Pasquier, Miriella; Galliverti, Gabriele; Piemonti, Lorenzo; Berishvili, Ekaterine; Burri, Olivier; Wiederkehr, Andreas; Hubbell, Jeffrey A.; Baekkeskov, Steinunn

2017-01-01

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells. PMID:28401888

Adaptive optics improves multiphoton super-resolution imaging

NASA Astrophysics Data System (ADS)

Zheng, Wei; Wu, Yicong; Winter, Peter; Shroff, Hari

2018-02-01

Three dimensional (3D) fluorescence microscopy has been essential for biological studies. It allows interrogation of structure and function at spatial scales spanning the macromolecular, cellular, and tissue levels. Critical factors to consider in 3D microscopy include spatial resolution, signal-to-noise (SNR), signal-to-background (SBR), and temporal resolution. Maintaining high quality imaging becomes progressively more difficult at increasing depth (where optical aberrations, induced by inhomogeneities of refractive index in the sample, degrade resolution and SNR), and in thick or densely labeled samples (where out-of-focus background can swamp the valuable, in-focus-signal from each plane). In this report, we introduce our new instrumentation to address these problems. A multiphoton structured illumination microscope was simply modified to integrate an adpative optics system for optical aberrations correction. Firstly, the optical aberrations are determined using direct wavefront sensing with a nonlinear guide star and subsequently corrected using a deformable mirror, restoring super-resolution information. We demonstrate the flexibility of our adaptive optics approach on a variety of semi-transparent samples, including bead phantoms, cultured cells in collagen gels and biological tissues. The performance of our super-resolution microscope is improved in all of these samples, as peak intensity is increased (up to 40-fold) and resolution recovered (up to 176+/-10 nm laterally and 729+/-39 nm axially) at depths up to 250 μm from the coverslip surface.

A fixable probe for visualizing flagella and plasma membranes of the African trypanosome.

PubMed

Wiedeman, Justin; Mensa-Wilmot, Kojo

2018-01-01

The protozoan Trypanosoma brucei sp. cause diseases in humans and animals. Studies of T. brucei cell biology have revealed unique features, such as major endocytic events being limited to a single region, and mitochondrial genome segregation mediated via basal bodies. Further understanding of trypanosome cell biology can be facilitated with super-resolution fluorescence microscopy. Lack of a plasma membrane probe for fixed trypanosomes remains a persistent problem in need of a working solution. Herein, we report protocols developed using mCLING in super-resolution structured illumination fluorescence microscopy (SR-SIM). mCLING comprehensively labels flagellar membranes, including nascent intracellular stages. To extend its usefulness for trypanosome biology we optimized mCLING in combination with organelle-specific antibodies for immunofluorescence of basal bodies or mitochondria. Then in work with live trypanosomes, we demonstrated internalization of mCLING into endocytic stations that overlap with LysoTracker in acidic organelles. Greater detail of the intracellular location of mCLING was obtained with SR-SIM after pulsing trypanosomes with the probe, and allowing continuous uptake of fluorescent concanavalin A (ConA) destined for lysosomes. In most cases, ConA and mCLING vesicles were juxtaposed but not coincident. A video of the complete image stack at the 15 min time point shows zones of mCLING staining surrounding patches of ConA, consistent with persistence of mCLING in membranes of compartments that contain luminal ConA. In summary, these studies establish mCLING as a versatile trypanosome membrane probe compatible with super-resolution microscopy that can be used for detailed analysis of flagellar membrane biogenesis. In addition, mCLING can be used for immunofluorescence in fixed, permeabilized trypanosomes. Its robust staining of the plasma membrane eliminates a need to overlay transmitted light images on fluorescence pictures obtained from widefield, confocal, or super-resolution microscopy.

Nobel Prize Recipient Eric Betzig Presents Lecture on Efforts to Improve High-Resolution Microscopy | Poster

Cancer.gov

Eric Betzig, Ph.D., a 2014 recipient of the Nobel Prize in Chemistry and a scientist at Janelia Research Campus (JRC), Howard Hughes Medical Institute, in Ashburn, Va., visited NCI at Frederick on Sept. 10 to present a Distinguished Scientist lecture and discuss the latest high-resolution microscopy techniques. Betzig co-invented photoactivation localization microscopy (PALM) in collaboration with scientists at NIH. PALM achieves 10-fold improvement in spatial resolution of cells, going from the resolution limit of approximately 250 nm in standard optical microscopy down to approximately 20 nm, thus producing a so-called “super-resolution” image. Spatial resolution refers to the clarity of an image or, in other words, the smallest details that can be observed from an image.

Super-Resolution Microscopy of Cerebrospinal Fluid Biomarkers as a Tool for Alzheimer's Disease Diagnostics.

PubMed

Zhang, William I; Antonios, Gregory; Rabano, Alberto; Bayer, Thomas A; Schneider, Anja; Rizzoli, Silvio O

2015-01-01

Alzheimer's disease (AD) is neuropathologically characterized by aggregates of amyloid-β peptides (Aβ) and tau proteins. The consensus in the AD field is that Aβ and tau should serve as diagnostic biomarkers for AD. However, their aggregates have been difficult to investigate by conventional fluorescence microscopy, since their size is below the diffraction limit (∼200 nm). To solve this, we turned to a super-resolution imaging technique, stimulated emission depletion (STED) microscopy, which has a high enough precision to allow the discrimination of low- and high-molecular weight aggregates prepared in vitro. We used STED to analyze the structural organization of Aβ and tau in cerebrospinal fluid (CSF) from 36 AD patients, 11 patients with mild cognitive impairment (MCI), and 21 controls. We measured the numbers of aggregates in the CSF samples, and the aggregate sizes and intensities. These parameters enabled us to distinguish AD patients from controls with a specificity of ∼87% and a sensitivity of ∼79% . In addition, the aggregate parameters determined with STED microscopy correlated with the severity of cognitive impairment in AD patients. Finally, these parameters may be useful as predictive tools for MCI cases. The STED parameters of two MCI patients who developed AD during the course of the study, as well as of MCI patients whose Aβ ELISA values fall within the accepted range for AD, placed them close to the AD averages. We suggest that super-resolution imaging is a promising tool for AD diagnostics.

Nanoscale Membrane Curvature detected by Polarized Localization Microscopy

NASA Astrophysics Data System (ADS)

Kelly, Christopher; Maarouf, Abir; Woodward, Xinxin

Nanoscale membrane curvature is a necessary component of countless cellular processes. Here we present Polarized Localization Microscopy (PLM), a super-resolution optical imaging technique that enables the detection of nanoscale membrane curvature with order-of-magnitude improvements over comparable optical techniques. PLM combines the advantages of polarized total internal reflection fluorescence microscopy and fluorescence localization microscopy to reveal single-fluorophore locations and orientations without reducing localization precision by point spread function manipulation. PLM resolved nanoscale membrane curvature of a supported lipid bilayer draped over polystyrene nanoparticles on a glass coverslip, thus creating a model membrane with coexisting flat and curved regions and membrane radii of curvature as small as 20 nm. Further, PLM provides single-molecule trajectories and the aggregation of curvature-inducing proteins with super-resolution to reveal the correlated effects of membrane curvature, dynamics, and molecular sorting. For example, cholera toxin subunit B has been observed to induce nanoscale membrane budding and concentrate at the bud neck. PLM reveals a previously hidden and critical information of membrane topology.

TestSTORM: Simulator for optimizing sample labeling and image acquisition in localization based super-resolution microscopy

PubMed Central

Sinkó, József; Kákonyi, Róbert; Rees, Eric; Metcalf, Daniel; Knight, Alex E.; Kaminski, Clemens F.; Szabó, Gábor; Erdélyi, Miklós

2014-01-01

Localization-based super-resolution microscopy image quality depends on several factors such as dye choice and labeling strategy, microscope quality and user-defined parameters such as frame rate and number as well as the image processing algorithm. Experimental optimization of these parameters can be time-consuming and expensive so we present TestSTORM, a simulator that can be used to optimize these steps. TestSTORM users can select from among four different structures with specific patterns, dye and acquisition parameters. Example results are shown and the results of the vesicle pattern are compared with experimental data. Moreover, image stacks can be generated for further evaluation using localization algorithms, offering a tool for further software developments. PMID:24688813

Stochastic Optical Reconstruction Microscopy (STORM).

PubMed

Xu, Jianquan; Ma, Hongqiang; Liu, Yang

2017-07-05

Super-resolution (SR) fluorescence microscopy, a class of optical microscopy techniques at a spatial resolution below the diffraction limit, has revolutionized the way we study biology, as recognized by the Nobel Prize in Chemistry in 2014. Stochastic optical reconstruction microscopy (STORM), a widely used SR technique, is based on the principle of single molecule localization. STORM routinely achieves a spatial resolution of 20 to 30 nm, a ten-fold improvement compared to conventional optical microscopy. Among all SR techniques, STORM offers a high spatial resolution with simple optical instrumentation and standard organic fluorescent dyes, but it is also prone to image artifacts and degraded image resolution due to improper sample preparation or imaging conditions. It requires careful optimization of all three aspects-sample preparation, image acquisition, and image reconstruction-to ensure a high-quality STORM image, which will be extensively discussed in this unit. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

A general strategy for developing cell-permeable photo-modulatable organic fluorescent probes for live-cell super-resolution imaging.

PubMed

Pan, Deng; Hu, Zhe; Qiu, Fengwu; Huang, Zhen-Li; Ma, Yilong; Wang, Yina; Qin, Lingsong; Zhang, Zhihong; Zeng, Shaoqun; Zhang, Yu-Hui

2014-11-20

Single-molecule localization microscopy (SMLM) achieves super-resolution imaging beyond the diffraction limit but critically relies on the use of photo-modulatable fluorescent probes. Here we report a general strategy for constructing cell-permeable photo-modulatable organic fluorescent probes for live-cell SMLM by exploiting the remarkable cytosolic delivery ability of a cell-penetrating peptide (rR)3R2. We develop photo-modulatable organic fluorescent probes consisting of a (rR)3R2 peptide coupled to a cell-impermeable organic fluorophore and a recognition unit. Our results indicate that these organic probes are not only cell permeable but can also specifically and directly label endogenous targeted proteins. Using the probes, we obtain super-resolution images of lysosomes and endogenous F-actin under physiological conditions. We resolve the dynamics of F-actin with 10 s temporal resolution in live cells and discern fine F-actin structures with diameters of ~80 nm. These results open up new avenues in the design of fluorescent probes for live-cell super-resolution imaging.

Super-resolution microscopy as a potential approach to diagnosis of platelet granule disorders.

PubMed

Westmoreland, D; Shaw, M; Grimes, W; Metcalf, D J; Burden, J J; Gomez, K; Knight, A E; Cutler, D F

2016-04-01

Many platelet functions are dependent on bioactive molecules released from their granules. Deficiencies of these granules in number, shape or content are associated with bleeding. The small size of these granules is such that imaging them for diagnosis has traditionally required electron microscopy. However, recently developed super-resolution microscopes provide sufficient spatial resolution to effectively image platelet granules. When combined with automated image analysis, these methods provide a quantitative, unbiased, rapidly acquired dataset that can readily and reliably reveal differences in platelet granules between individuals. To demonstrate the ability of structured illumination microscopy (SIM) to efficiently differentiate between healthy volunteers and three patients with Hermansky-Pudlak syndrome. Blood samples were taken from three patients with Hermansky-Pudlak syndrome and seven controls. Patients 1-3 have gene defects in HPS1, HPS6 and HPS5, respectively; all controls were healthy volunteers. Platelet-rich plasma was isolated from blood and the platelets fixed, stained for CD63 and processed for analysis by immunofluorescence microscopy, using a custom-built SIM microscope. SIM can successfully resolve CD63-positive structures in fixed platelets. A determination of the number of CD63-positive structures per platelet allowed us to conclude that each patient was significantly different from all of the controls with 99% confidence. A super-resolution imaging approach is effective and rapid in objectively differentiating between patients with a platelet bleeding disorder and healthy volunteers. CD63 is a useful marker for predicting Hermansky-Pudlak syndrome and could be used in the diagnosis of patients suspected of other platelet granule disorders. © 2016 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging

NASA Astrophysics Data System (ADS)

Wei, Lu; Zhu, Xinxin; Chen, Zhixing; Min, Wei

2014-02-01

Two-photon excited fluorescence microscopy (TPFM) offers the highest penetration depth with subcellular resolution in light microscopy, due to its unique advantage of nonlinear excitation. However, a fundamental imaging-depth limit, accompanied by a vanishing signal-to-background contrast, still exists for TPFM when imaging deep into scattering samples. Formally, the focusing depth, at which the in-focus signal and the out-of-focus background are equal to each other, is defined as the fundamental imaging-depth limit. To go beyond this imaging-depth limit of TPFM, we report a new class of super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging, including multiphoton activation and imaging (MPAI) harnessing novel photo-activatable fluorophores, stimulated emission reduced fluorescence (SERF) microscopy by adding a weak laser beam for stimulated emission, and two-photon induced focal saturation imaging with preferential depletion of ground-state fluorophores at focus. The resulting image contrasts all exhibit a higher-order (third- or fourth- order) nonlinear signal dependence on laser intensity than that in the standard TPFM. Both the physical principles and the imaging demonstrations will be provided for each super-nonlinear microscopy. In all these techniques, the created super-nonlinearity significantly enhances the imaging contrast and concurrently extends the imaging depth-limit of TPFM. Conceptually different from conventional multiphoton processes mediated by virtual states, our strategy constitutes a new class of fluorescence microscopy where high-order nonlinearity is mediated by real population transfer.

Painting Supramolecular Polymers in Organic Solvents by Super-resolution Microscopy

PubMed Central

2018-01-01

Despite the rapid development of complex functional supramolecular systems, visualization of these architectures under native conditions at high resolution has remained a challenging endeavor. Super-resolution microscopy was recently proposed as an effective tool to unveil one-dimensional nanoscale structures in aqueous media upon chemical functionalization with suitable fluorescent probes. Building upon our previous work, which enabled photoactivation localization microscopy in organic solvents, herein, we present the imaging of one-dimensional supramolecular polymers in their native environment by interface point accumulation for imaging in nanoscale topography (iPAINT). The noncovalent staining, typical of iPAINT, allows the investigation of supramolecular polymers’ structure in situ without any chemical modification. The quasi-permanent adsorption of the dye to the polymer is exploited to identify block-like arrangements within supramolecular fibers, which were obtained upon mixing homopolymers that were prestained with different colors. The staining of the blocks, maintained by the lack of exchange of the dyes, permits the imaging of complex structures for multiple days. This study showcases the potential of PAINT-like strategies such as iPAINT to visualize multicomponent dynamic systems in their native environment with an easy, synthesis-free approach and high spatial resolution. PMID:29697958

The internal architecture of dendritic spines revealed by super-resolution imaging: What did we learn so far?

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

MacGillavry, Harold D., E-mail: h.d.macgillavry@uu.nl; Hoogenraad, Casper C., E-mail: c.hoogenraad@uu.nl

2015-07-15

The molecular architecture of dendritic spines defines the efficiency of signal transmission across excitatory synapses. It is therefore critical to understand the mechanisms that control the dynamic localization of the molecular constituents within spines. However, because of the small scale at which most processes within spines take place, conventional light microscopy techniques are not adequate to provide the necessary level of resolution. Recently, super-resolution imaging techniques have overcome the classical barrier imposed by the diffraction of light, and can now resolve the localization and dynamic behavior of proteins within small compartments with nanometer precision, revolutionizing the study of dendritic spinemore » architecture. Here, we highlight exciting new findings from recent super-resolution studies on neuronal spines, and discuss how these studies revealed important new insights into how protein complexes are assembled and how their dynamic behavior shapes the efficiency of synaptic transmission.« less

Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.

PubMed

Subach, Fedor V; Patterson, George H; Renz, Malte; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav V

2010-05-12

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.

Resolution enhancement in deep-tissue nanoparticle imaging based on plasmonic saturated excitation microscopy

NASA Astrophysics Data System (ADS)

Deka, Gitanjal; Nishida, Kentaro; Mochizuki, Kentaro; Ding, Hou-Xian; Fujita, Katsumasa; Chu, Shi-Wei

2018-03-01

Recently, many resolution enhancing techniques are demonstrated, but most of them are severely limited for deep tissue applications. For example, wide-field based localization techniques lack the ability of optical sectioning, and structured light based techniques are susceptible to beam distortion due to scattering/aberration. Saturated excitation (SAX) microscopy, which relies on temporal modulation that is less affected when penetrating into tissues, should be the best candidate for deep-tissue resolution enhancement. Nevertheless, although fluorescence saturation has been successfully adopted in SAX, it is limited by photobleaching, and its practical resolution enhancement is less than two-fold. Recently, we demonstrated plasmonic SAX which provides bleaching-free imaging with three-fold resolution enhancement. Here we show that the three-fold resolution enhancement is sustained throughout the whole working distance of an objective, i.e., 200 μm, which is the deepest super-resolution record to our knowledge, and is expected to extend into deeper tissues. In addition, SAX offers the advantage of background-free imaging by rejecting unwanted scattering background from biological tissues. This study provides an inspirational direction toward deep-tissue super-resolution imaging and has the potential in tumor monitoring and beyond.

Super-Resolution Image Reconstruction by Nonlocal Means Applied to High-Angle Annular Darkfield Scanning Transmission Electron Microscopy (HAADF-STEM)

DTIC Science & Technology

2009-10-06

When talking about superresolution we always mean to recover the level of resolution set by the microscope, but by using a time series of low...on low resolution possibly very noisy data, is not feasible. Thus, standard superresolution concepts as described above that are based on registration

Far-field optical imaging with subdiffraction resolution enabled by nonlinear saturation absorption

NASA Astrophysics Data System (ADS)

Ding, Chenliang; Wei, Jingsong

2016-01-01

The resolution of far-field optical imaging is required to improve beyond the Abbe limit to the subdiffraction or even the nanoscale. In this work, inspired by scanning electronic microscopy (SEM) imaging, in which carbon (or Au) thin films are usually required to be coated on the sample surface before imaging to remove the charging effect while imaging by electrons. We propose a saturation-absorption-induced far-field super-resolution optical imaging method (SAI-SRIM). In the SAI-SRIM, the carbon (or Au) layers in SEM imaging are replaced by nonlinear-saturation-absorption (NSA) thin films, which are directly coated onto the sample surfaces using advanced thin film deposition techniques. The surface fluctuant morphologies are replicated to the NSA thin films, accordingly. The coated sample surfaces are then imaged using conventional laser scanning microscopy. Consequently, the imaging resolution is greatly improved, and subdiffraction-resolved optical images are obtained theoretically and experimentally. The SAI-SRIM provides an effective and easy way to achieve far-field super-resolution optical imaging for sample surfaces with geometric fluctuant morphology characteristics.

Hyperlens-array-implemented optical microscopy

NASA Astrophysics Data System (ADS)

Iwanaga, Masanobu

2014-08-01

Limit of resolution of conventional optical microscopes has never reached below 100 nm under visible light illumination. We show that numerically designed high-transmittance hyperlens array (HLA) is implemented in an optical microscope and works in practice for achieving one-shot-recording optical images of in-situ placed objects with sub 50 nm resolution in lateral direction. Direct resolution test employing well-defined nanopatterns proves that the HLA-implemented imaging is super-resolution optical microscopy, which works even under nW/mm2 visible illumination for objects. The HLA implementation makes the resolution of conventional microscopes one-scale higher, leading to the 1/10 illumination wavelength range, that is, mesoscopic range.

3D high- and super-resolution imaging using single-objective SPIM.

PubMed

Galland, Remi; Grenci, Gianluca; Aravind, Ajay; Viasnoff, Virgile; Studer, Vincent; Sibarita, Jean-Baptiste

2015-07-01

Single-objective selective-plane illumination microscopy (soSPIM) is achieved with micromirrored cavities combined with a laser beam-steering unit installed on a standard inverted microscope. The illumination and detection are done through the same objective. soSPIM can be used with standard sample preparations and features high background rejection and efficient photon collection, allowing for 3D single-molecule-based super-resolution imaging of whole cells or cell aggregates. Using larger mirrors enabled us to broaden the capabilities of our system to image Drosophila embryos.

Watching entangled circular DNA in real time with super-resolution

NASA Astrophysics Data System (ADS)

Jee, Ah-Young; Kim, Hyeongju; Granick, Steve

In this talk, we will show how we unraveled the conformational dynamics of entangled ring-shaped polymers in network, which is one of the most well-known problems in polymer physics, using deep imaging based on super-resolution fluorescence imaging, stimulated emission depletion (STED) microscopy. By using home-written software, we obtained the statistics of each of the hundreds of molecules, mapping out a large statistical distribution. Through inspection we not only found some aspects of the classic understanding of polymers, but some surprising aspects as well.

Time multiplexing super-resolution nanoscopy based on the Brownian motion of gold nanoparticles

NASA Astrophysics Data System (ADS)

Ilovitsh, Tali; Ilovitsh, Asaf; Wagner, Omer; Zalevsky, Zeev

2017-02-01

Super-resolution localization microscopy can overcome the diffraction limit and achieve a tens of order improvement in resolution. It requires labeling the sample with fluorescent probes followed with their repeated cycles of activation and photobleaching. This work presents an alternative approach that is free from direct labeling and does not require the activation and photobleaching cycles. Fluorescently labeled gold nanoparticles in a solution are distributed on top of the sample. The nanoparticles move in a random Brownian motion, and interact with the sample. By obscuring different areas in the sample, the nanoparticles encode the sub-wavelength features. A sequence of images of the sample is captured and decoded by digital post processing to create the super-resolution image. The achievable resolution is limited by the additive noise and the size of the nanoparticles. Regular nanoparticles with diameter smaller than 100nm are barely seen in a conventional bright field microscope, thus fluorescently labeled gold nanoparticles were used, with proper

From single-molecule spectroscopy to super-resolution imaging of the neuron: a review

PubMed Central

Laine, Romain F; Kaminski Schierle, Gabriele S; van de Linde, Sebastian; Kaminski, Clemens F

2016-01-01

Abstract For more than 20 years, single-molecule spectroscopy has been providing invaluable insights into nature at the molecular level. The field has received a powerful boost with the development of the technique into super-resolution imaging methods, ca. 10 years ago, which overcome the limitations imposed by optical diffraction. Today, single molecule super-resolution imaging is routinely used in the study of macromolecular function and structure in the cell. Concomitantly, computational methods have been developed that provide information on numbers and positions of molecules at the nanometer-scale. In this overview, we outline the technical developments that have led to the emergence of localization microscopy techniques from single-molecule spectroscopy. We then provide a comprehensive review on the application of the technique in the field of neuroscience research. PMID:28809165

3-D Cellular Ultrastructure Can Be Resolved by X-ray Microscopy | Center for Cancer Research

Cancer.gov

X-ray microscopy (XRM) is more rapid than cryoelectron tomography or super-resolution fluorescence microscopy and could fill an important gap in current technologies used to investigate in situ three-dimensional structure of cells. New XRM methods developed by first author Gerd Schneider, Ph.D., working with James McNally. Ph.D., and a team of colleagues, is capable of

Review of combined isotopic and optical nanoscopy

PubMed Central

Richter, Katharina N.; Rizzoli, Silvio O.; Jähne, Sebastian; Vogts, Angela; Lovric, Jelena

2017-01-01

Abstract. Investigating the detailed substructure of the cell is beyond the ability of conventional optical microscopy. Electron microscopy, therefore, has been the only option for such studies for several decades. The recent implementation of several super-resolution optical microscopy techniques has rendered the investigation of cellular substructure easier and more efficient. Nevertheless, optical microscopy only provides an image of the present structure of the cell, without any information on its long-temporal changes. These can be investigated by combining super-resolution optics with a nonoptical imaging technique, nanoscale secondary ion mass spectrometry, which investigates the isotopic composition of the samples. The resulting technique, combined isotopic and optical nanoscopy, enables the investigation of both the structure and the “history” of the cellular elements. The age and the turnover of cellular organelles can be read by isotopic imaging, while the structure can be analyzed by optical (fluorescence) approaches. We present these technologies, and we discuss their implementation for the study of biological samples. We conclude that, albeit complex, this type of technology is reliable enough for mass application to cell biology. PMID:28466025

Demosaiced pixel super-resolution in digital holography for multiplexed computational color imaging on-a-chip (Conference Presentation)

NASA Astrophysics Data System (ADS)

Wu, Yichen; Zhang, Yibo; Luo, Wei; Ozcan, Aydogan

2017-03-01

Digital holographic on-chip microscopy achieves large space-bandwidth-products (e.g., >1 billion) by making use of pixel super-resolution techniques. To synthesize a digital holographic color image, one can take three sets of holograms representing the red (R), green (G) and blue (B) parts of the spectrum and digitally combine them to synthesize a color image. The data acquisition efficiency of this sequential illumination process can be improved by 3-fold using wavelength-multiplexed R, G and B illumination that simultaneously illuminates the sample, and using a Bayer color image sensor with known or calibrated transmission spectra to digitally demultiplex these three wavelength channels. This demultiplexing step is conventionally used with interpolation-based Bayer demosaicing methods. However, because the pixels of different color channels on a Bayer image sensor chip are not at the same physical location, conventional interpolation-based demosaicing process generates strong color artifacts, especially at rapidly oscillating hologram fringes, which become even more pronounced through digital wave propagation and phase retrieval processes. Here, we demonstrate that by merging the pixel super-resolution framework into the demultiplexing process, such color artifacts can be greatly suppressed. This novel technique, termed demosaiced pixel super-resolution (D-PSR) for digital holographic imaging, achieves very similar color imaging performance compared to conventional sequential R,G,B illumination, with 3-fold improvement in image acquisition time and data-efficiency. We successfully demonstrated the color imaging performance of this approach by imaging stained Pap smears. The D-PSR technique is broadly applicable to high-throughput, high-resolution digital holographic color microscopy techniques that can be used in resource-limited-settings and point-of-care offices.

Computational-optical microscopy for 3D biological imaging beyond the diffraction limit

NASA Astrophysics Data System (ADS)

Grover, Ginni

In recent years, super-resolution imaging has become an important fluorescent microscopy tool. It has enabled imaging of structures smaller than the optical diffraction limit with resolution less than 50 nm. Extension to high-resolution volume imaging has been achieved by integration with various optical techniques. In this thesis, development of a fluorescent microscope to enable high resolution, extended depth, three dimensional (3D) imaging is discussed; which is achieved by integration of computational methods with optical systems. In the first part of the thesis, point spread function (PSF) engineering for volume imaging is discussed. A class of PSFs, referred to as double-helix (DH) PSFs, is generated. The PSFs exhibit two focused spots in the image plane which rotate about the optical axis, encoding depth in rotation of the image. These PSFs extend the depth-of-field up to a factor of ˜5. Precision performance of the DH-PSFs, based on an information theoretical analysis, is compared with other 3D methods with conclusion that the DH-PSFs provide the best precision and the longest depth-of-field. Out of various possible DH-PSFs, a suitable PSF is obtained for super-resolution microscopy. The DH-PSFs are implemented in imaging systems, such as a microscope, with a special phase modulation at the pupil plane. Surface-relief elements which are polarization-insensitive and ˜90% light efficient are developed for phase modulation. The photon-efficient DH-PSF microscopes thus developed are used, along with optimal position estimation algorithms, for tracking and super-resolution imaging in 3D. Imaging at depths-of-field of up to 2.5 microm is achieved without focus scanning. Microtubules were imaged with 3D resolution of (6, 9, 39) nm, which is in close agreement with the theoretical limit. A quantitative study of co-localization of two proteins in volume was conducted in live bacteria. In the last part of the thesis practical aspects of the DH-PSF microscope are discussed. A method to stabilize it, for extended periods of time, with 3-4 nm precision in 3D is developed. 3D Super-resolution is demonstrated without drift. A PSF correction algorithm is demonstrated to improve characteristics of the DH-PSF in an experiment, where it is implemented with a polarization-insensitive liquid crystal spatial light modulator.

Sensorless adaptive optics for isoSTED nanoscopy

NASA Astrophysics Data System (ADS)

Antonello, Jacopo; Hao, Xiang; Allgeyer, Edward S.; Bewersdorf, Joerg; Rittscher, Jens; Booth, Martin J.

2018-02-01

The presence of aberrations is a major concern when using fluorescence microscopy to image deep inside tissue. Aberrations due to refractive index mismatch and heterogeneity of the specimen under investigation cause severe reduction in the amount of fluorescence emission that is collected by the microscope. Furthermore, aberrations adversely affect the resolution, leading to loss of fine detail in the acquired images. These phenomena are particularly troublesome for super-resolution microscopy techniques such as isotropic stimulated-emission-depletion microscopy (isoSTED), which relies on accurate control of the shape and co-alignment of multiple excitation and depletion foci to operate as expected and to achieve the super-resolution effect. Aberrations can be suppressed by implementing sensorless adaptive optics techniques, whereby aberration correction is achieved by maximising a certain image quality metric. In confocal microscopy for example, one can employ the total image brightness as an image quality metric. Aberration correction is subsequently achieved by iteratively changing the settings of a wavefront corrector device until the metric is maximised. This simplistic approach has limited applicability to isoSTED microscopy where, due to the complex interplay between the excitation and depletion foci, maximising the total image brightness can lead to introducing aberrations in the depletion foci. In this work we first consider the effects that different aberration modes have on isoSTED microscopes. We then propose an iterative, wavelet-based aberration correction algorithm and evaluate its benefits.

A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags.

PubMed

Müller, Andreas; Neukam, Martin; Ivanova, Anna; Sönmez, Anke; Münster, Carla; Kretschmar, Susanne; Kalaidzidis, Yannis; Kurth, Thomas; Verbavatz, Jean-Marc; Solimena, Michele

2017-02-02

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

Coherent total internal reflection dark-field microscopy: label-free imaging beyond the diffraction limit.

PubMed

von Olshausen, Philipp; Rohrbach, Alexander

2013-10-15

Coherent imaging is barely applicable in life-science microscopy due to multiple interference artifacts. Here, we show how these interferences can be used to improve image resolution and contrast. We present a dark-field microscopy technique with evanescent illumination via total internal reflection that delivers high-contrast images of coherently scattering samples. By incoherent averaging of multiple coherent images illuminated from different directions we can resolve image structures that remain unresolved by conventional (incoherent) fluorescence microscopy. We provide images of 190 nm beads revealing resolution beyond the diffraction limit and slightly increased object distances. An analytical model is introduced that accounts for the observed effects and which is confirmed by numerical simulations. Our approach may be a route to fast, label-free, super-resolution imaging in live-cell microscopy.

Qualitative and quantitative analysis of PMN/T-cell interactions by InFlow and super-resolution microscopy.

PubMed

Balta, Emre; Stopp, Julian; Castelletti, Laura; Kirchgessner, Henning; Samstag, Yvonne; Wabnitz, Guido H

2017-01-01

Neutrophils or polymorphonuclear cells (PMN) eliminate bacteria via phagocytosis and/or NETosis. Apart from these conventional roles, PMN also have immune-regulatory functions. They can transdifferentiate and upregulate MHCII as well as ligands for costimulatory receptors which enables them to behave as antigen presenting cells (APC). The initial step for activating T-cells is the formation of an immune synapse between T-cells and antigen-presenting cells. However, the immune synapse that develops at the PMN/T-cell contact zone is as yet hardly investigated due to the non-availability of methods for analysis of large number of PMN interactions. In order to overcome these obstacles, we introduce here a workflow to analyse the immune synapse of primary human PMN and T-cells using multispectral imaging flow cytometry (InFlow microscopy) and super-resolution microscopy. For that purpose, we used CD3 and CD66b as the lineage markers for T-cells and PMN, respectively. Thereafter, we applied and critically discussed various "masks" for identification of T-cell PMN interactions. Using this approach, we found that a small fraction of transdifferentiated PMN (CD66b + CD86 high ) formed stable PMN/T-cell conjugates. Interestingly, while both CD3 and CD66b accumulation in the immune synapse was dependent on the maturation state of the PMN, only CD3 accumulation was greatly enhanced by the presence of superantigen. The actin cytoskeleton was weakly rearranged at the PMN side on the immune synapse upon contact with a T-cell in the presence of superantigen. A more detailed analysis using super-resolution microscopy (structured-illumination microscopy, SIM) confirmed this finding. Together, we present an InFlow microscopy based approach for the large scale analysis of PMN/T-cell interactions and - combined with SIM - a possibility for an in-depth analysis of protein translocation at the site of interactions. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Development of bimolecular fluorescence complementation using rsEGFP2 for detection and super-resolution imaging of protein-protein interactions in live cells

PubMed Central

Wang, Sheng; Ding, Miao; Chen, Xuanze; Chang, Lei; Sun, Yujie

2017-01-01

Direct visualization of protein-protein interactions (PPIs) at high spatial and temporal resolution in live cells is crucial for understanding the intricate and dynamic behaviors of signaling protein complexes. Recently, bimolecular fluorescence complementation (BiFC) assays have been combined with super-resolution imaging techniques including PALM and SOFI to visualize PPIs at the nanometer spatial resolution. RESOLFT nanoscopy has been proven as a powerful live-cell super-resolution imaging technique. With regard to the detection and visualization of PPIs in live cells with high temporal and spatial resolution, here we developed a BiFC assay using split rsEGFP2, a highly photostable and reversibly photoswitchable fluorescent protein previously developed for RESOLFT nanoscopy. Combined with parallelized RESOLFT microscopy, we demonstrated the high spatiotemporal resolving capability of a rsEGFP2-based BiFC assay by detecting and visualizing specifically the heterodimerization interactions between Bcl-xL and Bak as well as the dynamics of the complex on mitochondria membrane in live cells. PMID:28663931

Super-Resolution Imaging of a Dielectric Microsphere Is Governed by the Waist of Its Photonic Nanojet.

PubMed

Yang, Hui; Trouillon, Raphaël; Huszka, Gergely; Gijs, Martin A M

2016-08-10

Dielectric microspheres with appropriate refractive index can image objects with super-resolution, that is, with a precision well beyond the classical diffraction limit. A microsphere is also known to generate upon illumination a photonic nanojet, which is a scattered beam of light with a high-intensity main lobe and very narrow waist. Here, we report a systematic study of the imaging of water-immersed nanostructures by barium titanate glass microspheres of different size. A numerical study of the light propagation through a microsphere points out the light focusing capability of microspheres of different size and the waist of their photonic nanojet. The former correlates to the magnification factor of the virtual images obtained from linear test nanostructures, the biggest magnification being obtained with microspheres of ∼6-7 μm in size. Analyzing the light intensity distribution of microscopy images allows determining analytically the point spread function of the optical system and thereby quantifies its resolution. We find that the super-resolution imaging of a microsphere is dependent on the waist of its photonic nanojet, the best resolution being obtained with a 6 μm Ø microsphere, which generates the nanojet with the minimum waist. This comparison allows elucidating the super-resolution imaging mechanism.

Experimental assessment and analysis of super-resolution in fluorescence microscopy based on multiple-point spread function fitting of spectrally demultiplexed images

NASA Astrophysics Data System (ADS)

Nishimura, Takahiro; Kimura, Hitoshi; Ogura, Yusuke; Tanida, Jun

2018-06-01

This paper presents an experimental assessment and analysis of super-resolution microscopy based on multiple-point spread function fitting of spectrally demultiplexed images using a designed DNA structure as a test target. For the purpose, a DNA structure was designed to have binding sites at a certain interval that is smaller than the diffraction limit. The structure was labeled with several types of quantum dots (QDs) to acquire their spatial information as spectrally encoded images. The obtained images are analyzed with a point spread function multifitting algorithm to determine the QD locations that indicate the binding site positions. The experimental results show that the labeled locations can be observed beyond the diffraction-limited resolution using three-colored fluorescence images that were obtained with a confocal fluorescence microscope. Numerical simulations show that labeling with eight types of QDs enables the positions aligned at 27.2-nm pitches on the DNA structure to be resolved with high accuracy.

Super-resolution imaging and tracking of protein-protein interactions in sub-diffraction cellular space

NASA Astrophysics Data System (ADS)

Liu, Zhen; Xing, Dong; Su, Qian Peter; Zhu, Yun; Zhang, Jiamei; Kong, Xinyu; Xue, Boxin; Wang, Sheng; Sun, Hao; Tao, Yile; Sun, Yujie

2014-07-01

Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein-protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB-EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB-EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB-EF-Tu interactions.

Super-resolution imaging and tracking of protein–protein interactions in sub-diffraction cellular space

PubMed Central

Liu, Zhen; Xing, Dong; Su, Qian Peter; Zhu, Yun; Zhang, Jiamei; Kong, Xinyu; Xue, Boxin; Wang, Sheng; Sun, Hao; Tao, Yile; Sun, Yujie

2014-01-01

Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein–protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB–EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB–EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB–EF-Tu interactions. PMID:25030837

Super-resolution from single photon emission: toward biological application

NASA Astrophysics Data System (ADS)

Moreva, E.; Traina, P.; Forneris, J.; Ditalia Tchernij, S.; Guarina, L.; Franchino, C.; Picollo, F.; Ruo Berchera, I.; Brida, G.; Degiovanni, I. P.; Carabelli, V.; Olivero, P.; Genovese, M.

2017-08-01

Properties of quantum light represent a tool for overcoming limits of classical optics. Several experiments have demonstrated this advantage ranging from quantum enhanced imaging to quantum illumination. In this work, experimental demonstration of quantum-enhanced resolution in confocal fluorescence microscopy will be presented. This is achieved by exploiting the non-classical photon statistics of fluorescence emission of single nitrogen-vacancy (NV) color centers in diamond. By developing a general model of super-resolution based on the direct sampling of the kth-order autocorrelation function of the photoluminescence signal, we show the possibility to resolve, in principle, arbitrarily close emitting centers. Finally, possible applications of NV-based fluorescent nanodiamonds in biosensing and future developments will be presented.

Cassette Series Designed for Live-Cell Imaging of Proteins and High Resolution Techniques in Yeast

PubMed Central

Young, Carissa L.; Raden, David L.; Caplan, Jeffrey; Czymmek, Kirk; Robinson, Anne S.

2012-01-01

During the past decade, it has become clear that protein function and regulation are highly dependent upon intracellular localization. Although fluorescent protein variants are ubiquitously used to monitor protein dynamics, localization, and abundance; fluorescent light microscopy techniques often lack the resolution to explore protein heterogeneity and cellular ultrastructure. Several approaches have been developed to identify, characterize, and monitor the spatial localization of proteins and complexes at the sub-organelle level; yet, many of these techniques have not been applied to yeast. Thus, we have constructed a series of cassettes containing codon-optimized epitope tags, fluorescent protein variants that cover the full spectrum of visible light, a TetCys motif used for FlAsH-based localization, and the first evaluation in yeast of a photoswitchable variant – mEos2 – to monitor discrete subpopulations of proteins via confocal microscopy. This series of modules, complete with six different selection markers, provides the optimal flexibility during live-cell imaging and multicolor labeling in vivo. Furthermore, high-resolution imaging techniques include the yeast-enhanced TetCys motif that is compatible with diaminobenzidine photooxidation used for protein localization by electron microscopy and mEos2 that is ideal for super-resolution microscopy. We have examined the utility of our cassettes by analyzing all probes fused to the C-terminus of Sec61, a polytopic membrane protein of the endoplasmic reticulum of moderate protein concentration, in order to directly compare fluorescent probes, their utility and technical applications. Our series of cassettes expand the repertoire of molecular tools available to advance targeted spatiotemporal investigations using multiple live-cell, super-resolution or electron microscopy imaging techniques. PMID:22473760

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

Hierarchical super-structure identified by polarized light microscopy, electron microscopy and nanoindentation: Implications for the limits of biological control over the growth mode of abalone sea shells

PubMed Central

2012-01-01

Background Mollusc shells are commonly investigated using high-resolution imaging techniques based on cryo-fixation. Less detailed information is available regarding the light-optical properties. Sea shells of Haliotis pulcherina were embedded for polishing in defined orientations in order to investigate the interface between prismatic calcite and nacreous aragonite by standard materialographic methods. A polished thin section of the interface was prepared with a defined thickness of 60 μm for quantitative birefringence analysis using polarized light and LC-PolScope microscopy. Scanning electron microscopy images were obtained for comparison. In order to study structural-mechanical relationships, nanoindentation experiments were performed. Results Incident light microscopy revealed a super-structure in semi-transparent regions of the polished cross-section under a defined angle. This super-structure is not visible in transmitted birefringence analysis due to the blurred polarization of small nacre platelets and numerous organic interfaces. The relative orientation and homogeneity of calcite prisms was directly identified, some of them with their optical axes exactly normal to the imaging plane. Co-oriented "prism colonies" were identified by polarized light analyses. The nacreous super-structure was also visualized by secondary electron imaging under defined angles. The domains of the super-structure were interpreted to consist of crystallographically aligned platelet stacks. Nanoindentation experiments showed that mechanical properties changed with the same periodicity as the domain size. Conclusions In this study, we have demonstrated that insights into the growth mechanisms of nacre can be obtained by conventional light-optical methods. For example, we observed super-structures formed by co-oriented nacre platelets as previously identified using X-ray Photo-electron Emission Microscopy (X-PEEM) [Gilbert et al., Journal of the American Chemical Society 2008, 130:17519–17527]. Polarized optical microscopy revealed unprecedented super-structures in the calcitic shell part. This bears, in principle, the potential for in vivo studies, which might be useful for investigating the growth modes of nacre and other shell types. PMID:22967319

Comparison of Confocal and Super-Resolution Reflectance Imaging of Metal Oxide Nanoparticles

PubMed Central

Guggenheim, Emily J.; Khan, Abdullah; Pike, Jeremy; Chang, Lynne; Lynch, Iseult; Rappoport, Joshua Z.

2016-01-01

The potential for human exposure to manufactured nanoparticles (NPs) has increased in recent years, in part through the incorporation of engineered particles into a wide range of commercial goods and medical applications. NP are ideal candidates for use as therapeutic and diagnostic tools within biomedicine, however concern exists regarding their efficacy and safety. Thus, developing techniques for the investigation of NP uptake into cells is critically important. Current intracellular NP investigations rely on the use of either Transmission Electron Microscopy (TEM), which provides ultrahigh resolution, but involves cumbersome sample preparation rendering the technique incompatible with live cell imaging, or fluorescent labelling, which suffers from photobleaching, poor bioconjugation and, often, alteration of NP surface properties. Reflected light imaging provides an alternative non-destructive label free technique well suited, but not limited to, the visualisation of NP uptake within model systems, such as cells. Confocal reflectance microscopy provides optical sectioning and live imaging capabilities, with little sample preparation. However confocal microscopy is diffraction limited, thus the X-Y resolution is restricted to ~250 nm, substantially larger than the <100 nm size of NPs. Techniques such as super-resolution light microscopy overcome this fundamental limitation, providing increased X-Y resolution. The use of Reflectance SIM (R-SIM) for NP imaging has previously only been demonstrated on custom built microscopes, restricting the widespread use and limiting NP investigations. This paper demonstrates the use of a commercial SIM microscope for the acquisition of super-resolution reflectance data with X-Y resolution of 115 nm, a greater than two-fold increase compared to that attainable with RCM. This increase in resolution is advantageous for visualising small closely spaced structures, such as NP clusters, previously unresolvable by RCM. This is advantageous when investigating the subcellular trafficking of NP within fluorescently labelled cellular compartments. NP signal can be observed using RCM, R-SIM and TEM and a direct comparison is presented. Each of these techniques has its own benefits and limitations; RCM and R-SIM provide novel complementary information while the combination of modalities provides a unique opportunity to gain additional information regarding NP uptake. The use of multiple imaging methods therefore greatly enhances the range of NPs that can be studied under label-free conditions. PMID:27695038

SIL-STED microscopy technique enhancing super-resolution of fluorescence microscopy

NASA Astrophysics Data System (ADS)

Park, No-Cheol; Lim, Geon; Lee, Won-sup; Moon, Hyungbae; Choi, Guk-Jong; Park, Young-Pil

2017-08-01

We have characterized a new type STED microscope which combines a high numerical aperture (NA) optical head with a solid immersion lens (SIL), and we call it as SIL-STED microscope. The advantage of a SIL-STED microscope is that its high NA of the SIL makes it superior to a general STED microscope in lateral resolution, thus overcoming the optical diffraction limit at the macromolecular level and enabling advanced super-resolution imaging of cell surface or cell membrane structure and function Do. This study presents the first implementation of higher NA illumination in a STED microscope limiting the fluorescence lateral resolution to about 40 nm. The refractive index of the SIL which is made of material KTaO3 is about 2.23 and 2.20 at a wavelength of 633 nm and 780 nm which are used for excitation and depletion in STED imaging, respectively. Based on the vector diffraction theory, the electric field focused by the SILSTED microscope is numerically calculated so that the numerical results of the point dispersion function of the microscope and the expected resolution could be analyzed. For further investigation, fluorescence imaging of nano size fluorescent beads is fulfilled to show improved performance of the technique.

Portable lensless wide-field microscopy imaging platform based on digital inline holography and multi-frame pixel super-resolution

PubMed Central

Sobieranski, Antonio C; Inci, Fatih; Tekin, H Cumhur; Yuksekkaya, Mehmet; Comunello, Eros; Cobra, Daniel; von Wangenheim, Aldo; Demirci, Utkan

2017-01-01

In this paper, an irregular displacement-based lensless wide-field microscopy imaging platform is presented by combining digital in-line holography and computational pixel super-resolution using multi-frame processing. The samples are illuminated by a nearly coherent illumination system, where the hologram shadows are projected into a complementary metal-oxide semiconductor-based imaging sensor. To increase the resolution, a multi-frame pixel resolution approach is employed to produce a single holographic image from multiple frame observations of the scene, with small planar displacements. Displacements are resolved by a hybrid approach: (i) alignment of the LR images by a fast feature-based registration method, and (ii) fine adjustment of the sub-pixel information using a continuous optimization approach designed to find the global optimum solution. Numerical method for phase-retrieval is applied to decode the signal and reconstruct the morphological details of the analyzed sample. The presented approach was evaluated with various biological samples including sperm and platelets, whose dimensions are in the order of a few microns. The obtained results demonstrate a spatial resolution of 1.55 µm on a field-of-view of ≈30 mm2. PMID:29657866

Super-resolved Mirau digital holography by structured illumination

NASA Astrophysics Data System (ADS)

Ganjkhani, Yasaman; Charsooghi, Mohammad A.; Akhlaghi, Ehsan A.; Moradi, Ali-Reza

2017-12-01

In this paper, we apply structured illumination toward super-resolved 3D imaging in a common-path digital holography arrangement. Digital holographic microscopy (DHM) provides non-invasive 3D images of transparent samples as well as 3D profiles of reflective surfaces. A compact and vibration-immune arrangement for DHM may be obtained through the use of a Mirau microscope objective. However, high-magnification Mirau objectives have a low working distance and are expensive. Low-magnification ones, on the other hand, suffer from low lateral resolution. Structured illumination has been widely used for resolution improvement of intensity images, but the technique can also be readily applied to DHM. We apply structured illumination to Mirau DHM by implementing successive sinusoidal gratings with different orientations onto a spatial light modulator (SLM) and forming its image on the specimen. Moreover, we show that, instead of different orientations of 1D gratings, alternative single 2D gratings, e.g. checkerboard or hexagonal patterns, can provide resolution enhancement in multiple directions. Our results show a 35% improvement in the resolution power of the DHM. The presented arrangement has the potential to serve as a table-top device for high resolution holographic microscopy.

High-magnification super-resolution FINCH microscopy using birefringent crystal lens interferometers

NASA Astrophysics Data System (ADS)

Siegel, Nisan; Lupashin, Vladimir; Storrie, Brian; Brooker, Gary

2016-12-01

Fresnel incoherent correlation holography (FINCH) microscopy is a promising approach for high-resolution biological imaging but has so far been limited to use with low-magnification, low-numerical-aperture configurations. We report the use of in-line incoherent interferometers made from uniaxial birefringent α-barium borate (α-BBO) or calcite crystals that overcome the aberrations and distortions present with previous implementations that employed spatial light modulators or gradient refractive index lenses. FINCH microscopy incorporating these birefringent elements and high-numerical-aperture oil immersion objectives could outperform standard wide-field fluorescence microscopy, with, for example, a 149 nm lateral point spread function at a wavelength of 590 nm. Enhanced resolution was confirmed with sub-resolution fluorescent beads. Taking the Golgi apparatus as a biological example, three different proteins labelled with GFP and two other fluorescent dyes in HeLa cells were resolved with an image quality that is comparable to similar samples captured by structured illumination microscopy.

Advances in high-resolution imaging--techniques for three-dimensional imaging of cellular structures.

PubMed

Lidke, Diane S; Lidke, Keith A

2012-06-01

A fundamental goal in biology is to determine how cellular organization is coupled to function. To achieve this goal, a better understanding of organelle composition and structure is needed. Although visualization of cellular organelles using fluorescence or electron microscopy (EM) has become a common tool for the cell biologist, recent advances are providing a clearer picture of the cell than ever before. In particular, advanced light-microscopy techniques are achieving resolutions below the diffraction limit and EM tomography provides high-resolution three-dimensional (3D) images of cellular structures. The ability to perform both fluorescence and electron microscopy on the same sample (correlative light and electron microscopy, CLEM) makes it possible to identify where a fluorescently labeled protein is located with respect to organelle structures visualized by EM. Here, we review the current state of the art in 3D biological imaging techniques with a focus on recent advances in electron microscopy and fluorescence super-resolution techniques.

Introduction to Modern Methods in Light Microscopy.

PubMed

Ryan, Joel; Gerhold, Abby R; Boudreau, Vincent; Smith, Lydia; Maddox, Paul S

2017-01-01

For centuries, light microscopy has been a key method in biological research, from the early work of Robert Hooke describing biological organisms as cells, to the latest in live-cell and single-molecule systems. Here, we introduce some of the key concepts related to the development and implementation of modern microscopy techniques. We briefly discuss the basics of optics in the microscope, super-resolution imaging, quantitative image analysis, live-cell imaging, and provide an outlook on active research areas pertaining to light microscopy.

Super-resolution for asymmetric resolution of FIB-SEM 3D imaging using AI with deep learning.

PubMed

Hagita, Katsumi; Higuchi, Takeshi; Jinnai, Hiroshi

2018-04-12

Scanning electron microscopy equipped with a focused ion beam (FIB-SEM) is a promising three-dimensional (3D) imaging technique for nano- and meso-scale morphologies. In FIB-SEM, the specimen surface is stripped by an ion beam and imaged by an SEM installed orthogonally to the FIB. The lateral resolution is governed by the SEM, while the depth resolution, i.e., the FIB milling direction, is determined by the thickness of the stripped thin layer. In most cases, the lateral resolution is superior to the depth resolution; hence, asymmetric resolution is generated in the 3D image. Here, we propose a new approach based on an image-processing or deep-learning-based method for super-resolution of 3D images with such asymmetric resolution, so as to restore the depth resolution to achieve symmetric resolution. The deep-learning-based method learns from high-resolution sub-images obtained via SEM and recovers low-resolution sub-images parallel to the FIB milling direction. The 3D morphologies of polymeric nano-composites are used as test images, which are subjected to the deep-learning-based method as well as conventional methods. We find that the former yields superior restoration, particularly as the asymmetric resolution is increased. Our super-resolution approach for images having asymmetric resolution enables observation time reduction.

Synthesis of a Far-Red Photoactivatable Silicon-Containing Rhodamine for Super-Resolution Microscopy.

PubMed

Grimm, Jonathan B; Klein, Teresa; Kopek, Benjamin G; Shtengel, Gleb; Hess, Harald F; Sauer, Markus; Lavis, Luke D

2016-01-26

The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a "caged" Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Quantum correlation enhanced super-resolution localization microscopy enabled by a fibre bundle camera

PubMed Central

Israel, Yonatan; Tenne, Ron; Oron, Dan; Silberberg, Yaron

2017-01-01

Despite advances in low-light-level detection, single-photon methods such as photon correlation have rarely been used in the context of imaging. The few demonstrations, for example of subdiffraction-limited imaging utilizing quantum statistics of photons, have remained in the realm of proof-of-principle demonstrations. This is primarily due to a combination of low values of fill factors, quantum efficiencies, frame rates and signal-to-noise characteristic of most available single-photon sensitive imaging detectors. Here we describe an imaging device based on a fibre bundle coupled to single-photon avalanche detectors that combines a large fill factor, a high quantum efficiency, a low noise and scalable architecture. Our device enables localization-based super-resolution microscopy in a non-sparse non-stationary scene, utilizing information on the number of active emitters, as gathered from non-classical photon statistics. PMID:28287167

"Supertrap" at Work: Extremely Efficient Nonradiative Recombination Channels in MAPbI3 Perovskites Revealed by Luminescence Super-Resolution Imaging and Spectroscopy.

PubMed

Merdasa, Aboma; Tian, Yuxi; Camacho, Rafael; Dobrovolsky, Alexander; Debroye, Elke; Unger, Eva L; Hofkens, Johan; Sundström, Villy; Scheblykin, Ivan G

2017-06-27

Organo-metal halide perovskites are some of the most promising materials for the new generation of low-cost photovoltaic and light-emitting devices. Their solution processability is a beneficial trait, although it leads to a spatial inhomogeneity of perovskite films with a variation of the trap state density at the nanoscale. Comprehending their properties using traditional spectroscopy therefore becomes difficult, calling for a combination with microscopy in order to see beyond the ensemble-averaged response. We studied photoluminescence (PL) blinking of micrometer-sized individual methylammonium lead iodide (MAPbI 3 ) perovskite polycrystals, as well as monocrystalline microrods up to 10 μm long. We correlated their PL dynamics with structure employing scanning electron and optical super-resolution microscopy. Combining super-resolution localization imaging and super-resolution optical fluctuation imaging (SOFI), we could detect and quantify preferential emitting regions in polycrystals exhibiting different types of blinking. We propose that blinking in MAPbI 3 occurs by the activation/passivation of a "supertrap" which presumably is a donor-acceptor pair able to trap both electrons and holes. As such, nonradiative recombination via supertraps, in spite being present at a rather low concentrations (10 12 -10 15 cm -3 ), is much more efficient than via all other defect states present in the material at higher concentrations (10 16 -10 18 cm -3 ). We speculate that activation/deactivation of a supertrap occurs by its temporary dissociation into free donor and acceptor impurities. We found that supertraps are most efficient in structurally homogeneous and large MAPbI 3 crystals where carrier diffusion is efficient, which may therefore pose limitations on the efficiency of perovskite-based devices.

Breaking the diffraction barrier using coherent anti-Stokes Raman scattering difference microscopy.

PubMed

Wang, Dong; Liu, Shuanglong; Chen, Yue; Song, Jun; Liu, Wei; Xiong, Maozhen; Wang, Guangsheng; Peng, Xiao; Qu, Junle

2017-05-01

We propose a method to improve the resolution of coherent anti-Stokes Raman scattering microscopy (CARS), and present a theoretical model. The proposed method, coherent anti-Stokes Raman scattering difference microscopy (CARS-D), is based on the intensity difference between two differently acquired images. One being the conventional CARS image, and the other obtained when the sample is illuminated by a doughnut shaped spot. The final super-resolution CARS-D image is constructed by intensity subtraction of these two images. However, there is a subtractive factor between them, and the theoretical model sets this factor to obtain the best imaging effect.

Resolution enhancement in a double-helix phase engineered scanning microscope (RESCH microscope) (Presentation Recording)

NASA Astrophysics Data System (ADS)

Jesacher, Alexander; Ritsch-Marte, Monika; Piestun, Rafael

2015-08-01

Recently we introduced RESCH microscopy [1] - a scanning microscope that allows slightly refocusing the sample after the acquisition has been performed, solely by performing appropriate data post-processing. The microscope features a double-helix phase-engineered emission point spread function in combination with camera-based detection. Based on the principle of transverse resolution enhancement in Image Scanning Microscopy [2,3], we demonstrate similar resolution improvement in RESCH. Furthermore, we outline a pathway for how the collected 3D sample information can be used to construct sharper optical sections. [1] A. Jesacher, M. Ritsch-Marte and R. Piestun, accepted for Optica. [2] C.J.R. Sheppard, "Super-resolution in Confocal imaging," Optik, 80, 53-54 (1988). [3] C.B. Müller and J. Enderlein "Image Scanning Microscopy," Phys. Rev. Lett. 104, 198101 (2010).

Single-exposure super-resolved interferometric microscopy by RGB multiplexing in lensless configuration

NASA Astrophysics Data System (ADS)

Granero, Luis; Ferreira, Carlos; Zalevsky, Zeev; García, Javier; Micó, Vicente

2016-07-01

Single-Exposure Super-Resolved Interferometric Microscopy (SESRIM) reports on a way to achieve one-dimensional (1-D) superresolved imaging in digital holographic microscopy (DHM) by a single illumination shot and digital recording. SESRIM provides color-coded angular multiplexing of the accessible sample's range of spatial frequencies and it allows their recording in a single CCD (color or monochrome) snapshot by adding 3 RGB coherent reference beams at the output plane. In this manuscript, we extend the applicability of SESRIM to the field of digital in-line holographic microscopy (DIHM), that is, working without lenses. As consequence of the in-line configuration, an additional restriction concerning the object field of view (FOV) must be imposed to the technique. Experimental results are reported for both a synthetic object (USAF resolution test target) and a biological sample (swine sperm sample) validating this new kind of superresolution imaging method named as lensless SESRIM (L-SESRIM).

Estimation of reactogenicity of preparations produced on the basis of photoinactivated live vaccines against brucellosis and tularaemia on the organismic level.2. Using the method of speckle-microscopy with high spatial resolution

NASA Astrophysics Data System (ADS)

Ulianova, O. V.; Uianov, S. S.; Li, Pengcheng; Luo, Qingming

2011-04-01

The method of speckle microscopy was adapted to estimate the reactogenicity of the prototypes of vaccine preparations against extremely dangerous infections. The theory is proposed to describe the mechanism of formation of the output signal from the super-high spatial resolution speckle microscope. The experimental studies show that bacterial suspensions, irradiated in different regimes of inactivation, do not exert negative influence on the blood microcirculations in laboratory animals.

Three-dimensional wide-field pump-probe structured illumination microscopy

PubMed Central

Kim, Yang-Hyo; So, Peter T.C.

2017-01-01

We propose a new structured illumination scheme for achieving depth resolved wide-field pump-probe microscopy with sub-diffraction limit resolution. By acquiring coherent pump-probe images using a set of 3D structured light illumination patterns, a 3D super-resolution pump-probe image can be reconstructed. We derive the theoretical framework to describe the coherent image formation and reconstruction scheme for this structured illumination pump-probe imaging system and carry out numerical simulations to investigate its imaging performance. The results demonstrate a lateral resolution improvement by a factor of three and providing 0.5 µm level axial optical sectioning. PMID:28380860

Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns.

PubMed

Hennig, Simon; van de Linde, Sebastian; Bergmann, Stephan; Huser, Thomas; Sauer, Markus

2015-08-25

We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ∼100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3-168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.

Application of spectroscopy and super-resolution microscopy: Excited state

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

Bhattacharjee, Ujjal

Photophysics of inorganic materials and organic molecules in complex systems have been extensively studied with absorption and emission spectroscopy.1-4 Steady-state and time-resolved fluorescence studies are commonly carried out to characterize excited-state properties of fluorophores. Although steady-state fluorescence measurements are widely used for analytical applications, time-resolved fluorescence measurements provide more detailed information about excited-state properties and the environment in the vicinity of the fluorophore. Many photophysical processes, such as photoinduced electron transfer (PET), rotational reorientation, solvent relaxation, and energy transfer, occur on a nanosecond (10 -9 s) timescale, thus affecting the lifetime of the fluorophores. Moreover, time-resolved microscopy methods, such asmore » lifetimeimaging, combine the benefits of the microscopic measurement and information-rich, timeresolved data. Thus, time-resolved fluorescence spectroscopy combined with microscopy can be used to quantify these processes and to obtain a deeper understanding of the chemical surroundings of the fluorophore in a small area under investigation. This thesis discusses various photophysical and super-resolution microscopic studies of organic and inorganic materials, which have been outlined below.« less

Developing Photo Activated Localization Microscopy

NASA Astrophysics Data System (ADS)

Hess, Harald

2015-03-01

Photo Activated Localization Microscopy, PALM, acquires super-resolution images by activating a subset of activatable fluorescent labels and estimating the center of the each molecular label to sub-diffractive accuracy. When this process is repeated thousands of times for different subsets of molecules, then an image can be rendered from all the center coordinates of the molecules. I will describe the circuitous story of its development that began with another super-resolution technique, NSOM, developed by my colleague Eric Betzig, who imaged single molecules at room temperature, and later we spectrally resolved individual luminescent centers of quantum wells. These two observations inspired a generalized path to localization microscopy, but that path was abandoned because no really useful fluorescent labels were available. After a decade of nonacademic industrial pursuits and the subsequent freedom of unemployment, we came across a class of genetically expressible fluorescent proteins that were switchable or convertible that enabled the concept to be implemented and be biologically promising. The past ten years have been very active with many groups exploring applications and enhancements of this concept. Demonstrating significant biological relevance will be the metric if its success.

Genetically encoded sensors and fluorescence microscopy for anticancer research

NASA Astrophysics Data System (ADS)

Zagaynova, Elena V.; Shirmanova, Marina V.; Sergeeva, Tatiana F.; Klementieva, Natalia V.; Mishin, Alexander S.; Gavrina, Alena I.; Zlobovskay, Olga A.; Furman, Olga E.; Dudenkova, Varvara V.; Perelman, Gregory S.; Lukina, Maria M.; Lukyanov, Konstantin A.

2017-02-01

Early response of cancer cells to chemical compounds and chemotherapeutic drugs were studied using novel fluorescence tools and microscopy techniques. We applied confocal microscopy, two-photon fluorescence lifetime imaging microscopy and super-resolution localization-based microscopy to assess structural and functional changes in cancer cells in vitro. The dynamics of energy metabolism, intracellular pH, caspase-3 activation during staurosporine-induced apoptosis as well as actin cytoskeleton rearrangements under chemotherapy were evaluated. We have showed that new genetically encoded sensors and advanced fluorescence microscopy methods provide an efficient way for multiparameter analysis of cell activities

3-D Cellular Ultrastructure Can Be Resolved by X-ray Microscopy | Center for Cancer Research

Cancer.gov

X-ray microscopy (XRM) is more rapid than cryoelectron tomography or super-resolution fluorescence microscopy and could fill an important gap in current technologies used to investigate in situ three-dimensional structure of cells. New XRM methods developed by first author Gerd Schneider, Ph.D., working with James McNally. Ph.D., and a team of colleagues, is capable of revealing full cellular ultrastructure without requiring fixation, staining, or sectioning.

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

Alpha particle spectroscopy using FNTD and SIM super-resolution microscopy.

PubMed

Kouwenberg, J J M; Kremers, G J; Slotman, J A; Wolterbeek, H T; Houtsmuller, A B; Denkova, A G; Bos, A J J

2018-06-01

Structured illumination microscopy (SIM) for the imaging of alpha particle tracks in fluorescent nuclear track detectors (FNTD) was evaluated and compared to confocal laser scanning microscopy (CLSM). FNTDs were irradiated with an external alpha source and imaged using both methodologies. SIM imaging resulted in improved resolution, without increase in scan time. Alpha particle energy estimation based on the track length, direction and intensity produced results in good agreement with the expected alpha particle energy distribution. A pronounced difference was seen in the spatial scattering of alpha particles in the detectors, where SIM showed an almost 50% reduction compared to CLSM. The improved resolution of SIM allows for more detailed studies of the tracks induced by ionising particles. The combination of SIM and FNTDs for alpha radiation paves the way for affordable and fast alpha spectroscopy and dosimetry. © 2018 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Stimulated Emission Depletion Nanoscopy Reveals Time-Course of Human Immunodeficiency Virus Proteolytic Maturation.

PubMed

Hanne, Janina; Göttfert, Fabian; Schimer, Jiří; Anders-Össwein, Maria; Konvalinka, Jan; Engelhardt, Johann; Müller, Barbara; Hell, Stefan W; Kräusslich, Hans-Georg

2016-09-27

Concomitant with human immunodeficiency virus type 1 (HIV-1) budding from a host cell, cleavage of the structural Gag polyproteins by the viral protease (PR) triggers complete remodeling of virion architecture. This maturation process is essential for virus infectivity. Electron tomography provided structures of immature and mature HIV-1 with a diameter of 120-140 nm, but information about the sequence and dynamics of structural rearrangements is lacking. Here, we employed super-resolution STED (stimulated emission depletion) fluorescence nanoscopy of HIV-1 carrying labeled Gag to visualize the virion architecture. The incomplete Gag lattice of immature virions was clearly distinguishable from the condensed distribution of mature protein subunits. Synchronized activation of PR within purified particles by photocleavage of a caged PR inhibitor enabled time-resolved in situ observation of the induction of proteolysis and maturation by super-resolution microscopy. This study shows the rearrangement of subviral structures in a super-resolution light microscope over time, outwitting phototoxicity and fluorophore bleaching through synchronization of a biological process by an optical switch.

MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions, Dynamics and Formation of Multi-protein Structures.

PubMed

Caetano, Fabiana A; Dirk, Brennan S; Tam, Joshua H K; Cavanagh, P Craig; Goiko, Maria; Ferguson, Stephen S G; Pasternak, Stephen H; Dikeakos, Jimmy D; de Bruyn, John R; Heit, Bryan

2015-12-01

Our current understanding of the molecular mechanisms which regulate cellular processes such as vesicular trafficking has been enabled by conventional biochemical and microscopy techniques. However, these methods often obscure the heterogeneity of the cellular environment, thus precluding a quantitative assessment of the molecular interactions regulating these processes. Herein, we present Molecular Interactions in Super Resolution (MIiSR) software which provides quantitative analysis tools for use with super-resolution images. MIiSR combines multiple tools for analyzing intermolecular interactions, molecular clustering and image segmentation. These tools enable quantification, in the native environment of the cell, of molecular interactions and the formation of higher-order molecular complexes. The capabilities and limitations of these analytical tools are demonstrated using both modeled data and examples derived from the vesicular trafficking system, thereby providing an established and validated experimental workflow capable of quantitatively assessing molecular interactions and molecular complex formation within the heterogeneous environment of the cell.

Dynamic placement of plasmonic hotspots for super-resolution surface-enhanced Raman scattering.

PubMed

Ertsgaard, Christopher T; McKoskey, Rachel M; Rich, Isabel S; Lindquist, Nathan C

2014-10-28

In this paper, we demonstrate dynamic placement of locally enhanced plasmonic fields using holographic laser illumination of a silver nanohole array. To visualize these focused "hotspots", the silver surface was coated with various biological samples for surface-enhanced Raman spectroscopy (SERS) imaging. Due to the large field enhancements, blinking behavior of the SERS hotspots was observed and processed using a stochastic optical reconstruction microscopy algorithm enabling super-resolution localization of the hotspots to within 10 nm. These hotspots were then shifted across the surface in subwavelength (<100 nm for a wavelength of 660 nm) steps using holographic illumination from a spatial light modulator. This created a dynamic imaging and sensing surface, whereas static illumination would only have produced stationary hotspots. Using this technique, we also show that such subwavelength shifting and localization of plasmonic hotspots has potential for imaging applications. Interestingly, illuminating the surface with randomly shifting SERS hotspots was sufficient to completely fill in a wide field of view for super-resolution chemical imaging.

Three-dimensional refractive index and fluorescence tomography using structured illumination (Conference Presentation)

NASA Astrophysics Data System (ADS)

Park, GwangSik; Shin, SeungWoo; Kim, Kyoohyun; Park, YongKeun

2017-02-01

Optical diffraction tomography (ODT) has been an emerging optical technique for label-free imaging of three-dimensional (3-D) refractive index (RI) distribution of biological samples. ODT employs interferometric microscopy for measuring multiple holograms of samples with various incident angles, from which the Fourier diffraction theorem reconstructs the 3-D RI distribution of samples from retrieved complex optical fields. Since the RI value is linearly proportional to the protein concentration of biological samples where the proportional coefficient is called as refractive index increment (RII), reconstructed 3-D RI tomograms provide precise structural and biochemical information of individual biological samples. Because most proteins have similar RII value, however, ODT has limited molecular specificity, especially for imaging eukaryotic cells having various types of proteins and subcellular organelles. Here, we present an ODT system combined with structured illumination microscopy which can measure the 3-D RI distribution of biological samples as well as 3-D super-resolution fluorescent images in the same optical setup. A digital micromirror device (DMD) controls the incident angle of the illumination beam for tomogram reconstruction, and the same DMD modulates the structured illumination pattern of the excitation beam for super-resolution fluorescent imaging. We first validate the proposed method for simultaneous optical diffraction tomographic imaging and super-resolution fluorescent imaging of fluorescent beads. The proposed method is also exploited for various biological samples.

Advanced Methods in Fluorescence Microscopy

PubMed Central

Fritzky, Luke; Lagunoff, David

2013-01-01

It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbé limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres. PMID:23271142

Advanced methods in fluorescence microscopy.

PubMed

Fritzky, Luke; Lagunoff, David

2013-01-01

It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbe limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres.

Advanced methods in fluorescence microscopy.

PubMed

Fritzky, Luke; Lagunoff, David

2013-01-01

It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbé limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres.

Super-resolved microsphere-assisted Mirau digital holography by oblique illumination

NASA Astrophysics Data System (ADS)

Abbasian, Vahid; Ganjkhani, Yasaman; Akhlaghi, Ehsan A.; Anand, Arun; Javidi, Bahram; Moradi, Ali-Reza

2018-06-01

In this paper, oblique illumination is used to improve the lateral resolution and edge sharpness in microsphere (MS)-assisted Mirau digital holographic microscopy (Mirau-DHM). Abbe showed that tilting the illumination light allows entrance of higher spatial frequencies into the imaging system thus increasing the resolution power. We extended the idea to common-path DHM, based on Mirau objective, toward super-resolved 3D imaging. High magnification Mirau objectives are very expensive and low-magnification ones suffer from low resolution, therefore, any attempt to increase the effective resolution of the system may be of a great interest. We have already demonstrated the effective resolution increasing of a Mirau-DHM system by incorporating a transparent MS within the working distance of the objective. Here, we show that by integrating a MS-assisted Mirau-DHM with the oblique illumination even higher resolutions can be achieved. We have applied the technique for various samples and have shown the increase in the lateral resolution for the both cases of Mirau-DHM with and without the MS.

Antimicrobial agent triclosan disrupts mitochondrial structure, revealed by super-resolution microscopy, and inhibits mast cell signaling via calcium modulation.

PubMed

Weatherly, Lisa M; Nelson, Andrew J; Shim, Juyoung; Riitano, Abigail M; Gerson, Erik D; Hart, Andrew J; de Juan-Sanz, Jaime; Ryan, Timothy A; Sher, Roger; Hess, Samuel T; Gosse, Julie A

2018-06-15

The antimicrobial agent triclosan (TCS) is used in products such as toothpaste and surgical soaps and is readily absorbed into oral mucosa and human skin. These and many other tissues contain mast cells, which are involved in numerous physiologies and diseases. Mast cells release chemical mediators through a process termed degranulation, which is inhibited by TCS. Investigation into the underlying mechanisms led to the finding that TCS is a mitochondrial uncoupler at non-cytotoxic, low-micromolar doses in several cell types and live zebrafish. Our aim was to determine the mechanisms underlying TCS disruption of mitochondrial function and of mast cell signaling. We combined super-resolution (fluorescence photoactivation localization) microscopy and multiple fluorescence-based assays to detail triclosan's effects in living mast cells, fibroblasts, and primary human keratinocytes. TCS disrupts mitochondrial nanostructure, causing mitochondria to undergo fission and to form a toroidal, "donut" shape. TCS increases reactive oxygen species production, decreases mitochondrial membrane potential, and disrupts ER and mitochondrial Ca 2+ levels, processes that cause mitochondrial fission. TCS is 60 × more potent than the banned uncoupler 2,4-dinitrophenol. TCS inhibits mast cell degranulation by decreasing mitochondrial membrane potential, disrupting microtubule polymerization, and inhibiting mitochondrial translocation, which reduces Ca 2+ influx into the cell. Our findings provide mechanisms for both triclosan's inhibition of mast cell signaling and its universal disruption of mitochondria. These mechanisms provide partial explanations for triclosan's adverse effects on human reproduction, immunology, and development. This study is the first to utilize super-resolution microscopy in the field of toxicology. Copyright © 2018 Elsevier Inc. All rights reserved.

Memory-effect based deconvolution microscopy for super-resolution imaging through scattering media

NASA Astrophysics Data System (ADS)

Edrei, Eitan; Scarcelli, Giuliano

2016-09-01

High-resolution imaging through turbid media is a fundamental challenge of optical sciences that has attracted a lot of attention in recent years for its wide range of potential applications. Here, we demonstrate that the resolution of imaging systems looking behind a highly scattering medium can be improved below the diffraction-limit. To achieve this, we demonstrate a novel microscopy technique enabled by the optical memory effect that uses a deconvolution image processing and thus it does not require iterative focusing, scanning or phase retrieval procedures. We show that this newly established ability of direct imaging through turbid media provides fundamental and practical advantages such as three-dimensional refocusing and unambiguous object reconstruction.

Memory-effect based deconvolution microscopy for super-resolution imaging through scattering media.

PubMed

Edrei, Eitan; Scarcelli, Giuliano

2016-09-16

High-resolution imaging through turbid media is a fundamental challenge of optical sciences that has attracted a lot of attention in recent years for its wide range of potential applications. Here, we demonstrate that the resolution of imaging systems looking behind a highly scattering medium can be improved below the diffraction-limit. To achieve this, we demonstrate a novel microscopy technique enabled by the optical memory effect that uses a deconvolution image processing and thus it does not require iterative focusing, scanning or phase retrieval procedures. We show that this newly established ability of direct imaging through turbid media provides fundamental and practical advantages such as three-dimensional refocusing and unambiguous object reconstruction.

Estimation of reactogenicity of preparations produced on the basis of photoinactivated live vaccines against brucellosis and tularaemia on the organismic level. 2. Using the method of speckle-microscopy with high spatial resolution

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

Ulianova, O V; Uianov, S S; Li Pengcheng

2011-04-30

The method of speckle microscopy was adapted to estimate the reactogenicity of the prototypes of vaccine preparations against extremely dangerous infections. The theory is proposed to describe the mechanism of formation of the output signal from the super-high spatial resolution speckle microscope. The experimental studies show that bacterial suspensions, irradiated in different regimes of inactivation, do not exert negative influence on the blood microcirculations in laboratory animals. (optical technologies in biophysics and medicine)

Brain Slice Staining and Preparation for Three-Dimensional Super-Resolution Microscopy

PubMed Central

German, Christopher L.; Gudheti, Manasa V.; Fleckenstein, Annette E.; Jorgensen, Erik M.

2018-01-01

Localization microscopy techniques – such as photoactivation localization microscopy (PALM), fluorescent PALM (FPALM), ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM) – provide the highest precision for single molecule localization currently available. However, localization microscopy has been largely limited to cell cultures due to the difficulties that arise in imaging thicker tissue sections. Sample fixation and antibody staining, background fluorescence, fluorophore photoinstability, light scattering in thick sections, and sample movement create significant challenges for imaging intact tissue. We have developed a sample preparation and image acquisition protocol to address these challenges in rat brain slices. The sample preparation combined multiple fixation steps, saponin permeabilization, and tissue clarification. Together, these preserve intracellular structures, promote antibody penetration, reduce background fluorescence and light scattering, and allow acquisition of images deep in a 30 μm thick slice. Image acquisition challenges were resolved by overlaying samples with a permeable agarose pad and custom-built stainless steel imaging adapter, and sealing the imaging chamber. This approach kept slices flat, immobile, bathed in imaging buffer, and prevented buffer oxidation during imaging. Using this protocol, we consistently obtained single molecule localizations of synaptic vesicle and active zone proteins in three-dimensions within individual synaptic terminals of the striatum in rat brain slices. These techniques may be easily adapted to the preparation and imaging of other tissues, substantially broadening the application of super-resolution imaging. PMID:28924666

In-line FINCH super resolution digital holographic fluorescence microscopy using a high efficiency transmission liquid crystal GRIN lens.

PubMed

Brooker, Gary; Siegel, Nisan; Rosen, Joseph; Hashimoto, Nobuyuki; Kurihara, Makoto; Tanabe, Ayano

2013-12-15

We report a new optical arrangement that creates high-efficiency, high-quality Fresnel incoherent correlation holography (FINCH) holograms using polarization sensitive transmission liquid crystal gradient index (TLCGRIN) diffractive lenses. In contrast, current universal practice in the field employs a reflective spatial light modulator (SLM) to separate sample and reference beams. Polarization sensitive TLCGRIN lenses enable a straight optical path, have >90% transmission efficiency, are not pixilated, and are free of many limitations of reflective SLM devices. For each sample point, two spherical beams created by a glass lens in combination with a polarization sensitive TLCGRIN lens interfere and create a hologram and resultant super resolution image.

Correlation Functions Quantify Super-Resolution Images and Estimate Apparent Clustering Due to Over-Counting

PubMed Central

Veatch, Sarah L.; Machta, Benjamin B.; Shelby, Sarah A.; Chiang, Ethan N.; Holowka, David A.; Baird, Barbara A.

2012-01-01

We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy images of static surfaces in two dimensions. We use this method to quantify how over-counting of labeled molecules contributes to apparent self-clustering and to calculate the effective lateral resolution of an image. This treatment applies to distributions of proteins and lipids in cell membranes, where there is significant interest in using electron microscopy and super-resolution fluorescence localization techniques to probe membrane heterogeneity. When images are quantified using pair auto-correlation functions, the magnitude of apparent clustering arising from over-counting varies inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. In contrast, we demonstrate that over-counting does not give rise to apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (FcεRI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM/dSTORM) and scanning electron microscopy (SEM). We find that apparent clustering of FcεRI-bound IgE is dominated by over-counting labels on individual complexes when IgE is directly conjugated to organic fluorophores. We verify this observation by measuring pair cross-correlation functions between two distinguishably labeled pools of IgE-FcεRI on the cell surface using both imaging methods. After correcting for over-counting, we observe weak but significant self-clustering of IgE-FcεRI in fluorescence localization measurements, and no residual self-clustering as detected with SEM. We also apply this method to quantify IgE-FcεRI redistribution after deliberate clustering by crosslinking with two distinct trivalent ligands of defined architectures, and we evaluate contributions from both over-counting of labels and redistribution of proteins. PMID:22384026

Sub-diffraction nano manipulation using STED AFM.

PubMed

Chacko, Jenu Varghese; Canale, Claudio; Harke, Benjamin; Diaspro, Alberto

2013-01-01

In the last two decades, nano manipulation has been recognized as a potential tool of scientific interest especially in nanotechnology and nano-robotics. Contemporary optical microscopy (super resolution) techniques have also reached the nanometer scale resolution to visualize this and hence a combination of super resolution aided nano manipulation ineluctably gives a new perspective to the scenario. Here we demonstrate how specificity and rapid determination of structures provided by stimulated emission depletion (STED) microscope can aid another microscopic tool with capability of mechanical manoeuvring, like an atomic force microscope (AFM) to get topological information or to target nano scaled materials. We also give proof of principle on how high-resolution real time visualization can improve nano manipulation capability within a dense sample, and how STED-AFM is an optimal combination for this job. With these evidences, this article points to future precise nano dissections and maybe even to a nano-snooker game with an AFM tip and fluorospheres.

Achieving high-efficiency emission depletion nanoscopy by employing cross relaxation in upconversion nanoparticles.

PubMed

Zhan, Qiuqiang; Liu, Haichun; Wang, Baoju; Wu, Qiusheng; Pu, Rui; Zhou, Chao; Huang, Bingru; Peng, Xingyun; Ågren, Hans; He, Sailing

2017-10-20

Stimulated emission depletion microscopy provides a powerful sub-diffraction imaging modality for life science studies. Conventionally, stimulated emission depletion requires a relatively high light intensity to obtain an adequate depletion efficiency through only light-matter interaction. Here we show efficient emission depletion for a class of lanthanide-doped upconversion nanoparticles with the assistance of interionic cross relaxation, which significantly lowers the laser intensity requirements of optical depletion. We demonstrate two-color super-resolution imaging using upconversion nanoparticles (resolution ~ 66 nm) with a single pair of excitation/depletion beams. In addition, we show super-resolution imaging of immunostained cytoskeleton structures of fixed cells (resolution ~ 82 nm) using upconversion nanoparticles. These achievements provide a new perspective for the development of photoswitchable luminescent probes and will broaden the applications of lanthanide-doped nanoparticles for sub-diffraction microscopic imaging.

Super-resolution microscopy reveals LINC complex recruitment at nuclear indentation sites.

PubMed

Versaevel, Marie; Braquenier, Jean-Baptiste; Riaz, Maryam; Grevesse, Thomas; Lantoine, Joséphine; Gabriele, Sylvain

2014-12-08

Increasing evidences show that the actin cytoskeleton is a key parameter of the nuclear remodeling process in response to the modifications of cellular morphology. However, detailed information on the interaction between the actin cytoskeleton and the nuclear lamina was still lacking. We addressed this question by constraining endothelial cells on rectangular fibronectin-coated micropatterns and then using Structured Illumination Microscopy (SIM) to observe the interactions between actin stress fibers, nuclear lamina and LINC complexes at a super-resolution scale. Our results show that tension in apical actin stress fibers leads to deep nuclear indentations that significantly deform the nuclear lamina. Interestingly, indented nuclear zones are characterized by a local enrichment of LINC complexes, which anchor apical actin fibers to the nuclear lamina. Moreover, our findings indicate that nuclear indentations induce the formation of segregated domains of condensed chromatin. However, nuclear indentations and condensed chromatin domains are not irreversible processes and both can relax in absence of tension in apical actin stress fibers.

Super-Resolution Microscopy Unveils Dynamic Heterogeneities in Nanoparticle Protein Corona.

PubMed

Feiner-Gracia, Natalia; Beck, Michaela; Pujals, Sílvia; Tosi, Sébastien; Mandal, Tamoghna; Buske, Christian; Linden, Mika; Albertazzi, Lorenzo

2017-11-01

The adsorption of serum proteins, leading to the formation of a biomolecular corona, is a key determinant of the biological identity of nanoparticles in vivo. Therefore, gaining knowledge on the formation, composition, and temporal evolution of the corona is of utmost importance for the development of nanoparticle-based therapies. Here, it is shown that the use of super-resolution optical microscopy enables the imaging of the protein corona on mesoporous silica nanoparticles with single protein sensitivity. Particle-by-particle quantification reveals a significant heterogeneity in protein absorption under native conditions. Moreover, the diversity of the corona evolves over time depending on the surface chemistry and degradability of the particles. This paper investigates the consequences of protein adsorption for specific cell targeting by antibody-functionalized nanoparticles providing a detailed understanding of corona-activity relations. The methodology is widely applicable to a variety of nanostructures and complements the existing ensemble approaches for protein corona study. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking

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

Rossier, Olivier; Giannone, Grégory; CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux

Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements andmore » interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.« less

Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope

PubMed Central

Otsuka, Shotaro; Bui, Khanh Huy; Schorb, Martin; Hossain, M Julius; Politi, Antonio Z; Koch, Birgit; Eltsov, Mikhail; Beck, Martin; Ellenberg, Jan

2016-01-01

The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM. DOI: http://dx.doi.org/10.7554/eLife.19071.001 PMID:27630123

The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking.

PubMed

Rossier, Olivier; Giannone, Grégory

2016-04-10

Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells. Copyright © 2015. Published by Elsevier Inc.

Tracking individual membrane proteins and their biochemistry: The power of direct observation.

PubMed

Barden, Adam O; Goler, Adam S; Humphreys, Sara C; Tabatabaei, Samaneh; Lochner, Martin; Ruepp, Marc-David; Jack, Thomas; Simonin, Jonathan; Thompson, Andrew J; Jones, Jeffrey P; Brozik, James A

2015-11-01

The advent of single molecule fluorescence microscopy has allowed experimental molecular biophysics and biochemistry to transcend traditional ensemble measurements, where the behavior of individual proteins could not be precisely sampled. The recent explosion in popularity of new super-resolution and super-localization techniques coupled with technical advances in optical designs and fast highly sensitive cameras with single photon sensitivity and millisecond time resolution have made it possible to track key motions, reactions, and interactions of individual proteins with high temporal resolution and spatial resolution well beyond the diffraction limit. Within the purview of membrane proteins and ligand gated ion channels (LGICs), these outstanding advances in single molecule microscopy allow for the direct observation of discrete biochemical states and their fluctuation dynamics. Such observations are fundamentally important for understanding molecular-level mechanisms governing these systems. Examples reviewed here include the effects of allostery on the stoichiometry of ligand binding in the presence of fluorescent ligands; the observation of subdomain partitioning of membrane proteins due to microenvironment effects; and the use of single particle tracking experiments to elucidate characteristics of membrane protein diffusion and the direct measurement of thermodynamic properties, which govern the free energy landscape of protein dimerization. The review of such characteristic topics represents a snapshot of efforts to push the boundaries of fluorescence microscopy of membrane proteins to the absolute limit. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'. Copyright © 2015 Elsevier Ltd. All rights reserved.

Single molecules, cells, and super-resolution optics (Presentation Video)

NASA Astrophysics Data System (ADS)

Betzig, Eric

2015-03-01

In this plenary presentation, Eric Betzig talks about his scientific journey that led to the Nobel Prize. He made waves early in his career by helping to develop a technique known as near-field microscopy, which brought into focus structures that scientists had long considered too small to see with a light microscope. Eric Betzig is a group leader at Janelia Research Campus of the Howard Hughes Medical Institute (HHMI) in Ashburn, VA. He recieved a BS in physics from California Institute of Technology and a PhD in applied and engineering physics from Cornell University. Betzig received the 2014 Nobel Prize in Chemistry, along with William Moerner and Stefan Hell, for their development of super-resolved fluorescence microscopy.

A graphene oxide-carbon nanotube grid for high-resolution transmission electron microscopy of nanomaterials.

PubMed

Zhang, Lina; Zhang, Haoxu; Zhou, Ruifeng; Chen, Zhuo; Li, Qunqing; Fan, Shoushan; Ge, Guanglu; Liu, Renxiao; Jiang, Kaili

2011-09-23

A novel grid for use in transmission electron microscopy is developed. The supporting film of the grid is composed of thin graphene oxide films overlying a super-aligned carbon nanotube network. The composite film combines the advantages of graphene oxide and carbon nanotube networks and has the following properties: it is ultra-thin, it has a large flat and smooth effective supporting area with a homogeneous amorphous appearance, high stability, and good conductivity. The graphene oxide-carbon nanotube grid has a distinct advantage when characterizing the fine structure of a mass of nanomaterials over conventional amorphous carbon grids. Clear high-resolution transmission electron microscopy images of various nanomaterials are obtained easily using the new grids.

Towards real-time image deconvolution: application to confocal and STED microscopy

PubMed Central

Zanella, R.; Zanghirati, G.; Cavicchioli, R.; Zanni, L.; Boccacci, P.; Bertero, M.; Vicidomini, G.

2013-01-01

Although deconvolution can improve the quality of any type of microscope, the high computational time required has so far limited its massive spreading. Here we demonstrate the ability of the scaled-gradient-projection (SGP) method to provide accelerated versions of the most used algorithms in microscopy. To achieve further increases in efficiency, we also consider implementations on graphic processing units (GPUs). We test the proposed algorithms both on synthetic and real data of confocal and STED microscopy. Combining the SGP method with the GPU implementation we achieve a speed-up factor from about a factor 25 to 690 (with respect the conventional algorithm). The excellent results obtained on STED microscopy images demonstrate the synergy between super-resolution techniques and image-deconvolution. Further, the real-time processing allows conserving one of the most important property of STED microscopy, i.e the ability to provide fast sub-diffraction resolution recordings. PMID:23982127

Single cell genomic quantification by non-fluorescence nonlinear microscopy

NASA Astrophysics Data System (ADS)

Kota, Divya; Liu, Jing

2017-02-01

Human epidermal growth receptor 2 (Her2) is a gene which plays a major role in breast cancer development. The quantification of Her2 expression in single cells is limited by several drawbacks in existing fluorescence-based single molecule techniques, such as low signal-to-noise ratio (SNR), strong autofluorescence and background signals from biological components. For rigorous genomic quantification, a robust method of orthogonal detection is highly desirable and we demonstrated it by two non-fluorescent imaging techniques -transient absorption microscopy (TAM) and second harmonic generation (SHG). In TAM, gold nanoparticles (AuNPs) are chosen as an orthogonal probes for detection of single molecules which gives background-free quantifications of single mRNA transcript. In SHG, emission from barium titanium oxide (BTO) nanoprobes was demonstrated which allows stable signal beyond the autofluorescence window. Her2 mRNA was specifically labeled with nanoprobes which are conjugated with antibodies or oligonucleotides and quantified at single copy sensitivity in the cancer cells and tissues. Furthermore, a non-fluorescent super-resolution concept, named as second harmonic super-resolution microscopy (SHaSM), was proposed to quantify individual Her2 transcripts in cancer cells beyond the diffraction limit. These non-fluorescent imaging modalities will provide new dimensions in biomarker quantification at single molecule sensitivity in turbid biological samples, offering a strong cross-platform strategy for clinical monitoring at single cell resolution.

Development of targeted STORM for super resolution imaging of biological samples using digital micro-mirror device

NASA Astrophysics Data System (ADS)

Valiya Peedikakkal, Liyana; Steventon, Victoria; Furley, Andrew; Cadby, Ashley J.

2017-12-01

We demonstrate a simple illumination system based on a digital mirror device which allows for fine control over the power and pattern of illumination. We apply this to localization microscopy (LM), specifically stochastic optical reconstruction microscopy (STORM). Using this targeted STORM, we were able to image a selected area of a labelled cell without causing photo-damage to the surrounding areas of the cell.

Capturing the Surface Texture and Shape of Pollen: A Comparison of Microscopy Techniques

PubMed Central

Sivaguru, Mayandi; Mander, Luke; Fried, Glenn; Punyasena, Surangi W.

2012-01-01

Research on the comparative morphology of pollen grains depends crucially on the application of appropriate microscopy techniques. Information on the performance of microscopy techniques can be used to inform that choice. We compared the ability of several microscopy techniques to provide information on the shape and surface texture of three pollen types with differing morphologies. These techniques are: widefield, apotome, confocal and two-photon microscopy (reflected light techniques), and brightfield and differential interference contrast microscopy (DIC) (transmitted light techniques). We also provide a first view of pollen using super-resolution microscopy. The three pollen types used to contrast the performance of each technique are: Croton hirtus (Euphorbiaceae), Mabea occidentalis (Euphorbiaceae) and Agropyron repens (Poaceae). No single microscopy technique provided an adequate picture of both the shape and surface texture of any of the three pollen types investigated here. The wavelength of incident light, photon-collection ability of the optical technique, signal-to-noise ratio, and the thickness and light absorption characteristics of the exine profoundly affect the recovery of morphological information by a given optical microscopy technique. Reflected light techniques, particularly confocal and two-photon microscopy, best capture pollen shape but provide limited information on very fine surface texture. In contrast, transmitted light techniques, particularly differential interference contrast microscopy, can resolve very fine surface texture but provide limited information on shape. Texture comprising sculptural elements that are spaced near the diffraction limit of light (∼250 nm; NDL) presents an acute challenge to optical microscopy. Super-resolution structured illumination microscopy provides data on the NDL texture of A. repens that is more comparable to textural data from scanning electron microscopy than any other optical microscopy technique investigated here. Maximizing the recovery of morphological information from pollen grains should lead to more robust classifications, and an increase in the taxonomic precision with which ancient vegetation can be reconstructed. PMID:22720050

Design of discrete and continuous super-resolving Toraldo pupils in the microwave range.

PubMed

Olmi, Luca; Bolli, Pietro; Mugnai, Daniela

2018-03-20

The concept of super-resolution refers to various methods for improving the angular resolution of an optical imaging system beyond the classical diffraction limit. In optical microscopy, several techniques have been successfully developed with the aim of narrowing the central lobe of the illumination point spread function. In astronomy, however, no similar techniques can be used. A feasible method to design antennas and telescopes with angular resolution better than the diffraction limit consists of using variable transmittance pupils. In particular, discrete binary phase masks (0 or π ) with finite phase-jump positions, known as Toraldo pupils (TPs), have the advantage of being easy to fabricate but offer relatively little flexibility in terms of achieving specific trade-offs between design parameters, such as the angular width of the main lobe and the intensity of sidelobes. In this paper, we show that a complex transmittance filter (equivalent to a continuous TP, i.e., consisting of infinitely narrow concentric rings) can achieve more easily the desired trade-off between design parameters. We also show how the super-resolution effect can be generated with both amplitude- and phase-only masks and confirm the expected performance with electromagnetic numerical simulations in the microwave range.

Demosaiced pixel super-resolution for multiplexed holographic color imaging

PubMed Central

Wu, Yichen; Zhang, Yibo; Luo, Wei; Ozcan, Aydogan

2016-01-01

To synthesize a holographic color image, one can sequentially take three holograms at different wavelengths, e.g., at red (R), green (G) and blue (B) parts of the spectrum, and digitally merge them. To speed up the imaging process by a factor of three, a Bayer color sensor-chip can also be used to demultiplex three wavelengths that simultaneously illuminate the sample and digitally retrieve individual set of holograms using the known transmission spectra of the Bayer color filters. However, because the pixels of different channels (R, G, B) on a Bayer color sensor are not at the same physical location, conventional demosaicing techniques generate color artifacts in holographic imaging using simultaneous multi-wavelength illumination. Here we demonstrate that pixel super-resolution can be merged into the color de-multiplexing process to significantly suppress the artifacts in wavelength-multiplexed holographic color imaging. This new approach, termed Demosaiced Pixel Super-Resolution (D-PSR), generates color images that are similar in performance to sequential illumination at three wavelengths, and therefore improves the speed of holographic color imaging by 3-fold. D-PSR method is broadly applicable to holographic microscopy applications, where high-resolution imaging and multi-wavelength illumination are desired. PMID:27353242

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.

Bimetallic Effect of Single Nanocatalysts Visualized by Super-Resolution Catalysis Imaging

DOE PAGES

Chen, Guanqun; Zou, Ningmu; Chen, Bo; ...

2017-11-01

Compared with their monometallic counterparts, bimetallic nanoparticles often show enhanced catalytic activity associated with the bimetallic interface. Direct quantitation of catalytic activity at the bimetallic interface is important for understanding the enhancement mechanism, but challenging experimentally. Here using single-molecule super-resolution catalysis imaging in correlation with electron microscopy, we report the first quantitative visualization of enhanced bimetallic activity within single bimetallic nanoparticles. We focus on heteronuclear bimetallic PdAu nanoparticles that present a well-defined Pd–Au bimetallic interface in catalyzing a photodriven fluorogenic disproportionation reaction. Our approach also enables a direct comparison between the bimetallic and monometallic regions within the same nanoparticle. Theoreticalmore » calculations further provide insights into the electronic nature of N–O bond activation of the reactant (resazurin) adsorbed on bimetallic sites. Subparticle activity correlation between bimetallic enhancement and monometallic activity suggests that the favorable locations to construct bimetallic sites are those monometallic sites with higher activity, leading to a strategy for making effective bimetallic nanocatalysts. Furthermore, the results highlight the power of super-resolution catalysis imaging in gaining insights that could help improve nanocatalysts.« less

Axial tomography in live cell laser microscopy

NASA Astrophysics Data System (ADS)

Richter, Verena; Bruns, Sarah; Bruns, Thomas; Weber, Petra; Wagner, Michael; Cremer, Christoph; Schneckenburger, Herbert

2017-09-01

Single cell microscopy in a three-dimensional (3-D) environment is reported. Cells are grown in an agarose culture gel, located within microcapillaries and observed from different sides after adaptation of an innovative device for sample rotation. Thus, z-stacks can be recorded by confocal microscopy in different directions and used for illustration in 3-D. This gives additional information, since cells or organelles that appear superimposed in one direction, may be well resolved in another one. The method is tested and validated with single cells expressing a membrane or a mitochondrially associated green fluorescent protein, or cells accumulating fluorescent quantum dots. In addition, axial tomography supports measurements of cellular uptake and distribution of the anticancer drug doxorubicin in the nucleus (2 to 6 h after incubation) or the cytoplasm (24 h). This paper discusses that upon cell rotation an enhanced optical resolution in lateral direction compared to axial direction can be utilized to obtain an improved effective 3-D resolution, which represents an important step toward super-resolution microscopy of living cells.

Parallel detecting super-resolution microscopy using correlation based image restoration

NASA Astrophysics Data System (ADS)

Yu, Zhongzhi; Liu, Shaocong; Zhu, Dazhao; Kuang, Cuifang; Liu, Xu

2017-12-01

A novel approach to achieve the image restoration is proposed in which each detector's relative position in the detector array is no longer a necessity. We can identify each detector's relative location by extracting a certain area from one of the detector's image and scanning it on other detectors' images. According to this location, we can generate the point spread functions (PSF) for each detector and perform deconvolution for image restoration. Equipped with this method, the microscope with discretionally designed detector array can be easily constructed without the concern of exact relative locations of detectors. The simulated results and experimental results show the total improvement in resolution with a factor of 1.7 compared to conventional confocal fluorescence microscopy. With the significant enhancement in resolution and easiness for application of this method, this novel method should have potential for a wide range of application in fluorescence microscopy based on parallel detecting.

Self-interference 3D super-resolution microscopy for deep tissue investigations.

PubMed

Bon, Pierre; Linarès-Loyez, Jeanne; Feyeux, Maxime; Alessandri, Kevin; Lounis, Brahim; Nassoy, Pierre; Cognet, Laurent

2018-06-01

Fluorescence localization microscopy has achieved near-molecular resolution capable of revealing ultra-structures, with a broad range of applications, especially in cellular biology. However, it remains challenging to attain such resolution in three dimensions and inside biological tissues beyond the first cell layer. Here we introduce SELFI, a framework for 3D single-molecule localization within multicellular specimens and tissues. The approach relies on self-interference generated within the microscope's point spread function (PSF) to simultaneously encode equiphase and intensity fluorescence signals, which together provide the 3D position of an emitter. We combined SELFI with conventional localization microscopy to visualize F-actin 3D filament networks and reveal the spatial distribution of the transcription factor OCT4 in human induced pluripotent stem cells at depths up to 50 µm inside uncleared tissue spheroids. SELFI paves the way to nanoscale investigations of native cellular processes in intact tissues.

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.

Improving multiphoton STED nanoscopy with separation of photons by LIfetime Tuning (SPLIT)

NASA Astrophysics Data System (ADS)

Coto Hernández, Iván.; Lanzano, Luca; Castello, Marco; Jowett, Nate; Tortarolo, Giorgio; Diaspro, Alberto; Vicidomini, Giuseppe

2018-02-01

Stimulated emission depletion (STED) microscopy is a powerful bio-imaging technique since it provides molecular spatial resolution whilst preserving the most important assets of fluorescence microscopy. When combined with twophoton excitation (2PE) microscopy (2PE-STED), the sub-diffraction imaging ability of STED microscopy can be achieved also on thick biological samples. The most straightforward implementation of 2PE-STED microscopy is obtained by introducing a STED beam operating in continuous wave (CW) into a conventional Ti:Sapphire based 2PE microscope (2PE-CW-STED). In this implementation, an effective resolution enhancement is mainly obtained implementing a time-gated detection scheme, which however can drastically reduce the signal-to-noise/background ratio of the final image. Herein, we combine the lifetime tuning (SPLIT) approach with 2PE-CW-STED to overcome this limitation. The SPLIT approach is employed to discard fluorescence photons lacking super-resolution information, by means of a pixel-by-pixel phasor approach. Combining the SPLIT approach with image deconvolution further optimizes the signal-to-noise/background ratio.

A multi-emitter fitting algorithm for potential live cell super-resolution imaging over a wide range of molecular densities.

PubMed

Takeshima, T; Takahashi, T; Yamashita, J; Okada, Y; Watanabe, S

2018-05-25

Multi-emitter fitting algorithms have been developed to improve the temporal resolution of single-molecule switching nanoscopy, but the molecular density range they can analyse is narrow and the computation required is intensive, significantly limiting their practical application. Here, we propose a computationally fast method, wedged template matching (WTM), an algorithm that uses a template matching technique to localise molecules at any overlapping molecular density from sparse to ultrahigh density with subdiffraction resolution. WTM achieves the localization of overlapping molecules at densities up to 600 molecules μm -2 with a high detection sensitivity and fast computational speed. WTM also shows localization precision comparable with that of DAOSTORM (an algorithm for high-density super-resolution microscopy), at densities up to 20 molecules μm -2 , and better than DAOSTORM at higher molecular densities. The application of WTM to a high-density biological sample image demonstrated that it resolved protein dynamics from live cell images with subdiffraction resolution and a temporal resolution of several hundred milliseconds or less through a significant reduction in the number of camera images required for a high-density reconstruction. WTM algorithm is a computationally fast, multi-emitter fitting algorithm that can analyse over a wide range of molecular densities. The algorithm is available through the website. https://doi.org/10.17632/bf3z6xpn5j.1. © 2018 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Modeling of the axon membrane skeleton structure and implications for its mechanical properties

PubMed Central

Tzingounis, Anastasios V.

2017-01-01

Super-resolution microscopy recently revealed that, unlike the soma and dendrites, the axon membrane skeleton is structured as a series of actin rings connected by spectrin filaments that are held under tension. Currently, the structure-function relationship of the axonal structure is unclear. Here, we used atomic force microscopy (AFM) to show that the stiffness of the axon plasma membrane is significantly higher than the stiffnesses of dendrites and somata. To examine whether the structure of the axon plasma membrane determines its overall stiffness, we introduced a coarse-grain molecular dynamics model of the axon membrane skeleton that reproduces the structure identified by super-resolution microscopy. Our proposed computational model accurately simulates the median value of the Young’s modulus of the axon plasma membrane determined by atomic force microscopy. It also predicts that because the spectrin filaments are under entropic tension, the thermal random motion of the voltage-gated sodium channels (Nav), which are bound to ankyrin particles, a critical axonal protein, is reduced compared to the thermal motion when spectrin filaments are held at equilibrium. Lastly, our model predicts that because spectrin filaments are under tension, any axonal injuries that lacerate spectrin filaments will likely lead to a permanent disruption of the membrane skeleton due to the inability of spectrin filaments to spontaneously form their initial under-tension configuration. PMID:28241082

Non-heuristic automatic techniques for overcoming low signal-to-noise-ratio bias of localization microscopy and multiple signal classification algorithm.

PubMed

Agarwal, Krishna; Macháň, Radek; Prasad, Dilip K

2018-03-21

Localization microscopy and multiple signal classification algorithm use temporal stack of image frames of sparse emissions from fluorophores to provide super-resolution images. Localization microscopy localizes emissions in each image independently and later collates the localizations in all the frames, giving same weight to each frame irrespective of its signal-to-noise ratio. This results in a bias towards frames with low signal-to-noise ratio and causes cluttered background in the super-resolved image. User-defined heuristic computational filters are employed to remove a set of localizations in an attempt to overcome this bias. Multiple signal classification performs eigen-decomposition of the entire stack, irrespective of the relative signal-to-noise ratios of the frames, and uses a threshold to classify eigenimages into signal and null subspaces. This results in under-representation of frames with low signal-to-noise ratio in the signal space and over-representation in the null space. Thus, multiple signal classification algorithms is biased against frames with low signal-to-noise ratio resulting into suppression of the corresponding fluorophores. This paper presents techniques to automatically debias localization microscopy and multiple signal classification algorithm of these biases without compromising their resolution and without employing heuristics, user-defined criteria. The effect of debiasing is demonstrated through five datasets of invitro and fixed cell samples.

Modeling of the axon membrane skeleton structure and implications for its mechanical properties.

PubMed

Zhang, Yihao; Abiraman, Krithika; Li, He; Pierce, David M; Tzingounis, Anastasios V; Lykotrafitis, George

2017-02-01

Super-resolution microscopy recently revealed that, unlike the soma and dendrites, the axon membrane skeleton is structured as a series of actin rings connected by spectrin filaments that are held under tension. Currently, the structure-function relationship of the axonal structure is unclear. Here, we used atomic force microscopy (AFM) to show that the stiffness of the axon plasma membrane is significantly higher than the stiffnesses of dendrites and somata. To examine whether the structure of the axon plasma membrane determines its overall stiffness, we introduced a coarse-grain molecular dynamics model of the axon membrane skeleton that reproduces the structure identified by super-resolution microscopy. Our proposed computational model accurately simulates the median value of the Young's modulus of the axon plasma membrane determined by atomic force microscopy. It also predicts that because the spectrin filaments are under entropic tension, the thermal random motion of the voltage-gated sodium channels (Nav), which are bound to ankyrin particles, a critical axonal protein, is reduced compared to the thermal motion when spectrin filaments are held at equilibrium. Lastly, our model predicts that because spectrin filaments are under tension, any axonal injuries that lacerate spectrin filaments will likely lead to a permanent disruption of the membrane skeleton due to the inability of spectrin filaments to spontaneously form their initial under-tension configuration.

Super-resolution photoacoustic microscopy using joint sparsity

NASA Astrophysics Data System (ADS)

Burgholzer, P.; Haltmeier, M.; Berer, T.; Leiss-Holzinger, E.; Murray, T. W.

2017-07-01

We present an imaging method that uses the random optical speckle patterns that naturally emerge as light propagates through strongly scattering media as a structured illumination source for photoacoustic imaging. Our approach, termed blind structured illumination photoacoustic microscopy (BSIPAM), was inspired by recent work in fluorescence microscopy where super-resolution imaging was demonstrated using multiple unknown speckle illumination patterns. We extend this concept to the multiple scattering domain using photoacoustics (PA), with the speckle pattern serving to generate ultrasound. The optical speckle pattern that emerges as light propagates through diffuse media provides structured illumination to an object placed behind a scattering wall. The photoacoustic signal produced by such illumination is detected using a focused ultrasound transducer. We demonstrate through both simulation and experiment, that by acquiring multiple photoacoustic images, each produced by a different random and unknown speckle pattern, an image of an absorbing object can be reconstructed with a spatial resolution far exceeding that of the ultrasound transducer. We experimentally and numerically demonstrate a gain in resolution of more than a factor of two by using multiple speckle illuminations. The variations in the photoacoustic signals generated with random speckle patterns are utilized in BSIPAM using a novel reconstruction algorithm. Exploiting joint sparsity, this algorithm is capable of reconstructing the absorbing structure from measured PA signals with a resolution close to the speckle size. Another way to excite random excitation for photoacoustic imaging are small absorbing particles, including contrast agents, which flow through small vessels. For such a set-up, the joint-sparsity is generated by the fact that all the particles move in the same vessels. Structured illumination in that case is not necessary.

Super-resolution Imaging of Chemical Synapses in the Brain

PubMed Central

Dani, Adish; Huang, Bo; Bergan, Joseph; Dulac, Catherine; Zhuang, Xiaowei

2010-01-01

Determination of the molecular architecture of synapses requires nanoscopic image resolution and specific molecular recognition, a task that has so far defied many conventional imaging approaches. Here we present a super-resolution fluorescence imaging method to visualize the molecular architecture of synapses in the brain. Using multicolor, three-dimensional stochastic optical reconstruction microscopy, the distributions of synaptic proteins can be measured with nanometer precision. Furthermore, the wide-field, volumetric imaging method enables high-throughput, quantitative analysis of a large number of synapses from different brain regions. To demonstrate the capabilities of this approach, we have determined the organization of ten protein components of the presynaptic active zone and the postsynaptic density. Variations in synapse morphology, neurotransmitter receptor composition, and receptor distribution were observed both among synapses and across different brain regions. Combination with optogenetics further allowed molecular events associated with synaptic plasticity to be resolved at the single-synapse level. PMID:21144999

NicoLase—An open-source diode laser combiner, fiber launch, and sequencing controller for fluorescence microscopy

PubMed Central

Walsh, James; Böcking, Till; Gaus, Katharina

2017-01-01

Modern fluorescence microscopy requires software-controlled illumination sources with high power across a wide range of wavelengths. Diode lasers meet the power requirements and combining multiple units into a single fiber launch expands their capability across the required spectral range. We present the NicoLase, an open-source diode laser combiner, fiber launch, and software sequence controller for fluorescence microscopy and super-resolution microscopy applications. Two configurations are described, giving four or six output wavelengths and one or two single-mode fiber outputs, with all CAD files, machinist drawings, and controller source code openly available. PMID:28301563

Super-resolution optical microscopy resolves network morphology of smart colloidal microgels.

PubMed

Bergmann, Stephan; Wrede, Oliver; Huser, Thomas; Hellweg, Thomas

2018-02-14

We present a new method to resolve the network morphology of colloidal particles in an aqueous environment via super-resolution microscopy. By localization of freely diffusing fluorophores inside the particle network we can resolve the three dimensional structure of one species of colloidal particles (thermoresponsive microgels) without altering their chemical composition through copolymerization with fluorescent monomers. Our approach utilizes the interaction of the fluorescent dye rhodamine 6G with the polymer network to achieve an indirect labeling. We calculate the 3D structure from the 2D images and compare the structure to previously published models for the microgel morphology, e.g. the fuzzy sphere model. To describe the differences in the data an extension of this model is suggested. Our method enables the tailor-made fabrication of colloidal particles which are used in various applications, such as paints or cosmetics, and are promising candidates for drug delivery, smart surface coatings, and nanocatalysis. With the precise knowledge of the particle morphology an understanding of the underlying structure-property relationships for various colloidal systems is possible.

Common fluorescent proteins for single-molecule localization microscopy

NASA Astrophysics Data System (ADS)

Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

2015-07-01

Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

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.

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

PubMed Central

Young, Laurence J.; Ströhl, Florian; Kaminski, Clemens F.

2016-01-01

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells. PMID:27285848

Super-Resolution Imaging of the Golgi in Live Cells with a Bio-orthogonal Ceramide Probe**

PubMed Central

Erdmann, Roman S.; Takakura, Hideo; Thompson, Alexander D.; Rivera-Molina, Felix; Allgeyer, Edward S.; Bewersdorf, Joerg; Toomre, Derek K.; Schepartz, Alanna

2014-01-01

We report a lipid-based strategy to visualize Golgi structure and dynamics at super-resolution in live cells. The method is based on two novel reagents: a trans-cyclooctene-containing ceramide lipid (Cer-TCO) and a highly reactive, tetrazine-tagged near-IR dye (SiR-Tz). These reagents assemble via an extremely rapid ‘tetrazine-click’ reaction into Cer-SiR, a highly photostable ‘vital dye’ that enables prolonged live cell imaging of the Golgi apparatus by 3D confocal and STED microscopy. Cer-SiR is non-toxic at concentrations as high as 2 μM and does not perturb the mobility of Golgi-resident enzymes or the traffic of cargo from the endoplasmic reticulum through the Golgi and to the plasma membrane. PMID:25081303

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

Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.

Here, we have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single moleculemore » super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.« less

Macrophages in the Human Cochlea: Saviors or Predators—A Study Using Super-Resolution Immunohistochemistry

PubMed Central

Liu, Wei; Molnar, Matyas; Garnham, Carolyn; Benav, Heval; Rask-Andersen, Helge

2018-01-01

The human inner ear, which is segregated by a blood/labyrinth barrier, contains resident macrophages [CD163, ionized calcium-binding adaptor molecule 1 (IBA1)-, and CD68-positive cells] within the connective tissue, neurons, and supporting cells. In the lateral wall of the cochlea, these cells frequently lie close to blood vessels as perivascular macrophages. Macrophages are also shown to be recruited from blood-borne monocytes to damaged and dying hair cells induced by noise, ototoxic drugs, aging, and diphtheria toxin-induced hair cell degeneration. Precise monitoring may be crucial to avoid self-targeting. Macrophage biology has recently shown that populations of resident tissue macrophages may be fundamentally different from circulating macrophages. We removed uniquely preserved human cochleae during surgery for treating petroclival meningioma compressing the brain stem, after ethical consent. Molecular and cellular characterization using immunofluorescence with antibodies against IBA1, TUJ1, CX3CL1, and type IV collagen, and super-resolution structured illumination microscopy (SR-SIM) were made together with transmission electron microscopy. The super-resolution microscopy disclosed remarkable phenotypic variants of IBA1 cells closely associated with the spiral ganglion cells. Monitoring cells adhered to neurons with “synapse-like” specializations and protrusions. Active macrophages migrated occasionally nearby damaged hair cells. Results suggest that the human auditory nerve is under the surveillance and possible neurotrophic stimulation of a well-developed resident macrophage system. It may be alleviated by the non-myelinated nerve soma partly explaining why, in contrary to most mammals, the human’s auditory nerve is conserved following deafferentiation. It makes cochlear implantation possible, for the advantage of the profoundly deaf. The IBA1 cells may serve additional purposes such as immune modulation, waste disposal, and nerve regeneration. Their role in future stem cell-based therapy needs further exploration. PMID:29487598

Visualization of early influenza A virus trafficking in human dendritic cells using STED microscopy.

PubMed

Baharom, Faezzah; Thomas, Oliver S; Lepzien, Rico; Mellman, Ira; Chalouni, Cécile; Smed-Sörensen, Anna

2017-01-01

Influenza A viruses (IAV) primarily target respiratory epithelial cells, but can also replicate in immune cells, including human dendritic cells (DCs). Super-resolution microscopy provides a novel method of visualizing viral trafficking by overcoming the resolution limit imposed by conventional light microscopy, without the laborious sample preparation of electron microscopy. Using three-color Stimulated Emission Depletion (STED) microscopy, we visualized input IAV nucleoprotein (NP), early and late endosomal compartments (EEA1 and LAMP1 respectively), and HLA-DR (DC membrane/cytosol) by immunofluorescence in human DCs. Surface bound IAV were internalized within 5 min of infection. The association of virus particles with early endosomes peaked at 5 min when 50% of NP+ signals were also EEA1+. Peak association with late endosomes occurred at 15 min when 60% of NP+ signals were LAMP1+. At 30 min of infection, the majority of NP signals were in the nucleus. Our findings illustrate that early IAV trafficking in human DCs proceeds via the classical endocytic pathway.

Technical Review: Microscopy and Image Processing Tools to Analyze Plant Chromatin: Practical Considerations.

PubMed

Baroux, Célia; Schubert, Veit

2018-01-01

In situ nucleus and chromatin analyses rely on microscopy imaging that benefits from versatile, efficient fluorescent probes and proteins for static or live imaging. Yet the broad choice in imaging instruments offered to the user poses orientation problems. Which imaging instrument should be used for which purpose? What are the main caveats and what are the considerations to best exploit each instrument's ability to obtain informative and high-quality images? How to infer quantitative information on chromatin or nuclear organization from microscopy images? In this review, we present an overview of common, fluorescence-based microscopy systems and discuss recently developed super-resolution microscopy systems, which are able to bridge the resolution gap between common fluorescence microscopy and electron microscopy. We briefly present their basic principles and discuss their possible applications in the field, while providing experience-based recommendations to guide the user toward best-possible imaging. In addition to raw data acquisition methods, we discuss commercial and noncommercial processing tools required for optimal image presentation and signal evaluation in two and three dimensions.

Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe.

PubMed

Żurek-Biesiada, Dominika; Szczurek, Aleksander T; Prakash, Kirti; Best, Gerrit; Mohana, Giriram K; Lee, Hyun-Keun; Roignant, Jean-Yves; Dobrucki, Jurek W; Cremer, Christoph; Birk, Udo

2016-06-01

Single Molecule Localization Microscopy (SMLM) is a recently emerged optical imaging method that was shown to achieve a resolution in the order of tens of nanometers in intact cells. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of DNA and proteins, is arranged in the eukaryotic cell nucleus. Such an approach utilizing switching of a fluorescent, DNA-binding dye Vybrant® DyeCycle™ Violet has been previously demonstrated by us (Żurek-Biesiada et al., 2015) [1]. Here we provide quantitative information on the influence of the chemical environment on the behavior of the dye, discuss the variability in the DNA-associated signal density, and demonstrate direct proof of enhanced structural resolution. Furthermore, we compare different visualization approaches. Finally, we describe various opportunities of multicolor DNA/SMLM imaging in eukaryotic cell nuclei.

Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe

PubMed Central

Żurek-Biesiada, Dominika; Szczurek, Aleksander T.; Prakash, Kirti; Best, Gerrit; Mohana, Giriram K.; Lee, Hyun-Keun; Roignant, Jean-Yves; Dobrucki, Jurek W.; Cremer, Christoph; Birk, Udo

2016-01-01

Single Molecule Localization Microscopy (SMLM) is a recently emerged optical imaging method that was shown to achieve a resolution in the order of tens of nanometers in intact cells. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of DNA and proteins, is arranged in the eukaryotic cell nucleus. Such an approach utilizing switching of a fluorescent, DNA-binding dye Vybrant® DyeCycle™ Violet has been previously demonstrated by us (Żurek-Biesiada et al., 2015) [1]. Here we provide quantitative information on the influence of the chemical environment on the behavior of the dye, discuss the variability in the DNA-associated signal density, and demonstrate direct proof of enhanced structural resolution. Furthermore, we compare different visualization approaches. Finally, we describe various opportunities of multicolor DNA/SMLM imaging in eukaryotic cell nuclei. PMID:27054149

Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopy

NASA Astrophysics Data System (ADS)

Clausen, M. P.; Colin-York, H.; Schneider, F.; Eggeling, C.; Fritzsche, M.

2017-02-01

Nanoscale spacing between the plasma membrane and the underlying cortical actin cytoskeleton profoundly modulates cellular morphology, mechanics, and function. Measuring this distance has been a key challenge in cell biology. Current methods for dissecting the nanoscale spacing either limit themselves to complex survey design using fixed samples or rely on diffraction-limited fluorescence imaging whose spatial resolution is insufficient to quantify distances on the nanoscale. Using dual-color super-resolution STED (stimulated-emission-depletion) microscopy, we here overcome this challenge and accurately measure the density distribution of the cortical actin cytoskeleton and the distance between the actin cortex and the membrane in live Jurkat T-cells. We found an asymmetric cortical actin density distribution with a mean width of 230 (+105/-125) nm. The spatial distances measured between the maximum density peaks of the cortex and the membrane were bi-modally distributed with mean values of 50  ±  15 nm and 120  ±  40 nm, respectively. Taken together with the finite width of the cortex, our results suggest that in some regions the cortical actin is closer than 10 nm to the membrane and a maximum of 20 nm in others.

In Situ Molecular Imaging of the Biofilm and Its Matrix.

PubMed

Ding, Yuanzhao; Zhou, Yufan; Yao, Juan; Szymanski, Craig; Fredrickson, James; Shi, Liang; Cao, Bin; Zhu, Zihua; Yu, Xiao-Ying

2016-11-15

Molecular mapping of live biofilms at submicrometer resolution presents a grand challenge. Here, we present the first chemical mapping results of biofilm extracellular polymeric substance (EPS) in biofilms using correlative imaging between super resolution fluorescence microscopy and liquid time-of-flight secondary ion mass spectrometry (TOF-SIMS). Shewanella oneidensis is used as a model organism. Heavy metal chromate (Cr 2 O 7 2- ) anions consisting of chromium Cr(VI) was used as a model environmental stressor to treat the biofilms. Of particular interest, biologically relevant water clusters have been first observed in the biofilms. Characteristic fragments of biofilm matrix components such as proteins, polysaccharides, and lipids can be spatially imaged. Furthermore, characteristic fatty acids (e.g., palmitic acid), quinolone signal, and riboflavin fragments were found to respond after the biofilm is treated with Cr(VI), leading to biofilm dispersal. Significant changes in water clusters and quorum sensing signals indicative of intercellular communication in the aqueous environment were observed, suggesting that they might result in fatty acid synthesis and inhibition of riboflavin production. The Cr(VI) reduction seems to follow the Mtr pathway leading to Cr(III) formation. Our approach potentially opens a new avenue for mechanistic insight of microbial community processes and communications using in situ imaging mass spectrometry and super resolution optical microscopy.

Aptamer-recognized carbohydrates on the cell membrane revealed by super-resolution microscopy.

PubMed

Jing, Yingying; Cai, Mingjun; Xu, Haijiao; Zhou, Lulu; Yan, Qiuyan; Gao, Jing; Wang, Hongda

2018-04-26

Carbohydrates are one of the most important components on the cell membrane, which participate in various physiological activities, and their aberrant expression is a consequence of pathological changes. In previous studies, carbohydrate analysis basically relied on lectins. However, discrimination between lectins still exists due to their multivalent character. Furthermore, the structures obtained by carbohydrate-lectin crosslinking confuse our direct observation to some extent. Fortunately, the emergence of aptamers, which are smaller and more flexible, has provided us an unprecedented choice. Herein, an aptamer recognition method with high precise localization was developed for imaging membrane-bound N-acetylgalactosamine (GalNAc). By using direct stochastic optical reconstruction microscopy (dSTORM), we compared this aptamer recognition method with the lectin recognition method for visualizing the detailed structure of GalNAc at the nanometer scale. The results indicated that GalNAc forms irregular clusters on the cell membrane with a resolution of 23 ± 7 nm by aptamer recognition. Additionally, when treated with N-acetylgalactosidase, the aptamer-recognized GalNAc shows a more significant decrease in cluster size and localization density, thus verifying better specificity of aptamers than lectins. Collectively, our study suggests that aptamers can act as perfect substitutes for lectins in carbohydrate labeling, which will be of great potential value in the field of super-resolution fluorescence imaging.

Holographic pixel super-resolution in portable lensless on-chip microscopy using a fiber-optic array.

PubMed

Bishara, Waheb; Sikora, Uzair; Mudanyali, Onur; Su, Ting-Wei; Yaglidere, Oguzhan; Luckhart, Shirley; Ozcan, Aydogan

2011-04-07

We report a portable lensless on-chip microscope that can achieve <1 µm resolution over a wide field-of-view of ∼ 24 mm(2) without the use of any mechanical scanning. This compact on-chip microscope weighs ∼ 95 g and is based on partially coherent digital in-line holography. Multiple fiber-optic waveguides are butt-coupled to light emitting diodes, which are controlled by a low-cost micro-controller to sequentially illuminate the sample. The resulting lensfree holograms are then captured by a digital sensor-array and are rapidly processed using a pixel super-resolution algorithm to generate much higher resolution holographic images (both phase and amplitude) of the objects. This wide-field and high-resolution on-chip microscope, being compact and light-weight, would be important for global health problems such as diagnosis of infectious diseases in remote locations. Toward this end, we validate the performance of this field-portable microscope by imaging human malaria parasites (Plasmodium falciparum) in thin blood smears. Our results constitute the first-time that a lensfree on-chip microscope has successfully imaged malaria parasites.

SuperSegger: robust image segmentation, analysis and lineage tracking of bacterial cells.

PubMed

Stylianidou, Stella; Brennan, Connor; Nissen, Silas B; Kuwada, Nathan J; Wiggins, Paul A

2016-11-01

Many quantitative cell biology questions require fast yet reliable automated image segmentation to identify and link cells from frame-to-frame, and characterize the cell morphology and fluorescence. We present SuperSegger, an automated MATLAB-based image processing package well-suited to quantitative analysis of high-throughput live-cell fluorescence microscopy of bacterial cells. SuperSegger incorporates machine-learning algorithms to optimize cellular boundaries and automated error resolution to reliably link cells from frame-to-frame. Unlike existing packages, it can reliably segment microcolonies with many cells, facilitating the analysis of cell-cycle dynamics in bacteria as well as cell-contact mediated phenomena. This package has a range of built-in capabilities for characterizing bacterial cells, including the identification of cell division events, mother, daughter and neighbouring cells, and computing statistics on cellular fluorescence, the location and intensity of fluorescent foci. SuperSegger provides a variety of postprocessing data visualization tools for single cell and population level analysis, such as histograms, kymographs, frame mosaics, movies and consensus images. Finally, we demonstrate the power of the package by analyzing lag phase growth with single cell resolution. © 2016 John Wiley & Sons Ltd.

Aberrations in stimulated emission depletion (STED) microscopy

NASA Astrophysics Data System (ADS)

Antonello, Jacopo; Burke, Daniel; Booth, Martin J.

2017-12-01

Like all methods of super-resolution microscopy, stimulated emission depletion (STED) microscopy can suffer from the effects of aberrations. The most important aspect of a STED microscope is that the depletion focus maintains a minimum, ideally zero, intensity point that is surrounded by a region of higher intensity. It follows that aberrations that cause a non-zero value of this minimum intensity are the most detrimental, as they inhibit fluorescence emission even at the centre of the depletion focus. We present analysis that elucidates the nature of these effects in terms of the different polarisation components at the focus for two-dimensional and three-dimensional STED resolution enhancement. It is found that only certain low-order aberration modes can affect the minimum intensity at the Gaussian focus. This has important consequences for the design of adaptive optics aberration correction systems.

Auto-calibrated scanning-angle prism-type total internal reflection microscopy for nanometer-precision axial position determination and optional variable-illumination-depth pseudo total internal reflection microscopy

DOEpatents

Fang, Ning; Sun, Wei

2015-04-21

A method, apparatus, and system for improved VA-TIRFM microscopy. The method comprises automatically controlled calibration of one or more laser sources by precise control of presentation of each laser relative a sample for small incremental changes of incident angle over a range of critical TIR angles. The calibration then allows precise scanning of the sample for any of those calibrated angles for higher and more accurate resolution, and better reconstruction of the scans for super resolution reconstruction of the sample. Optionally the system can be controlled for incident angles of the excitation laser at sub-critical angles for pseudo TIRFM. Optionally both above-critical angle and sub critical angle measurements can be accomplished with the same system.

The ultrastructure of mono- and holocentric plant centromeres: an immunological investigation by structured illumination microscopy and scanning electron microscopy.

PubMed

Wanner, Gerhard; Schroeder-Reiter, Elizabeth; Ma, Wei; Houben, Andreas; Schubert, Veit

2015-12-01

The spatial distribution of the three centromere-associated proteins α-tubulin, CENH3, and phosphorylated histone H2A (at threonine 120, H2AThr120ph) was analysed by indirect immunodetection at monocentric cereal chromosomes and at the holocentric chromosomes of Luzula elegans by super-resolution light microscopy and scanning electron microscopy (SEM). Using structured illumination microscopy (SIM) as the super-resolution technique on squashed specimens and SEM on uncoated isolated specimens, the three-dimensional (3D) distribution of the proteins was visualized at the centromeres. Technical aspects of 3D SEM are explained in detail. We show that CENH3 forms curved "pads" mainly around the lateral centromeric region in the primary constriction of metacentric chromosomes. H2AThr120ph is present in both the primary constriction and in the pericentromere. α-tubulin-labeled microtubule bundles attach to CENH3-containing chromatin structures, either in single bundles with a V-shaped attachment to the centromere or in split bundles to bordering pericentromeric flanks. In holocentric L. elegans chromosomes, H2AThr120ph is located predominantly in the centromeric groove of each chromatid as proven by subsequent FIB/FESEM ablation and 3D reconstruction. α-tubulin localizes to the edges of the groove. In both holocentric and monocentric chromosomes, no additional intermediate structures between microtubules and the centromere were observed. We established models of the distribution of CENH3, H2AThr120ph and the attachment sites of microtubules for metacentric and holocentric plant chromosomes.

Periodic diffraction correlation imaging without a beam-splitter.

PubMed

Li, Hu; Chen, Zhipeng; Xiong, Jin; Zeng, Guihua

2012-01-30

In this paper, we proposed and demonstrated a new correlation imaging mechanism based on the periodic diffraction effect. In this effect, a periodic intensity pattern is generated at the output surface of a periodic point source array. This novel correlation imaging mechanism can realize super-resolution imaging, Nth-order ghost imaging without a beam-splitter and correlation microscopy.

Aberration control in 4Pi nanoscopy: definitions, properties, and applications (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hao, Xiang; Allgeyer, Edward S.; Velasco, Mary Grace M.; Booth, Martin J.; Bewersdorf, Joerg

2016-03-01

The development of fluorescence microscopy, which allows live-cell imaging with high labeling specificity, has made the visualization of cellular architecture routine. However, for centuries, the spatial resolution of optical microscopy was fundamentally limited by diffraction. The past two decades have seen a revolution in far-field optical nanoscopy (or "super-resolution" microscopy). The best 3D resolution is achieved by optical nanoscopes like the isoSTED or the iPALM/4Pi-SMS, which utilize two opposing objective lenses in a coherent manner. These system are, however, also more complex and the required interference conditions demand precise aberration control. Our research involves developing novel adaptive optics techniques that enable high spatial and temporal resolution imaging for biological applications. In this talk, we will discuss how adaptive optics can enhance dual-objective lens nanoscopes. We will demonstrate how adaptive optics devices provide unprecedented freedom to manipulate the light field in isoSTED nanoscopy, allow to realize automatic beam alignment, suppress the inherent side-lobes of the point-spread function, and dynamically compensate for sample-induced aberrations. We will present both the theoretical groundwork and the experimental confirmations.

Role of coherence in microsphere-assisted nanoscopy

NASA Astrophysics Data System (ADS)

Perrin, Stephane; Lecler, Sylvain; Leong-Hoi, Audrey; Montgomery, Paul C.

2017-06-01

The loss of the information, due to the diffraction and the evanescent waves, limits the resolving power of classical optical microscopy. In air, the lateral resolution of an optical microscope can approximated at half of the wavelength using a low-coherence illumination. Recently, several methods have been developed in order to overcome this limitation and, in 2011, a new far-field and full-field imaging technique was proposed where a sub-diffraction-limit resolution has been achieved using a transparent microsphere. In this article, the phenomenon of super-resolution using microsphere-assisted microscopy is analysed through rigorous electro-magnetic simulations. The performances of the imaging technique are estimated as function of optical and geometrical parameters. Furthermore, the role of coherence is introduced through the temporal coherence of the light source and the phase response of the object.

Analysing intracellular deformation of polymer capsules using structured illumination microscopy

NASA Astrophysics Data System (ADS)

Chen, Xi; Cui, Jiwei; Sun, Huanli; Müllner, Markus; Yan, Yan; Noi, Ka Fung; Ping, Yuan; Caruso, Frank

2016-06-01

Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties.Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties. Electronic supplementary information (ESI) available: Additional figures. See DOI: 10.1039/c6nr02151d

Portable microscopy platform for the clinical and environmental monitoring

NASA Astrophysics Data System (ADS)

Wang, Weiming; Yu, Yan; Huang, Hui; Ou, Jinping

2016-04-01

Light microscopy can not only address various diagnosis needs such as aquatic parasites and bacteria such as E. coli in water, but also provide a method for the screening of red tide. Traditional microscope based on the smartphone created by adding lens couldn't keep the tradeoff between field-of-view(FOV) and the resolution. In this paper, we demonstrate a non-contact, light and cost-effective microscope platform, that can image highly dense samples with a spatial resolution of ~0.8um over a field-of-view(FOV) of >1mm2. After captured the direct images, we performed the pixel super-resolution algorithm to improve the image resolution and overcome the hardware interference. The system would be a good point-of-care diagnostic solution in resource limited settings. We validated the performance of the system by imaging resolution test targets, the squamous cell cancer(SqCC) and green algae that necessary to detect the squamous carcinoma and red tide

A STED-FLIM microscope applied to imaging the natural killer cell immune synapse

NASA Astrophysics Data System (ADS)

Lenz, M. O.; Brown, A. C. N.; Auksorius, E.; Davis, D. M.; Dunsby, C.; Neil, M. A. A.; French, P. M. W.

2011-03-01

We present a stimulated emission depletion (STED) fluorescence lifetime imaging (FLIM) microscope, excited by a microstructured optical fibre supercontinuum source that is pumped by a femtosecond Ti:Sapphire-laser, which is also used for depletion. Implemented using a piezo-scanning stage on a laser scanning confocal fluorescence microscope system with FLIM realised using time correlated single photon counting (TCSPC), this provides convenient switching between confocal and STED-FLIM with spatial resolution down to below 60 nm. We will present our design considerations to make a robust instrument for biological applications including a comparison between fixed phase plate and spatial light modulator (SLM) approaches to shape the STED beam and the correlation of STED and confocal FLIM microscopy. Following our previous application of FLIM-FRET to study intercellular signalling at the immunological synapse (IS), we are employing STED microscopy to characterize the spatial distribution of cellular molecules with subdiffraction resolution at the IS. In particular, we are imaging cytoskeletal structure at the Natural Killer cell activated immune synapse. We will also present our progress towards multilabel STED microscopy to determine how relative spatial molecular organization, previously undetectable by conventional microscopy techniques, is important for NK cell cytotoxic function. Keywords: STED, Stimulated Emission Depletion Microscopy, Natural Killer (NK) cell, Fluorescence lifetime imaging, FLIM, Super resolution microscopy.

Concepts in Light Microscopy of Viruses

PubMed Central

Witte, Robert; Georgi, Fanny

2018-01-01

Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research. PMID:29670029

Concepts in Light Microscopy of Viruses.

PubMed

Witte, Robert; Andriasyan, Vardan; Georgi, Fanny; Yakimovich, Artur; Greber, Urs F

2018-04-18

Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research.

Super-Resolution Imaging Reveals TCTN2 Depletion-Induced IFT88 Lumen Leakage and Ciliary Weakening.

PubMed

Weng, Rueyhung Roc; Yang, T Tony; Huang, Chia-En; Chang, Chih-Wei; Wang, Won-Jing; Liao, Jung-Chi

2018-06-01

The primary cilium is an essential organelle mediating key signaling activities, such as sonic hedgehog signaling. The molecular composition of the ciliary compartment is distinct from that of the cytosol, with the transition zone (TZ) gated the ciliary base. The TZ is a packed and organized protein complex containing multiple ciliopathy-associated protein species. Tectonic 2 (TCTN2) is one of the TZ proteins in the vicinity of the ciliary membrane, and its mutation is associated with Meckel syndrome. Despite its importance in ciliopathies, the role of TCTN2 in ciliary structure and molecules remains unclear. Here, we created a CRISPR/Cas9 TCTN2 knockout human retinal pigment epithelial cell line and conducted quantitative analysis of geometric localization using both wide-field and super-resolution microscopy techniques. We found that TCTN2 depletion resulted in partial TZ damage, loss of ciliary membrane proteins, leakage of intraflagellar transport protein IFT88 toward the basal body lumen, and cilium shortening and curving. The basal body lumen occupancy of IFT88 was also observed in si-RPGRIP1L cells and cytochalasin-D-treated wild-type cells, suggesting varying lumen accessibility for intraflagellar transport proteins under different perturbed conditions. Our findings support two possible models for the lumen leakage of IFT88, i.e., a tip leakage model and a misregulation model. Together, our quantitative image analysis augmented by super-resolution microscopy facilitates the observation of structural destruction and molecular redistribution in TCTN2 -/- cilia, shedding light on mechanistic understanding of TZ-protein-associated ciliopathies. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Giga-pixel lensfree holographic microscopy and tomography using color image sensors.

PubMed

Isikman, Serhan O; Greenbaum, Alon; Luo, Wei; Coskun, Ahmet F; Ozcan, Aydogan

2012-01-01

We report Giga-pixel lensfree holographic microscopy and tomography using color sensor-arrays such as CMOS imagers that exhibit Bayer color filter patterns. Without physically removing these color filters coated on the sensor chip, we synthesize pixel super-resolved lensfree holograms, which are then reconstructed to achieve ~350 nm lateral resolution, corresponding to a numerical aperture of ~0.8, across a field-of-view of ~20.5 mm(2). This constitutes a digital image with ~0.7 Billion effective pixels in both amplitude and phase channels (i.e., ~1.4 Giga-pixels total). Furthermore, by changing the illumination angle (e.g., ± 50°) and scanning a partially-coherent light source across two orthogonal axes, super-resolved images of the same specimen from different viewing angles are created, which are then digitally combined to synthesize tomographic images of the object. Using this dual-axis lensfree tomographic imager running on a color sensor-chip, we achieve a 3D spatial resolution of ~0.35 µm × 0.35 µm × ~2 µm, in x, y and z, respectively, creating an effective voxel size of ~0.03 µm(3) across a sample volume of ~5 mm(3), which is equivalent to >150 Billion voxels. We demonstrate the proof-of-concept of this lensfree optical tomographic microscopy platform on a color CMOS image sensor by creating tomograms of micro-particles as well as a wild-type C. elegans nematode.

Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy

PubMed Central

Nahidiazar, Leila; Agronskaia, Alexandra V.; Broertjes, Jorrit; van den Broek, Bram; Jalink, Kees

2016-01-01

Single Molecule Localization super-resolution Microscopy (SMLM) has become a powerful tool to study cellular architecture at the nanometer scale. In SMLM, single fluorophore labels are made to repeatedly switch on and off (“blink”), and their exact locations are determined by mathematically finding the centers of individual blinks. The image quality obtainable by SMLM critically depends on efficacy of blinking (brightness, fraction of molecules in the on-state) and on preparation longevity and labeling density. Recent work has identified several combinations of bright dyes and imaging buffers that work well together. Unfortunately, different dyes blink optimally in different imaging buffers, and acquisition of good quality 2- and 3-color images has therefore remained challenging. In this study we describe a new imaging buffer, OxEA, that supports 3-color imaging of the popular Alexa dyes. We also describe incremental improvements in preparation technique that significantly decrease lateral- and axial drift, as well as increase preparation longevity. We show that these improvements allow us to collect very large series of images from the same cell, enabling image stitching, extended 3D imaging as well as multi-color recording. PMID:27391487

Optimized two-color super resolution imaging of Drp1 during mitochondrial fission with a slow-switching Dronpa variant.

PubMed

Rosenbloom, Alyssa B; Lee, Sang-Hyuk; To, Milton; Lee, Antony; Shin, Jae Yen; Bustamante, Carlos

2014-09-09

We studied the single-molecule photo-switching properties of Dronpa, a green photo-switchable fluorescent protein and a popular marker for photoactivated localization microscopy. We found the excitation light photoactivates as well as deactivates Dronpa single molecules, hindering temporal separation and limiting super resolution. To resolve this limitation, we have developed a slow-switching Dronpa variant, rsKame, featuring a V157L amino acid substitution proximal to the chromophore. The increased steric hindrance generated by the substitution reduced the excitation light-induced photoactivation from the dark to fluorescent state. To demonstrate applicability, we paired rsKame with PAmCherry1 in a two-color photoactivated localization microscopy imaging method to observe the inner and outer mitochondrial membrane structures and selectively labeled dynamin related protein 1 (Drp1), responsible for membrane scission during mitochondrial fission. We determined the diameter and length of Drp1 helical rings encircling mitochondria during fission and showed that, whereas their lengths along mitochondria were not significantly changed, their diameters decreased significantly. These results suggest support for the twistase model of Drp1 constriction, with potential loss of subunits at the helical ends.

Analytical SuperSTEM for extraterrestrial materials research

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

Bradley, J P; Dai, Z R

2009-09-08

Electron-beam studies of extraterrestrial materials with significantly improved spatial resolution, energy resolution and sensitivity are enabled using a 300 keV SuperSTEM scanning transmission electron microscope with a monochromator and two spherical aberration correctors. The improved technical capabilities enable analyses previously not possible. Mineral structures can be directly imaged and analyzed with single-atomic-column resolution, liquids and implanted gases can be detected, and UV-VIS optical properties can be measured. Detection limits for minor/trace elements in thin (<100 nm thick) specimens are improved such that quantitative measurements of some extend to the sub-500 ppm level. Electron energy-loss spectroscopy (EELS) can be carried outmore » with 0.10-0.20 eV energy resolution and atomic-scale spatial resolution such that variations in oxidation state from one atomic column to another can be detected. Petrographic mapping is extended down to the atomic scale using energy-dispersive x-ray spectroscopy (EDS) and energy-filtered transmission electron microscopy (EFTEM) imaging. Technical capabilities and examples of the applications of SuperSTEM to extraterrestrial materials are presented, including the UV spectral properties and organic carbon K-edge fine structure of carbonaceous matter in interplanetary dust particles (IDPs), x-ray elemental maps showing the nanometer-scale distribution of carbon within GEMS (glass with embedded metal and sulfides), the first detection and quantification of trace Ti in GEMS using EDS, and detection of molecular H{sub 2}O in vesicles and implanted H{sub 2} and He in irradiated mineral and glass grains.« less

Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy

NASA Astrophysics Data System (ADS)

Liu, Yujia; Lu, Yiqing; Yang, Xusan; Zheng, Xianlin; Wen, Shihui; Wang, Fan; Vidal, Xavier; Zhao, Jiangbo; Liu, Deming; Zhou, Zhiguang; Ma, Chenshuo; Zhou, Jiajia; Piper, James A.; Xi, Peng; Jin, Dayong

2017-02-01

Lanthanide-doped glasses and crystals are attractive for laser applications because the metastable energy levels of the trivalent lanthanide ions facilitate the establishment of population inversion and amplified stimulated emission at relatively low pump power. At the nanometre scale, lanthanide-doped upconversion nanoparticles (UCNPs) can now be made with precisely controlled phase, dimension and doping level. When excited in the near-infrared, these UCNPs emit stable, bright visible luminescence at a variety of selectable wavelengths, with single-nanoparticle sensitivity, which makes them suitable for advanced luminescence microscopy applications. Here we show that UCNPs doped with high concentrations of thulium ions (Tm3+), excited at a wavelength of 980 nanometres, can readily establish a population inversion on their intermediate metastable 3H4 level: the reduced inter-emitter distance at high Tm3+ doping concentration leads to intense cross-relaxation, inducing a photon-avalanche-like effect that rapidly populates the metastable 3H4 level, resulting in population inversion relative to the 3H6 ground level within a single nanoparticle. As a result, illumination by a laser at 808 nanometres, matching the upconversion band of the 3H4 → 3H6 transition, can trigger amplified stimulated emission to discharge the 3H4 intermediate level, so that the upconversion pathway to generate blue luminescence can be optically inhibited. We harness these properties to realize low-power super-resolution stimulated emission depletion (STED) microscopy and achieve nanometre-scale optical resolution (nanoscopy), imaging single UCNPs; the resolution is 28 nanometres, that is, 1/36th of the wavelength. These engineered nanocrystals offer saturation intensity two orders of magnitude lower than those of fluorescent probes currently employed in stimulated emission depletion microscopy, suggesting a new way of alleviating the square-root law that typically limits the resolution that can be practically achieved by such techniques.

Radial super-resolution in digital holographic microscopy using structured illumination with circular symmetry

NASA Astrophysics Data System (ADS)

Yin, Yujian; Su, Ping; Ma, Jianshe

2018-01-01

A method to improve the radial resolution using special structured light is proposed in the field of digital holographic microscopy (DHM). A specimen is illuminated with circular symmetrical structured light that makes the spectrum have radial movement, so that high frequency components of the specimen are moved into the passband of the receiver to overcome the diffraction limit. In the DHM imaging system, Computer Generated Hologram (CGH) technology is used to generate the required structured light grating. Then the grating is loaded into a spatial light modulator (SLM) to obtain specific structured illumination. After recording the hologram, digital reconstruction, for the microstructure of a binary optical element that needs to observe radial distribution, the radial resolution of the specimen is improved experimentally compare it with the result of one-dimensional sinusoidal structured light imaging. And a method of designing structured light is presented.

Malic Acid Carbon Dots: From Super-resolution Live-Cell Imaging to Highly Efficient Separation.

PubMed

Zhi, Bo; Cui, Yi; Wang, Shengyang; Frank, Benjamin P; Williams, Denise N; Brown, Richard P; Melby, Eric S; Hamers, Robert J; Rosenzweig, Zeev; Fairbrother, D Howard; Orr, Galya; Haynes, Christy L

2018-06-15

As-synthesized malic acid carbon dots are found to possess photoblinking properties that are outstanding and superior compared to those of conventional dyes. Considering their excellent biocompatibility, malic acid carbon dots are suitable for super-resolution fluorescence localization microscopy under a variety of conditions, as we demonstrate in fixed and live trout gill epithelial cells. In addition, during imaging experiments, the so-called "excitation wavelength-dependent" emission was not observed for individual as-made malic acid carbon dots, which motivated us to develop a time-saving and high-throughput separation technique to isolate malic acid carbon dots into fractions of different particle size distributions using C 18 reversed-phase silica gel column chromatography. This post-treatment allowed us to determine how particle size distribution influences the optical properties of malic acid carbon dot fractions, that is, optical band gap energies and photoluminescence behaviors.

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

PubMed Central

von Diezmann, Alex; Shechtman, Yoav; Moerner, W. E.

2017-01-01

Single-molecule super-resolution fluorescence microscopy and single-particle tracking are two imaging modalities that illuminate the properties of cells and materials on spatial scales down to tens of nanometers, or with dynamical information about nanoscale particle motion in the millisecond range, respectively. These methods generally use wide-field microscopes and two-dimensional camera detectors to localize molecules to much higher precision than the diffraction limit. Given the limited total photons available from each single-molecule label, both modalities require careful mathematical analysis and image processing. Much more information can be obtained about the system under study by extending to three-dimensional (3D) single-molecule localization: without this capability, visualization of structures or motions extending in the axial direction can easily be missed or confused, compromising scientific understanding. A variety of methods for obtaining both 3D super-resolution images and 3D tracking information have been devised, each with their own strengths and weaknesses. These include imaging of multiple focal planes, point-spread-function engineering, and interferometric detection. These methods may be compared based on their ability to provide accurate and precise position information of single-molecule emitters with limited photons. To successfully apply and further develop these methods, it is essential to consider many practical concerns, including the effects of optical aberrations, field-dependence in the imaging system, fluorophore labeling density, and registration between different color channels. Selected examples of 3D super-resolution imaging and tracking are described for illustration from a variety of biological contexts and with a variety of methods, demonstrating the power of 3D localization for understanding complex systems. PMID:28151646

Modeling super-resolution SERS using a T-matrix method to elucidate molecule-nanoparticle coupling and the origins of localization errors

NASA Astrophysics Data System (ADS)

Heaps, Charles W.; Schatz, George C.

2017-06-01

A computational method to model diffraction-limited images from super-resolution surface-enhanced Raman scattering microscopy is introduced. Despite significant experimental progress in plasmon-based super-resolution imaging, theoretical predictions of the diffraction limited images remain a challenge. The method is used to calculate localization errors and image intensities for a single spherical gold nanoparticle-molecule system. The light scattering is calculated using a modification of generalized Mie (T-matrix) theory with a point dipole source and diffraction limited images are calculated using vectorial diffraction theory. The calculation produces the multipole expansion for each emitter and the coherent superposition of all fields. Imaging the constituent fields in addition to the total field provides new insight into the strong coupling between the molecule and the nanoparticle. Regardless of whether the molecular dipole moment is oriented parallel or perpendicular to the nanoparticle surface, the anisotropic excitation distorts the center of the nanoparticle as measured by the point spread function by approximately fifty percent of the particle radius toward to the molecule. Inspection of the nanoparticle multipoles reveals that distortion arises from a weak quadrupole resonance interfering with the dipole field in the nanoparticle. When the nanoparticle-molecule fields are in-phase, the distorted nanoparticle field dominates the observed image. When out-of-phase, the nanoparticle and molecule are of comparable intensity and interference between the two emitters dominates the observed image. The method is also applied to different wavelengths and particle radii. At off-resonant wavelengths, the method predicts images closer to the molecule not because of relative intensities but because of greater distortion in the nanoparticle. The method is a promising approach to improving the understanding of plasmon-enhanced super-resolution experiments.

Diverse Protocols for Correlative Super-Resolution Fluorescence Imaging and Electron Microscopy of Cells and Tissue

DTIC Science & Technology

2016-05-25

tissue is critical to biology. Many factors determine optimal experimental design, including attainable localization precision, ultrastructural...both imaging modalities. Examples include: weak tissue preservation protocols resulting in poor ultrastructure, e.g. mitochondrial cristae membranes...tension effects during sample drying that may result in artifacts44. Samples dried in the presence of polyvinyl alcohol do not have the haziness

The Histochemistry and Cell Biology pandect: the year 2014 in review.

PubMed

Taatjes, Douglas J; Roth, Jürgen

2015-04-01

This review encompasses a brief synopsis of the articles published in 2014 in Histochemistry and Cell Biology. Out of the total of 12 issues published in 2014, two special issues were devoted to "Single-Molecule Super-Resolution Microscopy." The present review is divided into 11 categories, providing an easy format for readers to quickly peruse topics of particular interest to them.

Optical sectioning using a digital Fresnel incoherent-holography-based confocal imaging system

PubMed Central

Kelner, Roy; Katz, Barak; Rosen, Joseph

2015-01-01

We propose a new type of confocal microscope using Fresnel incoherent correlation holography (FINCH). Presented here is a confocal configuration of FINCH using a phase pinhole and point illumination that is able to suppress out-of-focus information from the recorded hologram and hence combine the super-resolution capabilities of FINCH with the sectioning capabilities of confocal microscopy. PMID:26413560

Contrast enhancement of biological nanoporous materials with zinc oxide infiltration for electron and X-ray nanoscale microscopy

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

Ocola, L. E.; Sampathkumar, V.; Kasthuri, N.

Here, we show that using infiltration of ZnO metal oxide can be useful for high resolution imaging of biological samples in electron and X-ray microscopy. This method is compatible with standard fixation techniques that leave the sample dry, such as finishing with super critical CO 2 drying, or simple vacuum drying at 95°C. We demonstrate this technique can be applied on tooth and brain tissue samples. We also show that high resolution X-ray tomography can be performed on biological systems using Zn K edge (1s) absorption to enhance internal structures, and obtained the first nanoscale 10 KeV X-ray absorption imagesmore » of the interior regions of a tooth.« less

Contrast enhancement of biological nanoporous materials with zinc oxide infiltration for electron and X-ray nanoscale microscopy

DOE PAGES

Ocola, L. E.; Sampathkumar, V.; Kasthuri, N.; ...

2017-07-19

Here, we show that using infiltration of ZnO metal oxide can be useful for high resolution imaging of biological samples in electron and X-ray microscopy. This method is compatible with standard fixation techniques that leave the sample dry, such as finishing with super critical CO 2 drying, or simple vacuum drying at 95°C. We demonstrate this technique can be applied on tooth and brain tissue samples. We also show that high resolution X-ray tomography can be performed on biological systems using Zn K edge (1s) absorption to enhance internal structures, and obtained the first nanoscale 10 KeV X-ray absorption imagesmore » of the interior regions of a tooth.« less

Microscopy illumination engineering using a low-cost liquid crystal display.

PubMed

Guo, Kaikai; Bian, Zichao; Dong, Siyuan; Nanda, Pariksheet; Wang, Ying Min; Zheng, Guoan

2015-02-01

Illumination engineering is critical for obtaining high-resolution, high-quality images in microscope settings. In a typical microscope, the condenser lens provides sample illumination that is uniform and free from glare. The associated condenser diaphragm can be manually adjusted to obtain the optimal illumination numerical aperture. In this paper, we report a programmable condenser lens for active illumination control. In our prototype setup, we used a $15 liquid crystal display as a transparent spatial light modulator and placed it at the back focal plane of the condenser lens. By setting different binary patterns on the display, we can actively control the illumination and the spatial coherence of the microscope platform. We demonstrated the use of such a simple scheme for multimodal imaging, including bright-field microscopy, darkfield microscopy, phase-contrast microscopy, polarization microscopy, 3D tomographic imaging, and super-resolution Fourier ptychographic imaging. The reported illumination engineering scheme is cost-effective and compatible with most existing platforms. It enables a turnkey solution with high flexibility for researchers in various communities. From the engineering point-of-view, the reported illumination scheme may also provide new insights for the development of multimodal microscopy and Fourier ptychographic imaging.

Special issue on high-resolution optical imaging

NASA Astrophysics Data System (ADS)

Smith, Peter J. S.; Davis, Ilan; Galbraith, Catherine G.; Stemmer, Andreas

2013-09-01

The pace of development in the field of advanced microscopy is truly breath-taking, and is leading to major breakthroughs in our understanding of molecular machines and cell function. This special issue of Journal of Optics draws attention to a number of interesting approaches, ranging from fluorescence and imaging of unlabelled cells, to computational methods, all of which are describing the ever increasing detail of the dynamic behaviour of molecules in the living cell. This is a field which traditionally, and currently, demonstrates a marvellous interplay between the disciplines of physics, chemistry and biology, where apparent boundaries to resolution dissolve and living cells are viewed in ever more clarity. It is fertile ground for those interested in optics and non-conventional imaging to contribute high-impact outputs in the fields of cell biology and biomedicine. The series of articles presented here has been selected to demonstrate this interdisciplinarity and to encourage all those with a background in the physical sciences to 'dip their toes' into the exciting and dynamic discoveries surrounding cell function. Although single molecule super-resolution microscopy is commercially available, specimen preparation and interpretation of single molecule data remain a major challenge for scientists wanting to adopt the techniques. The paper by Allen and Davidson [1] provides a much needed detailed introduction to the practical aspects of stochastic optical reconstruction microscopy, including sample preparation, image acquisition and image analysis, as well as a brief description of the different variants of single molecule localization microscopy. Since super-resolution microscopy is no longer restricted to three-dimensional imaging of fixed samples, the review by Fiolka [2] is a timely introduction to techniques that have been successfully applied to four-dimensional live cell super-resolution microscopy. The combination of multiple high-resolution techniques, such as the combination of light sheet and structured illumination microscopy (SIM), which efficiently utilize photon budget and avoid illuminating regions of the specimen not currently being imaged, hold the greatest promise for future biological applications. Therefore, the combined setup for SIM and single molecule localization microscopy (SMLM) described by Rossberger et al [3] will be very helpful and stimulating to advanced microscopists in further modifying their setups. The SIM image helps in identifying artefacts in SMLM reconstruction, e.g. when two active fluorophores are close together and get rejected as 'out-of-focus'. This combined setup is another way to facilitate imaging live samples. The article by Thomas et al [4] presents another advance for biological super-resolution imaging with a new approach to reconstruct optically sectioned images using structured illumination. The method produces images with higher spatial resolution and greater signal to noise compared to existing approaches. This algorithm demonstrates great promise for reconstructing biological images where the signal intensities are inherently lower. Shevchuk et al [5] present a non-optic near field approach to imaging with a review of scanning ion-conductance microscopy. This is a powerful alternative approach for examining the surface dynamics of living cells including exo and endocytosis, unlabelled, and at the level of the single event. Here they present the first data on combining this approach with fluorescence confocal microscopy—adding that extra dimension. Different approaches to label-free live cell imaging are presented in the papers by Patel et al [6], Mehta and Oldenbourg [7], as well as Rogers and Zheludev [8]. All three papers bring home the excitement of looking at live cell dynamics without reporters—Patel et al [6] review both the potential of coherent anti-Stokes Raman scattering and biological applications, where specific biomolecules are detected on the basis of their biophysical properties. Polarized light microscopy as presented by Mehta and Oldenbourg [7], describe a novel implementation of this technology to detect dichroism, and demonstrate beautifully its use in imaging unlabelled microtubules, mitochondria and lipid droplets. Sub-wavelength light focusing provides another avenue to super-resolution, and this is presented by Rogers and Zheludev [8]. Speculating on further improvements, these authors expect a resolution of 0.15λ. To date, the method has not been applied to low contrast, squishy and motile biotargets, but is included here for the clear potential to drive label-free imaging in new directions. A similar logic lies behind the inclusion of Parsons et al [9] where ultraviolet coherent diffractive imaging is further developed. These authors have demonstrated a shrink-wrap technique which reduces the integration time by a factor of 5, bringing closer the time when we have lab based imaging systems based on extreme ultraviolet and soft x-ray sources using sophisticated phase retrieval algorithms. Real biological specimens have spatially varying refractive indices that inevitably lead to aberrations and image distortions. Global refractive index matching of the embedding medium has been an historic solution, but unfortunately is not practical for live cell imaging. Adaptive optics appears an attractive solution and Simmonds and Booth [10] demonstrate the theoretical benefits of applying several adaptive optical elements, placed in different conjugate planes, to create a kind of 'inverse specimen' that unwarps phase distortions of the sample—but these have yet to be tested on real specimens. A difficulty in single molecule localization microscopy has been the determination of whether or not two molecules are colocalized. Kim et al [11] present a method for correcting bleed-through during multi-colour, single molecule localization microscopy. Such methods are welcome standards when trying to quantifiably interpret how close two molecules actually are. Rees et al [12] provide an invaluable overview of key image processing steps in localization microscopy. This paper is an excellent starting point for anyone implementing localization algorithms and the Matlab software provided will be invaluable; a strong paper on which to conclude our overview of the excellent articles brought together in this issue. One aspect brought home in several of these articles is the volume of data now being collected by high resolution live cell imaging. Data processing and image reconstruction will continue to be pressure points in the further development of instrumentation and analyses. We would hope that the series of papers presented here will motivate software engineers, optical physicists and biologists to contribute to the further development of this exciting field. References [1] Allen J R et al 2013 J. Opt. 15 094001 [2] Fiolka R et al 2013 J. Opt. 15 094002 [3] Rossberger S et al 2013 J. Opt. 15 094003 [4] Thomas B et al 2013 J. Opt. 15 094004 [5] Shevchuk A et al 2013 J. Opt. 15 094005 [6] Patel I et al 2013 J. Opt. 15 094006 [7] Mehta S B et al 2013 J. Opt. 15 094007 [8] Rogers E T F et al 2013 J. Opt. 15 094008 [9] Parsons A D et al 2013 J. Opt. 15 094009 [10] Simmonds R et al 2013 J. Opt. 15 094010 [11] Kim D et al 2013 J. Opt. 15 094011 [12] Rees E J et al 2013 J. Opt. 15 094012

Axial Colocalization of Single Molecules with Nanometer Accuracy Using Metal-Induced Energy Transfer.

PubMed

Isbaner, Sebastian; Karedla, Narain; Kaminska, Izabela; Ruhlandt, Daja; Raab, Mario; Bohlen, Johann; Chizhik, Alexey; Gregor, Ingo; Tinnefeld, Philip; Enderlein, Jörg; Tsukanov, Roman

2018-04-11

Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups.

Peering into Cells One Molecule at a Time: Single-molecule and plasmon-enhanced fluorescence super-resolution imaging

NASA Astrophysics Data System (ADS)

Biteen, Julie

2013-03-01

Single-molecule fluorescence brings the resolution of optical microscopy down to the nanometer scale, allowing us to unlock the mysteries of how biomolecules work together to achieve the complexity that is a cell. This high-resolution, non-destructive method for examining subcellular events has opened up an exciting new frontier: the study of macromolecular localization and dynamics in living cells. We have developed methods for single-molecule investigations of live bacterial cells, and have used these techniques to investigate thee important prokaryotic systems: membrane-bound transcription activation in Vibrio cholerae, carbohydrate catabolism in Bacteroides thetaiotaomicron, and DNA mismatch repair in Bacillus subtilis. Each system presents unique challenges, and we will discuss the important methods developed for each system. Furthermore, we use the plasmon modes of bio-compatible metal nanoparticles to enhance the emissivity of single-molecule fluorophores. The resolution of single-molecule imaging in cells is generally limited to 20-40 nm, far worse than the 1.5-nm localization accuracies which have been attained in vitro. We use plasmonics to improve the brightness and stability of single-molecule probes, and in particular fluorescent proteins, which are widely used for bio-imaging. We find that gold-coupled fluorophores demonstrate brighter, longer-lived emission, yielding an overall enhancement in total photons detected. Ultimately, this results in increased localization accuracy for single-molecule imaging. Furthermore, since fluorescence intensity is proportional to local electromagnetic field intensity, these changes in decay intensity and rate serve as a nm-scale read-out of the field intensity. Our work indicates that plasmonic substrates are uniquely advantageous for super-resolution imaging, and that plasmon-enhanced imaging is a promising technique for improving live cell single-molecule microscopy.

Comparison of two structured illumination techniques based on different 3D illumination patterns

NASA Astrophysics Data System (ADS)

Shabani, H.; Patwary, N.; Doblas, A.; Saavedra, G.; Preza, C.

2017-02-01

Manipulating the excitation pattern in optical microscopy has led to several super-resolution techniques. Among different patterns, the lateral sinusoidal excitation was used for the first demonstration of structured illumination microscopy (SIM), which provides the fastest SIM acquisition system (based on the number of raw images required) compared to the multi-spot illumination approach. Moreover, 3D patterns that include lateral and axial variations in the illumination have attracted more attention recently as they address resolution enhancement in three dimensions. A threewave (3W) interference technique based on coherent illumination has already been shown to provide super-resolution and optical sectioning in 3D-SIM. In this paper, we investigate a novel tunable technique that creates a 3D pattern from a set of multiple incoherently illuminated parallel slits that act as light sources for a Fresnel biprism. This setup is able to modulate the illumination pattern in the object space both axially and laterally with adjustable modulation frequencies. The 3D forward model for the new system is developed here to consider the effect of the axial modulation due to the 3D patterned illumination. The performance of 3D-SIM based on 3W interference and the tunable system are investigated in simulation and compared based on two different criteria. First, restored images obtained for both 3D-SIM systems using a generalized Wiener filter are compared to determine the effect of the illumination pattern on the reconstruction. Second, the effective frequency response of both systems is studied to determine the axial and lateral resolution enhancement that is obtained in each case.

Image quality improvement in cone-beam CT using the super-resolution technique.

PubMed

Oyama, Asuka; Kumagai, Shinobu; Arai, Norikazu; Takata, Takeshi; Saikawa, Yusuke; Shiraishi, Kenshiro; Kobayashi, Takenori; Kotoku, Jun'ichi

2018-04-05

This study was conducted to improve cone-beam computed tomography (CBCT) image quality using the super-resolution technique, a method of inferring a high-resolution image from a low-resolution image. This technique is used with two matrices, so-called dictionaries, constructed respectively from high-resolution and low-resolution image bases. For this study, a CBCT image, as a low-resolution image, is represented as a linear combination of atoms, the image bases in the low-resolution dictionary. The corresponding super-resolution image was inferred by multiplying the coefficients and the high-resolution dictionary atoms extracted from planning CT images. To evaluate the proposed method, we computed the root mean square error (RMSE) and structural similarity (SSIM). The resulting RMSE and SSIM between the super-resolution images and the planning CT images were, respectively, as much as 0.81 and 1.29 times better than those obtained without using the super-resolution technique. We used super-resolution technique to improve the CBCT image quality.

Super-resolution imaging applied to moving object tracking

NASA Astrophysics Data System (ADS)

Swalaganata, Galandaru; Ratna Sulistyaningrum, Dwi; Setiyono, Budi

2017-10-01

Moving object tracking in a video is a method used to detect and analyze changes that occur in an object that being observed. Visual quality and the precision of the tracked target are highly wished in modern tracking system. The fact that the tracked object does not always seem clear causes the tracking result less precise. The reasons are low quality video, system noise, small object, and other factors. In order to improve the precision of the tracked object especially for small object, we propose a two step solution that integrates a super-resolution technique into tracking approach. First step is super-resolution imaging applied into frame sequences. This step was done by cropping the frame in several frame or all of frame. Second step is tracking the result of super-resolution images. Super-resolution image is a technique to obtain high-resolution images from low-resolution images. In this research single frame super-resolution technique is proposed for tracking approach. Single frame super-resolution was a kind of super-resolution that it has the advantage of fast computation time. The method used for tracking is Camshift. The advantages of Camshift was simple calculation based on HSV color that use its histogram for some condition and color of the object varies. The computational complexity and large memory requirements required for the implementation of super-resolution and tracking were reduced and the precision of the tracked target was good. Experiment showed that integrate a super-resolution imaging into tracking technique can track the object precisely with various background, shape changes of the object, and in a good light conditions.

Plasmon-Assisted Selective and Super-Resolving Excitation of Individual Quantum Emitters on a Metal Nanowire.

PubMed

Li, Qiang; Pan, Deng; Wei, Hong; Xu, Hongxing

2018-03-14

Hybrid systems composed of multiple quantum emitters coupled with plasmonic waveguides are promising building blocks for future integrated quantum nanophotonic circuits. The techniques that can super-resolve and selectively excite contiguous quantum emitters in a diffraction-limited area are of great importance for studying the plasmon-mediated interaction between quantum emitters and manipulating the single plasmon generation and propagation in plasmonic circuits. Here we show that multiple quantum dots coupled with a silver nanowire can be controllably excited by tuning the interference field of surface plasmons on the nanowire. Because of the period of the interference pattern is much smaller than the diffraction limit, we demonstrate the selective excitation of two quantum dots separated by a distance as short as 100 nm. We also numerically demonstrate a new kind of super-resolution imaging method that combines the tunable surface plasmon interference pattern on the NW with the structured illumination microscopy technique. Our work provides a novel high-resolution optical excitation and imaging method for the coupled systems of multiple quantum emitters and plasmonic waveguides, which adds a new tool for studying and manipulating single quantum emitters and single plasmons for quantum plasmonic circuitry applications.

Correlated Fluorescence-Atomic Force Microscopy Studies of the Clathrin Mediated Endocytosis in SKMEL Cells

NASA Astrophysics Data System (ADS)

Smith, Steve; Hor, Amy; Luu, Anh; Kang, Lin; Scott, Brandon; Bailey, Elizabeth; Hoppe, Adam

Clathrin-mediated endocytosis is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorescent fusion proteins (actin filaments labeled with green phalloidin-antibody and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. Results from our work are compared against dynamical polarized total internal fluorescence (TIRF), super-resolution photo-activated localization microscopy (PALM) and transmission electron microscopy (TEM) to draw conclusions regarding the prominent model of vesicle formation in clathrin-mediated endocytosis. Funding provided by NSF MPS/DMR/BMAT award # 1206908.

Labeling proteins inside living cells using external fluorophores for microscopy.

PubMed

Teng, Kai Wen; Ishitsuka, Yuji; Ren, Pin; Youn, Yeoan; Deng, Xiang; Ge, Pinghua; Lee, Sang Hak; Belmont, Andrew S; Selvin, Paul R

2016-12-09

Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.

Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging

PubMed Central

Dempsey, Graham T.; Vaughan, Joshua C.; Chen, Kok Hao; Bates, Mark; Zhuang, Xiaowei

2011-01-01

One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes — the properties of the probes, including photons per switching event, on/off duty cycle, photostability, and number of switching cycles, largely dictate the quality of super-resolution images. While many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resolution image quality has been described in only a few cases. Here, we quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images. This analysis provides a set of guidelines for characterization of super-resolution probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low crosstalk, four-color super-resolution imaging. PMID:22056676

A fluorescence in situ staining method for investigating spores and vegetative cells of Clostridia by confocal laser scanning microscopy and structured illuminated microscopy.

PubMed

D'Incecco, P; Ong, L; Gras, S; Pellegrino, L

2018-04-18

Non-pathogenic spore-forming Clostridia are of increasing interest due to their application in biogas production and their capability to spoil different food products. The life cycle for Clostridium includes a spore stage that can assist in survival under environmentally stressful conditions, such as extremes of temperature or pH. Due to their size, spores can be investigated by a range of microscopic techniques, many of which involve sample pre-treatment. We have developed a quick, simple and non-destructive fluorescent staining procedure that allows a clear differentiation between spores and vegetative cells and effectively stains spores, allowing recovery and tracking in subsequent experiments. Hoechst 34580, Propidium iodide and wheat germ agglutinin WGA 488 were used in combination to stain four strains of Clostridia at different life cycle stages. Staining was conducted without drying the sample, preventing changes induced by dehydration and cells observed by confocal laser scanner microscopy or using a super-resolution microscope equipped with a 3D-structured illumination module. Dual staining with Hoechst/Propidium iodide differentiated spores from vegetative cells, provided information on the viability of cells and was successfully applied to follow spore production induced by heating. Super-resolution microscopy of spores probed by Hoechst 34580 also allowed chromatin to be visualised. Direct staining of a cheese specimen using Nile Red and Fast Green allowed in situ observation of spores within the cheese and their position within the cheese matrix. The proposed staining method has broad applicability and can potentially be applied to follow Clostridium spore behaviour in a range of different environments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Probing nano-organization of astroglia with multi-color super-resolution microscopy.

PubMed

Heller, Janosch P; Michaluk, Piotr; Sugao, Kohtaroh; Rusakov, Dmitri A

2017-11-01

Astroglia are essential for brain development, homeostasis, and metabolic support. They also contribute actively to the formation and regulation of synaptic circuits, by successfully handling, integrating, and propagating physiological signals of neural networks. The latter occurs mainly by engaging a versatile mechanism of internal Ca 2+ fluctuations and regenerative waves prompting targeted release of signaling molecules into the extracellular space. Astroglia also show substantial structural plasticity associated with age- and use-dependent changes in neural circuitry. However, the underlying cellular mechanisms are poorly understood, mainly because of the extraordinary complex morphology of astroglial compartments on the nanoscopic scale. This complexity largely prevents direct experimental access to astroglial processes, most of which are beyond the diffraction limit of optical microscopy. Here we employed super-resolution microscopy (direct stochastic optical reconstruction microscopy; dSTORM), to visualize astroglial organization on the nanoscale, in culture and in thin brain slices, as an initial step to understand the structural basis of astrocytic nano-physiology. We were able to follow nanoscopic morphology of GFAP-enriched astrocytes, which adapt a flattened shape in culture and a sponge-like structure in situ, with GFAP fibers of varied diameters. We also visualized nanoscopic astrocytic processes using the ubiquitous cytosolic astrocyte marker proteins S100β and glutamine synthetase. Finally, we overexpressed and imaged membrane-targeted pHluorin and lymphocyte-specific protein tyrosine kinase (N-terminal domain) -green fluorescent protein (lck-GFP), to better understand the molecular cascades underlying some common astroglia-targeted fluorescence imaging techniques. The results provide novel, albeit initial, insights into the cellular organization of astroglia on the nanoscale, paving the way for function-specific studies. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Scattering-type scanning near-field optical microscopy with reconstruction of vertical interaction

PubMed Central

Wang, Le; Xu, Xiaoji G.

2015-01-01

Scattering-type scanning near-field optical microscopy provides access to super-resolution spectroscopic imaging of the surfaces of a variety of materials and nanostructures. In addition to chemical identification, it enables observations of nano-optical phenomena, such as mid-infrared plasmons in graphene and phonon polaritons in boron nitride. Despite the high lateral spatial resolution, scattering-type near-field optical microscopy is not able to provide characteristics of near-field responses in the vertical dimension, normal to the sample surface. Here, we present an accurate and fast reconstruction method to obtain vertical characteristics of near-field interactions. For its first application, we investigated the bound electromagnetic field component of surface phonon polaritons on the surface of boron nitride nanotubes and found that it decays within 20 nm with a considerable phase change in the near-field signal. The method is expected to provide characterization of the vertical field distribution of a wide range of nano-optical materials and structures. PMID:26592949

Importing super-resolution imaging into nanoscale puzzles of materials dynamics

NASA Astrophysics Data System (ADS)

King, John; Tsang, Chi Hang Boyce; Wilson, William; Granick, Steve

2014-03-01

A limitation of the exciting recent advances in sub-diffraction microscopy is that they focus on imaging rather than dynamical changes. We are engaged in extending this technique beyond the usual biological applications to address materials problems instead. To this end, we employ stimulated emission depletion (STED) microscopy, which relies on selectively turning off fluorescence emitters through stimulated emission, allowing only a small subset of emitters to be detected, such that the excitation spot size can be downsized to tens of nanometers. By coupling the STED excitation scheme to fluorescence correlation spectroscopy (FCS), diffusive processes are studied with nanoscale resolution. Here, we demonstrate the benefits of such experimental capabilities in a diverse range of complex systems, ranging from the diffusion of nano-objects in crowded 3D environments to the study of polymer diffusion on 2D surfaces.

Three-dimensional nanoscale imaging by plasmonic Brownian microscopy

NASA Astrophysics Data System (ADS)

Labno, Anna; Gladden, Christopher; Kim, Jeongmin; Lu, Dylan; Yin, Xiaobo; Wang, Yuan; Liu, Zhaowei; Zhang, Xiang

2017-12-01

Three-dimensional (3D) imaging at the nanoscale is a key to understanding of nanomaterials and complex systems. While scanning probe microscopy (SPM) has been the workhorse of nanoscale metrology, its slow scanning speed by a single probe tip can limit the application of SPM to wide-field imaging of 3D complex nanostructures. Both electron microscopy and optical tomography allow 3D imaging, but are limited to the use in vacuum environment due to electron scattering and to optical resolution in micron scales, respectively. Here we demonstrate plasmonic Brownian microscopy (PBM) as a way to improve the imaging speed of SPM. Unlike photonic force microscopy where a single trapped particle is used for a serial scanning, PBM utilizes a massive number of plasmonic nanoparticles (NPs) under Brownian diffusion in solution to scan in parallel around the unlabeled sample object. The motion of NPs under an evanescent field is three-dimensionally localized to reconstruct the super-resolution topology of 3D dielectric objects. Our method allows high throughput imaging of complex 3D structures over a large field of view, even with internal structures such as cavities that cannot be accessed by conventional mechanical tips in SPM.

Assembly and microscopic characterization of DNA origami structures.

PubMed

Scheible, Max; Jungmann, Ralf; Simmel, Friedrich C

2012-01-01

DNA origami is a revolutionary method for the assembly of molecular nanostructures from DNA with precisely defined dimensions and with an unprecedented yield. This can be utilized to arrange nanoscale components such as proteins or nanoparticles into pre-defined patterns. For applications it will now be of interest to arrange such components into functional complexes and study their geometry-dependent interactions. While commonly DNA nanostructures are characterized by atomic force microscopy or electron microscopy, these techniques often lack the time-resolution to study dynamic processes. It is therefore of considerable interest to also apply fluorescence microscopic techniques to DNA nanostructures. Of particular importance here is the utilization of novel super-resolved microscopy methods that enable imaging beyond the classical diffraction limit.

Adaptive Markov Random Fields for Example-Based Super-resolution of Faces

NASA Astrophysics Data System (ADS)

Stephenson, Todd A.; Chen, Tsuhan

2006-12-01

Image enhancement of low-resolution images can be done through methods such as interpolation, super-resolution using multiple video frames, and example-based super-resolution. Example-based super-resolution, in particular, is suited to images that have a strong prior (for those frameworks that work on only a single image, it is more like image restoration than traditional, multiframe super-resolution). For example, hallucination and Markov random field (MRF) methods use examples drawn from the same domain as the image being enhanced to determine what the missing high-frequency information is likely to be. We propose to use even stronger prior information by extending MRF-based super-resolution to use adaptive observation and transition functions, that is, to make these functions region-dependent. We show with face images how we can adapt the modeling for each image patch so as to improve the resolution.

Nanoscale surface characterization using laser interference microscopy

NASA Astrophysics Data System (ADS)

Ignatyev, Pavel S.; Skrynnik, Andrey A.; Melnik, Yury A.

2018-03-01

Nanoscale surface characterization is one of the most significant parts of modern materials development and application. The modern microscopes are expensive and complicated tools, and its use for industrial tasks is limited due to laborious sample preparation, measurement procedures, and low operation speed. The laser modulation interference microscopy method (MIM) for real-time quantitative and qualitative analysis of glass, metals, ceramics, and various coatings has a spatial resolution of 0.1 nm for vertical and up to 100 nm for lateral. It is proposed as an alternative to traditional scanning electron microscopy (SEM) and atomic force microscopy (AFM) methods. It is demonstrated that in the cases of roughness metrology for super smooth (Ra >1 nm) surfaces the application of a laser interference microscopy techniques is more optimal than conventional SEM and AFM. The comparison of semiconductor test structure for lateral dimensions measurements obtained with SEM and AFM and white light interferometer also demonstrates the advantages of MIM technique.

A novel super-resolution camera model

NASA Astrophysics Data System (ADS)

Shao, Xiaopeng; Wang, Yi; Xu, Jie; Wang, Lin; Liu, Fei; Luo, Qiuhua; Chen, Xiaodong; Bi, Xiangli

2015-05-01

Aiming to realize super resolution(SR) to single image and video reconstruction, a super resolution camera model is proposed for the problem that the resolution of the images obtained by traditional cameras behave comparatively low. To achieve this function we put a certain driving device such as piezoelectric ceramics in the camera. By controlling the driving device, a set of continuous low resolution(LR) images can be obtained and stored instantaneity, which reflect the randomness of the displacements and the real-time performance of the storage very well. The low resolution image sequences have different redundant information and some particular priori information, thus it is possible to restore super resolution image factually and effectively. The sample method is used to derive the reconstruction principle of super resolution, which analyzes the possible improvement degree of the resolution in theory. The super resolution algorithm based on learning is used to reconstruct single image and the variational Bayesian algorithm is simulated to reconstruct the low resolution images with random displacements, which models the unknown high resolution image, motion parameters and unknown model parameters in one hierarchical Bayesian framework. Utilizing sub-pixel registration method, a super resolution image of the scene can be reconstructed. The results of 16 images reconstruction show that this camera model can increase the image resolution to 2 times, obtaining images with higher resolution in currently available hardware levels.

The super-resolution debate

NASA Astrophysics Data System (ADS)

Won, Rachel

2018-05-01

In the quest for nanoscopy with super-resolution, consensus from the imaging community is that super-resolution is not always needed and that scientists should choose an imaging technique based on their specific application.

Multiplane and Spectrally-Resolved Single Molecule Localization Microscopy with Industrial Grade CMOS cameras.

PubMed

Babcock, Hazen P

2018-01-29

This work explores the use of industrial grade CMOS cameras for single molecule localization microscopy (SMLM). We show that industrial grade CMOS cameras approach the performance of scientific grade CMOS cameras at a fraction of the cost. This makes it more economically feasible to construct high-performance imaging systems with multiple cameras that are capable of a diversity of applications. In particular we demonstrate the use of industrial CMOS cameras for biplane, multiplane and spectrally resolved SMLM. We also provide open-source software for simultaneous control of multiple CMOS cameras and for the reduction of the movies that are acquired to super-resolution images.

Super-resolution in a defocused plenoptic camera: a wave-optics-based approach.

PubMed

Sahin, Erdem; Katkovnik, Vladimir; Gotchev, Atanas

2016-03-01

Plenoptic cameras enable the capture of a light field with a single device. However, with traditional light field rendering procedures, they can provide only low-resolution two-dimensional images. Super-resolution is considered to overcome this drawback. In this study, we present a super-resolution method for the defocused plenoptic camera (Plenoptic 1.0), where the imaging system is modeled using wave optics principles and utilizing low-resolution depth information of the scene. We are particularly interested in super-resolution of in-focus and near in-focus scene regions, which constitute the most challenging cases. The simulation results show that the employed wave-optics model makes super-resolution possible for such regions as long as sufficiently accurate depth information is available.

Adaptive pixel-super-resolved lensfree in-line digital holography for wide-field on-chip microscopy.

PubMed

Zhang, Jialin; Sun, Jiasong; Chen, Qian; Li, Jiaji; Zuo, Chao

2017-09-18

High-resolution wide field-of-view (FOV) microscopic imaging plays an essential role in various fields of biomedicine, engineering, and physical sciences. As an alternative to conventional lens-based scanning techniques, lensfree holography provides a new way to effectively bypass the intrinsical trade-off between the spatial resolution and FOV of conventional microscopes. Unfortunately, due to the limited sensor pixel-size, unpredictable disturbance during image acquisition, and sub-optimum solution to the phase retrieval problem, typical lensfree microscopes only produce compromised imaging quality in terms of lateral resolution and signal-to-noise ratio (SNR). Here, we propose an adaptive pixel-super-resolved lensfree imaging (APLI) method which can solve, or at least partially alleviate these limitations. Our approach addresses the pixel aliasing problem by Z-scanning only, without resorting to subpixel shifting or beam-angle manipulation. Automatic positional error correction algorithm and adaptive relaxation strategy are introduced to enhance the robustness and SNR of reconstruction significantly. Based on APLI, we perform full-FOV reconstruction of a USAF resolution target (~29.85 mm 2 ) and achieve half-pitch lateral resolution of 770 nm, surpassing 2.17 times of the theoretical Nyquist-Shannon sampling resolution limit imposed by the sensor pixel-size (1.67µm). Full-FOV imaging result of a typical dicot root is also provided to demonstrate its promising potential applications in biologic imaging.

Super-low friction and super-elastic hydrogenated carbon films originated from a unique fullerene-like nanostructure

NASA Astrophysics Data System (ADS)

Wang, Chengbing; Yang, Shengrong; Wang, Qi; Wang, Zhou; Zhang, Junyan

2008-06-01

Hydrogenated carbon films were grown by a plasma-enhanced chemical vapor deposition (PECVD) technique using CH4 and H2 as feedstock at ambient temperature. The microstructure of the films was characterized by high resolution transmission electron microscopy (HRTEM). The images showed the presence of curved basal planes in fullerene-like arrangements. An apparent amorphous graphene structure with nm-sized packages of basal planes in a turbostratic feature was observed. The fabricated fullerene-like hydrogenated carbon films (FL-C:H) possess superior mechanical properties, i.e. high hardness (19 GPa) and high elasticity (elastic recovery of 85%). More importantly, the films exhibit ultra-low friction (μ = 0.009) under ambient conditions with 20% relative humidity.

Super-low friction and super-elastic hydrogenated carbon films originated from a unique fullerene-like nanostructure.

PubMed

Wang, Chengbing; Yang, Shengrong; Wang, Qi; Wang, Zhou; Zhang, Junyan

2008-06-04

Hydrogenated carbon films were grown by a plasma-enhanced chemical vapor deposition (PECVD) technique using CH(4) and H(2) as feedstock at ambient temperature. The microstructure of the films was characterized by high resolution transmission electron microscopy (HRTEM). The images showed the presence of curved basal planes in fullerene-like arrangements. An apparent amorphous graphene structure with nm-sized packages of basal planes in a turbostratic feature was observed. The fabricated fullerene-like hydrogenated carbon films (FL-C:H) possess superior mechanical properties, i.e. high hardness (19 GPa) and high elasticity (elastic recovery of 85%). More importantly, the films exhibit ultra-low friction (μ = 0.009) under ambient conditions with 20% relative humidity.

Fullerene-like hydrogenated carbon films with super-low friction and wear, and low sensitivity to environment

NASA Astrophysics Data System (ADS)

Ji, Li; Li, Hongxuan; Zhao, Fei; Quan, Weilong; Chen, Jianmin; Zhou, Huidi

2010-01-01

A novel hydrogenated carbon film containing fullerene-like nanostructure was prepared by pulse bias-assisted plasma enhanced chemical vapour deposition, and the fullerene-like arrangement in the film was characterized by high resolution transmission electron microscopy. The as-prepared hydrogenated carbon film exhibited super-low friction and wear in both dry N2 and humid ambient atmospheres, and was superior to the conventional hydrogenated carbon films. These excellent tribological properties could be attributed to the unique fullerene-like nanostructure, which endows the film with some special chemical and physical features, such as high chemical inertness, hardness and elastic recovery owing to the closed, curved and caged graphite planes, and hence, improves the tribological properties of the hydrogenated carbon film.

Highly selective creation of hydrophilic micro-craters on super hydrophobic surface using electrohydrodynamic jet printing

NASA Astrophysics Data System (ADS)

Lee, Jaehyun; Hwang, Sangyeon; Prasetyo, Fariza Dian; Nguyen, Vu Dat; Hong, Jungwoo; Shin, Jennifer H.; Byun, Doyoung

2014-11-01

Selective surface modification is considered as an alternative to conventional printing techniques in high resolution patterning. Here, we present fabrication of hydrophilic patterns on the super hydrophobic surface, which makes structure on the hydrophilic region. The super hydrophobic surface is able to be chemically changed to hydrophilic with alcohols. As a consecutive process, electrohydrodynamic (EHD) jet printing was utilized to fabricate local hydrophilic craters with 30-200 μm sizes. 3 kinds of target liquids were deposited well on hydrophilic region; PEDOT (poly 3,4 ethylenediocythiophene), polystyrene nano-particles, and salmonella bacteria medium. Additionally, qualitative analysis were presented for modification mechanism and surface properties on super hydrophobic/hydrophilic by analysis of surface energy with contact angle, SEM (scanning electron microscopy) image, and SIMS (secondary ion mass spectroscopy) analysis. This new simple modification method provides possibility to be utilizing in bio-patterning engineering such as cell culturing microchip and lab on a chip. This research was supported by the Basi Science Research Program through the National Research Foundation of Korea (NRF) (Grand Number: 2014-023284).

Structural analysis of herpes simplex virus by optical super-resolution imaging

NASA Astrophysics Data System (ADS)

Laine, Romain F.; Albecka, Anna; van de Linde, Sebastian; Rees, Eric J.; Crump, Colin M.; Kaminski, Clemens F.

2015-01-01

Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.

Optimized protocol for combined PALM-dSTORM imaging.

PubMed

Glushonkov, O; Réal, E; Boutant, E; Mély, Y; Didier, P

2018-06-08

Multi-colour super-resolution localization microscopy is an efficient technique to study a variety of intracellular processes, including protein-protein interactions. This technique requires specific labels that display transition between fluorescent and non-fluorescent states under given conditions. For the most commonly used label types, photoactivatable fluorescent proteins and organic fluorophores, these conditions are different, making experiments that combine both labels difficult. Here, we demonstrate that changing the standard imaging buffer of thiols/oxygen scavenging system, used for organic fluorophores, to the commercial mounting medium Vectashield increased the number of photons emitted by the fluorescent protein mEos2 and enhanced the photoconversion rate between its green and red forms. In addition, the photophysical properties of organic fluorophores remained unaltered with respect to the standard imaging buffer. The use of Vectashield together with our optimized protocol for correction of sample drift and chromatic aberrations enabled us to perform two-colour 3D super-resolution imaging of the nucleolus and resolve its three compartments.

Correlative live and super-resolution imaging reveals the dynamic structure of replication domains.

PubMed

Xiang, Wanqing; Roberti, M Julia; Hériché, Jean-Karim; Huet, Sébastien; Alexander, Stephanie; Ellenberg, Jan

2018-06-04

Chromosome organization in higher eukaryotes controls gene expression, DNA replication, and DNA repair. Genome mapping has revealed the functional units of chromatin at the submegabase scale as self-interacting regions called topologically associating domains (TADs) and showed they correspond to replication domains (RDs). A quantitative structural and dynamic description of RD behavior in the nucleus is, however, missing because visualization of dynamic subdiffraction-sized RDs remains challenging. Using fluorescence labeling of RDs combined with correlative live and super-resolution microscopy in situ, we determined biophysical parameters to characterize the internal organization, spacing, and mechanical coupling of RDs. We found that RDs are typically 150 nm in size and contain four co-replicating regions spaced 60 nm apart. Spatially neighboring RDs are spaced 300 nm apart and connected by highly flexible linker regions that couple their motion only <550 nm. Our pipeline allows a robust quantitative characterization of chromosome structure in situ and provides important biophysical parameters to understand general principles of chromatin organization. © 2018 Xiang et al.

Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging.

PubMed

Jayasinghe, Isuru D; Munro, Michelle; Baddeley, David; Launikonis, Bradley S; Soeller, Christian

2014-10-06

Localization microscopy is a fairly recently introduced super-resolution fluorescence imaging modality capable of achieving nanometre-scale resolution. We have applied the dSTORM variation of this method to image intracellular molecular assemblies in skeletal muscle fibres which are large cells that critically rely on nanoscale signalling domains, the triads. Immunofluorescence staining in fixed adult rat skeletal muscle sections revealed clear differences between fast- and slow-twitch fibres in the molecular organization of ryanodine receptors (RyRs; the primary calcium release channels) within triads. With the improved resolution offered by dSTORM, abutting arrays of RyRs in transverse view of fast fibres were observed in contrast to the fragmented distribution on slow-twitch muscle that were approximately 1.8 times shorter and consisted of approximately 1.6 times fewer receptors. To the best of our knowledge, for the first time, we have quantified the nanometre-scale spatial association between triadic proteins using multi-colour super-resolution, an analysis difficult to conduct with electron microscopy. Our findings confirm that junctophilin-1 (JPH1), which tethers the sarcoplasmic reticulum ((SR) intracellular calcium store) to the tubular (t-) system at triads, was present throughout the RyR array, whereas JPH2 was contained within much smaller nanodomains. Similar imaging of the primary SR calcium buffer, calsequestrin (CSQ), detected less overlap of the triad with CSQ in slow-twitch muscle supporting greater spatial heterogeneity in the luminal Ca2+ buffering when compared with fast twitch muscle. Taken together, these nanoscale differences can explain the fundamentally different physiologies of fast- and slow-twitch muscle. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging

PubMed Central

Jayasinghe, Isuru D.; Munro, Michelle; Baddeley, David; Launikonis, Bradley S.; Soeller, Christian

2014-01-01

Localization microscopy is a fairly recently introduced super-resolution fluorescence imaging modality capable of achieving nanometre-scale resolution. We have applied the dSTORM variation of this method to image intracellular molecular assemblies in skeletal muscle fibres which are large cells that critically rely on nanoscale signalling domains, the triads. Immunofluorescence staining in fixed adult rat skeletal muscle sections revealed clear differences between fast- and slow-twitch fibres in the molecular organization of ryanodine receptors (RyRs; the primary calcium release channels) within triads. With the improved resolution offered by dSTORM, abutting arrays of RyRs in transverse view of fast fibres were observed in contrast to the fragmented distribution on slow-twitch muscle that were approximately 1.8 times shorter and consisted of approximately 1.6 times fewer receptors. To the best of our knowledge, for the first time, we have quantified the nanometre-scale spatial association between triadic proteins using multi-colour super-resolution, an analysis difficult to conduct with electron microscopy. Our findings confirm that junctophilin-1 (JPH1), which tethers the sarcoplasmic reticulum ((SR) intracellular calcium store) to the tubular (t-) system at triads, was present throughout the RyR array, whereas JPH2 was contained within much smaller nanodomains. Similar imaging of the primary SR calcium buffer, calsequestrin (CSQ), detected less overlap of the triad with CSQ in slow-twitch muscle supporting greater spatial heterogeneity in the luminal Ca2+ buffering when compared with fast twitch muscle. Taken together, these nanoscale differences can explain the fundamentally different physiologies of fast- and slow-twitch muscle. PMID:25100314

Analyzing blinking effects in super resolution localization microscopy with single-photon SPAD imagers

NASA Astrophysics Data System (ADS)

Antolovic, Ivan Michel; Burri, Samuel; Bruschini, Claudio; Hoebe, Ron; Charbon, Edoardo

2016-02-01

For many scientific applications, electron multiplying charge coupled devices (EMCCDs) have been the sensor of choice because of their high quantum efficiency and built-in electron amplification. Lately, many researchers introduced scientific complementary metal-oxide semiconductor (sCMOS) imagers in their instrumentation, so as to take advantage of faster readout and the absence of excess noise. Alternatively, single-photon avalanche diode (SPAD) imagers can provide even faster frame rates and zero readout noise. SwissSPAD is a 1-bit 512×128 SPAD imager, one of the largest of its kind, featuring a frame duration of 6.4 μs. Additionally, a gating mechanism enables photosensitive windows as short as 5 ns with a skew better than 150 ps across the entire array. The SwissSPAD photon detection efficiency (PDE) uniformity is very high, thanks on one side to a photon-to-digital conversion and on the other to a reduced fraction of "hot pixels" or "screamers", which would pollute the image with noise. A low native fill factor was recovered to a large extent using a microlens array, leading to a maximum PDE increase of 12×. This enabled us to detect single fluorophores, as required by ground state depletion followed by individual molecule return imaging microscopy (GSDIM). We show the first super resolution results obtained with a SPAD imager, with an estimated localization uncertainty of 30 nm and resolution of 100 nm. The high time resolution of 6.4 μs can be utilized to explore the dye's photophysics or for dye optimization. We also present the methodology for the blinking analysis on experimental data.

Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations

PubMed Central

Gustafsson, Nils; Culley, Siân; Ashdown, George; Owen, Dylan M.; Pereira, Pedro Matos; Henriques, Ricardo

2016-01-01

Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours. PMID:27514992

Super-resolution chemical imaging with dynamic placement of plasmonic hotspots

NASA Astrophysics Data System (ADS)

Olson, Aeli P.; Ertsgaard, Christopher T.; McKoskey, Rachel M.; Rich, Isabel S.; Lindquist, Nathan C.

2015-08-01

We demonstrate dynamic placement of plasmonic "hotspots" for super-resolution chemical imaging via Surface Enhanced Raman Spectroscopy (SERS). A silver nanohole array surface was coated with biological samples and illuminated with a laser. Due to the large plasmonic field enhancements, blinking behavior of the SERS hotspots was observed and processed using a Stochastic Optical Reconstruction Microscopy (STORM) algorithm enabling localization to within 10 nm. However, illumination of the sample with a single static laser beam (i.e., a slightly defocused Gaussian beam) only produced SERS hotspots in fixed locations on the surface, leaving noticeable gaps in any final image. But, by using a spatial light modulator (SLM), the illumination profile of the beam could be altered, shifting any hotspots across the nanohole array surface in sub-wavelength steps. Therefore, by properly structuring an illuminating light field with the SLM, we show the possibility of positioning plasmonic hotspots over a metallic nanohole surface on-the-fly. Using this and our SERS-STORM imaging technique, we show potential for high-resolution chemical imaging without the noticeable gaps that were present with static laser illumination. Interestingly, even illuminating the surface with randomly shifting SLM phase profiles was sufficient to completely fill in a wide field of view for super-resolution SERS imaging of a single strand of 100-nm thick collagen protein fibrils. Images were then compared to those obtained with a scanning electron microscope (SEM). Additionally, we explored alternative methods of phase shifting other than holographic illumination through the SLM to create localization of hotspots necessary for SERS-STORM imaging.

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies.

PubMed

Hoischen, Christian; Monajembashi, Shamci; Weisshart, Klaus; Hemmerich, Peter

2018-01-01

The promyelocytic leukemia ( pml ) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology.

Characterization and improvement of highly inclined optical sheet microscopy

NASA Astrophysics Data System (ADS)

Vignolini, T.; Curcio, V.; Gardini, L.; Capitanio, M.; Pavone, F. S.

2018-02-01

Highly Inclined and Laminated Optical sheet (HILO) microscopy is an optical technique that employs a highly inclined laser beam to illuminate the sample with a thin sheet of light that can be scanned through the sample volume1 . HILO is an efficient illumination technique when applied to fluorescence imaging of thick samples owing to the confined illumination volume that allows high contrast imaging while retaining deep scanning capability in a wide-field configuration. The restricted illumination volume is crucial to limit background fluorescence originating from portions of the sample far from the focal plane, especially in applications such as single molecule localization and super-resolution imaging2-4. Despite its widespread use, current literature lacks comprehensive reports of the actual advantages of HILO in these kinds of microscopies. Here, we thoroughly characterize the propagation of a highly inclined beam through fluorescently labeled samples and implement appropriate beam shaping for optimal application to single molecule and super-resolution imaging. We demonstrate that, by reducing the beam size along the refracted axis only, the excitation volume is consequently reduced while maintaining a field of view suitable for single cell imaging. We quantify the enhancement in signal-tobackground ratio with respect to the standard HILO technique and apply our illumination method to dSTORM superresolution imaging of the actin and vimentin cytoskeleton. We define the conditions to achieve localization precisions comparable to state-of-the-art reports, obtain a significant improvement in the image contrast, and enhanced plane selectivity within the sample volume due to the further confinement of the inclined beam.

Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA

NASA Astrophysics Data System (ADS)

Lin, Chenxiang; Jungmann, Ralf; Leifer, Andrew M.; Li, Chao; Levner, Daniel; Church, George M.; Shih, William M.; Yin, Peng

2012-10-01

The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here, we used (deoxy)ribonucleic acid (DNA)-origami technology to construct submicrometre nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be decoded unambiguously using epifluorescence or total internal reflection fluorescence microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ˜40 nm. One species of the barcodes was used to tag yeast surface receptors, which suggests their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments.

fastSIM: a practical implementation of fast structured illumination microscopy.

PubMed

Lu-Walther, Hui-Wen; Kielhorn, Martin; Förster, Ronny; Jost, Aurélie; Wicker, Kai; Heintzmann, Rainer

2015-01-16

A significant improvement in acquisition speed of structured illumination microscopy (SIM) opens a new field of applications to this already well-established super-resolution method towards 3D scanning real-time imaging of living cells. We demonstrate a method of increased acquisition speed on a two-beam SIM fluorescence microscope with a lateral resolution of ~100 nm at a maximum raw data acquisition rate of 162 frames per second (fps) with a region of interest of 16.5  ×  16.5 µm 2 , free of mechanically moving components. We use a programmable spatial light modulator (ferroelectric LCOS) which promises precise and rapid control of the excitation pattern in the sample plane. A passive Fourier filter and a segmented azimuthally patterned polarizer are used to perform structured illumination with maximum contrast. Furthermore, the free running mode in a modern sCMOS camera helps to achieve faster data acquisition.

fastSIM: a practical implementation of fast structured illumination microscopy

NASA Astrophysics Data System (ADS)

Lu-Walther, Hui-Wen; Kielhorn, Martin; Förster, Ronny; Jost, Aurélie; Wicker, Kai; Heintzmann, Rainer

2015-03-01

A significant improvement in acquisition speed of structured illumination microscopy (SIM) opens a new field of applications to this already well-established super-resolution method towards 3D scanning real-time imaging of living cells. We demonstrate a method of increased acquisition speed on a two-beam SIM fluorescence microscope with a lateral resolution of ~100 nm at a maximum raw data acquisition rate of 162 frames per second (fps) with a region of interest of 16.5  ×  16.5 µm2, free of mechanically moving components. We use a programmable spatial light modulator (ferroelectric LCOS) which promises precise and rapid control of the excitation pattern in the sample plane. A passive Fourier filter and a segmented azimuthally patterned polarizer are used to perform structured illumination with maximum contrast. Furthermore, the free running mode in a modern sCMOS camera helps to achieve faster data acquisition.

Sub-micrometer Geometrically Encoded Fluorescent Barcodes Self-Assembled from DNA

PubMed Central

Lin, Chenxiang; Jungmann, Ralf; Leifer, Andrew M.; Li, Chao; Levner, Daniel; Church, George M.; Shih, William M.; Yin, Peng

2012-01-01

The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here we use DNA-origami technology to construct sub-micrometer nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be unambiguously decoded using epifluorescence or total internal reflection fluorescence (TIRF) microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ~40 nm. One species of the barcodes was used to tag yeast surface receptors, suggesting their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments. PMID:23000997

Optimizing single-nanoparticle two-photon microscopy by in situ adaptive control of femtosecond pulses

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

Li, Donghai; Deng, Yongkai; Chu, Saisai

2016-07-11

Single-nanoparticle two-photon microscopy shows great application potential in super-resolution cell imaging. Here, we report in situ adaptive optimization of single-nanoparticle two-photon luminescence signals by phase and polarization modulations of broadband laser pulses. For polarization-independent quantum dots, phase-only optimization was carried out to compensate the phase dispersion at the focus of the objective. Enhancement of the two-photon excitation fluorescence intensity under dispersion-compensated femtosecond pulses was achieved. For polarization-dependent single gold nanorod, in situ polarization optimization resulted in further enhancement of two-photon photoluminescence intensity than phase-only optimization. The application of in situ adaptive control of femtosecond pulse provides a way for object-orientedmore » optimization of single-nanoparticle two-photon microscopy for its future applications.« less

Image reconstructions from super-sampled data sets with resolution modeling in PET imaging.

PubMed

Li, Yusheng; Matej, Samuel; Metzler, Scott D

2014-12-01

Spatial resolution in positron emission tomography (PET) is still a limiting factor in many imaging applications. To improve the spatial resolution for an existing scanner with fixed crystal sizes, mechanical movements such as scanner wobbling and object shifting have been considered for PET systems. Multiple acquisitions from different positions can provide complementary information and increased spatial sampling. The objective of this paper is to explore an efficient and useful reconstruction framework to reconstruct super-resolution images from super-sampled low-resolution data sets. The authors introduce a super-sampling data acquisition model based on the physical processes with tomographic, downsampling, and shifting matrices as its building blocks. Based on the model, we extend the MLEM and Landweber algorithms to reconstruct images from super-sampled data sets. The authors also derive a backprojection-filtration-like (BPF-like) method for the super-sampling reconstruction. Furthermore, they explore variant methods for super-sampling reconstructions: the separate super-sampling resolution-modeling reconstruction and the reconstruction without downsampling to further improve image quality at the cost of more computation. The authors use simulated reconstruction of a resolution phantom to evaluate the three types of algorithms with different super-samplings at different count levels. Contrast recovery coefficient (CRC) versus background variability, as an image-quality metric, is calculated at each iteration for all reconstructions. The authors observe that all three algorithms can significantly and consistently achieve increased CRCs at fixed background variability and reduce background artifacts with super-sampled data sets at the same count levels. For the same super-sampled data sets, the MLEM method achieves better image quality than the Landweber method, which in turn achieves better image quality than the BPF-like method. The authors also demonstrate that the reconstructions from super-sampled data sets using a fine system matrix yield improved image quality compared to the reconstructions using a coarse system matrix. Super-sampling reconstructions with different count levels showed that the more spatial-resolution improvement can be obtained with higher count at a larger iteration number. The authors developed a super-sampling reconstruction framework that can reconstruct super-resolution images using the super-sampling data sets simultaneously with known acquisition motion. The super-sampling PET acquisition using the proposed algorithms provides an effective and economic way to improve image quality for PET imaging, which has an important implication in preclinical and clinical region-of-interest PET imaging applications.

Enhanced simulator software for image validation and interpretation for multimodal localization super-resolution fluorescence microscopy

NASA Astrophysics Data System (ADS)

Erdélyi, Miklós; Sinkó, József; Gajdos, Tamás.; Novák, Tibor

2017-02-01

Optical super-resolution techniques such as single molecule localization have become one of the most dynamically developed areas in optical microscopy. These techniques routinely provide images of fixed cells or tissues with sub-diffraction spatial resolution, and can even be applied for live cell imaging under appropriate circumstances. Localization techniques are based on the precise fitting of the point spread functions (PSF) to the measured images of stochastically excited, identical fluorescent molecules. These techniques require controlling the rate between the on, off and the bleached states, keeping the number of active fluorescent molecules at an optimum value, so their diffraction limited images can be detected separately both spatially and temporally. Because of the numerous (and sometimes unknown) parameters, the imaging system can only be handled stochastically. For example, the rotation of the dye molecules obscures the polarization dependent PSF shape, and only an averaged distribution - typically estimated by a Gaussian function - is observed. TestSTORM software was developed to generate image stacks for traditional localization microscopes, where localization meant the precise determination of the spatial position of the molecules. However, additional optical properties (polarization, spectra, etc.) of the emitted photons can be used for further monitoring the chemical and physical properties (viscosity, pH, etc.) of the local environment. The image stack generating program was upgraded by several new features, such as: multicolour, polarization dependent PSF, built-in 3D visualization, structured background. These features make the program an ideal tool for optimizing the imaging and sample preparation conditions.

Gradation (approx. 10 size states) of synaptic strength by quantal addition of structural modules

PubMed Central

2017-01-01

Memory storage involves activity-dependent strengthening of synaptic transmission, a process termed long-term potentiation (LTP). The late phase of LTP is thought to encode long-term memory and involves structural processes that enlarge the synapse. Hence, understanding how synapse size is graded provides fundamental information about the information storage capability of synapses. Recent work using electron microscopy (EM) to quantify synapse dimensions has suggested that synapses may structurally encode as many as 26 functionally distinct states, which correspond to a series of proportionally spaced synapse sizes. Other recent evidence using super-resolution microscopy has revealed that synapses are composed of stereotyped nanoclusters of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and scaffolding proteins; furthermore, synapse size varies linearly with the number of nanoclusters. Here we have sought to develop a model of synapse structure and growth that is consistent with both the EM and super-resolution data. We argue that synapses are composed of modules consisting of matrix material and potentially one nanocluster. LTP induction can add a trans-synaptic nanocluster to a module, thereby converting a silent module to an AMPA functional module. LTP can also add modules by a linear process, thereby producing an approximately 10-fold gradation in synapse size and strength. This article is part of the themed issue ‘Integrating Hebbian and homeostatic plasticity’. PMID:28093559

Gradation (approx. 10 size states) of synaptic strength by quantal addition of structural modules.

PubMed

Liu, Kang K L; Hagan, Michael F; Lisman, John E

2017-03-05

Memory storage involves activity-dependent strengthening of synaptic transmission, a process termed long-term potentiation (LTP). The late phase of LTP is thought to encode long-term memory and involves structural processes that enlarge the synapse. Hence, understanding how synapse size is graded provides fundamental information about the information storage capability of synapses. Recent work using electron microscopy (EM) to quantify synapse dimensions has suggested that synapses may structurally encode as many as 26 functionally distinct states, which correspond to a series of proportionally spaced synapse sizes. Other recent evidence using super-resolution microscopy has revealed that synapses are composed of stereotyped nanoclusters of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and scaffolding proteins; furthermore, synapse size varies linearly with the number of nanoclusters. Here we have sought to develop a model of synapse structure and growth that is consistent with both the EM and super-resolution data. We argue that synapses are composed of modules consisting of matrix material and potentially one nanocluster. LTP induction can add a trans-synaptic nanocluster to a module, thereby converting a silent module to an AMPA functional module. LTP can also add modules by a linear process, thereby producing an approximately 10-fold gradation in synapse size and strength.This article is part of the themed issue 'Integrating Hebbian and homeostatic plasticity'. © 2017 The Author(s).

Efficient super-resolution image reconstruction applied to surveillance video captured by small unmanned aircraft systems

NASA Astrophysics Data System (ADS)

He, Qiang; Schultz, Richard R.; Chu, Chee-Hung Henry

2008-04-01

The concept surrounding super-resolution image reconstruction is to recover a highly-resolved image from a series of low-resolution images via between-frame subpixel image registration. In this paper, we propose a novel and efficient super-resolution algorithm, and then apply it to the reconstruction of real video data captured by a small Unmanned Aircraft System (UAS). Small UAS aircraft generally have a wingspan of less than four meters, so that these vehicles and their payloads can be buffeted by even light winds, resulting in potentially unstable video. This algorithm is based on a coarse-to-fine strategy, in which a coarsely super-resolved image sequence is first built from the original video data by image registration and bi-cubic interpolation between a fixed reference frame and every additional frame. It is well known that the median filter is robust to outliers. If we calculate pixel-wise medians in the coarsely super-resolved image sequence, we can restore a refined super-resolved image. The primary advantage is that this is a noniterative algorithm, unlike traditional approaches based on highly-computational iterative algorithms. Experimental results show that our coarse-to-fine super-resolution algorithm is not only robust, but also very efficient. In comparison with five well-known super-resolution algorithms, namely the robust super-resolution algorithm, bi-cubic interpolation, projection onto convex sets (POCS), the Papoulis-Gerchberg algorithm, and the iterated back projection algorithm, our proposed algorithm gives both strong efficiency and robustness, as well as good visual performance. This is particularly useful for the application of super-resolution to UAS surveillance video, where real-time processing is highly desired.

Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells.

PubMed

Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

2017-07-01

Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only a few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. 3D-magnetic twisting cytometry (3D-MTC) is a technique for applying local mechanical stresses to living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors, followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic-field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super-resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real-time acquisition of a living cell's mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC-microscopy platform takes ∼20 d to construct, and the experimental procedures require ∼4 d when carried out by a life sciences graduate student.

Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells

PubMed Central

Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

2017-01-01

Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. Three dimensional-Magnetic Twisting Cytometry (3D-MTC) is a technique for applying local mechanical stresses on living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real time acquisition of a living cell’s mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC – microscopy platform takes around 20 days to construct and the experimental procedures require ~4 days when carried out by a life sciences graduate student. PMID:28686583

Temporally flickering nanoparticles for compound cellular imaging and super resolution

NASA Astrophysics Data System (ADS)

Ilovitsh, Tali; Danan, Yossef; Meir, Rinat; Meiri, Amihai; Zalevsky, Zeev

2016-03-01

This work presents the use of flickering nanoparticles for imaging biological samples. The method has high noise immunity, and it enables the detection of overlapping types of GNPs, at significantly sub-diffraction distances, making it attractive for super resolving localization microscopy techniques. The method utilizes a lock-in technique at which the imaging of the sample is done using a time-modulated laser beam that match the number of the types of gold nanoparticles (GNPs) that label a given sample, and resulting in the excitation of the temporal flickering of the scattered light at known temporal frequencies. The final image where the GNPs are spatially separated is obtained using post processing where the proper spectral components corresponding to the different modulation frequencies are extracted. This allows the simultaneous super resolved imaging of multiple types of GNPs that label targets of interest within biological samples. Additionally applying the post-processing algorithm of the K-factor image decomposition algorithm can further improve the performance of the proposed approach.

Infrared super-resolution imaging based on compressed sensing

NASA Astrophysics Data System (ADS)

Sui, Xiubao; Chen, Qian; Gu, Guohua; Shen, Xuewei

2014-03-01

The theoretical basis of traditional infrared super-resolution imaging method is Nyquist sampling theorem. The reconstruction premise is that the relative positions of the infrared objects in the low-resolution image sequences should keep fixed and the image restoration means is the inverse operation of ill-posed issues without fixed rules. The super-resolution reconstruction ability of the infrared image, algorithm's application area and stability of reconstruction algorithm are limited. To this end, we proposed super-resolution reconstruction method based on compressed sensing in this paper. In the method, we selected Toeplitz matrix as the measurement matrix and realized it by phase mask method. We researched complementary matching pursuit algorithm and selected it as the recovery algorithm. In order to adapt to the moving target and decrease imaging time, we take use of area infrared focal plane array to acquire multiple measurements at one time. Theoretically, the method breaks though Nyquist sampling theorem and can greatly improve the spatial resolution of the infrared image. The last image contrast and experiment data indicate that our method is effective in improving resolution of infrared images and is superior than some traditional super-resolution imaging method. The compressed sensing super-resolution method is expected to have a wide application prospect.

Color digital lensless holographic microscopy: laser versus LED illumination.

PubMed

Garcia-Sucerquia, Jorge

2016-08-20

A comparison of the performance of color digital lensless holographic microscopy (CDLHM) as utilized for illumination of RGB lasers or a super-bright white-light LED with a set of spectral filters is presented. As the use of lasers in CDLHM conceals the possibility of having a compact, lightweight, portable, and low cost microscope, and additionally the limited available laser radiation wavelengths limit a real multispectral imaging microscope, here we present the use of super-bright white-light LED and spectral filters for illuminating the sample. The performance of RGB laser-CDLHM and LED-CDLHM is evaluated on imaging a section of the head of a Drosophila melanogaster fly. This comparison shows that there is trade-off between the spatial resolution of the microscope and the light sources utilized, which can be understood with regard to the coherence properties of the illuminating light. Despite the smaller spatial coherence features of LED-CDLHM in comparison with laser-CDLHM, the former shows promise as a portable RGB digital lensless holographic microscope that could be extended to other wavelengths by the use of different spectral filters.

Silicon and aluminum doping effects on the microstructure and properties of polymeric amorphous carbon films

NASA Astrophysics Data System (ADS)

Liu, Xiaoqiang; Hao, Junying; Xie, Yuntao

2016-08-01

Polymeric amorphous carbon films were prepared by radio frequency (R.F. 13.56 MHz) magnetron sputtering deposition. The microstructure evolution of the deposited polymeric films induced by silicon (Si) and aluminum(Al) doping were scrutinized through infrared spectroscopy, multi-wavelength Raman spectroscopy, scanning electron microscopy (SEM) and high resolution transmission electron microscopy (HRTEM). The comparative results show that Si doping can enhance polymerization and Al doping results in an increase in the ordered carbon clusters. Si and Al co-doping into polymeric films leads to the formation of an unusual dual nanostructure consisting of cross-linked polymer-like hydrocarbon chains and fullerene-like carbon clusters. The super-high elasticity and super-low friction coefficients (<0.002) under a high vacuum were obtained through Si and Al co-doping into the films. Unconventionally, the co-doped polymeric films exhibited a superior wear resistance even though they were very soft. The relationship between the microstructure and properties of the polymeric amorphous carbon films with different elements doping are also discussed in detail.

A journey through the microscopic ages of DNA replication.

PubMed

Reinhart, Marius; Cardoso, M Cristina

2017-05-01

Scientific discoveries and technological advancements are inseparable but not always take place in a coherent chronological manner. In the next, we will provide a seemingly unconnected and serendipitous series of scientific facts that, in the whole, converged to unveil DNA and its duplication. We will not cover here the many and fundamental contributions from microbial genetics and in vitro biochemistry. Rather, in this journey, we will emphasize the interplay between microscopy development culminating on super resolution fluorescence microscopy (i.e., nanoscopy) and digital image analysis and its impact on our understanding of DNA duplication. We will interlace the journey with landmark concepts and experiments that have brought the cellular DNA replication field to its present state.

Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision.

PubMed

Tuijtel, Maarten W; Mulder, Aat A; Posthuma, Clara C; van der Hoeven, Barbara; Koster, Abraham J; Bárcena, Montserrat; Faas, Frank G A; Sharp, Thomas H

2017-09-05

Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron microscopy (TEM) with the capability of fluorescence light microscopy (FLM) to locate rare or transient cellular events within a large field of view. CLEM is therefore a powerful technique to study cellular processes. Aligning images derived from both imaging modalities is a prerequisite to correlate the two microscopy data sets, and poor alignment can limit interpretability of the data. Here, we describe how uranyl acetate, a commonly-used contrast agent for TEM, can be induced to fluoresce brightly at cryogenic temperatures (-195 °C) and imaged by cryoFLM using standard filter sets. This dual-purpose contrast agent can be used as a general tool for CLEM, whereby the equivalent staining allows direct correlation between fluorescence and TEM images. We demonstrate the potential of this approach by performing multi-colour CLEM of cells containing equine arteritis virus proteins tagged with either green- or red-fluorescent protein, and achieve high-precision localization of virus-induced intracellular membrane modifications. Using uranyl acetate as a dual-purpose contrast agent, we achieve an image alignment precision of ~30 nm, twice as accurate as when using fiducial beads, which will be essential for combining TEM with the evolving field of super-resolution light microscopy.

Super-resolved refocusing with a plenoptic camera

NASA Astrophysics Data System (ADS)

Zhou, Zhiliang; Yuan, Yan; Bin, Xiangli; Qian, Lulu

2011-03-01

This paper presents an approach to enhance the resolution of refocused images by super resolution methods. In plenoptic imaging, we demonstrate that the raw sensor image can be divided to a number of low-resolution angular images with sub-pixel shifts between each other. The sub-pixel shift, which defines the super-resolving ability, is mathematically derived by considering the plenoptic camera as equivalent camera arrays. We implement simulation to demonstrate the imaging process of a plenoptic camera. A high-resolution image is then reconstructed using maximum a posteriori (MAP) super resolution algorithms. Without other degradation effects in simulation, the super resolved image achieves a resolution as high as predicted by the proposed model. We also build an experimental setup to acquire light fields. With traditional refocusing methods, the image is rendered at a rather low resolution. In contrast, we implement the super-resolved refocusing methods and recover an image with more spatial details. To evaluate the performance of the proposed method, we finally compare the reconstructed images using image quality metrics like peak signal to noise ratio (PSNR).

A Super-Resolution Algorithm for Enhancement of FLASH LIDAR Data: Flight Test Results

NASA Technical Reports Server (NTRS)

Bulyshev, Alexander; Amzajerdian, Farzin; Roback, Eric; Reisse Robert

2014-01-01

This paper describes the results of a 3D super-resolution algorithm applied to the range data obtained from a recent Flash Lidar helicopter flight test. The flight test was conducted by the NASA's Autonomous Landing and Hazard Avoidance Technology (ALHAT) project over a simulated lunar terrain facility at NASA Kennedy Space Center. ALHAT is developing the technology for safe autonomous landing on the surface of celestial bodies: Moon, Mars, asteroids. One of the test objectives was to verify the ability of 3D super-resolution technique to generate high resolution digital elevation models (DEMs) and to determine time resolved relative positions and orientations of the vehicle. 3D super-resolution algorithm was developed earlier and tested in computational modeling, and laboratory experiments, and in a few dynamic experiments using a moving truck. Prior to the helicopter flight test campaign, a 100mX100m hazard field was constructed having most of the relevant extraterrestrial hazard: slopes, rocks, and craters with different sizes. Data were collected during the flight and then processed by the super-resolution code. The detailed DEM of the hazard field was constructed using independent measurement to be used for comparison. ALHAT navigation system data were used to verify abilities of super-resolution method to provide accurate relative navigation information. Namely, the 6 degree of freedom state vector of the instrument as a function of time was restored from super-resolution data. The results of comparisons show that the super-resolution method can construct high quality DEMs and allows for identifying hazards like rocks and craters within the accordance of ALHAT requirements.

A super-resolution algorithm for enhancement of flash lidar data: flight test results

NASA Astrophysics Data System (ADS)

Bulyshev, Alexander; Amzajerdian, Farzin; Roback, Eric; Reisse, Robert

2013-03-01

This paper describes the results of a 3D super-resolution algorithm applied to the range data obtained from a recent Flash Lidar helicopter flight test. The flight test was conducted by the NASA's Autonomous Landing and Hazard Avoidance Technology (ALHAT) project over a simulated lunar terrain facility at NASA Kennedy Space Center. ALHAT is developing the technology for safe autonomous landing on the surface of celestial bodies: Moon, Mars, asteroids. One of the test objectives was to verify the ability of 3D super-resolution technique to generate high resolution digital elevation models (DEMs) and to determine time resolved relative positions and orientations of the vehicle. 3D super-resolution algorithm was developed earlier and tested in computational modeling, and laboratory experiments, and in a few dynamic experiments using a moving truck. Prior to the helicopter flight test campaign, a 100mX100m hazard field was constructed having most of the relevant extraterrestrial hazard: slopes, rocks, and craters with different sizes. Data were collected during the flight and then processed by the super-resolution code. The detailed DEM of the hazard field was constructed using independent measurement to be used for comparison. ALHAT navigation system data were used to verify abilities of super-resolution method to provide accurate relative navigation information. Namely, the 6 degree of freedom state vector of the instrument as a function of time was restored from super-resolution data. The results of comparisons show that the super-resolution method can construct high quality DEMs and allows for identifying hazards like rocks and craters within the accordance of ALHAT requirements.

Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging

NASA Astrophysics Data System (ADS)

Errico, Claudia; Pierre, Juliette; Pezet, Sophie; Desailly, Yann; Lenkei, Zsolt; Couture, Olivier; Tanter, Mickael

2015-11-01

Non-invasive imaging deep into organs at microscopic scales remains an open quest in biomedical imaging. Although optical microscopy is still limited to surface imaging owing to optical wave diffusion and fast decorrelation in tissue, revolutionary approaches such as fluorescence photo-activated localization microscopy led to a striking increase in resolution by more than an order of magnitude in the last decade. In contrast with optics, ultrasonic waves propagate deep into organs without losing their coherence and are much less affected by in vivo decorrelation processes. However, their resolution is impeded by the fundamental limits of diffraction, which impose a long-standing trade-off between resolution and penetration. This limits clinical and preclinical ultrasound imaging to a sub-millimetre scale. Here we demonstrate in vivo that ultrasound imaging at ultrafast frame rates (more than 500 frames per second) provides an analogue to optical localization microscopy by capturing the transient signal decorrelation of contrast agents—inert gas microbubbles. Ultrafast ultrasound localization microscopy allowed both non-invasive sub-wavelength structural imaging and haemodynamic quantification of rodent cerebral microvessels (less than ten micrometres in diameter) more than ten millimetres below the tissue surface, leading to transcranial whole-brain imaging within short acquisition times (tens of seconds). After intravenous injection, single echoes from individual microbubbles were detected through ultrafast imaging. Their localization, not limited by diffraction, was accumulated over 75,000 images, yielding 1,000,000 events per coronal plane and statistically independent pixels of ten micrometres in size. Precise temporal tracking of microbubble positions allowed us to extract accurately in-plane velocities of the blood flow with a large dynamic range (from one millimetre per second to several centimetres per second). These results pave the way for deep non-invasive microscopy in animals and humans using ultrasound. We anticipate that ultrafast ultrasound localization microscopy may become an invaluable tool for the fundamental understanding and diagnostics of various disease processes that modify the microvascular blood flow, such as cancer, stroke and arteriosclerosis.

Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging.

PubMed

Errico, Claudia; Pierre, Juliette; Pezet, Sophie; Desailly, Yann; Lenkei, Zsolt; Couture, Olivier; Tanter, Mickael

2015-11-26

Non-invasive imaging deep into organs at microscopic scales remains an open quest in biomedical imaging. Although optical microscopy is still limited to surface imaging owing to optical wave diffusion and fast decorrelation in tissue, revolutionary approaches such as fluorescence photo-activated localization microscopy led to a striking increase in resolution by more than an order of magnitude in the last decade. In contrast with optics, ultrasonic waves propagate deep into organs without losing their coherence and are much less affected by in vivo decorrelation processes. However, their resolution is impeded by the fundamental limits of diffraction, which impose a long-standing trade-off between resolution and penetration. This limits clinical and preclinical ultrasound imaging to a sub-millimetre scale. Here we demonstrate in vivo that ultrasound imaging at ultrafast frame rates (more than 500 frames per second) provides an analogue to optical localization microscopy by capturing the transient signal decorrelation of contrast agents--inert gas microbubbles. Ultrafast ultrasound localization microscopy allowed both non-invasive sub-wavelength structural imaging and haemodynamic quantification of rodent cerebral microvessels (less than ten micrometres in diameter) more than ten millimetres below the tissue surface, leading to transcranial whole-brain imaging within short acquisition times (tens of seconds). After intravenous injection, single echoes from individual microbubbles were detected through ultrafast imaging. Their localization, not limited by diffraction, was accumulated over 75,000 images, yielding 1,000,000 events per coronal plane and statistically independent pixels of ten micrometres in size. Precise temporal tracking of microbubble positions allowed us to extract accurately in-plane velocities of the blood flow with a large dynamic range (from one millimetre per second to several centimetres per second). These results pave the way for deep non-invasive microscopy in animals and humans using ultrasound. We anticipate that ultrafast ultrasound localization microscopy may become an invaluable tool for the fundamental understanding and diagnostics of various disease processes that modify the microvascular blood flow, such as cancer, stroke and arteriosclerosis.

Super-resolved linear fluorescence localization microscopy using photostable fluorophores: A virtual microscopy study

NASA Astrophysics Data System (ADS)

Birk, Udo; Szczurek, Aleksander; Cremer, Christoph

2017-12-01

Current approaches to overcome the conventional limit of the resolution potential of light microscopy (of about 200 nm for visible light), often suffer from non-linear effects, which render the quantification of the image intensities in the reconstructions difficult, and also affect the quantification of the biological structure under investigation. As an attempt to face these difficulties, we discuss a particular method of localization microscopy which is based on photostable fluorescent dyes. The proposed method can potentially be implemented as a fast alternative for quantitative localization microscopy, circumventing the need for the acquisition of thousands of image frames and complex, highly dye-specific imaging buffers. Although the need for calibration remains in order to extract quantitative data (such as the number of emitters), multispectral approaches are largely facilitated due to the much less stringent requirements on imaging buffers. Furthermore, multispectral acquisitions can be readily obtained using commercial instrumentation such as e.g. the conventional confocal laser scanning microscope.

Using 3D Super-Resolution Microscopy to Probe Breast Cancer Stem Cells and Their Microenvironment

DTIC Science & Technology

2014-05-01

cultured on gelatin substrates. This imaging was the first of its kind for breast cancer. We also small controllable microenvironments, in which we can...pyruvate at 37 °C and 5% CO2. Cleaned coverslips were coated with gelatin attachment factor from Invitrogen and incubated at room temperature for 2 hours...or 37C for 30-45 min, and washed with media. The MCF7 cells were dissociated from their culture plate using enzyme-free dissociation buffer from

A fast- and positively photoswitchable fluorescent protein for ultralow-laser-power RESOLFT nanoscopy.

PubMed

Tiwari, Dhermendra K; Arai, Yoshiyuki; Yamanaka, Masahito; Matsuda, Tomoki; Agetsuma, Masakazu; Nakano, Masahiro; Fujita, Katsumasa; Nagai, Takeharu

2015-06-01

Fluorescence nanoscopy has revolutionized our ability to visualize biological structures not resolvable by conventional microscopy. However, photodamage induced by intense light exposure has limited its use in live specimens. Here we describe Kohinoor, a fast-switching, positively photoswitchable fluorescent protein, and show that it has high photostability over many switching repeats. With Kohinoor, we achieved super-resolution imaging of live HeLa cells using biocompatible, ultralow laser intensity (0.004 J/cm(2)) in reversible saturable optical fluorescence transition (RESOLFT) nanoscopy.

Single-shot and single-sensor high/super-resolution microwave imaging based on metasurface.

PubMed

Wang, Libo; Li, Lianlin; Li, Yunbo; Zhang, Hao Chi; Cui, Tie Jun

2016-06-01

Real-time high-resolution (including super-resolution) imaging with low-cost hardware is a long sought-after goal in various imaging applications. Here, we propose broadband single-shot and single-sensor high-/super-resolution imaging by using a spatio-temporal dispersive metasurface and an imaging reconstruction algorithm. The metasurface with spatio-temporal dispersive property ensures the feasibility of the single-shot and single-sensor imager for super- and high-resolution imaging, since it can convert efficiently the detailed spatial information of the probed object into one-dimensional time- or frequency-dependent signal acquired by a single sensor fixed in the far-field region. The imaging quality can be improved by applying a feature-enhanced reconstruction algorithm in post-processing, and the desired imaging resolution is related to the distance between the object and metasurface. When the object is placed in the vicinity of the metasurface, the super-resolution imaging can be realized. The proposed imaging methodology provides a unique means to perform real-time data acquisition, high-/super-resolution images without employing expensive hardware (e.g. mechanical scanner, antenna array, etc.). We expect that this methodology could make potential breakthroughs in the areas of microwave, terahertz, optical, and even ultrasound imaging.

Nanoscale Spatial Organization of Prokaryotic Cells Studied by Super-Resolution Optical Microscopy

NASA Astrophysics Data System (ADS)

McEvoy, Andrea Lynn

All cells spatially organize their interiors, and this arrangement is necessary for cell viability. Until recently, it was believed that only eukaryotic cells spatially segregate their components. However, it is becoming increasingly clear that bacteria also assemble their proteins into complex patterns. In eukaryotic cells, spatial organization arises from membrane bound organelles as well as motor transport proteins which can move cargos within the cell. To date, there are no known motor transport proteins in bacteria and most microbes lack membrane bound organelles, so it remains a mystery how bacterial spatial organization emerges. In hind-sight it is not surprising that bacteria also exhibit complex spatial organization considering much of what we have learned about the basic processes that take place in all cells, such as transcription and translation was first discovered in prokaryotic cells. Perhaps the fundamental principles that govern spatial organization in prokaryotic cells may be applicable in eukaryotic cells as well. In addition, bacteria are attractive model organism for spatial organization studies because they are genetically tractable, grow quickly and much biochemical and structural data is known about them. A powerful tool for observing spatial organization in cells is the fluorescence microscope. By specifically tagging a protein of interest with a fluorescent probe, it is possible to examine how proteins organize and dynamically assemble inside cells. A significant disadvantage of this technology is its spatial resolution (approximately 250 nm laterally and 500 nm axially). This limitation on resolution causes closely spaced proteins to look blurred making it difficult to observe the fine structure within the complexes. This resolution limit is especially problematic within small cells such as bacteria. With the recent invention of new optical microscopies, we now can surpass the existing limits of fluorescence imaging. In some cases, we can now see individual proteins inside of large complexes or observe structures with ten times the resolution of conventional imaging. These techniques are known as super-resolution microscopes. In this dissertation, I use super-resolution microscopes to understand how a model microbe, Escherichia coli, assembles complex protein structures. I focus on two spatially organized systems, the chemotaxis network and the cell division machinery. These assembly mechanisms could be general mechanisms for protein assembly in all organisms. I also characterize new fluorescent probes for use in multiple super-resolution imaging modalities and discuss the practicalities of using different super-resolution microscopes. The chemotaxis network in E. coli is the best understood signal transduction network in biology. Chemotaxis receptors cluster into complexes of thousands of proteins located at the cell poles and are used to move bacteria towards favorable stimuli in the environment. In these dense clusters, the receptors can bind each other and communicate to filter out noise and amplify weak signals. It is surprising that chemotaxis receptors are spatially segregated and the mechanism for polar localization of these complexes remains unclear. Using data from PALM images, we develop a model to understand how bacteria organize their receptors into large clusters. The model, stochastic cluster nucleation, is surprising in that is generates micron-scale periodic patterns without the need for accessory proteins to provide scaffolding or active transport. This model may be a general mechanism that cells utilize to organize small and large complexes of proteins. During cell division, E. coli must elongate, replicate its DNA and position its components properly prior to binary fission. Prior to septum formation, a ubiquitous protein called FtsZ, assembles into a ring at mid-cell (Z-ring) which constricts during cell division and recruits the remaining proteins necessary for cytokinesis. Though many details have been revealed about FtsZ, the detailed in vivo structure of the Z-ring is not well understood, and many questions remain about how ring constriction occurs. Using multiple super-resolution imaging modalities, in combination with conventional time-lapse fluorescence imaging, we show that the Z-ring does not form a long uniform filament around the circumference of the bacterium. We detail how this structure changes during division and how removal of proteins that help to position FtsZ affects the Z-ring as it proceeds through cytokinesis. Ultimately we present a simple model for Z-ring constriction during division.

Selective suppression of CARS signal with three-beam competing stimulated Raman scattering processes.

PubMed

Choi, Dae Sik; Rao, B Jayachander; Kim, Doyeon; Shim, Sang-Hee; Rhee, Hanju; Cho, Minhaeng

2018-06-14

Coherent Raman scattering spectroscopy and microscopy are useful methods for studying the chemical and biological structures of molecules with Raman-active modes. In particular, coherent anti-Stokes Raman scattering (CARS) microscopy, which is a label-free method capable of imaging structures by displaying the vibrational contrast of the molecules, has been widely used. However, the lack of a technique for switching-off the CARS signal has prevented the development of the super-resolution Raman imaging method. Here, we demonstrate that a selective suppression of the CARS signal is possible by using a three-beam double stimulated Raman scattering (SRS) scheme; the three beams are the pump, Stokes, and depletion lights in order of frequency. Both pump-Stokes and pump-depletion beam pairs can generate SRS processes by tuning their beat frequencies to match two different vibrational modes, then two CARS signals induced by pump-Stokes-pump and pump-depletion-pump interactions can be generated, where the two CARS signals are coupled with each other because they both involve interactions with the common pump beam. Herein, we show that as the intensity of the depletion beam is increased, one can selectively suppress the pump-Stokes-pump CARS signal because the pump-depletion SRS depletes the pump photons. A detailed theoretical description of the coupled differential equations for the three incident fields and the generated CARS signal fields is presented. Taking benzene as a molecular system, we obtained a maximum CARS suppression efficiency of about 97% with our experimental scheme, where the ring breathing mode of the benzene is associated with pump-Stokes-pump CARS, while the C-H stretching mode is associated with the competing pump-depletion SRS process. We anticipate that this selective switching-off scheme will be of use in developing super-resolution label-free CARS microscopy.

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies

PubMed Central

Hoischen, Christian; Monajembashi, Shamci; Weisshart, Klaus; Hemmerich, Peter

2018-01-01

The promyelocytic leukemia (pml) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology. PMID:29888200

ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging

PubMed Central

Ovesný, Martin; Křížek, Pavel; Borkovec, Josef; Švindrych, Zdeněk; Hagen, Guy M.

2014-01-01

Summary: ThunderSTORM is an open-source, interactive and modular plug-in for ImageJ designed for automated processing, analysis and visualization of data acquired by single-molecule localization microscopy methods such as photo-activated localization microscopy and stochastic optical reconstruction microscopy. ThunderSTORM offers an extensive collection of processing and post-processing methods so that users can easily adapt the process of analysis to their data. ThunderSTORM also offers a set of tools for creation of simulated data and quantitative performance evaluation of localization algorithms using Monte Carlo simulations. Availability and implementation: ThunderSTORM and the online documentation are both freely accessible at https://code.google.com/p/thunder-storm/ Contact: guy.hagen@lf1.cuni.cz Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24771516

The nano-architecture of the axonal cytoskeleton.

PubMed

Leterrier, Christophe; Dubey, Pankaj; Roy, Subhojit

2017-12-01

The corporeal beauty of the neuronal cytoskeleton has captured the imagination of generations of scientists. One of the easiest cellular structures to visualize by light microscopy, its existence has been known for well over 100 years, yet we have only recently begun to fully appreciate its intricacy and diversity. Recent studies combining new probes with super-resolution microscopy and live imaging have revealed surprising details about the axonal cytoskeleton and, in particular, have discovered previously unknown actin-based structures. Along with traditional electron microscopy, these newer techniques offer a nanoscale view of the axonal cytoskeleton, which is important for our understanding of neuronal form and function, and lay the foundation for future studies. In this Review, we summarize existing concepts in the field and highlight contemporary discoveries that have fundamentally altered our perception of the axonal cytoskeleton.

On Super-Resolution and the MUSIC Algorithm,

DTIC Science & Technology

1985-05-01

SUPER-RESOLUTION AND THE MUSIC ALGORITHM AUTHOR: G D de Villiers DATE: May 1985 SUMMARY Simulation results for phased array signal processing using...the MUSIC algorithm are presented. The model used is more realistic than previous ones and it gives an indication as to how the algorithm would perform...resolution ON SUPER-RESOLUTION AND THE MUSIC ALGORITHM 1. INTRODUCTION At present there is a considerable amount of interest in "high-resolution" b

Image resolution enhancement via image restoration using neural network

NASA Astrophysics Data System (ADS)

Zhang, Shuangteng; Lu, Yihong

2011-04-01

Image super-resolution aims to obtain a high-quality image at a resolution that is higher than that of the original coarse one. This paper presents a new neural network-based method for image super-resolution. In this technique, the super-resolution is considered as an inverse problem. An observation model that closely follows the physical image acquisition process is established to solve the problem. Based on this model, a cost function is created and minimized by a Hopfield neural network to produce high-resolution images from the corresponding low-resolution ones. Not like some other single frame super-resolution techniques, this technique takes into consideration point spread function blurring as well as additive noise and therefore generates high-resolution images with more preserved or restored image details. Experimental results demonstrate that the high-resolution images obtained by this technique have a very high quality in terms of PSNR and visually look more pleasant.

Tackling the Challenges of Dynamic Experiments Using Liquid-Cell Transmission Electron Microscopy.

PubMed

Parent, Lucas R; Bakalis, Evangelos; Proetto, Maria; Li, Yiwen; Park, Chiwoo; Zerbetto, Francesco; Gianneschi, Nathan C

2018-01-16

Revolutions in science and engineering frequently result from the development, and wide adoption, of a new, powerful characterization or imaging technique. Beginning with the first glass lenses and telescopes in astronomy, to the development of visual-light microscopy, staining techniques, confocal microscopy, and fluorescence super-resolution microscopy in biology, and most recently aberration-corrected, cryogenic, and ultrafast (4D) electron microscopy, X-ray microscopy, and scanning probe microscopy in nanoscience. Through these developments, our perception and understanding of the physical nature of matter at length-scales beyond ordinary perception have been fundamentally transformed. Despite this progression in microscopy, techniques for observing nanoscale chemical processes and solvated/hydrated systems are limited, as the necessary spatial and temporal resolution presents significant technical challenges. However, the standard reliance on indirect or bulk phase characterization of nanoscale samples in liquids is undergoing a shift in recent times with the realization ( Williamson et al. Nat. Mater . 2003 , 2 , 532 - 536 ) of liquid-cell (scanning) transmission electron microscopy, LC(S)TEM, where picoliters of solution are hermetically sealed between electron-transparent "windows," which can be directly imaged or videoed at the nanoscale using conventional transmission electron microscopes. This Account seeks to open a discussion on the topic of standardizing strategies for conducting imaging experiments with a view to characterizing dynamics and motion of nanoscale materials. This is a challenge that could be described by critics and proponents alike, as analogous to doing chemistry in a lightning storm; where the nature of the solution, the nanomaterial, and the dynamic behaviors are all potentially subject to artifactual influence by the very act of our observation.

Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex.

PubMed

Ma, Jiong; Kelich, Joseph M; Junod, Samuel L; Yang, Weidong

2017-04-01

The nuclear pore complex (NPC), composed of ∼30 different nucleoporins (Nups), is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. However, the precise copy number and the spatial location of each Nup in the native NPC remain obscure due to the inherent difficulty of counting and localizing proteins inside of the sub-micrometer supramolecular complex. Here, we combined super-resolution single-point edge-excitation subdiffraction (SPEED) microscopy and nanobody-specific labeling to reveal the spatial distribution of scaffold Nups within three separate layers in the native NPC with a precision of ∼3 nm. Our data reveal both the radial and axial spatial distributions for Pom121, Nup37 and Nup35 and provide evidence for their copy numbers of 8, 32 and 16, respectively, per NPC. This approach can help pave the path for mapping the entirety of Nups in native NPCs and also other structural components of macromolecular complexes. © 2017. Published by The Company of Biologists Ltd.

Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex

PubMed Central

Ma, Jiong; Kelich, Joseph M.; Junod, Samuel L.

2017-01-01

ABSTRACT The nuclear pore complex (NPC), composed of ∼30 different nucleoporins (Nups), is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. However, the precise copy number and the spatial location of each Nup in the native NPC remain obscure due to the inherent difficulty of counting and localizing proteins inside of the sub-micrometer supramolecular complex. Here, we combined super-resolution single-point edge-excitation subdiffraction (SPEED) microscopy and nanobody-specific labeling to reveal the spatial distribution of scaffold Nups within three separate layers in the native NPC with a precision of ∼3 nm. Our data reveal both the radial and axial spatial distributions for Pom121, Nup37 and Nup35 and provide evidence for their copy numbers of 8, 32 and 16, respectively, per NPC. This approach can help pave the path for mapping the entirety of Nups in native NPCs and also other structural components of macromolecular complexes. PMID:28202688

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

Chen, Guanqun; Zou, Ningmu; Chen, Bo

Compared with their monometallic counterparts, bimetallic nanoparticles often show enhanced catalytic activity associated with the bimetallic interface. Direct quantitation of catalytic activity at the bimetallic interface is important for understanding the enhancement mechanism, but challenging experimentally. Here using single-molecule super-resolution catalysis imaging in correlation with electron microscopy, we report the first quantitative visualization of enhanced bimetallic activity within single bimetallic nanoparticles. We focus on heteronuclear bimetallic PdAu nanoparticles that present a well-defined Pd–Au bimetallic interface in catalyzing a photodriven fluorogenic disproportionation reaction. Our approach also enables a direct comparison between the bimetallic and monometallic regions within the same nanoparticle. Theoreticalmore » calculations further provide insights into the electronic nature of N–O bond activation of the reactant (resazurin) adsorbed on bimetallic sites. Subparticle activity correlation between bimetallic enhancement and monometallic activity suggests that the favorable locations to construct bimetallic sites are those monometallic sites with higher activity, leading to a strategy for making effective bimetallic nanocatalysts. Furthermore, the results highlight the power of super-resolution catalysis imaging in gaining insights that could help improve nanocatalysts.« less

Super-Resolution Enhancement From Multiple Overlapping Images: A Fractional Area Technique

NASA Astrophysics Data System (ADS)

Michaels, Joshua A.

With the availability of large quantities of relatively low-resolution data from several decades of space borne imaging, methods of creating an accurate, higher-resolution image from the multiple lower-resolution images (i.e. super-resolution), have been developed almost since such imagery has been around. The fractional-area super-resolution technique developed in this thesis has never before been documented. Satellite orbits, like Landsat, have a quantifiable variation, which means each image is not centered on the exact same spot more than once and the overlapping information from these multiple images may be used for super-resolution enhancement. By splitting a single initial pixel into many smaller, desired pixels, a relationship can be created between them using the ratio of the area within the initial pixel. The ideal goal for this technique is to obtain smaller pixels with exact values and no error, yielding a better potential result than those methods that yield interpolated pixel values with consequential loss of spatial resolution. A Fortran 95 program was developed to perform all calculations associated with the fractional-area super-resolution technique. The fractional areas are calculated using traditional trigonometry and coordinate geometry and Linear Algebra Package (LAPACK; Anderson et al., 1999) is used to solve for the higher-resolution pixel values. In order to demonstrate proof-of-concept, a synthetic dataset was created using the intrinsic Fortran random number generator and Adobe Illustrator CS4 (for geometry). To test the real-life application, digital pictures from a Sony DSC-S600 digital point-and-shoot camera with a tripod were taken of a large US geological map under fluorescent lighting. While the fractional-area super-resolution technique works in perfect synthetic conditions, it did not successfully produce a reasonable or consistent solution in the digital photograph enhancement test. The prohibitive amount of processing time (up to 60 days for a relatively small enhancement area) severely limits the practical usefulness of fraction-area super-resolution. Fractional-area super-resolution is very sensitive to relative input image co-registration, which must be accurate to a sub-pixel degree. However, use of this technique, if input conditions permit, could be applied as a "pinpoint" super-resolution technique. Such an application could be possible by only applying it to only very small areas with very good input image co-registration.

Signal Characteristics of Super-Resolution Near-Field Structure Disks with 100 GB Capacity

NASA Astrophysics Data System (ADS)

Kim, Jooho; Hwang, Inoh; Kim, Hyunki; Park, Insik; Tominaga, Junji

2005-05-01

We report the basic characteristics of super resolution near-field structure (Super-RENS) media at a blue laser optical system (laser wavelength 405 nm, numerical aperture 0.85). Using a novel write once read many (WORM) structure for a blue laser system, we obtained a carrier-to-noise ratio (CNR) above 33 dB from the signal of the 37.5 nm mark length, which is equivalent to a 100 GB capacity with a 0.32 micrometer track pitch, and an eye pattern for 50 GB (2T: 75 nm) capacity using a patterned signal. Using a novel super-resolution material (tellurium, Te) with low super-resolution readout power, we also improved the read stability.

High Resolution Bathymetry Estimation Improvement with Single Image Super-Resolution Algorithm Super-Resolution Forests

DTIC Science & Technology

2017-01-26

Naval Research Laboratory Washington, DC 20375-5320 NRL/MR/5514--17-9692 High Resolution Bathymetry Estimation Improvement with Single Image Super...collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources...gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate

Single-shot and single-sensor high/super-resolution microwave imaging based on metasurface

PubMed Central

Wang, Libo; Li, Lianlin; Li, Yunbo; Zhang, Hao Chi; Cui, Tie Jun

2016-01-01

Real-time high-resolution (including super-resolution) imaging with low-cost hardware is a long sought-after goal in various imaging applications. Here, we propose broadband single-shot and single-sensor high-/super-resolution imaging by using a spatio-temporal dispersive metasurface and an imaging reconstruction algorithm. The metasurface with spatio-temporal dispersive property ensures the feasibility of the single-shot and single-sensor imager for super- and high-resolution imaging, since it can convert efficiently the detailed spatial information of the probed object into one-dimensional time- or frequency-dependent signal acquired by a single sensor fixed in the far-field region. The imaging quality can be improved by applying a feature-enhanced reconstruction algorithm in post-processing, and the desired imaging resolution is related to the distance between the object and metasurface. When the object is placed in the vicinity of the metasurface, the super-resolution imaging can be realized. The proposed imaging methodology provides a unique means to perform real-time data acquisition, high-/super-resolution images without employing expensive hardware (e.g. mechanical scanner, antenna array, etc.). We expect that this methodology could make potential breakthroughs in the areas of microwave, terahertz, optical, and even ultrasound imaging. PMID:27246668

Introduction to the virtual special issue on super-resolution imaging techniques

NASA Astrophysics Data System (ADS)

Cao, Liangcai; Liu, Zhengjun

2017-12-01

Until quite recently, the resolution of optical imaging instruments, including telescopes, cameras and microscopes, was considered to be limited by the diffraction of light and by image sensors. In the past few years, many exciting super-resolution approaches have emerged that demonstrate intriguing ways to bypass the classical limit in optics and detectors. More and more research groups are engaged in the study of advanced super-resolution schemes, devices, algorithms, systems, and applications [1-6]. Super-resolution techniques involve new methods in science and engineering of optics [7,8], measurements [9,10], chemistry [11,12] and information [13,14]. Promising applications, particularly in biomedical research and semiconductor industry, have been successfully demonstrated.

Measuring the performance of super-resolution reconstruction algorithms

NASA Astrophysics Data System (ADS)

Dijk, Judith; Schutte, Klamer; van Eekeren, Adam W. M.; Bijl, Piet

2012-06-01

For many military operations situational awareness is of great importance. This situational awareness and related tasks such as Target Acquisition can be acquired using cameras, of which the resolution is an important characteristic. Super resolution reconstruction algorithms can be used to improve the effective sensor resolution. In order to judge these algorithms and the conditions under which they operate best, performance evaluation methods are necessary. This evaluation, however, is not straightforward for several reasons. First of all, frequency-based evaluation techniques alone will not provide a correct answer, due to the fact that they are unable to discriminate between structure-related and noise-related effects. Secondly, most super-resolution packages perform additional image enhancement techniques such as noise reduction and edge enhancement. As these algorithms improve the results they cannot be evaluated separately. Thirdly, a single high-resolution ground truth is rarely available. Therefore, evaluation of the differences in high resolution between the estimated high resolution image and its ground truth is not that straightforward. Fourth, different artifacts can occur due to super-resolution reconstruction, which are not known on forehand and hence are difficult to evaluate. In this paper we present a set of new evaluation techniques to assess super-resolution reconstruction algorithms. Some of these evaluation techniques are derived from processing on dedicated (synthetic) imagery. Other evaluation techniques can be evaluated on both synthetic and natural images (real camera data). The result is a balanced set of evaluation algorithms that can be used to assess the performance of super-resolution reconstruction algorithms.

Oblique reconstructions in tomosynthesis. II. Super-resolution

PubMed Central

Acciavatti, Raymond J.; Maidment, Andrew D. A.

2013-01-01

Purpose: In tomosynthesis, super-resolution has been demonstrated using reconstruction planes parallel to the detector. Super-resolution allows for subpixel resolution relative to the detector. The purpose of this work is to develop an analytical model that generalizes super-resolution to oblique reconstruction planes. Methods: In a digital tomosynthesis system, a sinusoidal test object is modeled along oblique angles (i.e., “pitches”) relative to the plane of the detector in a 3D divergent-beam acquisition geometry. To investigate the potential for super-resolution, the input frequency is specified to be greater than the alias frequency of the detector. Reconstructions are evaluated in an oblique plane along the extent of the object using simple backprojection (SBP) and filtered backprojection (FBP). By comparing the amplitude of the reconstruction against the attenuation coefficient of the object at various frequencies, the modulation transfer function (MTF) is calculated to determine whether modulation is within detectable limits for super-resolution. For experimental validation of super-resolution, a goniometry stand was used to orient a bar pattern phantom along various pitches relative to the breast support in a commercial digital breast tomosynthesis system. Results: Using theoretical modeling, it is shown that a single projection image cannot resolve a sine input whose frequency exceeds the detector alias frequency. The high frequency input is correctly visualized in SBP or FBP reconstruction using a slice along the pitch of the object. The Fourier transform of this reconstructed slice is maximized at the input frequency as proof that the object is resolved. Consistent with the theoretical results, experimental images of a bar pattern phantom showed super-resolution in oblique reconstructions. At various pitches, the highest frequency with detectable modulation was determined by visual inspection of the bar patterns. The dependency of the highest detectable frequency on pitch followed the same trend as the analytical model. It was demonstrated that super-resolution is not achievable if the pitch of the object approaches 90°, corresponding to the case in which the test frequency is perpendicular to the breast support. Only low frequency objects are detectable at pitches close to 90°. Conclusions: This work provides a platform for investigating super-resolution in oblique reconstructions for tomosynthesis. In breast imaging, this study should have applications in visualizing microcalcifications and other subtle signs of cancer. PMID:24320445

Resolution enhancement of pump-probe microscope with an inverse-annular filter

NASA Astrophysics Data System (ADS)

Kobayashi, Takayoshi; Kawasumi, Koshi; Miyazaki, Jun; Nakata, Kazuaki

2018-04-01

Optical pump-probe microscopy can provide images by detecting changes in probe light intensity induced by stimulated emission, photoinduced absorbance change, or photothermal-induced refractive index change in either transmission or reflection mode. Photothermal microscopy, which is one type of optical pump-probe microscopy, has intrinsically super resolution capability due to the bilinear dependence of signal intensity of pump and probe. We introduce new techniques for further resolution enhancement and fast imaging in photothermal microscope. First, we introduce a new pupil filter, an inverse-annular pupil filter in a pump-probe photothermal microscope, which provides resolution enhancement in three dimensions. The resolutions are proved to be improved in lateral and axial directions by imaging experiment using 20-nm gold nanoparticles. The improvement in X (perpendicular to the common pump and probe polarization direction), Y (parallel to the polarization direction), and Z (axial direction) are by 15 ± 6, 8 ± 8, and 21 ± 2% from the resolution without a pupil filter. The resolution enhancement is even better than the calculation using vector field, which predicts the corresponding enhancement of 11, 8, and 6%. The discussion is made to explain the unexpected results. We also demonstrate the photothermal imaging of thick biological samples (cells from rabbit intestine and kidney) stained with hematoxylin and eosin dye with the inverse-annular filter. Second, a fast, high-sensitivity photothermal microscope is developed by implementing a spatially segmented balanced detection scheme into a laser scanning microscope using a Galvano mirror. We confirm a 4.9 times improvement in signal-to-noise ratio in the spatially segmented balanced detection compared with that of conventional detection. The system demonstrates simultaneous bi-modal photothermal and confocal fluorescence imaging of transgenic mouse brain tissue with a pixel dwell time of 20 µs. The fluorescence image visualizes neurons expressing yellow fluorescence proteins, while the photothermal signal detected endogenous chromophores in the mouse brain, allowing 3D visualization of the distribution of various features such as blood cells and fine structures most probably due to lipids. This imaging modality was constructed using compact and cost-effective laser diodes, and will thus be widely useful in the life and medical sciences. Third, we have made further resolution improvement of high-sensitivity laser scanning photothermal microscopy by applying non-linear detection. By this, the new method has super resolution with 61 and 42% enhancement from the diffraction limit values of the probe and pump wavelengths, respectively, by a second-order non-linear scheme and a high-frame rate in a laser scanning microscope. The maximum resolution is determined to be 160 nm in the second-order non-linear detection mode and 270 nm in the linear detection mode by the PT signal of GNPs. The pixel rate and frame rate for 300 × 300 pixel image are 50 µs and 4.5 s, respectively. The pixel and frame rate are shorter than the rates, those are 1 ms and 100 s, using the piezo-driven stage system.

Super-Resolution of Plant Disease Images for the Acceleration of Image-based Phenotyping and Vigor Diagnosis in Agriculture.

PubMed

Yamamoto, Kyosuke; Togami, Takashi; Yamaguchi, Norio

2017-11-06

Unmanned aerial vehicles (UAVs or drones) are a very promising branch of technology, and they have been utilized in agriculture-in cooperation with image processing technologies-for phenotyping and vigor diagnosis. One of the problems in the utilization of UAVs for agricultural purposes is the limitation in flight time. It is necessary to fly at a high altitude to capture the maximum number of plants in the limited time available, but this reduces the spatial resolution of the captured images. In this study, we applied a super-resolution method to the low-resolution images of tomato diseases to recover detailed appearances, such as lesions on plant organs. We also conducted disease classification using high-resolution, low-resolution, and super-resolution images to evaluate the effectiveness of super-resolution methods in disease classification. Our results indicated that the super-resolution method outperformed conventional image scaling methods in spatial resolution enhancement of tomato disease images. The results of disease classification showed that the accuracy attained was also better by a large margin with super-resolution images than with low-resolution images. These results indicated that our approach not only recovered the information lost in low-resolution images, but also exerted a beneficial influence on further image analysis. The proposed approach will accelerate image-based phenotyping and vigor diagnosis in the field, because it not only saves time to capture images of a crop in a cultivation field but also secures the accuracy of these images for further analysis.

Super-Resolution of Plant Disease Images for the Acceleration of Image-based Phenotyping and Vigor Diagnosis in Agriculture

PubMed Central

Togami, Takashi; Yamaguchi, Norio

2017-01-01

Unmanned aerial vehicles (UAVs or drones) are a very promising branch of technology, and they have been utilized in agriculture—in cooperation with image processing technologies—for phenotyping and vigor diagnosis. One of the problems in the utilization of UAVs for agricultural purposes is the limitation in flight time. It is necessary to fly at a high altitude to capture the maximum number of plants in the limited time available, but this reduces the spatial resolution of the captured images. In this study, we applied a super-resolution method to the low-resolution images of tomato diseases to recover detailed appearances, such as lesions on plant organs. We also conducted disease classification using high-resolution, low-resolution, and super-resolution images to evaluate the effectiveness of super-resolution methods in disease classification. Our results indicated that the super-resolution method outperformed conventional image scaling methods in spatial resolution enhancement of tomato disease images. The results of disease classification showed that the accuracy attained was also better by a large margin with super-resolution images than with low-resolution images. These results indicated that our approach not only recovered the information lost in low-resolution images, but also exerted a beneficial influence on further image analysis. The proposed approach will accelerate image-based phenotyping and vigor diagnosis in the field, because it not only saves time to capture images of a crop in a cultivation field but also secures the accuracy of these images for further analysis. PMID:29113104

High resolution computational on-chip imaging of biological samples using sparsity constraint (Conference Presentation)

NASA Astrophysics Data System (ADS)

Rivenson, Yair; Wu, Chris; Wang, Hongda; Zhang, Yibo; Ozcan, Aydogan

2017-03-01

Microscopic imaging of biological samples such as pathology slides is one of the standard diagnostic methods for screening various diseases, including cancer. These biological samples are usually imaged using traditional optical microscopy tools; however, the high cost, bulkiness and limited imaging throughput of traditional microscopes partially restrict their deployment in resource-limited settings. In order to mitigate this, we previously demonstrated a cost-effective and compact lens-less on-chip microscopy platform with a wide field-of-view of >20-30 mm^2. The lens-less microscopy platform has shown its effectiveness for imaging of highly connected biological samples, such as pathology slides of various tissue samples and smears, among others. This computational holographic microscope requires a set of super-resolved holograms acquired at multiple sample-to-sensor distances, which are used as input to an iterative phase recovery algorithm and holographic reconstruction process, yielding high-resolution images of the samples in phase and amplitude channels. Here we demonstrate that in order to reconstruct clinically relevant images with high resolution and image contrast, we require less than 50% of the previously reported nominal number of holograms acquired at different sample-to-sensor distances. This is achieved by incorporating a loose sparsity constraint as part of the iterative holographic object reconstruction. We demonstrate the success of this sparsity-based computational lens-less microscopy platform by imaging pathology slides of breast cancer tissue and Papanicolaou (Pap) smears.

Magnetic Resonance Super-resolution Imaging Measurement with Dictionary-optimized Sparse Learning

NASA Astrophysics Data System (ADS)

Li, Jun-Bao; Liu, Jing; Pan, Jeng-Shyang; Yao, Hongxun

2017-06-01

Magnetic Resonance Super-resolution Imaging Measurement (MRIM) is an effective way of measuring materials. MRIM has wide applications in physics, chemistry, biology, geology, medical and material science, especially in medical diagnosis. It is feasible to improve the resolution of MR imaging through increasing radiation intensity, but the high radiation intensity and the longtime of magnetic field harm the human body. Thus, in the practical applications the resolution of hardware imaging reaches the limitation of resolution. Software-based super-resolution technology is effective to improve the resolution of image. This work proposes a framework of dictionary-optimized sparse learning based MR super-resolution method. The framework is to solve the problem of sample selection for dictionary learning of sparse reconstruction. The textural complexity-based image quality representation is proposed to choose the optimal samples for dictionary learning. Comprehensive experiments show that the dictionary-optimized sparse learning improves the performance of sparse representation.

Breaking the acoustic diffraction barrier with localization optoacoustic tomography

NASA Astrophysics Data System (ADS)

Deán-Ben, X. Luís.; Razansky, Daniel

2018-02-01

Diffraction causes blurring of high-resolution features in images and has been traditionally associated to the resolution limit in light microscopy and other imaging modalities. The resolution of an imaging system can be generally assessed via its point spread function, corresponding to the image acquired from a point source. However, the precision in determining the position of an isolated source can greatly exceed the diffraction limit. By combining the estimated positions of multiple sources, localization-based imaging has resulted in groundbreaking methods such as super-resolution fluorescence optical microscopy and has also enabled ultrasound imaging of microvascular structures with unprecedented spatial resolution in deep tissues. Herein, we introduce localization optoacoustic tomography (LOT) and discuss on the prospects of using localization imaging principles in optoacoustic imaging. LOT was experimentally implemented by real-time imaging of flowing particles in 3D with a recently-developed volumetric optoacoustic tomography system. Provided the particles were separated by a distance larger than the diffraction-limited resolution, their individual locations could be accurately determined in each frame of the acquired image sequence and the localization image was formed by superimposing a set of points corresponding to the localized positions of the absorbers. The presented results demonstrate that LOT can significantly enhance the well-established advantages of optoacoustic imaging by breaking the acoustic diffraction barrier in deep tissues and mitigating artifacts due to limited-view tomographic acquisitions.

Detecting breast microcalcifications using super-resolution ultrasound imaging: a clinical study

NASA Astrophysics Data System (ADS)

Huang, Lianjie; Labyed, Yassin; Hanson, Kenneth; Sandoval, Daniel; Pohl, Jennifer; Williamson, Michael

2013-03-01

Imaging breast microcalcifications is crucial for early detection and diagnosis of breast cancer. It is challenging for current clinical ultrasound to image breast microcalcifications. However, new imaging techniques using data acquired with a synthetic-aperture ultrasound system have the potential to significantly improve ultrasound imaging. We recently developed a super-resolution ultrasound imaging method termed the phase-coherent multiple-signal classification (PC-MUSIC). This signal subspace method accounts for the phase response of transducer elements to improve image resolution. In this paper, we investigate the clinical feasibility of our super-resolution ultrasound imaging method for detecting breast microcalcifications. We use our custom-built, real-time synthetic-aperture ultrasound system to acquire breast ultrasound data for 40 patients whose mammograms show the presence of breast microcalcifications. We apply our super-resolution ultrasound imaging method to the patient data, and produce clear images of breast calcifications. Our super-resolution ultrasound PC-MUSIC imaging with synthetic-aperture ultrasound data can provide a new imaging modality for detecting breast microcalcifications in clinic without using ionizing radiation.

Demonstration of nanoimprinted hyperlens array for high-throughput sub-diffraction imaging

NASA Astrophysics Data System (ADS)

Byun, Minsueop; Lee, Dasol; Kim, Minkyung; Kim, Yangdoo; Kim, Kwan; Ok, Jong G.; Rho, Junsuk; Lee, Heon

2017-04-01

Overcoming the resolution limit of conventional optics is regarded as the most important issue in optical imaging science and technology. Although hyperlenses, super-resolution imaging devices based on highly anisotropic dispersion relations that allow the access of high-wavevector components, have recently achieved far-field sub-diffraction imaging in real-time, the previously demonstrated devices have suffered from the extreme difficulties of both the fabrication process and the non-artificial objects placement. This results in restrictions on the practical applications of the hyperlens devices. While implementing large-scale hyperlens arrays in conventional microscopy is desirable to solve such issues, it has not been feasible to fabricate such large-scale hyperlens array with the previously used nanofabrication methods. Here, we suggest a scalable and reliable fabrication process of a large-scale hyperlens device based on direct pattern transfer techniques. We fabricate a 5 cm × 5 cm size hyperlenses array and experimentally demonstrate that it can resolve sub-diffraction features down to 160 nm under 410 nm wavelength visible light. The array-based hyperlens device will provide a simple solution for much more practical far-field and real-time super-resolution imaging which can be widely used in optics, biology, medical science, nanotechnology and other closely related interdisciplinary fields.

Expansion Mini-Microscopy: An Enabling Alternative in Point-of-Care Diagnostics

PubMed Central

Zhang, Yu Shrike; Santiago, Grissel Trujillo-de; Alvarez, Mario Moisés; Schiff, Steven J.; Boyden, Edward S.; Khademhosseini, Ali

2017-01-01

Diagnostics play a significant role in health care. In the developing world and low-resource regions the utility for point-of-care (POC) diagnostics becomes even greater. This need has long been recognized, and diagnostic technology has seen tremendous progress with the development of portable instrumentation such as miniature imagers featuring low complexity and cost. However, such inexpensive devices have not been able to achieve a resolution sufficient for POC detection of pathogens at very small scales, such as single-cell parasites, bacteria, fungi, and viruses. To this end, expansion microscopy (ExM) is a recently developed technique that, by physically expanding preserved biological specimens through a chemical process, enables super-resolution imaging on conventional microscopes and improves imaging resolution of a given microscope without the need to modify the existing microscope hardware. Here we review recent advances in ExM and portable imagers, respectively, and discuss the rational combination of the two technologies, that we term expansion mini-microscopy (ExMM). In ExMM, the physical expansion of a biological sample followed by imaging on a mini-microscope achieves a resolution as high as that attainable by conventional high-end microscopes imaging non-expanded samples, at significant reduction in cost. We believe that this newly developed ExMM technique is likely to find widespread applications in POC diagnostics in resource-limited and remote regions by expanded-scale imaging of biological specimens that are otherwise not resolvable using low-cost imagers. PMID:29062977

Robust video super-resolution with registration efficiency adaptation

NASA Astrophysics Data System (ADS)

Zhang, Xinfeng; Xiong, Ruiqin; Ma, Siwei; Zhang, Li; Gao, Wen

2010-07-01

Super-Resolution (SR) is a technique to construct a high-resolution (HR) frame by fusing a group of low-resolution (LR) frames describing the same scene. The effectiveness of the conventional super-resolution techniques, when applied on video sequences, strongly relies on the efficiency of motion alignment achieved by image registration. Unfortunately, such efficiency is limited by the motion complexity in the video and the capability of adopted motion model. In image regions with severe registration errors, annoying artifacts usually appear in the produced super-resolution video. This paper proposes a robust video super-resolution technique that adapts itself to the spatially-varying registration efficiency. The reliability of each reference pixel is measured by the corresponding registration error and incorporated into the optimization objective function of SR reconstruction. This makes the SR reconstruction highly immune to the registration errors, as outliers with higher registration errors are assigned lower weights in the objective function. In particular, we carefully design a mechanism to assign weights according to registration errors. The proposed superresolution scheme has been tested with various video sequences and experimental results clearly demonstrate the effectiveness of the proposed method.

Structural and elemental changes in glioblastoma cells in situ: complementary imaging with high resolution visible light- and X-ray microscopy

DOE PAGES

Ducic, Tanja; Paunesku, Tatjana; Chen, Si; ...

2016-12-09

The glioblastoma (GBM) is characterized by a short median survival and an almost 100% tumor related mortality. GBM cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores application of X-ray and visible light microscopy to display the elemental and structural images of cells from 3 patient derived GMB samples and an established GMB cell line. Slight differences in elemental concentrations, in actin cytoskeleton organization and cell morphology were noted between all cells types by X-ray fluorescence and full field soft X-ray microscopy, as well as the Structured Illumination Super-resolution Microscope (SIM). Different samplemore » preparation approaches were used to match each imaging technique. While preparation for SIM included cell fixation and staining, intact frozen hydrated cells were used for the trace element imaging by hard X-ray fluorescence and exploration of the structural features by soft X-ray absorption tomography. In conclusion, each technique documented differences between samples with regard to morphology and elemental composition and underscored the importance of use of multiple patient derived samples for detailed GBM study.« less

Structural and elemental changes in glioblastoma cells in situ: complementary imaging with high resolution visible light- and X-ray microscopy

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

Ducic, Tanja; Paunesku, Tatjana; Chen, Si

The glioblastoma (GBM) is characterized by a short median survival and an almost 100% tumor related mortality. GBM cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores application of X-ray and visible light microscopy to display the elemental and structural images of cells from 3 patient derived GMB samples and an established GMB cell line. Slight differences in elemental concentrations, in actin cytoskeleton organization and cell morphology were noted between all cells types by X-ray fluorescence and full field soft X-ray microscopy, as well as the Structured Illumination Super-resolution Microscope (SIM). Different samplemore » preparation approaches were used to match each imaging technique. While preparation for SIM included cell fixation and staining, intact frozen hydrated cells were used for the trace element imaging by hard X-ray fluorescence and exploration of the structural features by soft X-ray absorption tomography. In conclusion, each technique documented differences between samples with regard to morphology and elemental composition and underscored the importance of use of multiple patient derived samples for detailed GBM study.« less

Imaging resolution and properties analysis of super resolution microscopy with parallel detection under different noise, detector and image restoration conditions

NASA Astrophysics Data System (ADS)

Yu, Zhongzhi; Liu, Shaocong; Sun, Shiyi; Kuang, Cuifang; Liu, Xu

2018-06-01

Parallel detection, which can use the additional information of a pinhole plane image taken at every excitation scan position, could be an efficient method to enhance the resolution of a confocal laser scanning microscope. In this paper, we discuss images obtained under different conditions and using different image restoration methods with parallel detection to quantitatively compare the imaging quality. The conditions include different noise levels and different detector array settings. The image restoration methods include linear deconvolution and pixel reassignment with Richard-Lucy deconvolution and with maximum-likelihood estimation deconvolution. The results show that the linear deconvolution share properties such as high-efficiency and the best performance under all different conditions, and is therefore expected to be of use for future biomedical routine research.

DNA origami-based standards for quantitative fluorescence microscopy.

PubMed

Schmied, Jürgen J; Raab, Mario; Forthmann, Carsten; Pibiri, Enrico; Wünsch, Bettina; Dammeyer, Thorben; Tinnefeld, Philip

2014-01-01

Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA origami is a new tool that provides a framework to place defined numbers of small molecules such as fluorescent dyes or proteins in a programmed geometry with nanometer precision. The flexibility and versatility in the design of DNA origami microscopy standards makes them ideally suited for the broad variety of emerging super-resolution microscopy methods. As DNA origami structures are durable and portable, they can become a universally available specimen to check the everyday functionality of a microscope. The standards are immobilized on a glass slide, and they can be imaged without further preparation and can be stored for up to 6 months. We describe a detailed protocol for the design, production and use of DNA origami microscopy standards, and we introduce a DNA origami rectangle, bundles and a nanopillar as fluorescent nanoscopic rulers. The protocol provides procedures for the design and realization of fluorescent marks on DNA origami structures, their production and purification, quality control, handling, immobilization, measurement and data analysis. The procedure can be completed in 1-2 d.

A smartphone-based chip-scale microscope using ambient illumination.

PubMed

Lee, Seung Ah; Yang, Changhuei

2014-08-21

Portable chip-scale microscopy devices can potentially address various imaging needs in mobile healthcare and environmental monitoring. Here, we demonstrate the adaptation of a smartphone's camera to function as a compact lensless microscope. Unlike other chip-scale microscopy schemes, this method uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is based on the shadow imaging technique where the sample is placed on the surface of the image sensor, which captures direct shadow images under illumination. To improve the image resolution beyond the pixel size, we perform pixel super-resolution reconstruction with multiple images at different angles of illumination, which are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. The lensless imaging scheme allows for sub-micron resolution imaging over an ultra-wide field-of-view (FOV). Image acquisition and reconstruction are performed on the device using a custom-built Android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.

A smartphone-based chip-scale microscope using ambient illumination

PubMed Central

Lee, Seung Ah; Yang, Changhuei

2014-01-01

Portable chip-scale microscopy devices can potentially address various imaging needs in mobile healthcare and environmental monitoring. Here, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope. Unlike other chip-scale microscopy schemes, this method uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is based on the shadow imaging technique where the sample is placed on the surface of the image sensor, which captures direct shadow images under illumination. To improve the imaging resolution beyond the pixel size, we perform pixel super-resolution reconstruction with multiple images at different angles of illumination, which are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. The lensless imaging scheme allows for sub-micron resolution imaging over an ultra-wide field-of-view (FOV). Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system. PMID:24964209

Computational microscopy: illumination coding and nonlinear optimization enables gigapixel 3D phase imaging

NASA Astrophysics Data System (ADS)

Tian, Lei; Waller, Laura

2017-05-01

Microscope lenses can have either large field of view (FOV) or high resolution, not both. Computational microscopy based on illumination coding circumvents this limit by fusing images from different illumination angles using nonlinear optimization algorithms. The result is a Gigapixel-scale image having both wide FOV and high resolution. We demonstrate an experimentally robust reconstruction algorithm based on a 2nd order quasi-Newton's method, combined with a novel phase initialization scheme. To further extend the Gigapixel imaging capability to 3D, we develop a reconstruction method to process the 4D light field measurements from sequential illumination scanning. The algorithm is based on a 'multislice' forward model that incorporates both 3D phase and diffraction effects, as well as multiple forward scatterings. To solve the inverse problem, an iterative update procedure that combines both phase retrieval and 'error back-propagation' is developed. To avoid local minimum solutions, we further develop a novel physical model-based initialization technique that accounts for both the geometric-optic and 1st order phase effects. The result is robust reconstructions of Gigapixel 3D phase images having both wide FOV and super resolution in all three dimensions. Experimental results from an LED array microscope were demonstrated.

Super-resolution image reconstruction from UAS surveillance video through affine invariant interest point-based motion estimation

NASA Astrophysics Data System (ADS)

He, Qiang; Schultz, Richard R.; Wang, Yi; Camargo, Aldo; Martel, Florent

2008-01-01

In traditional super-resolution methods, researchers generally assume that accurate subpixel image registration parameters are given a priori. In reality, accurate image registration on a subpixel grid is the single most critically important step for the accuracy of super-resolution image reconstruction. In this paper, we introduce affine invariant features to improve subpixel image registration, which considerably reduces the number of mismatched points and hence makes traditional image registration more efficient and more accurate for super-resolution video enhancement. Affine invariant interest points include those corners that are invariant to affine transformations, including scale, rotation, and translation. They are extracted from the second moment matrix through the integration and differentiation covariance matrices. Our tests are based on two sets of real video captured by a small Unmanned Aircraft System (UAS) aircraft, which is highly susceptible to vibration from even light winds. The experimental results from real UAS surveillance video show that affine invariant interest points are more robust to perspective distortion and present more accurate matching than traditional Harris/SIFT corners. In our experiments on real video, all matching affine invariant interest points are found correctly. In addition, for the same super-resolution problem, we can use many fewer affine invariant points than Harris/SIFT corners to obtain good super-resolution results.

Qdot Labeled Actin Super Resolution Motility Assay Measures Low Duty Cycle Muscle Myosin Step-Size

PubMed Central

Wang, Yihua; Ajtai, Katalin; Burghardt, Thomas P.

2013-01-01

Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves relative sliding of myosin and actin filaments. Skeletal actin filaments were fluorescence labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdot’s were imaged in time with total internal reflection fluorescence microscopy then spatially localized to 1-3 nanometers using a super-resolution algorithm as they translated with actin over a surface coated with skeletal heavy meromyosin (sHMM) or full length β-cardiac myosin (MYH7). Average Qdot-actin velocity matches measurements with rhodamine-phalloidin labeled actin. The sHMM Qdot-actin velocity histogram contains low velocity events corresponding to actin translation in quantized steps of ~5 nm. The MYH7 velocity histogram has quantized steps at 3 and 8 nm in addition to 5 nm, and, larger compliance than sHMM depending on MYH7 surface concentration. Low duty cycle skeletal and cardiac myosin present challenges for a single molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay has modestly more actomyosin interactions and methylcellulose inhibited diffusion to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space then characterized using super-resolution. The approach provides quick, quantitative, and inexpensive step-size measurement for low duty cycle muscle myosin. PMID:23383646

Multi-pulse pumping for far-field super-resolution imaging

NASA Astrophysics Data System (ADS)

Requena, Sebastian; Raut, Sangram; Doan, Hung; Kimball, Joe; Fudala, Rafal; Borejdo, Julian; Gryczynski, Ignacy; Strzhemechny, Yuri; Gryczynski, Zygmunt

2016-02-01

Recently, far-field optical imaging with a resolution significantly beyond diffraction limit has attracted tremendous attention allowing for high resolution imaging in living objects. Various methods have been proposed that are divided in to two basic approaches; deterministic super-resolution like STED or RESOLFT and stochastic super-resolution like PALM or STORM. We propose to achieve super-resolution in far-field fluorescence imaging by the use of controllable (on-demand) bursts of pulses that can change the fluorescence signal of long-lived component over one order of magnitude. We demonstrate that two beads, one labeled with a long-lived dye and another with a short-lived dye, separated by a distance lower than 100 nm can be easily resolved in a single experiment. The proposed method can be used to separate two biological structures in a cell by targeting them with two antibodies labeled with long-lived and short-lived fluorophores.

Texton-based super-resolution for achieving high spatiotemporal resolution in hybrid camera system

NASA Astrophysics Data System (ADS)

Kamimura, Kenji; Tsumura, Norimichi; Nakaguchi, Toshiya; Miyake, Yoichi

2010-05-01

Many super-resolution methods have been proposed to enhance the spatial resolution of images by using iteration and multiple input images. In a previous paper, we proposed the example-based super-resolution method to enhance an image through pixel-based texton substitution to reduce the computational cost. In this method, however, we only considered the enhancement of a texture image. In this study, we modified this texton substitution method for a hybrid camera to reduce the required bandwidth of a high-resolution video camera. We applied our algorithm to pairs of high- and low-spatiotemporal-resolution videos, which were synthesized to simulate a hybrid camera. The result showed that the fine detail of the low-resolution video can be reproduced compared with bicubic interpolation and the required bandwidth could be reduced to about 1/5 in a video camera. It was also shown that the peak signal-to-noise ratios (PSNRs) of the images improved by about 6 dB in a trained frame and by 1.0-1.5 dB in a test frame, as determined by comparison with the processed image using bicubic interpolation, and the average PSNRs were higher than those obtained by the well-known Freeman’s patch-based super-resolution method. Compared with that of the Freeman’s patch-based super-resolution method, the computational time of our method was reduced to almost 1/10.

Laser-assisted formation of micropores and nanobubbles in sclera promote stable normalization of intraocular pressure

NASA Astrophysics Data System (ADS)

Baum, Olga; Wachsmann-Hogiu, Sebastian; Milner, Thomas; Sobol, Emil

2017-06-01

Pores in sclera enhance uveoscleral water outflow and can normalize intraocular pressure in glaucomatous eyes. The aims of this study are to demonstrate laser-induced formation of pores with a dendritic structure and to answer the questions: How is a pore system stable and can laser treatment provide a long-lasting pressure stabilization effect? Effect of 1.56 µm laser radiation on porcine eye sclera was studied using atomic force microscopy and super resolution structured irradiation microscopy with fluorescent markers. Results suggest that the pores with a complex spatial configuration can arise as a result of laser irradiation and that laser-generated stable gas nanobubbles coated with calcium ions allow pore stabilization in the sclera. Our results support a laser based approach for treatment of glaucoma.

DNA probes for monitoring dynamic and transient molecular encounters on live cell membranes

NASA Astrophysics Data System (ADS)

You, Mingxu; Lyu, Yifan; Han, Da; Qiu, Liping; Liu, Qiaoling; Chen, Tao; Sam Wu, Cuichen; Peng, Lu; Zhang, Liqin; Bao, Gang; Tan, Weihong

2017-05-01

Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result in disease progression. Advances in super-resolution microscopy have allowed membrane encounters to be examined, however, these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules. Here, we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals. The probe, which translocates from one anchor site to another, mimicking motor proteins, is realized through a toehold-mediated DNA strand displacement reaction. Using this probe, we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy. Our results show a preference for encounters within the same lipid domains.

Chemical imaging of molecular changes in a hydrated single cell by dynamic secondary ion mass spectrometry and super-resolution microscopy

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

Hua, Xin; Szymanski, Craig; Wang, Zhaoying

2016-01-01

Chemical imaging of single cells is important in capturing biological dynamics. Single cell correlative imaging is realized between structured illumination microscopy (SIM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) using System for Analysis at the Liquid Vacuum Interface (SALVI), a multimodal microreactor. SIM characterized cells and guided subsequent ToF-SIMS analysis. Dynamic ToF-SIMS provided time- and space-resolved cell molecular mapping. Lipid fragments were identified in the hydrated cell membrane. Principal component analysis was used to elucidate chemical component differences among mouse lung cells that uptake zinc oxide nanoparticles. Our results provided submicron chemical spatial mapping for investigations of cell dynamics atmore » the molecular level.« less

Multi-frame super-resolution with quality self-assessment for retinal fundus videos.

PubMed

Köhler, Thomas; Brost, Alexander; Mogalle, Katja; Zhang, Qianyi; Köhler, Christiane; Michelson, Georg; Hornegger, Joachim; Tornow, Ralf P

2014-01-01

This paper proposes a novel super-resolution framework to reconstruct high-resolution fundus images from multiple low-resolution video frames in retinal fundus imaging. Natural eye movements during an examination are used as a cue for super-resolution in a robust maximum a-posteriori scheme. In order to compensate heterogeneous illumination on the fundus, we integrate retrospective illumination correction for photometric registration to the underlying imaging model. Our method utilizes quality self-assessment to provide objective quality scores for reconstructed images as well as to select regularization parameters automatically. In our evaluation on real data acquired from six human subjects with a low-cost video camera, the proposed method achieved considerable enhancements of low-resolution frames and improved noise and sharpness characteristics by 74%. In terms of image analysis, we demonstrate the importance of our method for the improvement of automatic blood vessel segmentation as an example application, where the sensitivity was increased by 13% using super-resolution reconstruction.

Super resolution terahertz imaging by subpixel estimation: application to hyperspectral beam profiling

NASA Astrophysics Data System (ADS)

Logofătu, Petre C.; Damian, Victor

2018-05-01

A super-resolution terahertz imaging technique based on subpixel estimation was applied to hyperspectral beam profiling. The topic of hyperspectral beam profiling was chosen because the beam profile and its dependence on wavelength are not well known and are important for imaging applications. Super-resolution is required here to avoid diffraction effects and to provide a stronger signal. Super-resolution usually adds supplementary information to the measurement, but in this case, it is a prerequisite for it. We report that the beam profile is almost Gaussian for many frequencies; the waist of the Gaussian profile increases with frequency while the center wobbles slightly. Knowledge of the beam profile may subsequently be used as reference for imaging.

Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx.

PubMed

Löschberger, Anna; Niehörster, Thomas; Sauer, Markus

2014-05-01

Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide-alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin.

PubMed

Chamma, Ingrid; Rossier, Olivier; Giannone, Grégory; Thoumine, Olivier; Sainlos, Matthieu

2017-04-01

Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM)). This method is complementary to the previously described anti-GFP-nanobody/SNAP-tag strategies, with the main advantage being that it requires only a short 15-amino-acid tag, and can thus be used with proteins resistant to fusion with large tags and for multicolor imaging. The protocol requires standard molecular biology/biochemistry equipment, making it easily accessible for laboratories with only basic skills in cell biology and biochemistry. The production/purification/conjugation steps take ∼5 d, and labeling takes a few minutes to an hour.

ON THE IMPACT OF SUPER RESOLUTION WSR-88D DOPPLER RADAR DATA ASSIMILATION ON HIGH RESOLUTION NUMERICAL MODEL FORECASTS

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

Chiswell, S

2009-01-11

Assimilation of radar velocity and precipitation fields into high-resolution model simulations can improve precipitation forecasts with decreased 'spin-up' time and improve short-term simulation of boundary layer winds (Benjamin, 2004 & 2007; Xiao, 2008) which is critical to improving plume transport forecasts. Accurate description of wind and turbulence fields is essential to useful atmospheric transport and dispersion results, and any improvement in the accuracy of these fields will make consequence assessment more valuable during both routine operation as well as potential emergency situations. During 2008, the United States National Weather Service (NWS) radars implemented a significant upgrade which increased the real-timemore » level II data resolution to 8 times their previous 'legacy' resolution, from 1 km range gate and 1.0 degree azimuthal resolution to 'super resolution' 250 m range gate and 0.5 degree azimuthal resolution (Fig 1). These radar observations provide reflectivity, velocity and returned power spectra measurements at a range of up to 300 km (460 km for reflectivity) at a frequency of 4-5 minutes and yield up to 13.5 million point observations per level in super-resolution mode. The migration of National Weather Service (NWS) WSR-88D radars to super resolution is expected to improve warning lead times by detecting small scale features sooner with increased reliability; however, current operational mesoscale model domains utilize grid spacing several times larger than the legacy data resolution, and therefore the added resolution of radar data is not fully exploited. The assimilation of super resolution reflectivity and velocity data into high resolution numerical weather model forecasts where grid spacing is comparable to the radar data resolution is investigated here to determine the impact of the improved data resolution on model predictions.« less

Real-time bacterial microcolony counting using on-chip microscopy

NASA Astrophysics Data System (ADS)

Jung, Jae Hee; Lee, Jung Eun

2016-02-01

Observing microbial colonies is the standard method for determining the microbe titer and investigating the behaviors of microbes. Here, we report an automated, real-time bacterial microcolony-counting system implemented on a wide field-of-view (FOV), on-chip microscopy platform, termed ePetri. Using sub-pixel sweeping microscopy (SPSM) with a super-resolution algorithm, this system offers the ability to dynamically track individual bacterial microcolonies over a wide FOV of 5.7 mm × 4.3 mm without requiring a moving stage or lens. As a demonstration, we obtained high-resolution time-series images of S. epidermidis at 20-min intervals. We implemented an image-processing algorithm to analyze the spatiotemporal distribution of microcolonies, the development of which could be observed from a single bacterial cell. Test bacterial colonies with a minimum diameter of 20 μm could be enumerated within 6 h. We showed that our approach not only provides results that are comparable to conventional colony-counting assays but also can be used to monitor the dynamics of colony formation and growth. This microcolony-counting system using on-chip microscopy represents a new platform that substantially reduces the detection time for bacterial colony counting. It uses chip-scale image acquisition and is a simple and compact solution for the automation of colony-counting assays and microbe behavior analysis with applications in antibacterial drug discovery.

Real-time bacterial microcolony counting using on-chip microscopy

PubMed Central

Jung, Jae Hee; Lee, Jung Eun

2016-01-01

Observing microbial colonies is the standard method for determining the microbe titer and investigating the behaviors of microbes. Here, we report an automated, real-time bacterial microcolony-counting system implemented on a wide field-of-view (FOV), on-chip microscopy platform, termed ePetri. Using sub-pixel sweeping microscopy (SPSM) with a super-resolution algorithm, this system offers the ability to dynamically track individual bacterial microcolonies over a wide FOV of 5.7 mm × 4.3 mm without requiring a moving stage or lens. As a demonstration, we obtained high-resolution time-series images of S. epidermidis at 20-min intervals. We implemented an image-processing algorithm to analyze the spatiotemporal distribution of microcolonies, the development of which could be observed from a single bacterial cell. Test bacterial colonies with a minimum diameter of 20 μm could be enumerated within 6 h. We showed that our approach not only provides results that are comparable to conventional colony-counting assays but also can be used to monitor the dynamics of colony formation and growth. This microcolony-counting system using on-chip microscopy represents a new platform that substantially reduces the detection time for bacterial colony counting. It uses chip-scale image acquisition and is a simple and compact solution for the automation of colony-counting assays and microbe behavior analysis with applications in antibacterial drug discovery. PMID:26902822

Near-field examination of perovskite-based superlenses and superlens-enhanced probe-object coupling.

PubMed

Kehr, S C; Liu, Y M; Martin, L W; Yu, P; Gajek, M; Yang, S-Y; Yang, C-H; Wenzel, M T; Jacob, R; von Ribbeck, H-G; Helm, M; Zhang, X; Eng, L M; Ramesh, R

2011-01-01

A planar slab of negative-index material works as a superlens with sub-diffraction-limited resolution, as propagating waves are focused and, moreover, evanescent waves are reconstructed in the image plane. Here we demonstrate a superlens for electric evanescent fields with low losses using perovskites in the mid-infrared regime. The combination of near-field microscopy with a tunable free-electron laser allows us to address precisely the polariton modes, which are critical for super-resolution imaging. We spectrally study the lateral and vertical distributions of evanescent waves around the image plane of such a lens, and achieve imaging resolution of λ/14 at the superlensing wavelength. Interestingly, at certain distances between the probe and sample surface, we observe a maximum of these evanescent fields. Comparisons with numerical simulations indicate that this maximum originates from an enhanced coupling between probe and object, which might be applicable for multifunctional circuits, infrared spectroscopy and thermal sensors.

Improved vocal tract reconstruction and modeling using an image super-resolution technique.

PubMed

Zhou, Xinhui; Woo, Jonghye; Stone, Maureen; Prince, Jerry L; Espy-Wilson, Carol Y

2013-06-01

Magnetic resonance imaging has been widely used in speech production research. Often only one image stack (sagittal, axial, or coronal) is used for vocal tract modeling. As a result, complementary information from other available stacks is not utilized. To overcome this, a recently developed super-resolution technique was applied to integrate three orthogonal low-resolution stacks into one isotropic volume. The results on vowels show that the super-resolution volume produces better vocal tract visualization than any of the low-resolution stacks. Its derived area functions generally produce formant predictions closer to the ground truth, particularly for those formants sensitive to area perturbations at constrictions.

Super resolution reconstruction of μ-CT image of rock sample using neighbour embedding algorithm

NASA Astrophysics Data System (ADS)

Wang, Yuzhu; Rahman, Sheik S.; Arns, Christoph H.

2018-03-01

X-ray computed tomography (μ-CT) is considered to be the most effective way to obtain the inner structure of rock sample without destructions. However, its limited resolution hampers its ability to probe sub-micro structures which is critical for flow transportation of rock sample. In this study, we propose an innovative methodology to improve the resolution of μ-CT image using neighbour embedding algorithm where low frequency information is provided by μ-CT image itself while high frequency information is supplemented by high resolution scanning electron microscopy (SEM) image. In order to obtain prior for reconstruction, a large number of image patch pairs contain high- and low- image patches are extracted from the Gaussian image pyramid generated by SEM image. These image patch pairs contain abundant information about tomographic evolution of local porous structures under different resolution spaces. Relying on the assumption of self-similarity of porous structure, this prior information can be used to supervise the reconstruction of high resolution μ-CT image effectively. The experimental results show that the proposed method is able to achieve the state-of-the-art performance.

Super-resolution reconstruction of hyperspectral images.

PubMed

Akgun, Toygar; Altunbasak, Yucel; Mersereau, Russell M

2005-11-01

Hyperspectral images are used for aerial and space imagery applications, including target detection, tracking, agricultural, and natural resource exploration. Unfortunately, atmospheric scattering, secondary illumination, changing viewing angles, and sensor noise degrade the quality of these images. Improving their resolution has a high payoff, but applying super-resolution techniques separately to every spectral band is problematic for two main reasons. First, the number of spectral bands can be in the hundreds, which increases the computational load excessively. Second, considering the bands separately does not make use of the information that is present across them. Furthermore, separate band super-resolution does not make use of the inherent low dimensionality of the spectral data, which can effectively be used to improve the robustness against noise. In this paper, we introduce a novel super-resolution method for hyperspectral images. An integral part of our work is to model the hyperspectral image acquisition process. We propose a model that enables us to represent the hyperspectral observations from different wavelengths as weighted linear combinations of a small number of basis image planes. Then, a method for applying super resolution to hyperspectral images using this model is presented. The method fuses information from multiple observations and spectral bands to improve spatial resolution and reconstruct the spectrum of the observed scene as a combination of a small number of spectral basis functions.

Single Image Super-Resolution Based on Multi-Scale Competitive Convolutional Neural Network

PubMed Central

Qu, Xiaobo; He, Yifan

2018-01-01

Deep convolutional neural networks (CNNs) are successful in single-image super-resolution. Traditional CNNs are limited to exploit multi-scale contextual information for image reconstruction due to the fixed convolutional kernel in their building modules. To restore various scales of image details, we enhance the multi-scale inference capability of CNNs by introducing competition among multi-scale convolutional filters, and build up a shallow network under limited computational resources. The proposed network has the following two advantages: (1) the multi-scale convolutional kernel provides the multi-context for image super-resolution, and (2) the maximum competitive strategy adaptively chooses the optimal scale of information for image reconstruction. Our experimental results on image super-resolution show that the performance of the proposed network outperforms the state-of-the-art methods. PMID:29509666

Single Image Super-Resolution Based on Multi-Scale Competitive Convolutional Neural Network.

PubMed

Du, Xiaofeng; Qu, Xiaobo; He, Yifan; Guo, Di

2018-03-06

Deep convolutional neural networks (CNNs) are successful in single-image super-resolution. Traditional CNNs are limited to exploit multi-scale contextual information for image reconstruction due to the fixed convolutional kernel in their building modules. To restore various scales of image details, we enhance the multi-scale inference capability of CNNs by introducing competition among multi-scale convolutional filters, and build up a shallow network under limited computational resources. The proposed network has the following two advantages: (1) the multi-scale convolutional kernel provides the multi-context for image super-resolution, and (2) the maximum competitive strategy adaptively chooses the optimal scale of information for image reconstruction. Our experimental results on image super-resolution show that the performance of the proposed network outperforms the state-of-the-art methods.

Super-resolution imaging of multiple cells by optimized flat-field epi-illumination

NASA Astrophysics Data System (ADS)

Douglass, Kyle M.; Sieben, Christian; Archetti, Anna; Lambert, Ambroise; Manley, Suliana

2016-11-01

Biological processes are inherently multi-scale, and supramolecular complexes at the nanoscale determine changes at the cellular scale and beyond. Single-molecule localization microscopy (SMLM) techniques have been established as important tools for studying cellular features with resolutions of the order of around 10 nm. However, in their current form these modalities are limited by a highly constrained field of view (FOV) and field-dependent image resolution. Here, we develop a low-cost microlens array (MLA)-based epi-illumination system—flat illumination for field-independent imaging (FIFI)—that can efficiently and homogeneously perform simultaneous imaging of multiple cells with nanoscale resolution. The optical principle of FIFI, which is an extension of the Köhler integrator, is further elucidated and modelled with a new, free simulation package. We demonstrate FIFI's capabilities by imaging multiple COS-7 and bacteria cells in 100 × 100 μm2 SMLM images—more than quadrupling the size of a typical FOV and producing near-gigapixel-sized images of uniformly high quality.

Column ratio mapping: a processing technique for atomic resolution high-angle annular dark-field (HAADF) images.

PubMed

Robb, Paul D; Craven, Alan J

2008-12-01

An image processing technique is presented for atomic resolution high-angle annular dark-field (HAADF) images that have been acquired using scanning transmission electron microscopy (STEM). This technique is termed column ratio mapping and involves the automated process of measuring atomic column intensity ratios in high-resolution HAADF images. This technique was developed to provide a fuller analysis of HAADF images than the usual method of drawing single intensity line profiles across a few areas of interest. For instance, column ratio mapping reveals the compositional distribution across the whole HAADF image and allows a statistical analysis and an estimation of errors. This has proven to be a very valuable technique as it can provide a more detailed assessment of the sharpness of interfacial structures from HAADF images. The technique of column ratio mapping is described in terms of a [110]-oriented zinc-blende structured AlAs/GaAs superlattice using the 1 angstroms-scale resolution capability of the aberration-corrected SuperSTEM 1 instrument.

Image inpainting and super-resolution using non-local recursive deep convolutional network with skip connections

NASA Astrophysics Data System (ADS)

Liu, Miaofeng

2017-07-01

In recent years, deep convolutional neural networks come into use in image inpainting and super-resolution in many fields. Distinct to most of the former methods requiring to know beforehand the local information for corrupted pixels, we propose a 20-depth fully convolutional network to learn an end-to-end mapping a dataset of damaged/ground truth subimage pairs realizing non-local blind inpainting and super-resolution. As there often exist image with huge corruptions or inpainting on a low-resolution image that the existing approaches unable to perform well, we also share parameters in local area of layers to achieve spatial recursion and enlarge the receptive field. To avoid the difficulty of training this deep neural network, skip-connections between symmetric convolutional layers are designed. Experimental results shows that the proposed method outperforms state-of-the-art methods for diverse corrupting and low-resolution conditions, it works excellently when realizing super-resolution and image inpainting simultaneously

Bayesian Deconvolution for Angular Super-Resolution in Forward-Looking Scanning Radar

PubMed Central

Zha, Yuebo; Huang, Yulin; Sun, Zhichao; Wang, Yue; Yang, Jianyu

2015-01-01

Scanning radar is of notable importance for ground surveillance, terrain mapping and disaster rescue. However, the angular resolution of a scanning radar image is poor compared to the achievable range resolution. This paper presents a deconvolution algorithm for angular super-resolution in scanning radar based on Bayesian theory, which states that the angular super-resolution can be realized by solving the corresponding deconvolution problem with the maximum a posteriori (MAP) criterion. The algorithm considers that the noise is composed of two mutually independent parts, i.e., a Gaussian signal-independent component and a Poisson signal-dependent component. In addition, the Laplace distribution is used to represent the prior information about the targets under the assumption that the radar image of interest can be represented by the dominant scatters in the scene. Experimental results demonstrate that the proposed deconvolution algorithm has higher precision for angular super-resolution compared with the conventional algorithms, such as the Tikhonov regularization algorithm, the Wiener filter and the Richardson–Lucy algorithm. PMID:25806871

Fluorescence lifetime imaging and its applications in cellular microenvironment measurement and auxiliary diagnosis

NASA Astrophysics Data System (ADS)

Luo, Teng; Levchenko, Svitlana M.; Pliss, Artem; Peng, Xiao; Yan, Wei; Prasad, Paras N.; Liu, Liwei; Qu, Junle

2018-02-01

We present our recent work on the applications of fluorescence lifetime imaging microscopy(FLIM), including the monitoring of macromolecule dynamic changes in the nucleolar compartments and the auxiliary diagnosis of H and E-stained sections. We demonstrated the capability of FLIM to measure protein concentration in the specific cellular compartments in live cells. We proposed to use FLIM to monitor changes in intracellular protein concentration caused by various factors e.g. cell cycle progression, drug treatment etc. In the future, FLIM technology is expected to be combined with super-resolution optical imaging. FLIM with molecular resolution will have the potential to serve as a powerful tool for discovering new phenomena and revealing new mechanisms in biomedical research, which will effectively promote the development of life science.

Optical Super-Resolution by High-Index Liquid-Immersed Microspheres

DTIC Science & Technology

2012-01-01

the BD without liquid can be achieved using microspheres with small-to-moderate index of refraction such as borosilicate glass (n 1.47), soda lime ...titanate glass microspheres with diameters (D) in the range 2–220 lm and with high refractive index (n 1.9–2.1) can be used for super-resolution...achieving optical super-resolution. It has been demonstrated10 that silica spheres with refractive index (n) about 1.46 and with diame- ters (D) in the

A robust statistical estimation (RoSE) algorithm jointly recovers the 3D location and intensity of single molecules accurately and precisely

NASA Astrophysics Data System (ADS)

Mazidi, Hesam; Nehorai, Arye; Lew, Matthew D.

2018-02-01

In single-molecule (SM) super-resolution microscopy, the complexity of a biological structure, high molecular density, and a low signal-to-background ratio (SBR) may lead to imaging artifacts without a robust localization algorithm. Moreover, engineered point spread functions (PSFs) for 3D imaging pose difficulties due to their intricate features. We develop a Robust Statistical Estimation algorithm, called RoSE, that enables joint estimation of the 3D location and photon counts of SMs accurately and precisely using various PSFs under conditions of high molecular density and low SBR.

Chronic 2P-STED imaging reveals high turnover of dendritic spines in the hippocampus in vivo.

PubMed

Pfeiffer, Thomas; Poll, Stefanie; Bancelin, Stephane; Angibaud, Julie; Inavalli, Vvg Krishna; Keppler, Kevin; Mittag, Manuel; Fuhrmann, Martin; Nägerl, U Valentin

2018-06-22

Rewiring neural circuits by the formation and elimination of synapses is thought to be a key cellular mechanism of learning and memory in the mammalian brain. Dendritic spines are the postsynaptic structural component of excitatory synapses, and their experience-dependent plasticity has been extensively studied in mouse superficial cortex using two-photon microscopy in vivo. By contrast, very little is known about spine plasticity in the hippocampus, which is the archetypical memory center of the brain, mostly because it is difficult to visualize dendritic spines in this deeply embedded structure with sufficient spatial resolution. We developed chronic 2P-STED microscopy in mouse hippocampus, using a 'hippocampal window' based on resection of cortical tissue and a long working distance objective for optical access. We observed a two-fold higher spine density than previous studies and measured a spine turnover of ~40% within 4 days, which depended on spine size. We thus provide direct evidence for a high level of structural rewiring of synaptic circuits and new insights into the structure-dynamics relationship of hippocampal spines. Having established chronic super-resolution microscopy in the hippocampus in vivo, our study enables longitudinal and correlative analyses of nanoscale neuroanatomical structures with genetic, molecular and behavioral experiments. © 2018, Pfeiffer et al.

Ultrafast photon counting applied to resonant scanning STED microscopy.

PubMed

Wu, Xundong; Toro, Ligia; Stefani, Enrico; Wu, Yong

2015-01-01

To take full advantage of fast resonant scanning in super-resolution stimulated emission depletion (STED) microscopy, we have developed an ultrafast photon counting system based on a multigiga sample per second analogue-to-digital conversion chip that delivers an unprecedented 450 MHz pixel clock (2.2 ns pixel dwell time in each scan). The system achieves a large field of view (∼50 × 50 μm) with fast scanning that reduces photobleaching, and advances the time-gated continuous wave STED technology to the usage of resonant scanning with hardware-based time-gating. The assembled system provides superb signal-to-noise ratio and highly linear quantification of light that result in superior image quality. Also, the system design allows great flexibility in processing photon signals to further improve the dynamic range. In conclusion, we have constructed a frontier photon counting image acquisition system with ultrafast readout rate, excellent counting linearity, and with the capacity of realizing resonant-scanning continuous wave STED microscopy with online time-gated detection. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

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.

RGB digital lensless holographic microscopy

NASA Astrophysics Data System (ADS)

Garcia-Sucerquia, Jorge

2013-11-01

The recent introduction of color digital lensless holographic microscopy (CDLHM) has shown the possibility of imaging microscopic specimens at full color without the need of lenses. Owing to the simplicity, robustness, and compactness of the digital lensless holographic microscopes (DLHM), they have been presented as the ideal candidates to being developed into portable holographic microscopes. However, in the case of CDLHM the utilization of three independent lasers hinders the portability option for this microscope. In this contribution an alternative to reduce the complexity of CDLHM aimed to recover the portability of this microscopy technology is presented. A super-bright white-light light-emitting diode (LED) is spectrally and spatially filtered to produce the needed illumination by CDLHM to work. CDLHM with LED illumination is used to image at full color a section of the head of a drosophila melanogaster fly (fruit fly). The LED-CDLHM method shows the capability of imaging objects of 2μm size in comparison with the micrometer resolution reported for LASER-CDLHM.

Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging

PubMed Central

Shcherbakova, Daria M.; Baloban, Mikhail; Emelyanov, Alexander V.; Brenowitz, Michael; Guo, Peng; Verkhusha, Vladislav V.

2016-01-01

Monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags and components of biosensors for deep-tissue imaging and multicolour microscopy. We report three bright and spectrally distinct monomeric NIR FPs, termed miRFPs, engineered from bacterial phytochrome, which can be used as easily as GFP-like FPs. miRFPs are 2–5-fold brighter in mammalian cells than other monomeric NIR FPs and perform well in protein fusions, allowing multicolour structured illumination microscopy. miRFPs enable development of several types of NIR biosensors, such as for protein–protein interactions, RNA detection, signalling cascades and cell fate. We demonstrate this by engineering the monomeric fluorescence complementation reporters, the IκBα reporter for NF-κB pathway and the cell cycle biosensor for detection of proliferation status of cells in culture and in animals. miRFPs allow non-invasive visualization and detection of biological processes at different scales, from super-resolution microscopy to in vivo imaging, using the same probes. PMID:27539380

Far-field nanoscopy on a semiconductor quantum dot via a rapid-adiabatic-passage-based switch

NASA Astrophysics Data System (ADS)

Kaldewey, Timo; Kuhlmann, Andreas V.; Valentin, Sascha R.; Ludwig, Arne; Wieck, Andreas D.; Warburton, Richard J.

2018-02-01

The diffraction limit prevents a conventional optical microscope from imaging at the nanoscale. However, nanoscale imaging of molecules is possible by exploiting an intensity-dependent molecular switch1-3. This switch is translated into a microscopy scheme, stimulated emission depletion microscopy4-7. Variants on this scheme exist3,8-13, yet all exploit an incoherent response to the lasers. We present a scheme that relies on a coherent response to a laser. Quantum control of a two-level system proceeds via rapid adiabatic passage, an ideal molecular switch. We implement this scheme on an ensemble of quantum dots. Each quantum dot results in a bright spot in the image with extent down to 30 nm (λ/31). There is no significant loss of intensity with respect to confocal microscopy, resulting in a factor of 10 improvement in emitter position determination. The experiments establish rapid adiabatic passage as a versatile tool in the super-resolution toolbox.

DNA probe for monitoring dynamic and transient molecular encounters on live cell membranes

PubMed Central

You, Mingxu; Lyu, Yifan; Han, Da; Qiu, Liping; Liu, Qiaoling; Chen, Tao; Wu, Cuichen Sam; Peng, Lu; Zhang, Liqin; Bao, Gang; Tan, Weihong

2017-01-01

Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result in disease progression. Advances in super-resolution microscopy have allowed membrane encounters to be examined, however, these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules. Here, we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals. The probe, which translocates from one anchor site to another, such as motor proteins, is realized through a toehold-mediated DNA strand displacement reaction. Using this probe, we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy. Our results show a preference for encounters within different lipid domains. PMID:28319616

Partially coherent lensfree tomographic microscopy⋄

PubMed Central

Isikman, Serhan O.; Bishara, Waheb; Ozcan, Aydogan

2012-01-01

Optical sectioning of biological specimens provides detailed volumetric information regarding their internal structure. To provide a complementary approach to existing three-dimensional (3D) microscopy modalities, we have recently demonstrated lensfree optical tomography that offers high-throughput imaging within a compact and simple platform. In this approach, in-line holograms of objects at different angles of partially coherent illumination are recorded using a digital sensor-array, which enables computing pixel super-resolved tomographic images of the specimen. This imaging modality, which forms the focus of this review, offers micrometer-scale 3D resolution over large imaging volumes of, for example, 10–15 mm3, and can be assembled in light weight and compact architectures. Therefore, lensfree optical tomography might be particularly useful for lab-on-a-chip applications as well as for microscopy needs in resource-limited settings. PMID:22193016

A Novel Azimuth Super-Resolution Method by Synthesizing Azimuth Bandwidth of Multiple Tracks of Airborne Stripmap SAR Data

PubMed Central

Wang, Yan; Li, Jingwen; Sun, Bing; Yang, Jian

2016-01-01

Azimuth resolution of airborne stripmap synthetic aperture radar (SAR) is restricted by the azimuth antenna size. Conventionally, a higher azimuth resolution should be achieved by employing alternate modes that steer the beam in azimuth to enlarge the synthetic antenna aperture. However, if a data set of a certain region, consisting of multiple tracks of airborne stripmap SAR data, is available, the azimuth resolution of specific small region of interest (ROI) can be conveniently improved by a novel azimuth super-resolution method as introduced by this paper. The proposed azimuth super-resolution method synthesize the azimuth bandwidth of the data selected from multiple discontinuous tracks and contributes to a magnifier-like function with which the ROI can be further zoomed in with a higher azimuth resolution than that of the original stripmap images. Detailed derivation of the azimuth super-resolution method, including the steps of two-dimensional dechirping, residual video phase (RVP) removal, data stitching and data correction, is provided. The restrictions of the proposed method are also discussed. Lastly, the presented approach is evaluated via both the single- and multi-target computer simulations. PMID:27304959

Patch-Based Super-Resolution of MR Spectroscopic Images: Application to Multiple Sclerosis

PubMed Central

Jain, Saurabh; Sima, Diana M.; Sanaei Nezhad, Faezeh; Hangel, Gilbert; Bogner, Wolfgang; Williams, Stephen; Van Huffel, Sabine; Maes, Frederik; Smeets, Dirk

2017-01-01

Purpose: Magnetic resonance spectroscopic imaging (MRSI) provides complementary information to conventional magnetic resonance imaging. Acquiring high resolution MRSI is time consuming and requires complex reconstruction techniques. Methods: In this paper, a patch-based super-resolution method is presented to increase the spatial resolution of metabolite maps computed from MRSI. The proposed method uses high resolution anatomical MR images (T1-weighted and Fluid-attenuated inversion recovery) to regularize the super-resolution process. The accuracy of the method is validated against conventional interpolation techniques using a phantom, as well as simulated and in vivo acquired human brain images of multiple sclerosis subjects. Results: The method preserves tissue contrast and structural information, and matches well with the trend of acquired high resolution MRSI. Conclusions: These results suggest that the method has potential for clinically relevant neuroimaging applications. PMID:28197066

In vivo structure of the E. coli FtsZ-ring revealed by photoactivated localization microscopy (PALM).

PubMed

Fu, Guo; Huang, Tao; Buss, Jackson; Coltharp, Carla; Hensel, Zach; Xiao, Jie

2010-09-13

The FtsZ protein, a tubulin-like GTPase, plays a pivotal role in prokaryotic cell division. In vivo it localizes to the midcell and assembles into a ring-like structure-the Z-ring. The Z-ring serves as an essential scaffold to recruit all other division proteins and generates contractile force for cytokinesis, but its supramolecular structure remains unknown. Electron microscopy (EM) has been unsuccessful in detecting the Z-ring due to the dense cytoplasm of bacterial cells, and conventional fluorescence light microscopy (FLM) has only provided images with limited spatial resolution (200-300 nm) due to the diffraction of light. Hence, given the small sizes of bacteria cells, identifying the in vivo structure of the Z-ring presents a substantial challenge. Here, we used photoactivated localization microscopy (PALM), a single molecule-based super-resolution imaging technique, to characterize the in vivo structure of the Z-ring in E. coli. We achieved a spatial resolution of ∼35 nm and discovered that in addition to the expected ring-like conformation, the Z-ring of E. coli adopts a novel compressed helical conformation with variable helical length and pitch. We measured the thickness of the Z-ring to be ∼110 nm and the packing density of FtsZ molecules inside the Z-ring to be greater than what is expected for a single-layered flat ribbon configuration. Our results strongly suggest that the Z-ring is composed of a loose bundle of FtsZ protofilaments that randomly overlap with each other in both longitudinal and radial directions of the cell. Our results provide significant insight into the spatial organization of the Z-ring and open the door for further investigations of structure-function relationships and cell cycle-dependent regulation of the Z-ring.

A straightforward approach for gated STED-FCS to investigate lipid membrane dynamics

PubMed Central

Clausen, Mathias P.; Sezgin, Erdinc; Bernardino de la Serna, Jorge; Waithe, Dominic; Lagerholm, B. Christoffer; Eggeling, Christian

2015-01-01

Recent years have seen the development of multiple technologies to investigate, with great spatial and temporal resolution, the dynamics of lipids in cellular and model membranes. One of these approaches is the combination of far-field super-resolution stimulated-emission-depletion (STED) microscopy with fluorescence correlation spectroscopy (FCS). STED-FCS combines the diffraction-unlimited spatial resolution of STED microscopy with the statistical accuracy of FCS to determine sub-millisecond-fast molecular dynamics with single-molecule sensitivity. A unique advantage of STED-FCS is that the observation spot for the FCS data recordings can be tuned to sub-diffraction scales, i.e. <200 nm in diameter, in a gradual manner to investigate fast diffusion of membrane-incorporated labelled entities. Unfortunately, so far the STED-FCS technology has mostly been applied on a few custom-built setups optimised for far-red fluorescent emitters. Here, we summarise the basics of the STED-FCS technology and highlight how it can give novel details into molecular diffusion modes. Most importantly, we present a straightforward way for performing STED-FCS measurements on an unmodified turnkey commercial system using a time-gated detection scheme. Further, we have evaluated the STED-FCS performance of different commonly used green emitting fluorescent dyes applying freely available, custom-written analysis software. PMID:26123184

Dual-color STED microscopy reveals a sandwich structure of Bassoon and Piccolo in active zones of adult and aged mice.

PubMed

Nishimune, Hiroshi; Badawi, Yomna; Mori, Shuuichi; Shigemoto, Kazuhiro

2016-06-20

Presynaptic active zones play a pivotal role as synaptic vesicle release sites for synaptic transmission, but the molecular architecture of active zones in mammalian neuromuscular junctions (NMJs) at sub-diffraction limited resolution remains unknown. Bassoon and Piccolo are active zone specific cytosolic proteins essential for active zone assembly in NMJs, ribbon synapses, and brain synapses. These proteins are thought to colocalize and share some functions at active zones. Here, we report an unexpected finding of non-overlapping localization of these two proteins in mouse NMJs revealed using dual-color stimulated emission depletion (STED) super resolution microscopy. Piccolo puncta sandwiched Bassoon puncta and aligned in a Piccolo-Bassoon-Piccolo structure in adult NMJs. P/Q-type voltage-gated calcium channel (VGCC) puncta colocalized with Bassoon puncta. The P/Q-type VGCC and Bassoon protein levels decreased significantly in NMJs from aged mouse. In contrast, the Piccolo levels in NMJs from aged mice were comparable to levels in adult mice. This study revealed the molecular architecture of active zones in mouse NMJs at sub-diffraction limited resolution, and described the selective degeneration mechanism of active zone proteins in NMJs from aged mice. Interestingly, the localization pattern of active zone proteins described herein is similar to active zone structures described using electron microscope tomography.

Single image super-resolution via an iterative reproducing kernel Hilbert space method.

PubMed

Deng, Liang-Jian; Guo, Weihong; Huang, Ting-Zhu

2016-11-01

Image super-resolution, a process to enhance image resolution, has important applications in satellite imaging, high definition television, medical imaging, etc. Many existing approaches use multiple low-resolution images to recover one high-resolution image. In this paper, we present an iterative scheme to solve single image super-resolution problems. It recovers a high quality high-resolution image from solely one low-resolution image without using a training data set. We solve the problem from image intensity function estimation perspective and assume the image contains smooth and edge components. We model the smooth components of an image using a thin-plate reproducing kernel Hilbert space (RKHS) and the edges using approximated Heaviside functions. The proposed method is applied to image patches, aiming to reduce computation and storage. Visual and quantitative comparisons with some competitive approaches show the effectiveness of the proposed method.

Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research

PubMed Central

Ilić, Nataša; Pilarczyk, Götz; Lee, Jin-Ho; Logeswaran, Abiramy; Borroni, Aurora Paola; Krufczik, Matthias; Theda, Franziska; Waltrich, Nadine; Bestvater, Felix; Hildenbrand, Georg; Cremer, Christoph; Blank, Michael

2017-01-01

Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine. PMID:28956810

Subnuclear localization, rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield, confocal and super-resolution microscopy.

PubMed

Pierzyńska-Mach, Agnieszka; Szczurek, Aleksander; Cella Zanacchi, Francesca; Pennacchietti, Francesca; Drukała, Justyna; Diaspro, Alberto; Cremer, Christoph; Darzynkiewicz, Zbigniew; Dobrucki, Jurek W

2016-01-01

Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2'-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts.

Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research.

PubMed

Hausmann, Michael; Ilić, Nataša; Pilarczyk, Götz; Lee, Jin-Ho; Logeswaran, Abiramy; Borroni, Aurora Paola; Krufczik, Matthias; Theda, Franziska; Waltrich, Nadine; Bestvater, Felix; Hildenbrand, Georg; Cremer, Christoph; Blank, Michael

2017-09-28

Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine.

Subnuclear localization, rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield, confocal and super-resolution microscopy

PubMed Central

Pierzyńska-Mach, Agnieszka; Szczurek, Aleksander; Cella Zanacchi, Francesca; Pennacchietti, Francesca; Drukała, Justyna; Diaspro, Alberto; Cremer, Christoph; Darzynkiewicz, Zbigniew; Dobrucki, Jurek W.

2016-01-01

ABSTRACT Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2′-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts. PMID:27097376

Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope

PubMed Central

Klauss, André; König, Marcelle; Hille, Carsten

2015-01-01

By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED) microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as “easy-STED”, achieving lateral resolution < λ/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating. PMID:26091552

A novel algorithm of super-resolution image reconstruction based on multi-class dictionaries for natural scene

NASA Astrophysics Data System (ADS)

Wu, Wei; Zhao, Dewei; Zhang, Huan

2015-12-01

Super-resolution image reconstruction is an effective method to improve the image quality. It has important research significance in the field of image processing. However, the choice of the dictionary directly affects the efficiency of image reconstruction. A sparse representation theory is introduced into the problem of the nearest neighbor selection. Based on the sparse representation of super-resolution image reconstruction method, a super-resolution image reconstruction algorithm based on multi-class dictionary is analyzed. This method avoids the redundancy problem of only training a hyper complete dictionary, and makes the sub-dictionary more representatives, and then replaces the traditional Euclidean distance computing method to improve the quality of the whole image reconstruction. In addition, the ill-posed problem is introduced into non-local self-similarity regularization. Experimental results show that the algorithm is much better results than state-of-the-art algorithm in terms of both PSNR and visual perception.

On the dynamic readout characteristic of nonlinear super-resolution optical storage

NASA Astrophysics Data System (ADS)

Wei, Jingsong

2013-03-01

Researchers have developed nonlinear super-resolution optical storage for the past twenty years. However, several concerns remain, including (1) the presence of readout threshold power; (2) the increase of threshold power with the reduction of the mark size, and (3) the increase of the carrier-to-noise ratio (CNR) at the initial stage and then decrease with the increase of readout laser power or laser irradiation time. The present work calculates and analyzes the super-resolution spot formed by the thin film masks and the readout threshold power characteristic according to the derived formula and based on the nonlinear saturable absorption characteristic and threshold of structural change. The obtained theoretical calculation and experimental data answer the concerns regarding the dynamic readout threshold characteristic and CNR dependence on laser power and irradiation time. The near-field optical spot scanning experiment further verifies the super-resolution spot formation produced through the nonlinear thin film masks.

Multiband super-resolution imaging of graded-index photonic crystal flat lens

NASA Astrophysics Data System (ADS)

Xie, Jianlan; Wang, Junzhong; Ge, Rui; Yan, Bei; Liu, Exian; Tan, Wei; Liu, Jianjun

2018-05-01

Multiband super-resolution imaging of point source is achieved by a graded-index photonic crystal flat lens. With the calculations of six bands in common photonic crystal (CPC) constructed with scatterers of different refractive indices, it can be found that the super-resolution imaging of point source can be realized by different physical mechanisms in three different bands. In the first band, the imaging of point source is based on far-field condition of spherical wave while in the second band, it is based on the negative effective refractive index and exhibiting higher imaging quality than that of the CPC. However, in the fifth band, the imaging of point source is mainly based on negative refraction of anisotropic equi-frequency surfaces. The novel method of employing different physical mechanisms to achieve multiband super-resolution imaging of point source is highly meaningful for the field of imaging.

Chip-based wide field-of-view nanoscopy

NASA Astrophysics Data System (ADS)

Diekmann, Robin; Helle, Øystein I.; Øie, Cristina I.; McCourt, Peter; Huser, Thomas R.; Schüttpelz, Mark; Ahluwalia, Balpreet S.

2017-04-01

Present optical nanoscopy techniques use a complex microscope for imaging and a simple glass slide to hold the sample. Here, we demonstrate the inverse: the use of a complex, but mass-producible optical chip, which hosts the sample and provides a waveguide for the illumination source, and a standard low-cost microscope to acquire super-resolved images via two different approaches. Waveguides composed of a material with high refractive-index contrast provide a strong evanescent field that is used for single-molecule switching and fluorescence excitation, thus enabling chip-based single-molecule localization microscopy. Additionally, multimode interference patterns induce spatial fluorescence intensity variations that enable fluctuation-based super-resolution imaging. As chip-based nanoscopy separates the illumination and detection light paths, total-internal-reflection fluorescence excitation is possible over a large field of view, with up to 0.5 mm × 0.5 mm being demonstrated. Using multicolour chip-based nanoscopy, we visualize fenestrations in liver sinusoidal endothelial cells.

Correction of a Depth-Dependent Lateral Distortion in 3D Super-Resolution Imaging

PubMed Central

Manley, Suliana

2015-01-01

Three-dimensional (3D) localization-based super-resolution microscopy (SR) requires correction of aberrations to accurately represent 3D structure. Here we show how a depth-dependent lateral shift in the apparent position of a fluorescent point source, which we term `wobble`, results in warped 3D SR images and provide a software tool to correct this distortion. This system-specific, lateral shift is typically > 80 nm across an axial range of ~ 1 μm. A theoretical analysis based on phase retrieval data from our microscope suggests that the wobble is caused by non-rotationally symmetric phase and amplitude aberrations in the microscope’s pupil function. We then apply our correction to the bacterial cytoskeletal protein FtsZ in live bacteria and demonstrate that the corrected data more accurately represent the true shape of this vertically-oriented ring-like structure. We also include this correction method in a registration procedure for dual-color, 3D SR data and show that it improves target registration error (TRE) at the axial limits over an imaging depth of 1 μm, yielding TRE values of < 20 nm. This work highlights the importance of correcting aberrations in 3D SR to achieve high fidelity between the measurements and the sample. PMID:26600467

Multiframe super resolution reconstruction method based on light field angular images

NASA Astrophysics Data System (ADS)

Zhou, Shubo; Yuan, Yan; Su, Lijuan; Ding, Xiaomin; Wang, Jichao

2017-12-01

The plenoptic camera can directly obtain 4-dimensional light field information from a 2-dimensional sensor. However, based on the sampling theorem, the spatial resolution is greatly limited by the microlenses. In this paper, we present a method of reconstructing high-resolution images from the angular images. First, the ray tracing method is used to model the telecentric-based light field imaging process. Then, we analyze the subpixel shifts between the angular images extracted from the defocused light field data and the blur in the angular images. According to the analysis above, we construct the observation model from the ideal high-resolution image to the angular images. Applying the regularized super resolution method, we can obtain the super resolution result with a magnification ratio of 8. The results demonstrate the effectiveness of the proposed observation model.

ELM: super-resolution analysis of wide-field images of fluorescent shell structures

NASA Astrophysics Data System (ADS)

Manton, James D.; Xiao, Yao; Turner, Robert D.; Christie, Graham; Rees, Eric J.

2018-07-01

It is often necessary to precisely quantify the size of specimens in biological studies. When measuring feature size in fluorescence microscopy, significant biases can arise due to blurring of its edges if the feature is smaller than the diffraction limit of resolution. This problem is avoided if an equation describing the feature’s entire image is fitted to its image data. In this paper we present open-source software, ELM, which uses this approach to measure the size of spheroidal or cylindrical fluorescent shells with a precision of around 10 nm. This has been used to measure coat protein locations in bacterial spores and cell wall diameter in vegetative bacilli, and may also be valuable in microbiological studies of algae, fungi and viruses. ELM is available for download at https://github.com/quantitativeimaging/ELM.

ELM: super-resolution analysis of wide-field images of fluorescent shell structures.

PubMed

Manton, James; Xiao, Yao; Turner, Robert; Christie, Graham; Rees, Eric

2018-05-04

It is often necessary to precisely quantify the size of specimens in biological studies. When measuring feature size in fluorescence microscopy, significant biases can arise due to blurring of its edges if the feature is smaller than the diffraction limit of resolution. This problem is avoided if an equation describing the feature's entire image is fitted to its image data. In this paper we present open-source software, ELM, which uses this approach to measure the size of spheroidal or cylindrical fluorescent shells with a precision of around 10 nm. This has been used to measure coat protein locations in bacterial spores and cell wall diameter in vegetative bacilli, and may also be valuable in microbiological studies of algae, fungi and viruses. ELM is available for download at https://github.com/quantitativeimaging/ELM. Creative Commons Attribution license.

Underwater video enhancement using multi-camera super-resolution

NASA Astrophysics Data System (ADS)

Quevedo, E.; Delory, E.; Callicó, G. M.; Tobajas, F.; Sarmiento, R.

2017-12-01

Image spatial resolution is critical in several fields such as medicine, communications or satellite, and underwater applications. While a large variety of techniques for image restoration and enhancement has been proposed in the literature, this paper focuses on a novel Super-Resolution fusion algorithm based on a Multi-Camera environment that permits to enhance the quality of underwater video sequences without significantly increasing computation. In order to compare the quality enhancement, two objective quality metrics have been used: PSNR (Peak Signal-to-Noise Ratio) and the SSIM (Structural SIMilarity) index. Results have shown that the proposed method enhances the objective quality of several underwater sequences, avoiding the appearance of undesirable artifacts, with respect to basic fusion Super-Resolution algorithms.

Single image super-resolution using self-optimizing mask via fractional-order gradient interpolation and reconstruction.

PubMed

Yang, Qi; Zhang, Yanzhu; Zhao, Tiebiao; Chen, YangQuan

2017-04-04

Image super-resolution using self-optimizing mask via fractional-order gradient interpolation and reconstruction aims to recover detailed information from low-resolution images and reconstruct them into high-resolution images. Due to the limited amount of data and information retrieved from low-resolution images, it is difficult to restore clear, artifact-free images, while still preserving enough structure of the image such as the texture. This paper presents a new single image super-resolution method which is based on adaptive fractional-order gradient interpolation and reconstruction. The interpolated image gradient via optimal fractional-order gradient is first constructed according to the image similarity and afterwards the minimum energy function is employed to reconstruct the final high-resolution image. Fractional-order gradient based interpolation methods provide an additional degree of freedom which helps optimize the implementation quality due to the fact that an extra free parameter α-order is being used. The proposed method is able to produce a rich texture detail while still being able to maintain structural similarity even under large zoom conditions. Experimental results show that the proposed method performs better than current single image super-resolution techniques. Copyright © 2017 ISA. Published by Elsevier Ltd. All rights reserved.

Tuning donut profile for spatial resolution in stimulated emission depletion microscopy.

PubMed

Neupane, Bhanu; Chen, Fang; Sun, Wei; Chiu, Daniel T; Wang, Gufeng

2013-04-01

In stimulated emission depletion (STED)-based or up-conversion depletion-based super-resolution optical microscopy, the donut-shaped depletion beam profile is of critical importance to its resolution. In this study, we investigate the transformation of the donut-shaped depletion beam focused by a high numerical aperture (NA) microscope objective, and model STED point spread function (PSF) as a function of donut beam profile. We show experimentally that the intensity profile of the dark kernel of the donut can be approximated as a parabolic function, whose slope is determined by the donut beam size before the objective back aperture, or the effective NA. Based on this, we derive the mathematical expression for continuous wave (CW) STED PSF as a function of focal plane donut and excitation beam profiles, as well as dye properties. We find that the effective NA and the residual intensity at the center are critical factors for STED imaging quality and the resolution. The effective NA is critical for STED resolution in that it not only determines the donut shape but also the area the depletion laser power is dispersed. An improperly expanded depletion beam will have negligible improvement in resolution. The polarization of the depletion beam also plays an important role as it affects the residual intensity in the center of the donut. Finally, we construct a CW STED microscope operating at 488 nm excitation and 592 nm depletion with a resolution of 70 nm. Our study provides detailed insight to the property of donut beam, and parameters that are important for the optimal performance of STED microscopes. This paper will provide a useful guide for the construction and future development of STED microscopes.

Optimized computational imaging methods for small-target sensing in lens-free holographic microscopy

NASA Astrophysics Data System (ADS)

Xiong, Zhen; Engle, Isaiah; Garan, Jacob; Melzer, Jeffrey E.; McLeod, Euan

2018-02-01

Lens-free holographic microscopy is a promising diagnostic approach because it is cost-effective, compact, and suitable for point-of-care applications, while providing high resolution together with an ultra-large field-of-view. It has been applied to biomedical sensing, where larger targets like eukaryotic cells, bacteria, or viruses can be directly imaged without labels, and smaller targets like proteins or DNA strands can be detected via scattering labels like micro- or nano-spheres. Automated image processing routines can count objects and infer target concentrations. In these sensing applications, sensitivity and specificity are critically affected by image resolution and signal-to-noise ratio (SNR). Pixel super-resolution approaches have been shown to boost resolution and SNR by synthesizing a high-resolution image from multiple, partially redundant, low-resolution images. However, there are several computational methods that can be used to synthesize the high-resolution image, and previously, it has been unclear which methods work best for the particular case of small-particle sensing. Here, we quantify the SNR achieved in small-particle sensing using regularized gradient-descent optimization method, where the regularization is based on cardinal-neighbor differences, Bayer-pattern noise reduction, or sparsity in the image. In particular, we find that gradient-descent with sparsity-based regularization works best for small-particle sensing. These computational approaches were evaluated on images acquired using a lens-free microscope that we assembled from an off-the-shelf LED array and color image sensor. Compared to other lens-free imaging systems, our hardware integration, calibration, and sample preparation are particularly simple. We believe our results will help to enable the best performance in lens-free holographic sensing.

New learning based super-resolution: use of DWT and IGMRF prior.

PubMed

Gajjar, Prakash P; Joshi, Manjunath V

2010-05-01

In this paper, we propose a new learning-based approach for super-resolving an image captured at low spatial resolution. Given the low spatial resolution test image and a database consisting of low and high spatial resolution images, we obtain super-resolution for the test image. We first obtain an initial high-resolution (HR) estimate by learning the high-frequency details from the available database. A new discrete wavelet transform (DWT) based approach is proposed for learning that uses a set of low-resolution (LR) images and their corresponding HR versions. Since the super-resolution is an ill-posed problem, we obtain the final solution using a regularization framework. The LR image is modeled as the aliased and noisy version of the corresponding HR image, and the aliasing matrix entries are estimated using the test image and the initial HR estimate. The prior model for the super-resolved image is chosen as an Inhomogeneous Gaussian Markov random field (IGMRF) and the model parameters are estimated using the same initial HR estimate. A maximum a posteriori (MAP) estimation is used to arrive at the cost function which is minimized using a simple gradient descent approach. We demonstrate the effectiveness of the proposed approach by conducting the experiments on gray scale as well as on color images. The method is compared with the standard interpolation technique and also with existing learning-based approaches. The proposed approach can be used in applications such as wildlife sensor networks, remote surveillance where the memory, the transmission bandwidth, and the camera cost are the main constraints.

Single image super-resolution via regularized extreme learning regression for imagery from microgrid polarimeters

NASA Astrophysics Data System (ADS)

Sargent, Garrett C.; Ratliff, Bradley M.; Asari, Vijayan K.

2017-08-01

The advantage of division of focal plane imaging polarimeters is their ability to obtain temporally synchronized intensity measurements across a scene; however, they sacrifice spatial resolution in doing so due to their spatially modulated arrangement of the pixel-to-pixel polarizers and often result in aliased imagery. Here, we propose a super-resolution method based upon two previously trained extreme learning machines (ELM) that attempt to recover missing high frequency and low frequency content beyond the spatial resolution of the sensor. This method yields a computationally fast and simple way of recovering lost high and low frequency content from demosaicing raw microgrid polarimetric imagery. The proposed method outperforms other state-of-the-art single-image super-resolution algorithms in terms of structural similarity and peak signal-to-noise ratio.

Direct conversion of h-BN into c-BN and formation of epitaxial c-BN/diamond heterostructures

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

Narayan, Jagdish, E-mail: narayan@ncsu.edu; Bhaumik, Anagh; Xu, Weizong

2016-05-14

We have created a new state of BN (named Q-BN) through rapid melting and super undercooling and quenching by using nanosecond laser pulses. Phase pure c-BN is formed either by direct quenching of super undercooled liquid or by nucleation and growth from Q-BN. Thus, a direct conversion of hexagonal boron nitride (h-BN) into phase-pure cubic boron nitride (c-BN) is achieved by nanosecond pulsed laser melting at ambient temperatures and atmospheric pressure in air. According to the P-T phase diagram, the transformation from h-BN into c-BN under equilibrium processing can occur only at high temperatures and pressures, as the hBN-cBN-Liquid triplemore » point is at 3500 K/9.5 GPa or 3700 K/7.0 GPa with a recent theoretical refinement. Using nonequilibrium nanosecond laser melting, we have created super undercooled state and shifted this triple point to as low as 2800 K and atmospheric pressure. The rapid quenching from super undercooled state leads to the formation of a new phase, named as Q-BN. We present detailed characterization of Q-BN and c-BN layers by using Raman spectroscopy, high-resolution scanning electron microscopy, electron-back-scatter diffraction, high-resolution TEM, and electron energy loss spectroscopy, and discuss the mechanism of formation of nanodots, nanoneedles, microneedles, and single-crystal c-BN on sapphire substrate. We have also deposited diamond by pulsed laser deposition of carbon on c-BN and created c-BN/diamond heterostructures, where c-BN acts as a template for epitaxial diamond growth. We discuss the mechanism of epitaxial c-BN and diamond growth on lattice matching c-BN template under pulsed laser evaporation of amorphous carbon, and the impact of this discovery on a variety of applications.« less

Protein labeling for live cell fluorescence microscopy with a highly photostable renewable signal† †Electronic supplementary information (ESI) available: Supplementary methods, figures, movies, and data. See DOI: 10.1039/c7sc01628j

PubMed Central

Bozhanova, Nina G.; Baranov, Mikhail S.; Klementieva, Natalia V.; Sarkisyan, Karen S.; Gavrikov, Alexey S.; Yampolsky, Ilia V.; Zagaynova, Elena V.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

2017-01-01

We present protein-PAINT – the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes. We demonstrate an order of magnitude higher photostability of the fluorescence signal in comparison with spectrally similar fluorescent proteins. Protein-PAINT ensures prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes. PMID:29147545

Nanoscale invaginations of the nuclear envelope: Shedding new light on wormholes with elusive function.

PubMed

Schoen, Ingmar; Aires, Lina; Ries, Jonas; Vogel, Viola

2017-09-03

Recent advances in fluorescence microscopy have opened up new possibilities to investigate chromosomal and nuclear 3D organization on the nanoscale. We here discuss their potential for elucidating topographical details of the nuclear lamina. Single molecule localization microscopy (SMLM) in combination with immunostainings of lamina proteins readily reveals tube-like invaginations with a diameter of 100-500 nm. Although these invaginations have been established as a frequent and general feature of interphase nuclei across different cell types, their formation mechanism and function have remained largely elusive. We critically review the current state of research, propose possible connections to lamina associated domains (LADs), and revisit the discussion about the potential role of these invaginations for accelerating mRNA nuclear export. Illustrative studies using 3D super-resolution imaging are shown and will be instrumental to decipher the physiological role of these nanoscale invaginations.

Seeing and believing: recent advances in imaging cell-cell interactions

PubMed Central

Yap, Alpha S.; Michael, Magdalene; Parton, Robert G.

2015-01-01

Advances in cell and developmental biology have often been closely linked to advances in our ability to visualize structure and function at many length and time scales. In this review, we discuss how new imaging technologies and new reagents have provided novel insights into the biology of cadherin-based cell-cell junctions. We focus on three developments: the application of super-resolution optical technologies to characterize the nanoscale organization of cadherins at cell-cell contacts, new approaches to interrogate the mechanical forces that act upon junctions, and advances in electron microscopy which have the potential to transform our understanding of cell-cell junctions. PMID:26543555

Seeing and believing: recent advances in imaging cell-cell interactions.

PubMed

Yap, Alpha S; Michael, Magdalene; Parton, Robert G

2015-01-01

Advances in cell and developmental biology have often been closely linked to advances in our ability to visualize structure and function at many length and time scales. In this review, we discuss how new imaging technologies and new reagents have provided novel insights into the biology of cadherin-based cell-cell junctions. We focus on three developments: the application of super-resolution optical technologies to characterize the nanoscale organization of cadherins at cell-cell contacts, new approaches to interrogate the mechanical forces that act upon junctions, and advances in electron microscopy which have the potential to transform our understanding of cell-cell junctions.

Engineering of bacterial phytochromes for in vivo imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Verkhusha, Vladislav; Shcherbakova, Daria M.; Kaberniuk, Andrii A.; Baloban, Mikhail

2017-03-01

Genetically encoded probes with absorbance and fluorescence spectra within a near-infrared tissue transparency window are preferable for deep-tissue imaging. On the basis of bacterial phytochromes we engineered several types of near-infrared absorbing probes for photoacoustic tomography and fluorescent probes for purely optical imaging. They can be used as protein and cell labels and as building blocks for biosensors. The probes enabled imaging of tumors and metastases, protein-protein interactions, RNA visualization, detection of apoptosis, cellular metabolites, signaling pathways and cell proliferation. The developed probes allow non-invasive visualization of biological processes across scales, from super-resolution microscopy to tissue and whole-body animal imaging.

Convex relaxations of spectral sparsity for robust super-resolution and line spectrum estimation

NASA Astrophysics Data System (ADS)

Chi, Yuejie

2017-08-01

We consider recovering the amplitudes and locations of spikes in a point source signal from its low-pass spectrum that may suffer from missing data and arbitrary outliers. We first review and provide a unified view of several recently proposed convex relaxations that characterize and capitalize the spectral sparsity of the point source signal without discretization under the framework of atomic norms. Next we propose a new algorithm when the spikes are known a priori to be positive, motivated by applications such as neural spike sorting and fluorescence microscopy imaging. Numerical experiments are provided to demonstrate the effectiveness of the proposed approach.

Functional Scanning Probe Imaging of Nanostructured Solar Energy Materials.

PubMed

Giridharagopal, Rajiv; Cox, Phillip A; Ginger, David S

2016-09-20

From hybrid perovskites to semiconducting polymer/fullerene blends for organic photovoltaics, many new materials being explored for energy harvesting and storage exhibit performance characteristics that depend sensitively on their nanoscale morphology. At the same time, rapid advances in the capability and accessibility of scanning probe microscopy methods over the past decade have made it possible to study processing/structure/function relationships ranging from photocurrent collection to photocarrier lifetimes with resolutions on the scale of tens of nanometers or better. Importantly, such scanning probe methods offer the potential to combine measurements of local structure with local function, and they can be implemented to study materials in situ or devices in operando to better understand how materials evolve in time in response to an external stimulus or environmental perturbation. This Account highlights recent advances in the development and application of scanning probe microscopy methods that can help address such questions while filling key gaps between the capabilities of conventional electron microscopy and newer super-resolution optical methods. Focusing on semiconductor materials for solar energy applications, we highlight a range of electrical and optoelectronic scanning probe microscopy methods that exploit the local dynamics of an atomic force microscope tip to probe key properties of the solar cell material or device structure. We discuss how it is possible to extract relevant device properties using noncontact scanning probe methods as well as how these properties guide materials development. Specifically, we discuss intensity-modulated scanning Kelvin probe microscopy (IM-SKPM), time-resolved electrostatic force microscopy (trEFM), frequency-modulated electrostatic force microscopy (FM-EFM), and cantilever ringdown imaging. We explain these developments in the context of classic atomic force microscopy (AFM) methods that exploit the physics of cantilever motion and photocarrier generation to provide robust, nanoscale measurements of materials physics that are correlated with device operation. We predict that the multidimensional data sets made possible by these types of methods will become increasingly important as advances in data science expand capabilities and opportunities for image correlation and discovery.

Sparse super-resolution reconstructions of video from mobile devices in digital TV broadcast applications

NASA Astrophysics Data System (ADS)

Boon, Choong S.; Guleryuz, Onur G.; Kawahara, Toshiro; Suzuki, Yoshinori

2006-08-01

We consider the mobile service scenario where video programming is broadcast to low-resolution wireless terminals. In such a scenario, broadcasters utilize simultaneous data services and bi-directional communications capabilities of the terminals in order to offer substantially enriched viewing experiences to users by allowing user participation and user tuned content. While users immediately benefit from this service when using their phones in mobile environments, the service is less appealing in stationary environments where a regular television provides competing programming at much higher display resolutions. We propose a fast super-resolution technique that allows the mobile terminals to show a much enhanced version of the broadcast video on nearby high-resolution devices, extending the appeal and usefulness of the broadcast service. The proposed single frame super-resolution algorithm uses recent sparse recovery results to provide high quality and high-resolution video reconstructions based solely on individual decoded frames provided by the low-resolution broadcast.

Near-field examination of perovskite-based superlenses and superlens-enhanced probe-object coupling

PubMed Central

Kehr, S.C.; Liu, Y.M.; Martin, L.W.; Yu, P.; Gajek, M.; Yang, S.-Y.; Yang, C.-H.; Wenzel, M.T.; Jacob, R.; von Ribbeck, H.-G.; Helm, M.; Zhang, X.; Eng, L.M.; Ramesh, R.

2011-01-01

A planar slab of negative-index material works as a superlens with sub-diffraction-limited resolution, as propagating waves are focused and, moreover, evanescent waves are reconstructed in the image plane. Here we demonstrate a superlens for electric evanescent fields with low losses using perovskites in the mid-infrared regime. The combination of near-field microscopy with a tunable free-electron laser allows us to address precisely the polariton modes, which are critical for super-resolution imaging. We spectrally study the lateral and vertical distributions of evanescent waves around the image plane of such a lens, and achieve imaging resolution of λ/14 at the superlensing wavelength. Interestingly, at certain distances between the probe and sample surface, we observe a maximum of these evanescent fields. Comparisons with numerical simulations indicate that this maximum originates from an enhanced coupling between probe and object, which might be applicable for multifunctional circuits, infrared spectroscopy and thermal sensors. PMID:21427720

Resolution enhancement by extrapolation of coherent diffraction images: a quantitative study on the limits and a numerical study of nonbinary and phase objects.

PubMed

Latychevskaia, T; Chushkin, Y; Fink, H-W

2016-10-01

In coherent diffractive imaging, the resolution of the reconstructed object is limited by the numerical aperture of the experimental setup. We present here a theoretical and numerical study for achieving super-resolution by postextrapolation of coherent diffraction images, such as diffraction patterns or holograms. We demonstrate that a diffraction pattern can unambiguously be extrapolated from only a fraction of the entire pattern and that the ratio of the extrapolated signal to the originally available signal is linearly proportional to the oversampling ratio. Although there could be in principle other methods to achieve extrapolation, we devote our discussion to employing iterative phase retrieval methods and demonstrate their limits. We present two numerical studies; namely, the extrapolation of diffraction patterns of nonbinary and that of phase objects together with a discussion of the optimal extrapolation procedure. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Image super-resolution via sparse representation.

PubMed

Yang, Jianchao; Wright, John; Huang, Thomas S; Ma, Yi

2010-11-01

This paper presents a new approach to single-image super-resolution, based on sparse signal representation. Research on image statistics suggests that image patches can be well-represented as a sparse linear combination of elements from an appropriately chosen over-complete dictionary. Inspired by this observation, we seek a sparse representation for each patch of the low-resolution input, and then use the coefficients of this representation to generate the high-resolution output. Theoretical results from compressed sensing suggest that under mild conditions, the sparse representation can be correctly recovered from the downsampled signals. By jointly training two dictionaries for the low- and high-resolution image patches, we can enforce the similarity of sparse representations between the low resolution and high resolution image patch pair with respect to their own dictionaries. Therefore, the sparse representation of a low resolution image patch can be applied with the high resolution image patch dictionary to generate a high resolution image patch. The learned dictionary pair is a more compact representation of the patch pairs, compared to previous approaches, which simply sample a large amount of image patch pairs, reducing the computational cost substantially. The effectiveness of such a sparsity prior is demonstrated for both general image super-resolution and the special case of face hallucination. In both cases, our algorithm generates high-resolution images that are competitive or even superior in quality to images produced by other similar SR methods. In addition, the local sparse modeling of our approach is naturally robust to noise, and therefore the proposed algorithm can handle super-resolution with noisy inputs in a more unified framework.

Super-resolution imaging based on the temperature-dependent electron-phonon collision frequency effect of metal thin films

NASA Astrophysics Data System (ADS)

Ding, Chenliang; Wei, Jingsong; Xiao, Mufei

2018-05-01

We herein propose a far-field super-resolution imaging with metal thin films based on the temperature-dependent electron-phonon collision frequency effect. In the proposed method, neither fluorescence labeling nor any special properties are required for the samples. The 100 nm lands and 200 nm grooves on the Blu-ray disk substrates were clearly resolved and imaged through a laser scanning microscope of wavelength 405 nm. The spot size was approximately 0.80 μm , and the imaging resolution of 1/8 of the laser spot size was experimentally obtained. This work can be applied to the far-field super-resolution imaging of samples with neither fluorescence labeling nor any special properties.

Graphene quantum dots with visible light absorption of the carbon core: insights from single-particle spectroscopy and first principles based theory

NASA Astrophysics Data System (ADS)

Ghosh, Siddharth; Awasthi, Manohar; Ghosh, Moumita; Seibt, Michael; Niehaus, Thomas A.

2016-12-01

Luminescent carbon nanodots (CND) are a recent addition to the family of carbon nanostructures. Interestingly, a large group of CNDs are fluorescent in the visible spectrum and possess single dipole emitters with potential applications in super-resolution microscopy, quantum information science, and optoelectronics. There is a large diversity of CND’s size as well as a strong variability of edge topology and functional groups in real samples. This hampers a direct comparison of experimental and theoretical findings that is necessary to understand the unusual photophysics of these systems. Here, we derive atomistic models of finite sized (<2.5 nm) CNDs from high resolution transmission electron microscopy (HRTEM) which are studied using approximate time-dependent density functional theory. The atomistic models are found to be primarily two-dimensional (2D) and can hence be categorised as graphene quantum dots (GQD). The GQD model structures that are presented here show excitation energies in the visible spectrum matching previous single GQD level photoluminescence studies. We also present the effect of edge hydroxyl and carboxyl functional groups on the absorption spectrum. Overall, the study reveals the atomistic origin of CNDs photoluminescence in the visible range.

Breast Microcalcification Detection Using Super-Resolution Ultrasound Image Reconstruction

DTIC Science & Technology

2010-09-01

microcalcifications often occur as one of two types: calcium oxalate dihydrate or calcium hydroxyapatite. Their sizes range approximately from 0.1 mm to 0.5 mm...super-resolution imaging, ultrasound imaging, wave equation. 1. INTRODUCTION Microcalcifications, tiny specks of mineral deposits ( calcium ), are the

Super-resolution of fluorescence-free plasmonic nanoparticles using enhanced dark-field illumination based on wavelength-modulation

DOE PAGES

Zhang, Peng; Lee, Seungah; Yu, Hyunung; ...

2015-06-15

Super-resolution imaging of fluorescence-free plasmonic nanoparticles (NPs) was achieved using enhanced dark-field (EDF) illumination based on wavelength-modulation. Indistinguishable adjacent EDF images of 103-nm gold nanoparticles (GNPs), 40-nm gold nanorods (GNRs), and 80-nm silver nanoparticles (SNPs) were modulated at their wavelengths of specific localized surface plasmon scattering. The coordinates (x, y) of each NP were resolved by fitting their point spread functions with a two-dimensional Gaussian. The measured localization precisions of GNPs, GNRs, and SNPs were 2.5 nm, 5.0 nm, and 2.9 nm, respectively. From the resolved coordinates of NPs and the corresponding localization precisions, super-resolution images were reconstructed. Depending onmore » the spontaneous polarization of GNR scattering, the orientation angle of GNRs in two-dimensions was resolved and provided more elaborate localization information. This novel fluorescence-free super-resolution method was applied to live HeLa cells to resolve NPs and provided remarkable subdiffraction limit images.« less

Super-Resolution Reconstruction of Remote Sensing Images Using Multifractal Analysis

PubMed Central

Hu, Mao-Gui; Wang, Jin-Feng; Ge, Yong

2009-01-01

Satellite remote sensing (RS) is an important contributor to Earth observation, providing various kinds of imagery every day, but low spatial resolution remains a critical bottleneck in a lot of applications, restricting higher spatial resolution analysis (e.g., intra-urban). In this study, a multifractal-based super-resolution reconstruction method is proposed to alleviate this problem. The multifractal characteristic is common in Nature. The self-similarity or self-affinity presented in the image is useful to estimate details at larger and smaller scales than the original. We first look for the presence of multifractal characteristics in the images. Then we estimate parameters of the information transfer function and noise of the low resolution image. Finally, a noise-free, spatial resolution-enhanced image is generated by a fractal coding-based denoising and downscaling method. The empirical case shows that the reconstructed super-resolution image performs well in detail enhancement. This method is not only useful for remote sensing in investigating Earth, but also for other images with multifractal characteristics. PMID:22291530

A super resolution framework for low resolution document image OCR

NASA Astrophysics Data System (ADS)

Ma, Di; Agam, Gady

2013-01-01

Optical character recognition is widely used for converting document images into digital media. Existing OCR algorithms and tools produce good results from high resolution, good quality, document images. In this paper, we propose a machine learning based super resolution framework for low resolution document image OCR. Two main techniques are used in our proposed approach: a document page segmentation algorithm and a modified K-means clustering algorithm. Using this approach, by exploiting coherence in the document, we reconstruct from a low resolution document image a better resolution image and improve OCR results. Experimental results show substantial gain in low resolution documents such as the ones captured from video.

A highly sensitive room temperature H2S gas sensor based on SnO2 multi-tube arrays bio-templated from insect bristles.

PubMed

Tian, Junlong; Pan, Feng; Xue, Ruiyang; Zhang, Wang; Fang, Xiaotian; Liu, Qinglei; Wang, Yuhua; Zhang, Zhijian; Zhang, Di

2015-05-07

A tin oxide multi-tube array (SMTA) with a parallel effect was fabricated through a simple and promising method combining chemosynthesis and biomimetic techniques; a biomimetic template was derived from the bristles on the wings of the Alpine Black Swallowtail butterfly (Papilio maackii). SnO2 tubes are hollow and porous structures with micro-pores regularly distributed on the wall. The morphology, the delicate microstructure and the crystal structure of this SMTA were characterized by super resolution digital microscopy, scanning electron microscopy, transmission electron microscopy and X-ray diffraction. The SMTA exhibits a high sensitivity to H2S gas at room temperature. It also exhibits a short response/recovery time, with an average value of 14/30 s at 5 ppm. In particular, heating is not required for the SMTA in the gas sensitivity measurement process. On the basis of these results, SMTA is proposed as a suitable new material for the design and fabrication of room-temperature H2S gas sensors.

Construction of an instant structured illumination microscope

PubMed Central

Curd, Alistair; Cleasby, Alexa; Makowska, Katarzyna; York, Andrew; Shroff, Hari; Peckham, Michelle

2015-01-01

A challenge in biological imaging is to capture high-resolution images at fast frame rates in live cells. The “instant structured illumination microscope” (iSIM) is a system designed for this purpose. Similarly to standard structured illumination microscopy (SIM), an iSIM provides a twofold improvement over widefield microscopy, in x, y and z, but also allows much faster image acquisition, with real-time display of super-resolution images. The assembly of an iSIM is reasonably complex, involving the combination and alignment of many optical components, including three micro-optics arrays (two lenslet arrays and an array of pinholes, all with a pitch of 222 μm) and a double-sided scanning mirror. In addition, a number of electronic components must be correctly controlled. Construction of the system is therefore not trivial, but is highly desirable, particularly for live-cell imaging. We report, and provide instructions for, the construction of an iSIM, including minor modifications to a previous design in both hardware and software. The final instrument allows us to rapidly acquire fluorescence images at rates faster than 100 fps, with approximately twofold improvement in resolution in both x–y and z; sub-diffractive biological features have an apparent size (full width at half maximum) of 145 nm (lateral) and 320 nm (axial), using a 1.49 NA objective and 488 nm excitation. PMID:26210400

Super-Resolution Localization Microscopy of γ-H2AX and Heterochromatin after Folate Deficiency.

PubMed

Bach, Margund; Savini, Claudia; Krufczik, Matthias; Cremer, Christoph; Rösl, Frank; Hausmann, Michael

2017-08-08

Folate is an essential water-soluble vitamin in food and nutrition supplements. As a one-carbon source, it is involved in many central regulatory processes, such as DNA, RNA, and protein methylation as well as DNA synthesis and repair. Deficiency in folate is considered to be associated with an increased incidence of several malignancies, including cervical cancer that is etiologically linked to an infection with "high-risk" human papilloma viruses (HPV). However, it is still not known how a recommended increase in dietary folate after its deprivation affects the physiological status of cells. To study the impact of folate depletion and its subsequent reconstitution in single cells, we used quantitative chromatin conformation measurements obtained by super-resolution fluorescence microscopy, i.e., single molecule localization microscopy (SMLM). As a read-out, we examined the levels and the (re)positioning of γ-H2AX tags and histone H3K9me3 heterochromatin tags after immunostaining in three-dimensional (3D)-conserved cell nuclei. As model, we used HPV16 positive immortalized human keratinocytes that were cultivated under normal, folate deficient, and reconstituted conditions for different periods of time. The results were compared to cells continuously cultivated in standard folate medium. After 13 weeks in low folate, an increase in the phosphorylation of the histone H2AX was noted, indicative of an accumulation of DNA double strand breaks. DNA repair activity represented by the formation of those γ-H2AX clusters was maintained during the following 15 weeks of examination. However, the clustered arrangements of tags appeared to relax in a time-dependent manner. Parallel to the repair activity, the chromatin methylation activity increased as detected by H3K9me3 tags. The progress of DNA double strand repair was accompanied by a reduction of the detected nucleosome density around the γ-H2AX clusters, suggesting a shift from hetero- to euchromatin to allow access to the repair machinery. In conclusion, these data demonstrated a folate-dependent repair activity and chromatin re-organization on the SMLM nanoscale level. This offers new opportunities to further investigate folate-induced chromatin re-organization and the associated mechanisms.

Supporting lander and rover operation: a novel super-resolution restoration technique

NASA Astrophysics Data System (ADS)

Tao, Yu; Muller, Jan-Peter

2015-04-01

Higher resolution imaging data is always desirable to critical rover engineering operations, such as landing site selection, path planning, and optical localisation. For current Mars missions, 25cm HiRISE images have been widely used by the MER & MSL engineering team for rover path planning and location registration/adjustment. However, 25cm is not high enough resolution to be able to view individual rocks (≤2m in size) or visualise the types of sedimentary features that rover onboard cameras might observe. Nevertheless, due to various physical constraints (e.g. telescope size and mass) from the imaging instruments themselves, one needs to be able to tradeoff spatial resolution and bandwidth. This means that future imaging systems are likely to be limited to resolve features larger than 25cm. We have developed a novel super-resolution algorithm/pipeline to be able to restore higher resolution image from the non-redundant sub-pixel information contained in multiple lower resolution raw images [Tao & Muller 2015]. We will demonstrate with experiments performed using 5-10 overlapped 25cm HiRISE images for MER-A, MER-B & MSL to resolve 5-10cm super resolution images that can be directly compared to rover imagery at a range of 5 metres from the rover cameras but in our case can be used to visualise features many kilometres away from the actual rover traverse. We will demonstrate how these super-resolution images together with image understanding software can be used to quantify rock size-frequency distributions as well as measure sedimentary rock layers for several critical sites for comparison with rover orthorectified image mosaic to demonstrate optimality of using our super-resolution resolved image to better support future lander and rover operation in future. We present the potential of super-resolution for virtual exploration to the ˜400 HiRISE areas which have been viewed 5 or more times and the potential application of this technique to all of the ESA ExoMars Trace Gas orbiter CaSSiS stereo, multi-angle and colour camera images from 2017 onwards. Acknowledgements: The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement No.312377 PRoViDE.

Metabolic labelling of the carbohydrate core in bacterial peptidoglycan and its applications

PubMed Central

Liang, Hai; DeMeester, Kristen E.; Hou, Ching-Wen; Parent, Michelle A.; Caplan, Jeffrey L.; Grimes, Catherine L.

2017-01-01

Bacterial cells are surrounded by a polymer known as peptidoglycan (PG), which protects the cell from changes in osmotic pressure and small molecule insults. A component of this material, N-acetyl-muramic acid (NAM), serves as a core structural element for innate immune recognition of PG fragments. We report the synthesis of modifiable NAM carbohydrate derivatives and the installation of these building blocks into the backbone of Gram-positive and Gram-negative bacterial PG utilizing metabolic cell wall recycling and biosynthetic machineries. Whole cells are labelled via click chemistry and visualized using super-resolution microscopy, revealing higher resolution PG structural details and allowing the cell wall biosynthesis, as well as its destruction in immune cells, to be tracked. This study will assist in the future identification of mechanisms that the immune system uses to recognize bacteria, glean information about fundamental cell wall architecture and aid in the design of novel antibiotics. PMID:28425464

Polarization-controlled directional scattering for nanoscopic position sensing

PubMed Central

Neugebauer, Martin; Woźniak, Paweł; Bag, Ankan; Leuchs, Gerd; Banzer, Peter

2016-01-01

Controlling the propagation and coupling of light to sub-wavelength antennas is a crucial prerequisite for many nanoscale optical devices. Recently, the main focus of attention has been directed towards high-refractive-index materials such as silicon as an integral part of the antenna design. This development is motivated by the rich spectral properties of individual high-refractive-index nanoparticles. Here we take advantage of the interference of their magnetic and electric resonances to achieve strong lateral directionality. For controlled excitation of a spherical silicon nanoantenna, we use tightly focused radially polarized light. The resultant directional emission depends on the antenna's position relative to the focus. This approach finds application as a novel position sensing technique, which might be implemented in modern nanometrology and super-resolution microscopy set-ups. We demonstrate in a proof-of-concept experiment that a lateral resolution in the Ångström regime can be achieved. PMID:27095171

Super-resolution with an SLM and two intensity images

NASA Astrophysics Data System (ADS)

Alcalá Ochoa, Noé; de León, Y. Ponce

2018-06-01

It is reported a method which may simplify the optical setups used to achieve super-resolution through the amplitude multiplication of two waves. For this end we decompose a super-resolving pupil into two complex masks and with the aid of a Spatial Light Modulator (LCoS) we obtain two intensity images that are subtracted. With this proposal, the traditional experimental optical setups are considerably simplified, with the additional benefit that different masks can be utilized without needing to perform the setup alignment each time.

Super-resolution links vinculin localization to function in focal adhesions.

PubMed

Giannone, Grégory

2015-07-01

Integrin-based focal adhesions integrate biochemical and biomechanical signals from the extracellular matrix and the actin cytoskeleton. The combination of three-dimensional super-resolution imaging and loss- or gain-of-function protein mutants now links the nanoscale dynamic localization of proteins to their activation and function within focal adhesions.

A Bayesian Nonparametric Approach to Image Super-Resolution.

PubMed

Polatkan, Gungor; Zhou, Mingyuan; Carin, Lawrence; Blei, David; Daubechies, Ingrid

2015-02-01

Super-resolution methods form high-resolution images from low-resolution images. In this paper, we develop a new Bayesian nonparametric model for super-resolution. Our method uses a beta-Bernoulli process to learn a set of recurring visual patterns, called dictionary elements, from the data. Because it is nonparametric, the number of elements found is also determined from the data. We test the results on both benchmark and natural images, comparing with several other models from the research literature. We perform large-scale human evaluation experiments to assess the visual quality of the results. In a first implementation, we use Gibbs sampling to approximate the posterior. However, this algorithm is not feasible for large-scale data. To circumvent this, we then develop an online variational Bayes (VB) algorithm. This algorithm finds high quality dictionaries in a fraction of the time needed by the Gibbs sampler.

Optical super-resolution effect induced by nonlinear characteristics of graphene oxide films

NASA Astrophysics Data System (ADS)

Zhao, Yong-chuang; Nie, Zhong-quan; Zhai, Ai-ping; Tian, Yan-ting; Liu, Chao; Shi, Chang-kun; Jia, Bao-hua

2018-01-01

In this work, we focus on the optical super-resolution effect induced by strong nonlinear saturation absorption (NSA) of graphene oxide (GO) membranes. The third-order optical nonlinearities are characterized by the canonical Z-scan technique under femtosecond laser (wavelength: 800 nm, pulse width: 100 fs) excitation. Through controlling the applied femtosecond laser energy, NSA of the GO films can be tuned continuously. The GO film is placed at the focal plane as a unique amplitude filter to improve the resolution of the focused field. A multi-layer system model is proposed to present the generation of a deep sub-wavelength spot associated with the nonlinearity of GO films. Moreover, the parameter conditions to achieve the best resolution (˜λ/6) are determined entirely. The demonstrated results here are useful for high density optical recoding and storage, nanolithography, and super-resolution optical imaging.

An Example-Based Super-Resolution Algorithm for Selfie Images

PubMed Central

William, Jino Hans; Venkateswaran, N.; Narayanan, Srinath; Ramachandran, Sandeep

2016-01-01

A selfie is typically a self-portrait captured using the front camera of a smartphone. Most state-of-the-art smartphones are equipped with a high-resolution (HR) rear camera and a low-resolution (LR) front camera. As selfies are captured by front camera with limited pixel resolution, the fine details in it are explicitly missed. This paper aims to improve the resolution of selfies by exploiting the fine details in HR images captured by rear camera using an example-based super-resolution (SR) algorithm. HR images captured by rear camera carry significant fine details and are used as an exemplar to train an optimal matrix-value regression (MVR) operator. The MVR operator serves as an image-pair priori which learns the correspondence between the LR-HR patch-pairs and is effectively used to super-resolve LR selfie images. The proposed MVR algorithm avoids vectorization of image patch-pairs and preserves image-level information during both learning and recovering process. The proposed algorithm is evaluated for its efficiency and effectiveness both qualitatively and quantitatively with other state-of-the-art SR algorithms. The results validate that the proposed algorithm is efficient as it requires less than 3 seconds to super-resolve LR selfie and is effective as it preserves sharp details without introducing any counterfeit fine details. PMID:27064500

Framework for Detection and Localization of Extreme Climate Event with Pixel Recursive Super Resolution

NASA Astrophysics Data System (ADS)

Kim, S. K.; Lee, J.; Zhang, C.; Ames, S.; Williams, D. N.

2017-12-01

Deep learning techniques have been successfully applied to solve many problems in climate and geoscience using massive-scaled observed and modeled data. For extreme climate event detections, several models based on deep neural networks have been recently proposed and attend superior performance that overshadows all previous handcrafted expert based method. The issue arising, though, is that accurate localization of events requires high quality of climate data. In this work, we propose framework capable of detecting and localizing extreme climate events in very coarse climate data. Our framework is based on two models using deep neural networks, (1) Convolutional Neural Networks (CNNs) to detect and localize extreme climate events, and (2) Pixel recursive recursive super resolution model to reconstruct high resolution climate data from low resolution climate data. Based on our preliminary work, we have presented two CNNs in our framework for different purposes, detection and localization. Our results using CNNs for extreme climate events detection shows that simple neural nets can capture the pattern of extreme climate events with high accuracy from very coarse reanalysis data. However, localization accuracy is relatively low due to the coarse resolution. To resolve this issue, the pixel recursive super resolution model reconstructs the resolution of input of localization CNNs. We present a best networks using pixel recursive super resolution model that synthesizes details of tropical cyclone in ground truth data while enhancing their resolution. Therefore, this approach not only dramat- ically reduces the human effort, but also suggests possibility to reduce computing cost required for downscaling process to increase resolution of data.

Super-resolution for scanning light stimulation systems

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

Bitzer, L. A.; Neumann, K.; Benson, N., E-mail: niels.benson@uni-due.de

Super-resolution (SR) is a technique used in digital image processing to overcome the resolution limitation of imaging systems. In this process, a single high resolution image is reconstructed from multiple low resolution images. SR is commonly used for CCD and CMOS (Complementary Metal-Oxide-Semiconductor) sensor images, as well as for medical applications, e.g., magnetic resonance imaging. Here, we demonstrate that super-resolution can be applied with scanning light stimulation (LS) systems, which are common to obtain space-resolved electro-optical parameters of a sample. For our purposes, the Projection Onto Convex Sets (POCS) was chosen and modified to suit the needs of LS systems.more » To demonstrate the SR adaption, an Optical Beam Induced Current (OBIC) LS system was used. The POCS algorithm was optimized by means of OBIC short circuit current measurements on a multicrystalline solar cell, resulting in a mean square error reduction of up to 61% and improved image quality.« less

North Twin Peak in super resolution

NASA Technical Reports Server (NTRS)

1997-01-01

This pair of images shows the result of taking a sequence of 25 identical exposures from the Imager for Mars Pathfinder (IMP) of the northern Twin Peak, with small camera motions, and processing them with the Super-Resolution algorithm developed at NASA's Ames Research Center.

The upper image is a representative input image, scaled up by a factor of five, with the pixel edges smoothed out for a fair comparison. The lower image allows significantly finer detail to be resolved.

Mars Pathfinder is the second in NASA's Discovery program of low-cost spacecraft with highly focused science goals. The Jet Propulsion Laboratory, Pasadena, CA, developed and manages the Mars Pathfinder mission for NASA's Office of Space Science, Washington, D.C. JPL is an operating division of the California Institute of Technology (Caltech). The Imager for Mars Pathfinder (IMP) was developed by the University of Arizona Lunar and Planetary Laboratory under contract to JPL. Peter Smith is the Principal Investigator.

The super-resolution research was conducted by Peter Cheeseman, Bob Kanefsky, Robin Hanson, and John Stutz of NASA's Ames Research Center, Mountain View, CA. More information on this technology is available on the Ames Super Resolution home page at

http://ic-www.arc.nasa.gov/ic/projects/bayes-group/ group/super-res/

Simultaneous digital super-resolution and nonuniformity correction for infrared imaging systems.

PubMed

Meza, Pablo; Machuca, Guillermo; Torres, Sergio; Martin, Cesar San; Vera, Esteban

2015-07-20

In this article, we present a novel algorithm to achieve simultaneous digital super-resolution and nonuniformity correction from a sequence of infrared images. We propose to use spatial regularization terms that exploit nonlocal means and the absence of spatial correlation between the scene and the nonuniformity noise sources. We derive an iterative optimization algorithm based on a gradient descent minimization strategy. Results from infrared image sequences corrupted with simulated and real fixed-pattern noise show a competitive performance compared with state-of-the-art methods. A qualitative analysis on the experimental results obtained with images from a variety of infrared cameras indicates that the proposed method provides super-resolution images with significantly less fixed-pattern noise.

Super-resolution using a light inception layer in convolutional neural network

NASA Astrophysics Data System (ADS)

Mou, Qinyang; Guo, Jun

2018-04-01

Recently, several models based on CNN architecture have achieved great result on Single Image Super-Resolution (SISR) problem. In this paper, we propose an image super-resolution method (SR) using a light inception layer in convolutional network (LICN). Due to the strong representation ability of our well-designed inception layer that can learn richer representation with less parameters, we can build our model with shallow architecture that can reduce the effect of vanishing gradients problem and save computational costs. Our model strike a balance between computational speed and the quality of the result. Compared with state-of-the-art result, we produce comparable or better results with faster computational speed.

Determination of in vivo regulation kinetics of small non-coding RNA in bacteria

NASA Astrophysics Data System (ADS)

Fei, Jingyi

Small RNAs (sRNAs) play important roles in regulating gene expression through a variety of mechanisms. As one of the most common strategies, sRNA induced target messenger RNA (mRNA) includes two major steps: target search by base-pairing interactions with the and downstream execution by modulating translation or the stability of the mRNA. Here we describe a new imaging and analysis platform based on super-resolution fluorescence microscopy, which enabled the first in vivo kinetic measurement of sRNA-mediated gene regulation. Specifically, this platform was used to investigate a sugar-phosphate stress-induced bacterial sRNA that induces the degradation of target mRNAs. The data reveal that the sRNA binds to a primary target mRNA in a reversible and dynamic fashion, and that formation of the sRNA-mRNA complexes is the rate-limiting step, dictating the overall efficiency of regulation in vivo; whereas the downstream co-degradation of sRNA-mRNA complex can kinetically compete with the fast complex disassembly. Examination of a secondary target of this sRNA indicated that differences in the target search kinetics contribute to setting the regulation priority among different target mRNAs. This super-resolution imaging and analysis approach provides a conceptual framework that can be generalized to other sRNA systems and other target search processes.

HIV-1 Nef sequesters MHC-I intracellularly by targeting early stages of endocytosis and recycling

PubMed Central

Dirk, Brennan S.; Pawlak, Emily N.; Johnson, Aaron L.; Van Nynatten, Logan R.; Jacob, Rajesh A.; Heit, Bryan; Dikeakos, Jimmy D.

2016-01-01

A defining characteristic of HIV-1 infection is the ability of the virus to persist within the host. Specifically, MHC-I downregulation by the HIV-1 accessory protein Nef is of critical importance in preventing infected cells from cytotoxic T-cell mediated killing. Nef downregulates MHC-I by modulating the host membrane trafficking machinery, resulting in the endocytosis and eventual sequestration of MHC-I within the cell. In the current report, we utilized the intracellular protein-protein interaction reporter system, bimolecular fluorescence complementation (BiFC), in combination with super-resolution microscopy, to track the Nef/MHC-I interaction and determine its subcellular localization in cells. We demonstrate that this interaction occurs upon Nef binding the MHC-I cytoplasmic tail early during endocytosis in a Rab5-positive endosome. Disruption of early endosome regulation inhibited Nef-dependent MHC-I downregulation, demonstrating that Nef hijacks the early endosome to sequester MHC-I within the cell. Furthermore, super-resolution imaging identified that the Nef:MHC-I BiFC complex transits through both early and late endosomes before ultimately residing at the trans-Golgi network. Together we demonstrate the importance of the early stages of the endocytic network in the removal of MHC-I from the cell surface and its re-localization within the cell, which allows HIV-1 to optimally evade host immune responses. PMID:27841315

Advanced imaging of tau pathology in Alzheimer Disease: New perspectives from super resolution microscopy and label-free nanoscopy.

PubMed

Schierle, Gabriele S Kaminski; Michel, Claire H; Gasparini, Laura

2016-08-01

Alzheimer's disease (AD) is the main cause of dementia in the elderly population. Over 30 million people worldwide are living with dementia and AD prevalence is projected to increase dramatically in the next two decades. In terms of neuropathology, AD is characterized by two major cerebral hallmarks: extracellular β-amyloid (Aβ) plaques and intracellular Tau inclusions, which start accumulating in the brain 15-20 years before the onset of symptoms. Within this context, the scientific community worldwide is undertaking a wide research effort to detect AD pathology at its earliest, before symptoms appear. Neuroimaging of Aβ by positron emission tomography (PET) is clinically available and is a promising modality for early detection of Aβ pathology and AD diagnosis. Substantive efforts are ongoing to develop advanced imaging techniques for early detection of Tau pathology. Here, we will briefly describe the key features of Tau pathology and its heterogeneity across various neurodegenerative diseases bearing cerebral Tau inclusions (i.e., tauopathies). We will outline the current status of research on Tau-specific PET tracers and their clinical development. Finally, we will discuss the potential application of novel super-resolution and label-free techniques for investigating Tau pathology at the experimental level and their potential application for AD diagnosis. Microsc. Res. Tech. 79:677-683, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Barnacle Bill in Super Resolution from Super Panorama

NASA Image and Video Library

1998-07-03

"Barnacle Bill" is a small rock immediately west-northwest of the Mars Pathfinder lander and was the first rock visited by the Sojourner Rover's alpha proton X-ray spectrometer (APXS) instrument. This image shows super resolution techniques applied to the first APXS target rock, which was never imaged with the rover's forward cameras. Super resolution was applied to help to address questions about the texture of this rock and what it might tell us about its mode of origin. This view of Barnacle Bill was produced by combining the "Super Panorama" frames from the IMP camera. Super resolution was applied to help to address questions about the texture of these rocks and what it might tell us about their mode of origin. The composite color frames that make up this anaglyph were produced for both the right and left eye of the IMP. The composites consist of 7 frames in the right eye and 8 frames in the left eye, taken with different color filters that were enlarged by 500% and then co-added using Adobe Photoshop to produce, in effect, a super-resolution panchromatic frame that is sharper than an individual frame would be. These panchromatic frames were then colorized with the red, green, and blue filtered images from the same sequence. The color balance was adjusted to approximate the true color of Mars. The anaglyph view was produced by combining the left with the right eye color composite frames by assigning the left eye composite view to the red color plane and the right eye composite view to the green and blue color planes (cyan), to produce a stereo anaglyph mosaic. This mosaic can be viewed in 3-D on your computer monitor or in color print form by wearing red-blue 3-D glasses. http://photojournal.jpl.nasa.gov/catalog/PIA01409

Carbofluoresceins and Carborhodamines as Scaffolds for High-Contrast Fluorogenic Probes

PubMed Central

2013-01-01

Fluorogenic molecules are important tools for advanced biochemical and biological experiments. The extant collection of fluorogenic probes is incomplete, however, leaving regions of the electromagnetic spectrum unutilized. Here, we synthesize green-excited fluorescent and fluorogenic analogues of the classic fluorescein and rhodamine 110 fluorophores by replacement of the xanthene oxygen with a quaternary carbon. These anthracenyl “carbofluorescein” and “carborhodamine 110” fluorophores exhibit excellent fluorescent properties and can be masked with enzyme- and photolabile groups to prepare high-contrast fluorogenic molecules useful for live cell imaging experiments and super-resolution microscopy. Our divergent approach to these red-shifted dye scaffolds will enable the preparation of numerous novel fluorogenic probes with high biological utility. PMID:23557713