Visualizing Viral Protein Structures in Cells Using Genetic Probes for Correlated Light and Electron Microscopy

PubMed Central

Ou, Horng D.; Deerinck, Thomas J.; Bushong, Eric; Ellisman, Mark H.; O’Shea, Clodagh C.

2015-01-01

Structural studies of viral proteins most often use high-resolution techniques such as X-ray crystallography, nuclear magnetic resonance, single particle negative stain, or cryo-electron microscopy (EM) to reveal atomic interactions of soluble, homogeneous viral proteins or viral protein complexes. Once viral proteins or complexes are separated from their host’s cellular environment, their natural in-situ structure and details of how they interact with other cellular components may be lost. EM has been an invaluable tool in virology since its introduction in the late 1940’s and subsequent application to cells in the 1950’s. EM studies have expanded our knowledge of viral entry, viral replication, alteration of cellular components, and viral lysis. Most of these early studies were focused on conspicuous morphological cellular changes, because classic EM metal stains were designed to highlight classes of cellular structures rather than specific molecular structures. Much later, to identify viral proteins inducing specific structural configurations at the cellular level, immunostaining with a primary antibody followed by colloidal gold secondary antibody was employed to mark the location of specific viral proteins. This technique can suffer from artifacts in cellular ultrastructure due to compromises required to provide access to the immuno-reagents. Immunolocalization methods also require the generation of highly specific antibodies, which may not be available for every viral protein. Here we discuss new methods to visualize viral proteins and structures at high resolutions in-situ using correlated light and electron microscopy (CLEM). We discuss the use of genetically encoded protein fusions that oxidize diaminobenzidine (DAB) into an osmiophilic polymer that can be visualized by EM. Detailed protocols for applying the genetically encoded photo-oxidizing protein MiniSOG to a viral protein, photo-oxidation of the fusion protein to yield DAB polymer staining, and preparation of photo-oxidized samples for TEM and serial block-face scanning EM (SBEM) for large-scale volume EM data acquisition are also presented. As an example, we discuss the recent multi-scale analysis of Adenoviral protein E4-ORF3 that reveals a new type of multi-functional polymer that disrupts multiple cellular proteins. This new capability to visualize unambiguously specific viral protein structures at high resolutions in the native cellular environment is revealing new insights into how they usurp host proteins and functions to drive pathological viral replication. PMID:26066760

Visualizing viral protein structures in cells using genetic probes for correlated light and electron microscopy.

PubMed

Ou, Horng D; Deerinck, Thomas J; Bushong, Eric; Ellisman, Mark H; O'Shea, Clodagh C

2015-11-15

Structural studies of viral proteins most often use high-resolution techniques such as X-ray crystallography, nuclear magnetic resonance, single particle negative stain, or cryo-electron microscopy (EM) to reveal atomic interactions of soluble, homogeneous viral proteins or viral protein complexes. Once viral proteins or complexes are separated from their host's cellular environment, their natural in situ structure and details of how they interact with other cellular components may be lost. EM has been an invaluable tool in virology since its introduction in the late 1940's and subsequent application to cells in the 1950's. EM studies have expanded our knowledge of viral entry, viral replication, alteration of cellular components, and viral lysis. Most of these early studies were focused on conspicuous morphological cellular changes, because classic EM metal stains were designed to highlight classes of cellular structures rather than specific molecular structures. Much later, to identify viral proteins inducing specific structural configurations at the cellular level, immunostaining with a primary antibody followed by colloidal gold secondary antibody was employed to mark the location of specific viral proteins. This technique can suffer from artifacts in cellular ultrastructure due to compromises required to provide access to the immuno-reagents. Immunolocalization methods also require the generation of highly specific antibodies, which may not be available for every viral protein. Here we discuss new methods to visualize viral proteins and structures at high resolutions in situ using correlated light and electron microscopy (CLEM). We discuss the use of genetically encoded protein fusions that oxidize diaminobenzidine (DAB) into an osmiophilic polymer that can be visualized by EM. Detailed protocols for applying the genetically encoded photo-oxidizing protein MiniSOG to a viral protein, photo-oxidation of the fusion protein to yield DAB polymer staining, and preparation of photo-oxidized samples for TEM and serial block-face scanning EM (SBEM) for large-scale volume EM data acquisition are also presented. As an example, we discuss the recent multi-scale analysis of Adenoviral protein E4-ORF3 that reveals a new type of multi-functional polymer that disrupts multiple cellular proteins. This new capability to visualize unambiguously specific viral protein structures at high resolutions in the native cellular environment is revealing new insights into how they usurp host proteins and functions to drive pathological viral replication. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2015-01-01

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

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.

Imaging cells and sub-cellular structures with ultrahigh resolution full-field X-ray microscopy.

PubMed

Chien, C C; Tseng, P Y; Chen, H H; Hua, T E; Chen, S T; Chen, Y Y; Leng, W H; Wang, C H; Hwu, Y; Yin, G C; Liang, K S; Chen, F R; Chu, Y S; Yeh, H I; Yang, Y C; Yang, C S; Zhang, G L; Je, J H; Margaritondo, G

2013-01-01

Our experimental results demonstrate that full-field hard-X-ray microscopy is finally able to investigate the internal structure of cells in tissues. This result was made possible by three main factors: the use of a coherent (synchrotron) source of X-rays, the exploitation of contrast mechanisms based on the real part of the refractive index and the magnification provided by high-resolution Fresnel zone-plate objectives. We specifically obtained high-quality microradiographs of human and mouse cells with 29 nm Rayleigh spatial resolution and verified that tomographic reconstruction could be implemented with a final resolution level suitable for subcellular features. We also demonstrated that a phase retrieval method based on a wave propagation algorithm could yield good subcellular images starting from a series of defocused microradiographs. The concluding discussion compares cellular and subcellular hard-X-ray microradiology with other techniques and evaluates its potential impact on biomedical research. Copyright © 2012 Elsevier Inc. All rights reserved.

In vivo cellular imaging with microscopes enabled by MEMS scanners

NASA Astrophysics Data System (ADS)

Ra, Hyejun

High-resolution optical imaging plays an important role in medical diagnosis and biomedical research. Confocal microscopy is a widely used imaging method for obtaining cellular and sub-cellular images of biological tissue in reflectance and fluorescence modes. Its characteristic optical sectioning capability also enables three-dimensional (3-D) image reconstruction. However, its use has mostly been limited to excised tissues due to the requirement of high numerical aperture (NA) lenses for cellular resolution. Microscope miniaturization can enable in vivo imaging to make possible early cancer diagnosis and biological studies in the innate environment. In this dissertation, microscope miniaturization for in vivo cellular imaging is presented. The dual-axes confocal (DAC) architecture overcomes limitations of the conventional single-axis confocal (SAC) architecture to allow for miniaturization with high resolution. A microelectromechanical systems (MEMS) scanner is the central imaging component that is key in miniaturization of the DAC architecture. The design, fabrication, and characterization of the two-dimensional (2-D) MEMS scanner are presented. The gimbaled MEMS scanner is fabricated on a double silicon-on-insulator (SOI) wafer and is actuated by self-aligned vertical electrostatic combdrives. The imaging performance of the MEMS scanner in a DAC configuration is shown in a breadboard microscope setup, where reflectance and fluorescence imaging is demonstrated. Then, the MEMS scanner is integrated into a miniature DAC microscope. The whole imaging system is integrated into a portable unit for research in small animal models of human biology and disease. In vivo 3-D imaging is demonstrated on mouse skin models showing gene transfer and siRNA silencing. The siRNA silencing process is sequentially imaged in one mouse over time.

High-resolution fiber-optic microendoscopy for in situ cellular imaging.

PubMed

Pierce, Mark; Yu, Dihua; Richards-Kortum, Rebecca

2011-01-11

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user. We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture, in excised and living animal tissues, and in human tissues in vivo. Users have reported the use of several different fluorescent contrast agents, including proflavine, benzoporphyrin-derivative monoacid ring A (BPD-MA), and fluoroscein, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

Super-resolution optical microscopy for studying membrane structure and dynamics.

PubMed

Sezgin, Erdinc

2017-07-12

Investigation of cell membrane structure and dynamics requires high spatial and temporal resolution. The spatial resolution of conventional light microscopy is limited due to the diffraction of light. However, recent developments in microscopy enabled us to access the nano-scale regime spatially, thus to elucidate the nanoscopic structures in the cellular membranes. In this review, we will explain the resolution limit, address the working principles of the most commonly used super-resolution microscopy techniques and summarise their recent applications in the biomembrane field.

Revealing fine microstructural morphology in the living human retina using Optical Coherence Tomography with pancorrection

NASA Astrophysics Data System (ADS)

Torti, C.; Považay, B.; Hofer, B.; Unterhuber, A.; Hermann, B.; Drexler, W.

2008-09-01

Ultra-high speed optical coherence tomography employing an ultra-broadband light source has been combined with adaptive optics utilizing a single high stroke deformable mirror and chromatic aberration compensation. The reduction of motion artefacts, geometric and chromatic aberrations (pancorrection) permits to achieve an isotropic resolution of 2-3 μm in the human eye. The performance of this non-invasive imaging modality enables to resolve cellular structures including cone photoreceptors, nerve fibre bundles and collagenous plates of the lamina cribrosa, and retinal pigment epithelial (RPE) cells in the human retina in vivo with superior detail. Alterations of cellular morphology due to cone degeneration in a colour-blind subject are investigated in ultra-high resolution with selective depth sectioning for the first time.

FishFace: interactive atlas of zebrafish craniofacial development at cellular resolution

PubMed Central

2013-01-01

Background The vertebrate craniofacial skeleton may exhibit anatomical complexity and diversity, but its genesis and evolution can be understood through careful dissection of developmental programs at cellular resolution. Resources are lacking that include introductory overviews of skeletal anatomy coupled with descriptions of craniofacial development at cellular resolution. In addition to providing analytical guidelines for other studies, such an atlas would suggest cellular mechanisms underlying development. Description We present the Fish Face Atlas, an online, 3D-interactive atlas of craniofacial development in the zebrafish Danio rerio. Alizarin red-stained skulls scanned by fluorescent optical projection tomography and segmented into individual elements provide a resource for understanding the 3D structure of the zebrafish craniofacial skeleton. These data provide the user an anatomical entry point to confocal images of Alizarin red-stained zebrafish with transgenically-labelled pharyngeal arch ectomesenchyme, chondrocytes, and osteoblasts, which illustrate the appearance, morphogenesis, and growth of the mandibular and hyoid cartilages and bones, as viewed in live, anesthetized zebrafish during embryonic and larval development. Confocal image stacks at high magnification during the same stages provide cellular detail and suggest developmental and evolutionary hypotheses. Conclusion The FishFace Atlas is a novel learning tool for understanding craniofacial skeletal development, and can serve as a reference for a variety of studies, including comparative and mutational analyses. PMID:23714426

High Resolution, Large Deformation 3D Traction Force Microscopy

PubMed Central

López-Fagundo, Cristina; Reichner, Jonathan; Hoffman-Kim, Diane; Franck, Christian

2014-01-01

Traction Force Microscopy (TFM) is a powerful approach for quantifying cell-material interactions that over the last two decades has contributed significantly to our understanding of cellular mechanosensing and mechanotransduction. In addition, recent advances in three-dimensional (3D) imaging and traction force analysis (3D TFM) have highlighted the significance of the third dimension in influencing various cellular processes. Yet irrespective of dimensionality, almost all TFM approaches have relied on a linear elastic theory framework to calculate cell surface tractions. Here we present a new high resolution 3D TFM algorithm which utilizes a large deformation formulation to quantify cellular displacement fields with unprecedented resolution. The results feature some of the first experimental evidence that cells are indeed capable of exerting large material deformations, which require the formulation of a new theoretical TFM framework to accurately calculate the traction forces. Based on our previous 3D TFM technique, we reformulate our approach to accurately account for large material deformation and quantitatively contrast and compare both linear and large deformation frameworks as a function of the applied cell deformation. Particular attention is paid in estimating the accuracy penalty associated with utilizing a traditional linear elastic approach in the presence of large deformation gradients. PMID:24740435

Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane.

PubMed

Stone, Matthew B; Shelby, Sarah A; Veatch, Sarah L

2017-06-14

Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It

PubMed Central

Kraft, Mary L.

2017-01-01

Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with cholesterol or hemagglutinin. The alternate views of plasma membrane organization suggested by these findings are discussed. PMID:28119913

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It.

PubMed

Kraft, Mary L

2016-01-01

Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with cholesterol or hemagglutinin. The alternate views of plasma membrane organization suggested by these findings are discussed.

Imaging Cellular Architecture with X-rays

PubMed Central

Larabell, Carolyn A.; Nugent, Keith A.

2012-01-01

X-ray imaging of biological samples is progressing rapidly. In this paper we review the progress to date in high resolution imaging of cellular architecture. In particular we survey the progress in soft X-ray tomography and argue that the field is coming of age and that important biological insights are starting to emerge. We then review the new ideas based on coherent diffraction. These methods are at a much earlier stage of development but, as they eliminate the need for X-ray optics, have the capacity to provide substantially better spatial resolution than zone plate based methods. PMID:20869868

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

NASA Astrophysics Data System (ADS)

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

2016-03-01

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

Nature's crucible: Manufacturing optical nonlinearities for high resolution, high sensitivity encoding in the compound eye of the fly, Musca domestica

NASA Technical Reports Server (NTRS)

Wilcox, Mike

1993-01-01

The number of pixels per unit area sampling an image determines Nyquist resolution. Therefore, the highest pixel density is the goal. Unfortunately, as reduction in pixel size approaches the wavelength of light, sensitivity is lost and noise increases. Animals face the same problems and have achieved novel solutions. Emulating these solutions offers potentially unlimited sensitivity with detector size approaching the diffraction limit. Once an image is 'captured', cellular preprocessing of information allows extraction of high resolution information from the scene. Computer simulation of this system promises hyperacuity for machine vision.

Adaptive optics optical coherence tomography at 120,000 depth scans/s for non-invasive cellular phenotyping of the living human retina

PubMed Central

Torti, Cristiano; Považay, Boris; Hofer, Bernd; Unterhuber, Angelika; Carroll, Joseph; Ahnelt, Peter Kurt; Drexler, Wolfgang

2012-01-01

This paper presents a successful combination of ultra-high speed (120,000 depth scans/s), ultra-high resolution optical coherence tomography with adaptive optics and an achromatizing lens for compensation of monochromatic and longitudinal chromatic ocular aberrations, respectively, allowing for non-invasive volumetric imaging in normal and pathologic human retinas at cellular resolution. The capability of this imaging system is demonstrated here through preliminary studies by probing cellular intraretinal structures that have not been accessible so far with in vivo, non-invasive, label-free imaging techniques, including pigment epithelial cells, micro-vasculature of the choriocapillaris, single nerve fibre bundles and collagenous plates of the lamina cribrosa in the optic nerve head. In addition, the volumetric extent of cone loss in two colour-blinds could be quantified for the first time. This novel technique provides opportunities to enhance the understanding of retinal pathogenesis and early diagnosis of retinal diseases. PMID:19997159

An automated image processing routine for segmentation of cell cytoplasms in high-resolution autofluorescence images

NASA Astrophysics Data System (ADS)

Walsh, Alex J.; Skala, Melissa C.

2014-02-01

The heterogeneity of genotypes and phenotypes within cancers is correlated with disease progression and drug-resistant cellular sub-populations. Therefore, robust techniques capable of probing majority and minority cell populations are important both for cancer diagnostics and therapy monitoring. Herein, we present a modified CellProfiler routine to isolate cytoplasmic fluorescence signal on a single cell level from high resolution auto-fluorescence microscopic images.

Pollen structure visualization using high-resolution laboratory-based hard X-ray tomography

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

Li, Qiong; Gluch, Jürgen; Krüger, Peter

A laboratory-based X-ray microscope is used to investigate the 3D structure of unstained whole pollen grains. For the first time, high-resolution laboratory-based hard X-ray microscopy is applied to study pollen grains. Based on the efficient acquisition of statistically relevant information-rich images using Zernike phase contrast, both surface- and internal structures of pine pollen - including exine, intine and cellular structures - are clearly visualized. The specific volumes of these structures are calculated from the tomographic data. The systematic three-dimensional study of pollen grains provides morphological and structural information about taxonomic characters that are essential in palynology. Such studies have amore » direct impact on disciplines such as forestry, agriculture, horticulture, plant breeding and biodiversity. - Highlights: • The unstained whole pine pollen was visualized by high-resolution laboratory-based HXRM for the first time. • The comparison study of pollen grains by LM, SEM and high-resolution laboratory-based HXRM. • Phase contrast imaging provides significantly higher contrast of the raw images compared to absorption contrast imaging. • Surface and internal structure of the pine pollen including exine, intine and cellular structures are clearly visualized. • 3D volume data of unstained whole pollen grains are acquired and the specific volumes of the different layer are calculated.« less

Resolution of inflammation by interleukin-9-producing type 2 innate lymphoid cells

PubMed Central

Rauber, Simon; Luber, Markus; Weber, Stefanie; Maul, Lisa; Soare, Alina; Wohlfahrt, Thomas; Lin, Neng-Yu; Dietel, Katharina; Bozec, Aline; Herrmann, Martin; Kaplan, Mark H.; Weigmann, Benno; Zaiss, Mario M.; Fearon, Ursula; Veale, Douglas J.; Canete, Juan D.; Distler, Oliver; Rivellese, Felice; Pitzalis, Costantino; Neurath, Markus F.; McKenzie, Andrew N.J.; Wirtz, Stefan; Schett, Georg; Distler, Jörg H.W.; Ramming, Andreas

2017-01-01

Inflammatory diseases such as arthritis are chronic conditions that fail to resolve spontaneously. While the cytokine and cellular pathways triggering arthritis are well defined, those responsible for the resolution of inflammation are incompletely characterized. Here we identified IL-9-producing type 2 innate lymphoid cells (ILC2s) as a molecular and cellular pathway that orchestrates the resolution of chronic inflammation. In mice, the absence of IL-9 impaired ILC2 proliferation, activation of regulatory T cells (Treg) and resulted in chronic arthritis with excessive cartilage destruction and bone loss. In contrast, treatment with IL-9 promoted ILC2-dependent Treg activation and effectively induced resolution of inflammation and protection of bone. Rheumatoid arthritis patients in remission demonstrated high numbers of IL-9+ ILC2s in the joints and in the circulation. Hence, fostering IL-9-mediated ILC2 activation may offer a novel therapeutic approach inducing resolution of inflammation rather than suppression of inflammatory responses. PMID:28714991

High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events

PubMed Central

Suzuki, Yuki; Sakai, Nobuaki; Yoshida, Aiko; Uekusa, Yoshitsugu; Yagi, Akira; Imaoka, Yuka; Ito, Shuichi; Karaki, Koichi; Takeyasu, Kunio

2013-01-01

A hybrid atomic force microscopy (AFM)-optical fluorescence microscopy is a powerful tool for investigating cellular morphologies and events. However, the slow data acquisition rates of the conventional AFM unit of the hybrid system limit the visualization of structural changes during cellular events. Therefore, high-speed AFM units equipped with an optical/fluorescence detection device have been a long-standing wish. Here we describe the implementation of high-speed AFM coupled with an optical fluorescence microscope. This was accomplished by developing a tip-scanning system, instead of a sample-scanning system, which operates on an inverted optical microscope. This novel device enabled the acquisition of high-speed AFM images of morphological changes in individual cells. Using this instrument, we conducted structural studies of living HeLa and 3T3 fibroblast cell surfaces. The improved time resolution allowed us to image dynamic cellular events. PMID:23823461

High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events.

PubMed

Suzuki, Yuki; Sakai, Nobuaki; Yoshida, Aiko; Uekusa, Yoshitsugu; Yagi, Akira; Imaoka, Yuka; Ito, Shuichi; Karaki, Koichi; Takeyasu, Kunio

2013-01-01

A hybrid atomic force microscopy (AFM)-optical fluorescence microscopy is a powerful tool for investigating cellular morphologies and events. However, the slow data acquisition rates of the conventional AFM unit of the hybrid system limit the visualization of structural changes during cellular events. Therefore, high-speed AFM units equipped with an optical/fluorescence detection device have been a long-standing wish. Here we describe the implementation of high-speed AFM coupled with an optical fluorescence microscope. This was accomplished by developing a tip-scanning system, instead of a sample-scanning system, which operates on an inverted optical microscope. This novel device enabled the acquisition of high-speed AFM images of morphological changes in individual cells. Using this instrument, we conducted structural studies of living HeLa and 3T3 fibroblast cell surfaces. The improved time resolution allowed us to image dynamic cellular events.

Architectural transitions in Vibrio cholerae biofilms at single-cell resolution

PubMed Central

Drescher, Knut; Dunkel, Jörn; Nadell, Carey D.; van Teeffelen, Sven; Grnja, Ivan; Wingreen, Ned S.; Stone, Howard A.; Bassler, Bonnie L.

2016-01-01

Many bacterial species colonize surfaces and form dense 3D structures, known as biofilms, which are highly tolerant to antibiotics and constitute one of the major forms of bacterial biomass on Earth. Bacterial biofilms display remarkable changes during their development from initial attachment to maturity, yet the cellular architecture that gives rise to collective biofilm morphology during growth is largely unknown. Here, we use high-resolution optical microscopy to image all individual cells in Vibrio cholerae biofilms at different stages of development, including colonies that range in size from 2 to 4,500 cells. From these data, we extracted the precise 3D cellular arrangements, cell shapes, sizes, and global morphological features during biofilm growth on submerged glass substrates under flow. We discovered several critical transitions of the internal and external biofilm architectures that separate the major phases of V. cholerae biofilm growth. Optical imaging of biofilms with single-cell resolution provides a new window into biofilm formation that will prove invaluable to understanding the mechanics underlying biofilm development. PMID:26933214

Integrated micro-endoscopy system for simultaneous fluorescence and optical-resolution photoacoustic imaging.

PubMed

Shao, Peng; Shi, Wei; Hajireza, Parsin; Zemp, Roger J

2012-07-01

We present a new integrated micro-endoscopy system combining label-free, fiber-based, real-time C-scan optical-resolution photoacoustic microscopy (F-OR-PAM) and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the F-OR-PAM sub-system is able to reach a resolution of ∼7  μm. The fluorescence subsystem, which does not require any mechanical scanning, consists of a 447.5-nm-centered diode laser as the light source, an objective lens, and a CCD camera. Proflavine is used as the fluorescent contrast agent by topical application. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single-mode fibers. The absorption of proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural information given by F-OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping visualize angiogenesis and the effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.

Combined optical resolution photoacoustic and fluorescence micro-endoscopy

NASA Astrophysics Data System (ADS)

Shao, Peng; Shi, Wei; Hajireza, Parsin; Zemp, Roger J.

2012-02-01

We present a new micro-endoscopy system combining real-time C-scan optical-resolution photoacoustic micro-endoscopy (OR-PAME), and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the OR-PAM sub-system is capable of imaging with a resolution of ~ 7μm. The fluorescence sub-system consists of a diode laser with 445 nm-centered emissions as the light source, an objective lens and a CCD camera. Proflavine, a FDA approved drug for human use, is used as the fluorescent contrast agent by topical application. The fluorescence system does not require any mechanical scanning. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single mode fibers. The absorption of Proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural and functional information given by OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping researchers and clinicians visualize angiogenesis, effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.

Integrated micro-endoscopy system for simultaneous fluorescence and optical-resolution photoacoustic imaging

NASA Astrophysics Data System (ADS)

Shao, Peng; Shi, Wei; Hajireza, Parsin; Zemp, Roger J.

2012-07-01

We present a new integrated micro-endoscopy system combining label-free, fiber-based, real-time C-scan optical-resolution photoacoustic microscopy (F-OR-PAM) and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the F-OR-PAM sub-system is able to reach a resolution of ~7 μm. The fluorescence subsystem, which does not require any mechanical scanning, consists of a 447.5-nm-centered diode laser as the light source, an objective lens, and a CCD camera. Proflavine is used as the fluorescent contrast agent by topical application. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single-mode fibers. The absorption of proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural information given by F-OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping visualize angiogenesis and the effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.

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.

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.

Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy

NASA Astrophysics Data System (ADS)

Descloux, A.; Grußmayer, K. S.; Bostan, E.; Lukes, T.; Bouwens, A.; Sharipov, A.; Geissbuehler, S.; Mahul-Mellier, A.-L.; Lashuel, H. A.; Leutenegger, M.; Lasser, T.

2018-03-01

Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going `beyond the diffraction barrier' comes at a price, since most far-field super-resolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase imaging with fluorescence to provide high-speed imaging and spatial super-resolution. The non-iterative phase retrieval relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of eight planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument. The 4D microscope platform unifies the sensitivity and high temporal resolution of phase imaging with the specificity and high spatial resolution of fluorescence microscopy.

Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D)

PubMed Central

Li, Weizhe; Germain, Ronald N.

2017-01-01

Organ homeostasis, cellular differentiation, signal relay, and in situ function all depend on the spatial organization of cells in complex tissues. For this reason, comprehensive, high-resolution mapping of cell positioning, phenotypic identity, and functional state in the context of macroscale tissue structure is critical to a deeper understanding of diverse biological processes. Here we report an easy to use method, clearing-enhanced 3D (Ce3D), which generates excellent tissue transparency for most organs, preserves cellular morphology and protein fluorescence, and is robustly compatible with antibody-based immunolabeling. This enhanced signal quality and capacity for extensive probe multiplexing permits quantitative analysis of distinct, highly intermixed cell populations in intact Ce3D-treated tissues via 3D histo-cytometry. We use this technology to demonstrate large-volume, high-resolution microscopy of diverse cell types in lymphoid and nonlymphoid organs, as well as to perform quantitative analysis of the composition and tissue distribution of multiple cell populations in lymphoid tissues. Combined with histo-cytometry, Ce3D provides a comprehensive strategy for volumetric quantitative imaging and analysis that bridges the gap between conventional section imaging and disassociation-based techniques. PMID:28808033

New Frontiers and Challenges for Single-Cell Electrochemical Analysis.

PubMed

Zhang, Jingjing; Zhou, Junyu; Pan, Rongrong; Jiang, Dechen; Burgess, James D; Chen, Hong-Yuan

2018-02-23

Previous measurements of cell populations might obscure many important cellular differences, and new strategies for single-cell analyses are urgently needed to re-examine these fundamental biological principles for better diagnosis and treatment of diseases. Electrochemistry is a robust technique for the analysis of single living cells that has the advantages of minor interruption of cellular activity and provides the capability of high spatiotemporal resolution. The achievements of the past 30 years have revealed significant information about the exocytotic events of single cells to elucidate the mechanisms of cellular activity. Currently, the rapid developments of micro/nanofabrication and optoelectronic technologies drive the development of multifunctional electrodes and novel electrochemical approaches with higher resolution for single cells. In this Perspective, three new frontiers in this field, namely, electrochemical microscopy, intracellular analysis, and single-cell analysis in a biological system (i.e., neocortex and retina), are reviewed. The unique features and remaining challenges of these techniques are discussed.

A cellular perspective on brain energy metabolism and functional imaging.

PubMed

Magistretti, Pierre J; Allaman, Igor

2015-05-20

The energy demands of the brain are high: they account for at least 20% of the body's energy consumption. Evolutionary studies indicate that the emergence of higher cognitive functions in humans is associated with an increased glucose utilization and expression of energy metabolism genes. Functional brain imaging techniques such as fMRI and PET, which are widely used in human neuroscience studies, detect signals that monitor energy delivery and use in register with neuronal activity. Recent technological advances in metabolic studies with cellular resolution have afforded decisive insights into the understanding of the cellular and molecular bases of the coupling between neuronal activity and energy metabolism and point at a key role of neuron-astrocyte metabolic interactions. This article reviews some of the most salient features emerging from recent studies and aims at providing an integration of brain energy metabolism across resolution scales. Copyright © 2015 Elsevier Inc. All rights reserved.

Genetically targeted 3D visualisation of Drosophila neurons under Electron Microscopy and X-Ray Microscopy using miniSOG

PubMed Central

Ng, Julian; Browning, Alyssa; Lechner, Lorenz; Terada, Masako; Howard, Gillian; Jefferis, Gregory S. X. E.

2016-01-01

Large dimension, high-resolution imaging is important for neural circuit visualisation as neurons have both long- and short-range patterns: from axons and dendrites to the numerous synapses at terminal endings. Electron Microscopy (EM) is the favoured approach for synaptic resolution imaging but how such structures can be segmented from high-density images within large volume datasets remains challenging. Fluorescent probes are widely used to localise synapses, identify cell-types and in tracing studies. The equivalent EM approach would benefit visualising such labelled structures from within sub-cellular, cellular, tissue and neuroanatomical contexts. Here we developed genetically-encoded, electron-dense markers using miniSOG. We demonstrate their ability in 1) labelling cellular sub-compartments of genetically-targeted neurons, 2) generating contrast under different EM modalities, and 3) segmenting labelled structures from EM volumes using computer-assisted strategies. We also tested non-destructive X-ray imaging on whole Drosophila brains to evaluate contrast staining. This enabled us to target specific regions for EM volume acquisition. PMID:27958322

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

PubMed

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

2014-01-01

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

New Funding Opportunity from the Human Biomolecular Atlas Program (HuBMAP)! | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

The NIH Common Fund Human Biomolecular Atlas Program (HuBMAP) aims to develop a framework for functional mapping the human body with cellular resolution to enhance our understanding of cellular organization-function. HuBMAP will accelerate the development of the next generation of tools and techniques to generate 3D tissue maps using validated high-content, high-throughput imaging and omics assays, and establish an open data platform for integrating, visualizing data to build multi-dimensional maps.

High-resolution imaging of cellular processes across textured surfaces using an indexed-matched elastomer.

PubMed

Ravasio, Andrea; Vaishnavi, Sree; Ladoux, Benoit; Viasnoff, Virgile

2015-03-01

Understanding and controlling how cells interact with the microenvironment has emerged as a prominent field in bioengineering, stem cell research and in the development of the next generation of in vitro assays as well as organs on a chip. Changing the local rheology or the nanotextured surface of substrates has proved an efficient approach to improve cell lineage differentiation, to control cell migration properties and to understand environmental sensing processes. However, introducing substrate surface textures often alters the ability to image cells with high precision, compromising our understanding of molecular mechanisms at stake in environmental sensing. In this paper, we demonstrate how nano/microstructured surfaces can be molded from an elastomeric material with a refractive index matched to the cell culture medium. Once made biocompatible, contrast imaging (differential interference contrast, phase contrast) and high-resolution fluorescence imaging of subcellular structures can be implemented through the textured surface using an inverted microscope. Simultaneous traction force measurements by micropost deflection were also performed, demonstrating the potential of our approach to study cell-environment interactions, sensing processes and cellular force generation with unprecedented resolution. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Imaging of cellular spread on a three-dimensional scaffold by means of a novel cell-labeling technique for high-resolution computed tomography.

PubMed

Thimm, Benjamin W; Hofmann, Sandra; Schneider, Philipp; Carretta, Roberto; Müller, Ralph

2012-03-01

Computed tomography (CT) represents a truly three-dimensional (3D) imaging technique that can provide high-resolution images on the cellular level. Thus, one approach to detect single cells is X-ray absorption-based CT, where cells are labeled with a dense, opaque material providing the required contrast for CT imaging. Within the present work, a novel cell-labeling method has been developed showing the feasibility of labeling fixed cells with iron oxide (FeO) particles for subsequent CT imaging and quantitative morphometry. A biotin-streptavidin detection system was exploited to bind FeO particles to its target endothelial cells. The binding of the particles was predominantly close to the cell centers on 2D surfaces as shown by light microscopy, scanning electron microscopy, and CT. When cells were cultured on porous, 3D polyurethane surfaces, significantly more FeO particles were detected compared with surfaces without cells and FeO particle labeling using CT. Here, we report on the implementation and evaluation of a novel cell detection method based on high-resolution CT. This system has potential in cell tracking for 3D in vitro imaging in the future.

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

PubMed

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

2015-11-07

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

Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy.

PubMed

Martell, Jeffrey D; Deerinck, Thomas J; Sancak, Yasemin; Poulos, Thomas L; Mootha, Vamsi K; Sosinsky, Gina E; Ellisman, Mark H; Ting, Alice Y

2012-11-01

Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments or require light and can be difficult to use. Here we report the development of 'APEX', a genetically encodable EM tag that is active in all cellular compartments and does not require light. APEX is a monomeric 28-kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins using a simple and robust labeling procedure. We also fused APEX to the N or C terminus of the mitochondrial calcium uniporter (MCU), a recently identified channel whose topology is disputed. These fusions give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N and C termini of MCU face the matrix. Because APEX staining is not dependent on light activation, APEX should make EM imaging of any cellular protein straightforward, regardless of the size or thickness of the specimen.

High-speed Fourier ptychographic microscopy based on programmable annular illuminations.

PubMed

Sun, Jiasong; Zuo, Chao; Zhang, Jialin; Fan, Yao; Chen, Qian

2018-05-16

High-throughput quantitative phase imaging (QPI) is essential to cellular phenotypes characterization as it allows high-content cell analysis and avoids adverse effects of staining reagents on cellular viability and cell signaling. Among different approaches, Fourier ptychographic microscopy (FPM) is probably the most promising technique to realize high-throughput QPI by synthesizing a wide-field, high-resolution complex image from multiple angle-variably illuminated, low-resolution images. However, the large dataset requirement in conventional FPM significantly limits its imaging speed, resulting in low temporal throughput. Moreover, the underlying theoretical mechanism as well as optimum illumination scheme for high-accuracy phase imaging in FPM remains unclear. Herein, we report a high-speed FPM technique based on programmable annular illuminations (AIFPM). The optical-transfer-function (OTF) analysis of FPM reveals that the low-frequency phase information can only be correctly recovered if the LEDs are precisely located at the edge of the objective numerical aperture (NA) in the frequency space. By using only 4 low-resolution images corresponding to 4 tilted illuminations matching a 10×, 0.4 NA objective, we present the high-speed imaging results of in vitro Hela cells mitosis and apoptosis at a frame rate of 25 Hz with a full-pitch resolution of 655 nm at a wavelength of 525 nm (effective NA = 0.8) across a wide field-of-view (FOV) of 1.77 mm 2 , corresponding to a space-bandwidth-time product of 411 megapixels per second. Our work reveals an important capability of FPM towards high-speed high-throughput imaging of in vitro live cells, achieving video-rate QPI performance across a wide range of scales, both spatial and temporal.

Nanoscale live cell optical imaging of the dynamics of intracellular microvesicles in neural cells.

PubMed

Lee, Sohee; Heo, Chaejeong; Suh, Minah; Lee, Young Hee

2013-11-01

Recent advances in biotechnology and imaging technology have provided great opportunities to investigate cellular dynamics. Conventional imaging methods such as transmission electron microscopy, scanning electron microscopy, and atomic force microscopy are powerful techniques for cellular imaging, even at the nanoscale level. However, these techniques have limitations applications in live cell imaging because of the experimental preparation required, namely cell fixation, and the innately small field of view. In this study, we developed a nanoscale optical imaging (NOI) system that combines a conventional optical microscope with a high resolution dark-field condenser (Cytoviva, Inc.) and halogen illuminator. The NOI system's maximum resolution for live cell imaging is around 100 nm. We utilized NOI to investigate the dynamics of intracellular microvesicles of neural cells without immunocytological analysis. In particular, we studied direct, active random, and moderate random dynamic motions of intracellular microvesicles and visualized lysosomal vesicle changes after treatment of cells with a lysosomal inhibitor (NH4Cl). Our results indicate that the NOI system is a feasible, high-resolution optical imaging system for live small organelles that does not require complicated optics or immunocytological staining processes.

Detection and signal amplification in zebrafish RNA FISH.

PubMed

Hauptmann, Giselbert; Lauter, Gilbert; Söll, Iris

2016-04-01

In situ hybridization (ISH) has become an invaluable tool for the detection of RNA in cells, tissues and organisms. Due to improvements in target and signal amplification and in probe design remarkable progress has been made concerning sensitivity, specificity and resolution of chromogenic and fluorescent ISH (FISH). These advancements allow for exquisite cellular and sub-cellular resolution and for detecting multiple RNA species at a time by multiplexing. In zebrafish (F)ISH non-enzymatic and enzymatic amplification systems have been employed to obtain enhanced signal intensities and signal-to-noise ratios. These amplification strategies include branched DNA-based RNAscope and in situ hybridization chain reaction (HCR) techniques, as well as alkaline phosphatase (AP)- and horseradish peroxidase (PO)-based immunoassays. For practical application, we provide proven multiplex FISH protocols for AP- and PO-based visualization of mRNAs at high resolution. The protocols take advantage of optimized tyramide signal amplification (TSA) conditions of the PO assay and long-lasting high signal-to-noise ratio of the AP reaction, thereby enabling detection of less abundant transcripts. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Enhancing image contrast of carbon nanotubes on cellular background using helium ion microscope by varying helium ion fluence.

PubMed

Dykas, M M; Poddar, K; Yoong, S L; Viswanathan, V; Mathew, S; Patra, A; Saha, S; Pastorin, G; Venkatesan, T

2018-01-01

Carbon nanotubes (CNTs) have become an important nano entity for biomedical applications. Conventional methods of their imaging, often cannot be applied in biological samples due to an inadequate spatial resolution or poor contrast between the CNTs and the biological sample. Here we report a unique and effective detection method, which uses differences in conductivities of carbon nanotubes and HeLa cells. The technique involves the use of a helium ion microscope to image the sample with the surface charging artefacts created by the He + and neutralised by electron flood gun. This enables us to obtain a few nanometre resolution images of CNTs in HeLa Cells with high contrast, which was achieved by tailoring the He + fluence. Charging artefacts can be efficiently removed for conductive CNTs by a low amount of electrons, the fluence of which is not adequate to discharge the cell surface, resulting in high image contrast. Thus, this technique enables rapid detection of any conducting nano structures on insulating cellular background even in large fields of view and fine spatial resolution. The technique demonstrated has wider applications for researchers seeking enhanced contrast and high-resolution imaging of any conducting entity in a biological matrix - a commonly encountered issue of importance in drug delivery, tissue engineering and toxicological studies. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

High-resolution MALDI mass spectrometry imaging of gallotannins and monoterpene glucosides in the root of Paeonia lactiflora

NASA Astrophysics Data System (ADS)

Li, Bin; Bhandari, Dhaka Ram; Römpp, Andreas; Spengler, Bernhard

2016-10-01

High-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) at 10 μm pixel size was performed to unravel the spatio-chemical distribution of major secondary metabolites in the root of Paeonia lactiflora. The spatial distributions of two major classes of bioactive components, gallotannins and monoterpene glucosides, were investigated and visualized at the cellular level in tissue sections of P. lactiflora roots. Accordingly, other primary and secondary metabolites were imaged, including amino acids, carbohydrates, lipids and monoterpenes, indicating the capability of untargeted localization of metabolites by using high-resolution MSI platform. The employed AP-SMALDI MSI system provides significant technological advancement in the visualization of individual molecular species at the cellular level. In contrast to previous histochemical studies of tannins using unspecific staining reagents, individual gallotannin species were accurately localized and unequivocally discriminated from other phenolic components in the root tissues. High-quality ion images were obtained, providing significant clues for understanding the biosynthetic pathway of gallotannins and monoterpene glucosides and possibly helping to decipher the role of tannins in xylem cells differentiation and in the defence mechanisms of plants, as well as to investigate the interrelationship between tannins and lignins.

High-resolution MALDI mass spectrometry imaging of gallotannins and monoterpene glucosides in the root of Paeonia lactiflora.

PubMed

Li, Bin; Bhandari, Dhaka Ram; Römpp, Andreas; Spengler, Bernhard

2016-10-31

High-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) at 10 μm pixel size was performed to unravel the spatio-chemical distribution of major secondary metabolites in the root of Paeonia lactiflora. The spatial distributions of two major classes of bioactive components, gallotannins and monoterpene glucosides, were investigated and visualized at the cellular level in tissue sections of P. lactiflora roots. Accordingly, other primary and secondary metabolites were imaged, including amino acids, carbohydrates, lipids and monoterpenes, indicating the capability of untargeted localization of metabolites by using high-resolution MSI platform. The employed AP-SMALDI MSI system provides significant technological advancement in the visualization of individual molecular species at the cellular level. In contrast to previous histochemical studies of tannins using unspecific staining reagents, individual gallotannin species were accurately localized and unequivocally discriminated from other phenolic components in the root tissues. High-quality ion images were obtained, providing significant clues for understanding the biosynthetic pathway of gallotannins and monoterpene glucosides and possibly helping to decipher the role of tannins in xylem cells differentiation and in the defence mechanisms of plants, as well as to investigate the interrelationship between tannins and lignins.

Computational adaptive optics for broadband optical interferometric tomography of biological tissue

NASA Astrophysics Data System (ADS)

Boppart, Stephen A.

2015-03-01

High-resolution real-time tomography of biological tissues is important for many areas of biological investigations and medical applications. Cellular level optical tomography, however, has been challenging because of the compromise between transverse imaging resolution and depth-of-field, the system and sample aberrations that may be present, and the low imaging sensitivity deep in scattering tissues. The use of computed optical imaging techniques has the potential to address several of these long-standing limitations and challenges. Two related techniques are interferometric synthetic aperture microscopy (ISAM) and computational adaptive optics (CAO). Through three-dimensional Fourierdomain resampling, in combination with high-speed OCT, ISAM can be used to achieve high-resolution in vivo tomography with enhanced depth sensitivity over a depth-of-field extended by more than an order-of-magnitude, in realtime. Subsequently, aberration correction with CAO can be performed in a tomogram, rather than to the optical beam of a broadband optical interferometry system. Based on principles of Fourier optics, aberration correction with CAO is performed on a virtual pupil using Zernike polynomials, offering the potential to augment or even replace the more complicated and expensive adaptive optics hardware with algorithms implemented on a standard desktop computer. Interferometric tomographic reconstructions are characterized with tissue phantoms containing sub-resolution scattering particles, and in both ex vivo and in vivo biological tissue. This review will collectively establish the foundation for high-speed volumetric cellular-level optical interferometric tomography in living tissues.

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

PubMed Central

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

2009-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

Rapid, low dose X-ray diffractive imaging of the malaria parasite Plasmodium falciparum.

PubMed

Jones, Michael W M; Dearnley, Megan K; van Riessen, Grant A; Abbey, Brian; Putkunz, Corey T; Junker, Mark D; Vine, David J; McNulty, Ian; Nugent, Keith A; Peele, Andrew G; Tilley, Leann

2014-08-01

Phase-diverse X-ray coherent diffractive imaging (CDI) provides a route to high sensitivity and spatial resolution with moderate radiation dose. It also provides a robust solution to the well-known phase-problem, making on-line image reconstruction feasible. Here we apply phase-diverse CDI to a cellular sample, obtaining images of an erythrocyte infected by the sexual stage of the malaria parasite, Plasmodium falciparum, with a radiation dose significantly lower than the lowest dose previously reported for cellular imaging using CDI. The high sensitivity and resolution allow key biological features to be identified within intact cells, providing complementary information to optical and electron microscopy. This high throughput method could be used for fast tomographic imaging, or to generate multiple replicates in two-dimensions of hydrated biological systems without freezing or fixing. This work demonstrates that phase-diverse CDI is a valuable complementary imaging method for the biological sciences and ready for immediate application. © 2013 Elsevier B.V. All rights reserved.

Automated quantitative histology reveals vascular morphodynamics during Arabidopsis hypocotyl secondary growth.

PubMed

Sankar, Martial; Nieminen, Kaisa; Ragni, Laura; Xenarios, Ioannis; Hardtke, Christian S

2014-02-11

Among various advantages, their small size makes model organisms preferred subjects of investigation. Yet, even in model systems detailed analysis of numerous developmental processes at cellular level is severely hampered by their scale. For instance, secondary growth of Arabidopsis hypocotyls creates a radial pattern of highly specialized tissues that comprises several thousand cells starting from a few dozen. This dynamic process is difficult to follow because of its scale and because it can only be investigated invasively, precluding comprehensive understanding of the cell proliferation, differentiation, and patterning events involved. To overcome such limitation, we established an automated quantitative histology approach. We acquired hypocotyl cross-sections from tiled high-resolution images and extracted their information content using custom high-throughput image processing and segmentation. Coupled with automated cell type recognition through machine learning, we could establish a cellular resolution atlas that reveals vascular morphodynamics during secondary growth, for example equidistant phloem pole formation. DOI: http://dx.doi.org/10.7554/eLife.01567.001.

Automated quantitative histology reveals vascular morphodynamics during Arabidopsis hypocotyl secondary growth

PubMed Central

Sankar, Martial; Nieminen, Kaisa; Ragni, Laura; Xenarios, Ioannis; Hardtke, Christian S

2014-01-01

Among various advantages, their small size makes model organisms preferred subjects of investigation. Yet, even in model systems detailed analysis of numerous developmental processes at cellular level is severely hampered by their scale. For instance, secondary growth of Arabidopsis hypocotyls creates a radial pattern of highly specialized tissues that comprises several thousand cells starting from a few dozen. This dynamic process is difficult to follow because of its scale and because it can only be investigated invasively, precluding comprehensive understanding of the cell proliferation, differentiation, and patterning events involved. To overcome such limitation, we established an automated quantitative histology approach. We acquired hypocotyl cross-sections from tiled high-resolution images and extracted their information content using custom high-throughput image processing and segmentation. Coupled with automated cell type recognition through machine learning, we could establish a cellular resolution atlas that reveals vascular morphodynamics during secondary growth, for example equidistant phloem pole formation. DOI: http://dx.doi.org/10.7554/eLife.01567.001 PMID:24520159

3D Structure Determination of Native Mammalian Cells using Cryo-FIB and Cryo-electron Tomography

PubMed Central

Wang, Ke; Strunk, Korrinn; Zhao, Gongpu; Gray, Jennifer L.; Zhang, Peijun

2012-01-01

Cryo-electron tomography (cryo-ET) has enabled high resolution three-dimensional (3D) structural analysis of virus and host cell interactions and many cell signaling events; these studies, however, have largely been limited to very thin, peripheral regions of eukaryotic cells or to small prokaryotic cells. Recent efforts to make thin, vitreous sections using cryo-ultramicrotomy have been successful, however, this method is technically very challenging and with many artifacts. Here, we report a simple and robust method for creating in situ, frozen-hydrated cell lamellas using a focused ion beam at cryogenic temperature (cryo-FIB), allowing access to any interior cellular regions of interest. We demonstrate the utility of cryo-FIB with high resolution 3D cellular structures from both bacterial cells and large mammalian cells. The method will not only facilitate high-throughput 3D structural analysis of biological specimens, but is also broadly applicable to sample preparation of thin films and surface materials without the need for FIB “lift-out”. PMID:22796867

Investigation of Cellular Interactions of Nanoparticles by Helium Ion Microscopy

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

Arey, Bruce W.; Shutthanandan, V.; Xie, Yumei

The helium ion mircroscope (HIM) probes light elements (e.g. C, N, O, P) with high contrast due to the large variation in secondary electron yield, which minimizes the necessity of specimen staining. A defining characteristic of HIM is its remarkable capability to neutralize charge by the implementation of an electron flood gun, which eliminates the need for coating non-conductive specimens for imaging at high resolution. In addition, the small convergence angle in HeIM offers a large depth of field (~5x FE-SEM), enabling tall structures to be viewed in focus within a single image. Taking advantage of these capabilities, we investigatemore » the interactions of engineered nanoparticles (NPs) at the surface of alveolar type II epithelial cells grown at the air-liquid interface (ALI). The increasing use of nanomaterials in a wide range of commercial applications has the potential to increase human exposure to these materials, but the impact of such exposure on human health is still unclear. One of the main routs of exposure is the respiratory tract, where alveolar epithelial cells present a vulnerable target at the interface with ambient air. Since the cellular interactions of NPs govern the cellular response and ultimately determine the impact on human health, our studies will help delineating relationships between particle properties and cellular interactions and response to better evaluate NP toxicity or biocompatibility. The Rutherford backscattered ion (RBI) is a helium ions imaging mode, which backscatters helium ions from every element except hydrogen, with a backscatter yield that depends on the atomic number of the target. Energy-sensitive backscatter analysis is being developed, which when combined with RBI image information, supports elemental identification at helium ion nanometer resolution. This capability will enable distinguishing NPs from cell surface structures with nanometer resolution.« less

Investigation of cellular interactions of nanoparticles by helium ion microscopy

NASA Astrophysics Data System (ADS)

Arey, B. W.; Shutthanandan, V.; Xie, Y.; Tolic, A.; Williams, N.; Orr, G.

2011-06-01

The helium ion microscope (HIM) probes light elements (e.g. C, N, O, P) with high contrast due to the large variation in secondary electron yield, which minimizes the necessity of specimen staining. A defining characteristic of HIM is its remarkable capability to neutralize charge by the implementation of an electron flood gun, which eliminates the need for coating non-conductive specimens for imaging at high resolution. In addition, the small convergence angle in HeIM offers a large depth of field (~5× FE-SEM), enabling tall structures to be viewed in focus within a single image. Taking advantage of these capabilities, we investigate the interactions of engineered nanoparticles (NPs) at the surface of alveolar type II epithelial cells grown at the airliquid interface (ALI). The increasing use of nanomaterials in a wide range of commercial applications has the potential to increase human exposure to these materials, but the impact of such exposure on human health is still unclear. One of the main routs of exposure is the respiratory tract, where alveolar epithelial cells present a vulnerable target at the interface with ambient air. Since the cellular interactions of NPs govern the cellular response and ultimately determine the impact on human health, our studies will help delineating relationships between particle properties and cellular interactions and response to better evaluate NP toxicity or biocompatibility. The Rutherford backscattered ion (RBI) is a helium ions imaging mode, which backscatters helium ions from every element except hydrogen, with a backscatter yield that depends on the atomic number of the target. Energy-sensitive backscatter analysis is being developed, which when combined with RBI image information, supports elemental identification at helium ion nanometer resolution. This capability will enable distinguishing NPs from cell surface structures with nanometer resolution.

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

PubMed Central

Oheim, M

2007-01-01

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

First multiphoton tomography of brain in man

NASA Astrophysics Data System (ADS)

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

2016-03-01

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

In-vivo imaging of the palisades of Vogt and the limbal crypts with sub-micrometer axial resolution optical coherence tomography

PubMed Central

Bizheva, Kostadinka; Tan, Bingyao; MacLellan, Benjamin; Hosseinaee, Zohreh; Mason, Erik; Hileeto, Denise; Sorbara, Luigina

2017-01-01

A research-grade OCT system was used to image in-vivo and without contact with the tissue, the cellular structure and microvasculature of the healthy human corneo-scleral limbus. The OCT system provided 0.95 µm axial and 4 µm (2 µm) lateral resolution in biological tissue depending on the magnification of the imaging objective. Cross-sectional OCT images acquired tangentially from the inferior limbus showed reflective, loop-like features that correspond to the fibrous folds of the palisades of Vogt (POV). The high OCT resolution allowed for visualization of individual cells inside the limbal crypts, capillaries extending from the inside of the POV’s fibrous folds and connecting to a lateral grid of micro-vessels located in the connective tissue directly below the POV, as well as reflections from individual red blood cells inside the capillaries. Difference in the reflective properties of the POV was observed among subjects of various pigmentation levels of the POV. Morphological features observed in the high resolution OCT images correlated well with histology. The ability to visualize the limbal morphology and microvasculature in-vivo at cellular level can aid the diagnostics and treatment of limbal stem cell dysfunction and dystrophies. PMID:28966853

High-resolution x-ray absorption spectroscopy studies of metal compounds in neurodegenerative brain tissue

NASA Astrophysics Data System (ADS)

Collingwood, J. F.; Mikhaylova, A.; Davidson, M. R.; Batich, C.; Streit, W. J.; Eskin, T.; Terry, J.; Barrea, R.; Underhill, R. S.; Dobson, J.

2005-01-01

Fluorescence mapping and microfocus X-ray absorption spectroscopy are used to detect, locate and identify iron biominerals and other inorganic metal accumulations in neurodegenerative brain tissue at sub-cellular resolution (<5 microns). Recent progress in developing the technique is reviewed. Synchrotron X-rays are used to map tissue sections for metals of interest, and XANES and XAFS are used to characterise anomalous concentrations of the metals in-situ so that they can be correlated with tissue structures and disease pathology. Iron anomalies associated with biogenic magnetite, ferritin and haemoglobin are located and identified in an avian tissue model with a pixel resolution ~5 microns. Subsequent studies include brain tissue sections from transgenic Huntington's mice, and the first high-resolution mapping and identification of iron biominerals in human Alzheimer's and control autopsy brain tissue. Technical developments include use of microfocus diffraction to obtain structural information about biominerals in-situ, and depositing sample location grids by lithography for the location of anomalies by conventional microscopy. The combined techniques provide a breakthrough in the study of both intra- and extra-cellular iron compounds and related metals in tissue. The information to be gained from this approach has implications for future diagnosis and treatment of neurodegeneration, and for our understanding of the mechanisms involved.

Simple platform for chronic imaging of hippocampal activity during spontaneous behaviour in an awake mouse

PubMed Central

Villette, Vincent; Levesque, Mathieu; Miled, Amine; Gosselin, Benoit; Topolnik, Lisa

2017-01-01

Chronic electrophysiological recordings of neuronal activity combined with two-photon Ca2+ imaging give access to high resolution and cellular specificity. In addition, awake drug-free experimentation is required for investigating the physiological mechanisms that operate in the brain. Here, we developed a simple head fixation platform, which allows simultaneous chronic imaging and electrophysiological recordings to be obtained from the hippocampus of awake mice. We performed quantitative analyses of spontaneous animal behaviour, the associated network states and the cellular activities in the dorsal hippocampus as well as estimated the brain stability limits to image dendritic processes and individual axonal boutons. Ca2+ imaging recordings revealed a relatively stereotyped hippocampal activity despite a high inter-animal and inter-day variability in the mouse behavior. In addition to quiet state and locomotion behavioural patterns, the platform allowed the reliable detection of walking steps and fine speed variations. The brain motion during locomotion was limited to ~1.8 μm, thus allowing for imaging of small sub-cellular structures to be performed in parallel with recordings of network and behavioural states. This simple device extends the drug-free experimentation in vivo, enabling high-stability optophysiological experiments with single-bouton resolution in the mouse awake brain. PMID:28240275

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

PubMed

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

2017-01-01

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

Propagation-based phase-contrast tomography for high-resolution lung imaging with laboratory sources

NASA Astrophysics Data System (ADS)

Krenkel, Martin; Töpperwien, Mareike; Dullin, Christian; Alves, Frauke; Salditt, Tim

2016-03-01

We have performed high-resolution phase-contrast tomography on whole mice with a laboratory setup. Enabled by a high-brilliance liquid-metal-jet source, we show the feasibility of propagation-based phase contrast in local tomography even in the presence of strongly absorbing surrounding tissue as it is the case in small animal imaging of the lung. We demonstrate the technique by reconstructions of the mouse lung for two different fields of view, covering the whole organ, and a zoom to the local finer structure of terminal airways and alveoli. With a resolution of a few micrometers and the wide availability of the technique, studies of larger biological samples at the cellular level become possible.

Cellular imaging of deep organ using two-photon Bessel light-sheet nonlinear structured illumination microscopy

PubMed Central

Zhao, Ming; Zhang, Han; Li, Yu; Ashok, Amit; Liang, Rongguang; Zhou, Weibin; Peng, Leilei

2014-01-01

In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM. PMID:24876996

Avoiding Misannotation of In-Source Fragmentation Products as Cellular Metabolites in Liquid Chromatography–Mass Spectrometry-Based Metabolomics

DOE PAGES

Xu, Yi-Fan; Lu, Wenyun; Rabinowitz, Joshua D.

2015-01-15

Liquid chromatography–mass spectrometry (LC-MS) technology allows for rapid quantitation of cellular metabolites, with metabolites identified by mass spectrometry and chromatographic retention time. Recently, with the development of rapid scanning high-resolution high accuracy mass spectrometers and the desire for high throughput screening, minimal or no chromatographic separation has become increasingly popular. Furthermore, when analyzing complex cellular extracts, however, the lack of chromatographic separation could potentially result in misannotation of structurally related metabolites. Here, we show that, even using electrospray ionization, a soft ionization method, in-source fragmentation generates unwanted byproducts of identical mass to common metabolites. For example, nucleotide-triphosphates generate nucleotide-diphosphates, andmore » hexose-phosphates generate triose-phosphates. We also evaluated yeast intracellular metabolite extracts and found more than 20 cases of in-source fragments that mimic common metabolites. Finally and accordingly, chromatographic separation is required for accurate quantitation of many common cellular metabolites.« less

Texturing Silicon Nanowires for Highly Localized Optical Modulation of Cellular Dynamics.

PubMed

Fang, Yin; Jiang, Yuanwen; Acaron Ledesma, Hector; Yi, Jaeseok; Gao, Xiang; Weiss, Dara E; Shi, Fengyuan; Tian, Bozhi

2018-06-18

Engineered silicon-based materials can display photoelectric and photothermal responses under light illumination, which may lead to further innovations at the silicon-biology interfaces. Silicon nanowires have small radial dimensions, promising as highly localized cellular modulators, however the single crystalline form typically has limited photothermal efficacy due to the poor light absorption and fast heat dissipation. In this work, we report strategies to improve the photothermal response from silicon nanowires by introducing nanoscale textures on the surface and in the bulk. We next demonstrate high-resolution extracellular modulation of calcium dynamics in a number of mammalian cells including glial cells, neurons, and cancer cells. The new materials may be broadly used in probing and modulating electrical and chemical signals at the subcellular length scale, which is currently a challenge in the field of electrophysiology or cellular engineering.

Individual human cell responses to low doses of chemicals studied by synchrotron infrared spectromicroscopy

NASA Astrophysics Data System (ADS)

Holman, Hoi-Ying N.; Goth-Goldstein, Regine; Blakely, Elanor A.; Bjornstad, Kathy; Martin, Michael C.; McKinney, Wayne R.

2000-05-01

Vibrational spectroscopy, when combined with synchrotron radiation-based (SR) microscopy, is a powerful new analytical tool with high spatial resolution for detecting biochemical changes in the individual living cells. In contrast to other microscopy methods that require fixing, drying, staining or labeling, SR-FTIR microscopy probes intact living cells providing a composite view of all of the molecular response and the ability to monitor the response over time in the same cell. Observed spectral changes include all types of lesions induced in that cell as well as cellular responses to external and internal stresses. These spectral changes combined with other analytical tools may provide a fundamental understanding of the key molecular mechanisms induced in response to stresses created by low- doses of chemicals. In this study we used the high spatial - resolution SR-FTIR vibrational spectromicroscopy as a sensitive analytical tool to detect chemical- and radiation- induced changes in individual human cells. Our preliminary spectral measurements indicate that this technique is sensitive enough to detect changes in nucleic acids and proteins of cells treated with environmentally relevant concentrations of dioxin. This technique has the potential to distinguish changes from exogenous or endogenous oxidative processes. Future development of this technique will allow rapid monitoring of cellular processes such as drug metabolism, early detection of disease, bio- compatibility of implant materials, cellular repair mechanisms, self assembly of cellular apparatus, cell differentiation and fetal development.

Live animal myelin histomorphometry of the spinal cord with video-rate multimodal nonlinear microendoscopy

NASA Astrophysics Data System (ADS)

Bélanger, Erik; Crépeau, Joël; Laffray, Sophie; Vallée, Réal; De Koninck, Yves; Côté, Daniel

2012-02-01

In vivo imaging of cellular dynamics can be dramatically enabling to understand the pathophysiology of nervous system diseases. To fully exploit the power of this approach, the main challenges have been to minimize invasiveness and maximize the number of concurrent optical signals that can be combined to probe the interplay between multiple cellular processes. Label-free coherent anti-Stokes Raman scattering (CARS) microscopy, for example, can be used to follow demyelination in neurodegenerative diseases or after trauma, but myelin imaging alone is not sufficient to understand the complex sequence of events that leads to the appearance of lesions in the white matter. A commercially available microendoscope is used here to achieve minimally invasive, video-rate multimodal nonlinear imaging of cellular processes in live mouse spinal cord. The system allows for simultaneous CARS imaging of myelin sheaths and two-photon excitation fluorescence microendoscopy of microglial cells and axons. Morphometric data extraction at high spatial resolution is also described, with a technique for reducing motion-related imaging artifacts. Despite its small diameter, the microendoscope enables high speed multimodal imaging over wide areas of tissue, yet at resolution sufficient to quantify subtle differences in myelin thickness and microglial motility.

Clinical confocal microlaparoscope for real-time in vivo optical biopsies

NASA Astrophysics Data System (ADS)

Tanbakuchi, Anthony A.; Rouse, Andrew R.; Udovich, Joshua A.; Hatch, Kenneth D.; Gmitro, Arthur F.

2009-07-01

Successful treatment of cancer is highly dependent on the stage at which it is diagnosed. Early diagnosis, when the disease is still localized at its origin, results in very high cure rates-even for cancers that typically have poor prognosis. Biopsies are often used for diagnosis of disease. However, because biopsies are destructive, only a limited number can be taken. This leads to reduced sensitivity for detection due to sampling error. A real-time fluorescence confocal microlaparoscope has been developed that provides instant in vivo cellular images, comparable to those provided by histology, through a nondestructive procedure. The device includes an integrated contrast agent delivery mechanism and a computerized depth scan system. The instrument uses a fiber bundle to relay the image plane of a slit-scan confocal microlaparoscope into tissue. It has a 3-μm lateral resolution and a 25-μm axial resolution. Initial in vivo clinical testing using the device to image human ovaries has been done in 21 patients. Results indicate that the device can successfully image organs in vivo without complications. Results with excised tissue demonstrate that the instrument can resolve sufficient cellular detail to visualize the cellular changes associated with the onset of cancer.

Imaging of single cells and tissue using MeV ions

NASA Astrophysics Data System (ADS)

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

2009-06-01

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

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

PubMed Central

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-01-01

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications. PMID:26525841

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope.

PubMed

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-11-03

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

NASA Astrophysics Data System (ADS)

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-11-01

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

Analysis of antigen-specific B-cell memory directly ex vivo.

PubMed

McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

2004-01-01

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.

Towards a minimally invasive sampling tool for high resolution tissue analytical mapping

NASA Astrophysics Data System (ADS)

Gottardi, R.

2015-09-01

Multiple spatial mapping techniques of biological tissues have been proposed over the years, but all present limitations either in terms of resolution, analytical capacity or invasiveness. Ren et al (2015 Nanotechnology 26 284001) propose in their most recent work the use of a picosecond infrared laser (PIRL) under conditions of ultrafast desorption by impulsive vibrational excitation (DIVE) to extract small amounts of cellular and molecular components, conserving their viability, structure and activity. The PIRL DIVE technique would then work as a nanobiopsy with minimal damage to the surrounding tissues, which could potentially be applied for high resolution local structural characterization of tissues in health and disease with the spatial limit determined by the laser focus.

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.

On chip cryo-anesthesia of Drosophila larvae for high resolution in vivo imaging applications.

PubMed

Chaudhury, Amrita Ray; Insolera, Ryan; Hwang, Ran-Der; Fridell, Yih-Woei; Collins, Catherine; Chronis, Nikos

2017-06-27

We present a microfluidic chip for immobilizing Drosophila melanogaster larvae for high resolution in vivo imaging. The chip creates a low-temperature micro-environment that anaesthetizes and immobilizes the larva in under 3 minutes. We characterized the temperature distribution within the chip and analyzed the resulting larval body movement using high resolution fluorescence imaging. Our results indicate that the proposed method minimizes submicron movements of internal organs and tissue without affecting the larva physiology. It can be used to continuously immobilize larvae for short periods of time (minutes) or for longer periods (several hours) if used intermittently. The same chip can be used to accommodate and immobilize arvae across all developmental stages (1st instar to late 3rd instar), and loading larvae onto the chip does not require any specialized skills. To demonstrate the usability of the chip, we observed mitochondrial trafficking in neurons from the cell bodies to the axon terminals along with mitochondrial fusion and neuro-synaptic growth through time in intact larvae. Besides studying sub-cellular processes and cellular development, we envision the use of on chip cryo-anesthesia in a wide variety of biological in vivo imaging applications, including observing organ development of the salivary glands, fat bodies and body-wall muscles.

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

PubMed

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

2011-06-01

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

Spatial Mapping of Lipids at Cellular Resolution in Embryos of Cotton[W][OA

PubMed Central

Horn, Patrick J.; Korte, Andrew R.; Neogi, Purnima B.; Love, Ebony; Fuchs, Johannes; Strupat, Kerstin; Borisjuk, Ljudmilla; Shulaev, Vladimir; Lee, Young-Jin; Chapman, Kent D.

2012-01-01

Advances in mass spectrometry (MS) have made comprehensive lipidomics analysis of complex tissues relatively commonplace. These compositional analyses, although able to resolve hundreds of molecular species of lipids in single extracts, lose the original cellular context from which these lipids are derived. Recently, high-resolution MS of individual lipid droplets from seed tissues indicated organelle-to-organelle variation in lipid composition, suggesting that heterogeneity of lipid distributions at the cellular level may be prevalent. Here, we employed matrix-assisted laser desorption/ionization–MS imaging (MALDI-MSI) approaches to visualize lipid species directly in seed tissues of upland cotton (Gossypium hirsutum). MS imaging of cryosections of mature cotton embryos revealed a distinct, heterogeneous distribution of molecular species of triacylglycerols and phosphatidylcholines, the major storage and membrane lipid classes in cotton embryos. Other lipids were imaged, including phosphatidylethanolamines, phosphatidic acids, sterols, and gossypol, indicating the broad range of metabolites and applications for this chemical visualization approach. We conclude that comprehensive lipidomics images generated by MALDI-MSI report accurate, relative amounts of lipid species in plant tissues and reveal previously unseen differences in spatial distributions providing for a new level of understanding in cellular biochemistry. PMID:22337917

Investigation of tissue cellularity at the tip of the core biopsy needle with optical coherence tomography.

PubMed

Iftimia, Nicusor; Park, Jesung; Maguluri, Gopi; Krishnamurthy, Savitri; McWatters, Amanda; Sabir, Sharjeel H

2018-02-01

We report the development and the pre-clinical testing of a new technology based on optical coherence tomography (OCT) for investigating tissue composition at the tip of the core biopsy needle. While ultrasound, computed tomography, and magnetic resonance imaging are routinely used to guide needle placement within a tumor, they still do not provide the resolution needed to investigate tissue cellularity (ratio between viable tumor and benign stroma) at the needle tip prior to taking a biopsy core. High resolution OCT imaging, however, can be used to investigate tissue morphology at the micron scale, and thus to determine if the biopsy core would likely have the expected composition. Therefore, we implemented this capability within a custom-made biopsy gun and evaluated its capability for a correct estimation of tumor tissue cellularity. A pilot study on a rabbit model of soft tissue cancer has shown the capability of this technique to provide correct evaluation of tumor tissue cellularity in over 85% of the cases. These initial results indicate the potential benefit of the OCT-based approach for improving the success of the core biopsy procedures.

Multiplexed 3D FRET imaging in deep tissue of live embryos

PubMed Central

Zhao, Ming; Wan, Xiaoyang; Li, Yu; Zhou, Weibin; Peng, Leilei

2015-01-01

Current deep tissue microscopy techniques are mostly restricted to intensity mapping of fluorophores, which significantly limit their applications in investigating biochemical processes in vivo. We present a deep tissue multiplexed functional imaging method that probes multiple Förster resonant energy transfer (FRET) sensors in live embryos with high spatial resolution. The method simultaneously images fluorescence lifetimes in 3D with multiple excitation lasers. Through quantitative analysis of triple-channel intensity and lifetime images, we demonstrated that Ca2+ and cAMP levels of live embryos expressing dual FRET sensors can be monitored simultaneously at microscopic resolution. The method is compatible with a broad range of FRET sensors currently available for probing various cellular biochemical functions. It opens the door to imaging complex cellular circuitries in whole live organisms. PMID:26387920

Pan-neuronal calcium imaging with cellular resolution in freely swimming zebrafish.

PubMed

Kim, Dal Hyung; Kim, Jungsoo; Marques, João C; Grama, Abhinav; Hildebrand, David G C; Gu, Wenchao; Li, Jennifer M; Robson, Drew N

2017-11-01

Calcium imaging with cellular resolution typically requires an animal to be tethered under a microscope, which substantially restricts the range of behaviors that can be studied. To expand the behavioral repertoire amenable to imaging, we have developed a tracking microscope that enables whole-brain calcium imaging with cellular resolution in freely swimming larval zebrafish. This microscope uses infrared imaging to track a target animal in a behavior arena. On the basis of the predicted trajectory of the animal, we applied optimal control theory to a motorized stage system to cancel brain motion in three dimensions. We combined this motion-cancellation system with differential illumination focal filtering, a variant of HiLo microscopy, which enabled us to image the brain of a freely swimming larval zebrafish for more than an hour. This work expands the repertoire of natural behaviors that can be studied with cellular-resolution calcium imaging to potentially include spatial navigation, social behavior, feeding and reward.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

PubMed

Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

2017-06-28

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

A draft map of the mouse pluripotent stem cell spatial proteome

PubMed Central

Christoforou, Andy; Mulvey, Claire M.; Breckels, Lisa M.; Geladaki, Aikaterini; Hurrell, Tracey; Hayward, Penelope C.; Naake, Thomas; Gatto, Laurent; Viner, Rosa; Arias, Alfonso Martinez; Lilley, Kathryn S.

2016-01-01

Knowledge of the subcellular distribution of proteins is vital for understanding cellular mechanisms. Capturing the subcellular proteome in a single experiment has proven challenging, with studies focusing on specific compartments or assigning proteins to subcellular niches with low resolution and/or accuracy. Here we introduce hyperLOPIT, a method that couples extensive fractionation, quantitative high-resolution accurate mass spectrometry with multivariate data analysis. We apply hyperLOPIT to a pluripotent stem cell population whose subcellular proteome has not been extensively studied. We provide localization data on over 5,000 proteins with unprecedented spatial resolution to reveal the organization of organelles, sub-organellar compartments, protein complexes, functional networks and steady-state dynamics of proteins and unexpected subcellular locations. The method paves the way for characterizing the impact of post-transcriptional and post-translational modification on protein location and studies involving proteome-level locational changes on cellular perturbation. An interactive open-source resource is presented that enables exploration of these data. PMID:26754106

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

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

PubMed Central

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

2010-01-01

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

CAM: A high-performance cellular-automaton machine

NASA Astrophysics Data System (ADS)

Toffoli, Tommaso

1984-01-01

CAM is a high-performance machine dedicated to the simulation of cellular automata and other distributed dynamical systems. Its speed is about one-thousand times greater than that of a general-purpose computer programmed to do the same task; in practical terms, this means that CAM can show the evolution of cellular automata on a color monitor with an update rate, dynamic range, and spatial resolution comparable to those of a Super-8 movie, thus permitting intensive interactive experimentation. Machines of this kind can open up novel fields of research, and in this context it is important that results be easy to obtain, reproduce, and transmit. For these reasons, in designing CAM it was important to achieve functional simplicity, high flexibility, and moderate production cost. We expect that many research groups will be able to own their own copy of the machine to do research with.

Example-Based Super-Resolution Fluorescence Microscopy.

PubMed

Jia, Shu; Han, Boran; Kutz, J Nathan

2018-04-23

Capturing biological dynamics with high spatiotemporal resolution demands the advancement in imaging technologies. Super-resolution fluorescence microscopy offers spatial resolution surpassing the diffraction limit to resolve near-molecular-level details. While various strategies have been reported to improve the temporal resolution of super-resolution imaging, all super-resolution techniques are still fundamentally limited by the trade-off associated with the longer image acquisition time that is needed to achieve higher spatial information. Here, we demonstrated an example-based, computational method that aims to obtain super-resolution images using conventional imaging without increasing the imaging time. With a low-resolution image input, the method provides an estimate of its super-resolution image based on an example database that contains super- and low-resolution image pairs of biological structures of interest. The computational imaging of cellular microtubules agrees approximately with the experimental super-resolution STORM results. This new approach may offer potential improvements in temporal resolution for experimental super-resolution fluorescence microscopy and provide a new path for large-data aided biomedical imaging.

High-resolution measurements of the multilayer ultra-structure of articular cartilage and their translational potential

PubMed Central

2014-01-01

Current musculoskeletal imaging techniques usually target the macro-morphology of articular cartilage or use histological analysis. These techniques are able to reveal advanced osteoarthritic changes in articular cartilage but fail to give detailed information to distinguish early osteoarthritis from healthy cartilage, and this necessitates high-resolution imaging techniques measuring cells and the extracellular matrix within the multilayer structure of articular cartilage. This review provides a comprehensive exploration of the cellular components and extracellular matrix of articular cartilage as well as high-resolution imaging techniques, including magnetic resonance image, electron microscopy, confocal laser scanning microscopy, second harmonic generation microscopy, and laser scanning confocal arthroscopy, in the measurement of multilayer ultra-structures of articular cartilage. This review also provides an overview for micro-structural analysis of the main components of normal or osteoarthritic cartilage and discusses the potential and challenges associated with developing non-invasive high-resolution imaging techniques for both research and clinical diagnosis of early to late osteoarthritis. PMID:24946278

Correlative Single-Molecule Localization Microscopy and Confocal Microscopy.

PubMed

Soeller, Christian; Hou, Yufeng; Jayasinghe, Isuru D; Baddeley, David; Crossman, David

2017-01-01

Single-molecule localization microscopy allows the ability to image fluorescence labeled molecular targets at nanoscale resolution. However, for many biological questions the ability to provide tissue and cellular context in addition to these high resolution data is eminently informative. Here, we describe a procedure to achieve this aim by correlatively imaging human cardiac tissue first at the nanoscale with direct stochastic optical reconstruction microscopy (dSTORM) and then at the diffraction limit with conventional confocal microscopy.

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

PubMed Central

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

2018-01-01

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

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

PubMed

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

2011-08-15

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

X-ray phase-contrast tomography for high-spatial-resolution zebrafish muscle imaging

NASA Astrophysics Data System (ADS)

Vågberg, William; Larsson, Daniel H.; Li, Mei; Arner, Anders; Hertz, Hans M.

2015-11-01

Imaging of muscular structure with cellular or subcellular detail in whole-body animal models is of key importance for understanding muscular disease and assessing interventions. Classical histological methods for high-resolution imaging methods require excision, fixation and staining. Here we show that the three-dimensional muscular structure of unstained whole zebrafish can be imaged with sub-5 μm detail with X-ray phase-contrast tomography. Our method relies on a laboratory propagation-based phase-contrast system tailored for detection of low-contrast 4-6 μm subcellular myofibrils. The method is demonstrated on 20 days post fertilization zebrafish larvae and comparative histology confirms that we resolve individual myofibrils in the whole-body animal. X-ray imaging of healthy zebrafish show the expected structured muscle pattern while specimen with a dystrophin deficiency (sapje) displays an unstructured pattern, typical of Duchenne muscular dystrophy. The method opens up for whole-body imaging with sub-cellular detail also of other types of soft tissue and in different animal models.

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.

PubMed

Treweek, Jennifer B; Chan, Ken Y; Flytzanis, Nicholas C; Yang, Bin; Deverman, Benjamin E; Greenbaum, Alon; Lignell, Antti; Xiao, Cheng; Cai, Long; Ladinsky, Mark S; Bjorkman, Pamela J; Fowlkes, Charless C; Gradinaru, Viviana

2015-11-01

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1-2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.

Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

PubMed

De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M

2011-10-01

Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay

PubMed Central

De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M.

2011-01-01

Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin. PMID:22006542

3D wide field-of-view Gabor-domain optical coherence microscopy advancing real-time in-vivo imaging and metrology

NASA Astrophysics Data System (ADS)

Canavesi, Cristina; Cogliati, Andrea; Hayes, Adam; Tankam, Patrice; Santhanam, Anand; Rolland, Jannick P.

2017-02-01

Real-time volumetric high-definition wide-field-of-view in-vivo cellular imaging requires micron-scale resolution in 3D. Compactness of the handheld device and distortion-free images with cellular resolution are also critically required for onsite use in clinical applications. By integrating a custom liquid lens-based microscope and a dual-axis MEMS scanner in a compact handheld probe, Gabor-domain optical coherence microscopy (GD-OCM) breaks the lateral resolution limit of optical coherence tomography through depth, overcoming the tradeoff between numerical aperture and depth of focus, enabling advances in biotechnology. Furthermore, distortion-free imaging with no post-processing is achieved with a compact, lightweight handheld MEMS scanner that obtained a 12-fold reduction in volume and 17-fold reduction in weight over a previous dual-mirror galvanometer-based scanner. Approaching the holy grail of medical imaging - noninvasive real-time imaging with histologic resolution - GD-OCM demonstrates invariant resolution of 2 μm throughout a volume of 1 x 1 x 0.6 mm3, acquired and visualized in less than 2 minutes with parallel processing on graphics processing units. Results on the metrology of manufactured materials and imaging of human tissue with GD-OCM are presented.

Optical magnetic imaging of living cells

PubMed Central

Le Sage, D.; Arai, K.; Glenn, D. R.; DeVience, S. J.; Pham, L. M.; Rahn-Lee, L.; Lukin, M. D.; Yacoby, A.; Komeili, A.; Walsworth, R. L.

2013-01-01

Magnetic imaging is a powerful tool for probing biological and physical systems. However, existing techniques either have poor spatial resolution compared to optical microscopy and are hence not generally applicable to imaging of sub-cellular structure (e.g., magnetic resonance imaging [MRI]1), or entail operating conditions that preclude application to living biological samples while providing sub-micron resolution (e.g., scanning superconducting quantum interference device [SQUID] microscopy2, electron holography3, and magnetic resonance force microscopy [MRFM]4). Here we demonstrate magnetic imaging of living cells (magnetotactic bacteria) under ambient laboratory conditions and with sub-cellular spatial resolution (400 nm), using an optically-detected magnetic field imaging array consisting of a nanoscale layer of nitrogen-vacancy (NV) colour centres implanted at the surface of a diamond chip. With the bacteria placed on the diamond surface, we optically probe the NV quantum spin states and rapidly reconstruct images of the vector components of the magnetic field created by chains of magnetic nanoparticles (magnetosomes) produced in the bacteria, and spatially correlate these magnetic field maps with optical images acquired in the same apparatus. Wide-field sCMOS acquisition allows parallel optical and magnetic imaging of multiple cells in a population with sub-micron resolution and >100 micron field-of-view. Scanning electron microscope (SEM) images of the bacteria confirm that the correlated optical and magnetic images can be used to locate and characterize the magnetosomes in each bacterium. The results provide a new capability for imaging bio-magnetic structures in living cells under ambient conditions with high spatial resolution, and will enable the mapping of a wide range of magnetic signals within cells and cellular networks5, 6. PMID:23619694

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

PubMed

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

2014-12-01

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

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

PubMed

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

2014-01-28

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

Fiber optic in vivo imaging in the mammalian nervous system

PubMed Central

Mehta, Amit D; Jung, Juergen C; Flusberg, Benjamin A; Schnitzer, Mark J

2010-01-01

The compact size, mechanical flexibility, and growing functionality of optical fiber and fiber optic devices are enabling several new modalities for imaging the mammalian nervous system in vivo. Fluorescence microendoscopy is a minimally invasive fiber modality that provides cellular resolution in deep brain areas. Diffuse optical tomography is a non-invasive modality that uses assemblies of fiber optic emitters and detectors on the cranium for volumetric imaging of brain activation. Optical coherence tomography is a sensitive interferometric imaging technique that can be implemented in a variety of fiber based formats and that might allow intrinsic optical detection of brain activity at a high resolution. Miniaturized fiber optic microscopy permits cellular level imaging in the brains of behaving animals. Together, these modalities will enable new uses of imaging in the intact nervous system for both research and clinical applications. PMID:15464896

aMAP is a validated pipeline for registration and segmentation of high-resolution mouse brain data

PubMed Central

Niedworok, Christian J.; Brown, Alexander P. Y.; Jorge Cardoso, M.; Osten, Pavel; Ourselin, Sebastien; Modat, Marc; Margrie, Troy W.

2016-01-01

The validation of automated image registration and segmentation is crucial for accurate and reliable mapping of brain connectivity and function in three-dimensional (3D) data sets. While validation standards are necessarily high and routinely met in the clinical arena, they have to date been lacking for high-resolution microscopy data sets obtained from the rodent brain. Here we present a tool for optimized automated mouse atlas propagation (aMAP) based on clinical registration software (NiftyReg) for anatomical segmentation of high-resolution 3D fluorescence images of the adult mouse brain. We empirically evaluate aMAP as a method for registration and subsequent segmentation by validating it against the performance of expert human raters. This study therefore establishes a benchmark standard for mapping the molecular function and cellular connectivity of the rodent brain. PMID:27384127

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

NASA Astrophysics Data System (ADS)

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

2017-07-01

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

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

PubMed Central

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

2011-01-01

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

Confocal reflectance quantitative phase microscope system for cellular membranes dynamics study (Conference Presentation)

NASA Astrophysics Data System (ADS)

Singh, Vijay Raj; Yaqoob, Zahid; So, Peter T. C.

2017-02-01

Quantitative phase microscopy (QPM) techniques developed so far primarily belongs to high speed transmitted light based systems that has enough sensitivity to resolve membrane fluctuations and dynamics, but has no depth resolution. Therefore, most biomechanics studies using QPM today is confined to simple cells, such as RBCs, without internal organelles. An important instrument that will greatly extend the biomedical applications of QPM is to develop next generation microscope with 3D capability and sufficient temporal resolution to study biomechanics of complex eukaryotic cells including the mechanics of their internal compartments. For eukaryotic cells, the depth sectioning capability is critical and should be sufficient to distinguish nucleic membrane fluctuations from plasma membrane fluctuations. Further, this microscope must provide high temporal resolution since typical eukaryotes membranes are substantially stiffer than RBCs. A confocal reflectance quantitative phase microscope is presented based on multi-pinhole scanning, with the capabilities of higher temporal resolution and sensitivity for nucleic and plasma membranes of eukaryotic cells. System hardware is developed based on an array of confocal pinhole generated by using the `ON' state of subset of micro-mirrors of digital micro-mirror device (DMD, from Texas Instruments) and high-speed raster scanning provides 14ms imaging speed in wide-field mode. A common path interferometer is integrated at the imaging arm for detection of specimens' quantitative phase information. Theoretical investigation of quantitative phase reconstructed from system is investigated and application of system is presented for dimensional fluctuations measurements of both cellular plasma and nucleic membranes of embryonic stem cells.

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

PubMed

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

2015-06-10

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

Boron dipyrromethene (BODIPY) functionalized carbon nano-onions for high resolution cellular imaging

NASA Astrophysics Data System (ADS)

Bartelmess, Juergen; de Luca, Elisa; Signorelli, Angelo; Baldrighi, Michele; Becce, Michele; Brescia, Rosaria; Nardone, Valentina; Parisini, Emilio; Echegoyen, Luis; Pompa, Pier Paolo; Giordani, Silvia

2014-10-01

Carbon nano-onions (CNOs) are an exciting class of carbon nanomaterials, which have recently demonstrated a facile cell-penetration capability. In the present work, highly fluorescent boron dipyrromethene (BODIPY) dyes were covalently attached to the surface of CNOs. The introduction of this new carbon nanomaterial-based imaging platform, made of CNOs and BODIPY fluorophores, allows for the exploration of synergetic effects between the two building blocks and for the elucidation of its performance in biological applications. The high fluorescence intensity exhibited by the functionalized CNOs translates into an excellent in vitro probe for the high resolution imaging of MCF-7 human breast cancer cells. It was also found that the CNOs, internalized by the cells by endocytosis, localized in the lysosomes and did not show any cytotoxic effects. The presented results highlight CNOs as excellent platforms for biological and biomedical studies due to their low toxicity, efficient cellular uptake and low fluorescence quenching of attached probes.Carbon nano-onions (CNOs) are an exciting class of carbon nanomaterials, which have recently demonstrated a facile cell-penetration capability. In the present work, highly fluorescent boron dipyrromethene (BODIPY) dyes were covalently attached to the surface of CNOs. The introduction of this new carbon nanomaterial-based imaging platform, made of CNOs and BODIPY fluorophores, allows for the exploration of synergetic effects between the two building blocks and for the elucidation of its performance in biological applications. The high fluorescence intensity exhibited by the functionalized CNOs translates into an excellent in vitro probe for the high resolution imaging of MCF-7 human breast cancer cells. It was also found that the CNOs, internalized by the cells by endocytosis, localized in the lysosomes and did not show any cytotoxic effects. The presented results highlight CNOs as excellent platforms for biological and biomedical studies due to their low toxicity, efficient cellular uptake and low fluorescence quenching of attached probes. Electronic supplementary information (ESI) available: Additional experimental and crystallographic data, additional confocal microscopy and HR-TEM images and illustrations, EELS, TGA, DLS and Z-potential results. Movie M1. See DOI: 10.1039/c4nr04533e

High-resolution genome-wide scan of genes, gene-networks and cellular systems impacting the yeast ionome

USDA-ARS?s Scientific Manuscript database

To balance the demand for uptake of essential elements with their potential toxicity living cells have complex regulatory mechanisms. Here, we describe a genome-wide screen to identify genes that impact the elemental composition (‘ionome’) of yeast Saccharomyces cerevisiae. Using inductively coupled...

Whole-cell imaging of the budding yeast Saccharomyces cerevisiae by high-voltage scanning transmission electron tomography.

PubMed

Murata, Kazuyoshi; Esaki, Masatoshi; Ogura, Teru; Arai, Shigeo; Yamamoto, Yuta; Tanaka, Nobuo

2014-11-01

Electron tomography using a high-voltage electron microscope (HVEM) provides three-dimensional information about cellular components in sections thicker than 1 μm, although in bright-field mode image degradation caused by multiple inelastic scattering of transmitted electrons limit the attainable resolution. Scanning transmission electron microscopy (STEM) is believed to give enhanced contrast and resolution compared to conventional transmission electron microscopy (CTEM). Samples up to 1 μm in thickness have been analyzed with an intermediate-voltage electron microscope because inelastic scattering is not a critical limitation, and probe broadening can be minimized. Here, we employed STEM at 1 MeV high-voltage to extend the useful specimen thickness for electron tomography, which we demonstrate by a seamless tomographic reconstruction of a whole, budding Saccharomyces cerevisiae yeast cell, which is ~3 μm in thickness. High-voltage STEM tomography, especially in the bright-field mode, demonstrated sufficiently enhanced contrast and intensity, compared to CTEM tomography, to permit segmentation of major organelles in the whole cell. STEM imaging also reduced specimen shrinkage during tilt-series acquisition. The fidelity of structural preservation was limited by cytoplasmic extraction, and the spatial resolution was limited by the relatively large convergence angle of the scanning probe. However, the new technique has potential to solve longstanding problems of image blurring in biological specimens beyond 1 μm in thickness, and may facilitate new research in cellular structural biology. Copyright © 2014 Elsevier B.V. All rights reserved.

High-throughput isotropic mapping of whole mouse brain using multi-view light-sheet microscopy

NASA Astrophysics Data System (ADS)

Nie, Jun; Li, Yusha; Zhao, Fang; Ping, Junyu; Liu, Sa; Yu, Tingting; Zhu, Dan; Fei, Peng

2018-02-01

Light-sheet fluorescence microscopy (LSFM) uses an additional laser-sheet to illuminate selective planes of the sample, thereby enabling three-dimensional imaging at high spatial-temporal resolution. These advantages make LSFM a promising tool for high-quality brain visualization. However, even by the use of LSFM, the spatial resolution remains insufficient to resolve the neural structures across a mesoscale whole mouse brain in three dimensions. At the same time, the thick-tissue scattering prevents a clear observation from the deep of brain. Here we use multi-view LSFM strategy to solve this challenge, surpassing the resolution limit of standard light-sheet microscope under a large field-of-view (FOV). As demonstrated by the imaging of optically-cleared mouse brain labelled with thy1-GFP, we achieve a brain-wide, isotropic cellular resolution of 3μm. Besides the resolution enhancement, multi-view braining imaging can also recover complete signals from deep tissue scattering and attenuation. The identification of long distance neural projections across encephalic regions can be identified and annotated as a result.

Multiple-image hiding using super resolution reconstruction in high-frequency domains

NASA Astrophysics Data System (ADS)

Li, Xiao-Wei; Zhao, Wu-Xiang; Wang, Jun; Wang, Qiong-Hua

2017-12-01

In this paper, a robust multiple-image hiding method using the computer-generated integral imaging and the modified super-resolution reconstruction algorithm is proposed. In our work, the host image is first transformed into frequency domains by cellular automata (CA), to assure the quality of the stego-image, the secret images are embedded into the CA high-frequency domains. The proposed method has the following advantages: (1) robustness to geometric attacks because of the memory-distributed property of elemental images, (2) increasing quality of the reconstructed secret images as the scheme utilizes the modified super-resolution reconstruction algorithm. The simulation results show that the proposed multiple-image hiding method outperforms other similar hiding methods and is robust to some geometric attacks, e.g., Gaussian noise and JPEG compression attacks.

High-Throughput Single-Cell RNA Sequencing and Data Analysis.

PubMed

Sagar; Herman, Josip Stefan; Pospisilik, John Andrew; Grün, Dominic

2018-01-01

Understanding biological systems at a single cell resolution may reveal several novel insights which remain masked by the conventional population-based techniques providing an average readout of the behavior of cells. Single-cell transcriptome sequencing holds the potential to identify novel cell types and characterize the cellular composition of any organ or tissue in health and disease. Here, we describe a customized high-throughput protocol for single-cell RNA-sequencing (scRNA-seq) combining flow cytometry and a nanoliter-scale robotic system. Since scRNA-seq requires amplification of a low amount of endogenous cellular RNA, leading to substantial technical noise in the dataset, downstream data filtering and analysis require special care. Therefore, we also briefly describe in-house state-of-the-art data analysis algorithms developed to identify cellular subpopulations including rare cell types as well as to derive lineage trees by ordering the identified subpopulations of cells along the inferred differentiation trajectories.

A high-resolution, confocal laser-scanning microscope and flash photolysis system for physiological studies.

PubMed

Parker, I; Callamaras, N; Wier, W G

1997-06-01

We describe the construction of a high-resolution confocal laser-scanning microscope, and illustrate its use for studying elementary Ca2+ signalling events in cells. An avalanche photodiode module and simple optical path provide a high efficiency system for detection of fluorescence signals, allowing use of a small confocal aperture giving near diffraction-limited spatial resolution (< 300 nm lateral and < 400 nm axial). When operated in line-scan mode, the maximum temporal resolution is 1 ms, and the associated computer software allows complete flexibility to record line-scans continuously for long (minutes) periods or to obtain any desired pixel resolution in x-y scans. An independent UV irradiation system permits simultaneous photolysis of caged compounds over either a uniform, wide field (arc lamp source) or at a tightly focussed spot (frequency-tripled Nd:YAG laser). The microscope thus provides a versatile tool for optical studies of dynamic cellular processes, as well as excellent resolution for morphological studies. The confocal scanner can be added to virtually any inverted microscope for a component cost that is only a small fraction of that of comparable commercial instruments, yet offers better performance and greater versatility.

A 181 GOPS AKAZE Accelerator Employing Discrete-Time Cellular Neural Networks for Real-Time Feature Extraction.

PubMed

Jiang, Guangli; Liu, Leibo; Zhu, Wenping; Yin, Shouyi; Wei, Shaojun

2015-09-04

This paper proposes a real-time feature extraction VLSI architecture for high-resolution images based on the accelerated KAZE algorithm. Firstly, a new system architecture is proposed. It increases the system throughput, provides flexibility in image resolution, and offers trade-offs between speed and scaling robustness. The architecture consists of a two-dimensional pipeline array that fully utilizes computational similarities in octaves. Secondly, a substructure (block-serial discrete-time cellular neural network) that can realize a nonlinear filter is proposed. This structure decreases the memory demand through the removal of data dependency. Thirdly, a hardware-friendly descriptor is introduced in order to overcome the hardware design bottleneck through the polar sample pattern; a simplified method to realize rotation invariance is also presented. Finally, the proposed architecture is designed in TSMC 65 nm CMOS technology. The experimental results show a performance of 127 fps in full HD resolution at 200 MHz frequency. The peak performance reaches 181 GOPS and the throughput is double the speed of other state-of-the-art architectures.

Whole-body and Whole-Organ Clearing and Imaging Techniques with Single-Cell Resolution: Toward Organism-Level Systems Biology in Mammals.

PubMed

Susaki, Etsuo A; Ueda, Hiroki R

2016-01-21

Organism-level systems biology aims to identify, analyze, control and design cellular circuits in organisms. Many experimental and computational approaches have been developed over the years to allow us to conduct these studies. Some of the most powerful methods are based on using optical imaging in combination with fluorescent labeling, and for those one of the long-standing stumbling blocks has been tissue opacity. Recently, the solutions to this problem have started to emerge based on whole-body and whole-organ clearing techniques that employ innovative tissue-clearing chemistry. Here, we review these advancements and discuss how combining new clearing techniques with high-performing fluorescent proteins or small molecule tags, rapid volume imaging and efficient image informatics is resulting in comprehensive and quantitative organ-wide, single-cell resolution experimental data. These technologies are starting to yield information on connectivity and dynamics in cellular circuits at unprecedented resolution, and bring us closer to system-level understanding of physiology and diseases of complex mammalian systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

OPTICAL AND PHARMACOLOGICAL TOOLS TO INVESTIGATE THE ROLE OF MITOCHONDRIA DURING OXIDATIVE STRESS AND NEURODEGENERATION

PubMed Central

Foster, Kelley A.; Galeffi, Francesca; Gerich, Florian J.; Turner, Dennis A.; Müller, Michael

2007-01-01

Mitochondria are critical for cellular ATP production; however, recent studies suggest that these organelles fulfill a much broader range of tasks. For example, they are involved in the regulation of cytosolic Ca2+ levels, intracellular pH and apoptosis, and are the major source of reactive oxygen species (ROS). Various reactive molecules that originate from mitochondria, such as ROS, are critical in pathological events, such as ischemia, as well as in physiological events such as long-term potentiation, neuronal-vascular coupling and neuronal-glial interactions. Due to their key roles in the regulation of several cellular functions, the dysfunction of mitochondria may be critical in various brain disorders. There has been increasing interest in the development of tools that modulate mitochondrial function, and the refinement of techniques that allow for real time monitoring of mitochondria, particularly within their intact cellular environment. Innovative imaging techniques are especially powerful since they allow for mitochondrial visualization at high resolution, tracking of mitochondrial structures and optical real time monitoring of parameters of mitochondrial function. Among the techniques discussed are the uses of classic imaging techniques such as rhodamine-123, the highly advanced semi-conductor nanoparticles (quantum dots), and wide field microscopy as well as high-resolution multi-photon imaging. We have highlighted the use of these techniques to study mitochondrial function in brain tissue and have included studies from our laboratories in which these techniques have been successfully applied. PMID:16920246

Individual Human Cell Responses to Low Doses of Chemicals and Radiation Studied by Synchrotron Infrared Spectromicroscopy

NASA Astrophysics Data System (ADS)

Martin, Michael C.; Holman, Hoi-Ying N.; Blakely, Eleanor A.; Goth-Goldstein, Regine; McKinney, Wayne R.

2000-03-01

Vibrational spectroscopy, when combined with synchrotron radiation-based (SR) microscopy, is a powerful new analytical tool with high spatial resolution for detecting biochemical changes in individual living cells. In contrast to other microscopy methods that require fixing, drying, staining or labeling, SR FTIR microscopy probes intact living cells providing a composite view of all of the molecular responses and the ability to monitor the responses over time in the same cell. Observed spectral changes include all types of lesions induced in that cell as well as cellular responses to external and internal stresses. These spectral changes combined with other analytical tools may provide a fundamental understanding of the key molecular mechanisms induced in response to stresses created by low-doses of radiation and chemicals. In this study we used high spatial-resolution SR FTIR vibrational spectromicroscopy at ALS Beamline 1.4.3 as a sensitive analytical tool to detect chemical- and radiation-induced changes in individual human cells. Our preliminary spectral measurements indicate that this technique is sensitive enough to detect changes in nucleic acids and proteins of cells treated with environmentally relevant concentrations of oxidative stresses: bleomycin, hydrogen peroxide, and X-rays. We observe spectral changes that are unique to each exogenous stressor. This technique has the potential to distinguish changes from exogenous or endogenous oxidative processes. Future development of this technique will allow rapid monitoring of cellular processes such as drug metabolism, early detection of disease, bio-compatibility of implant materials, cellular repair mechanisms, self assembly of cellular apparatus, cell differentiation and fetal development.

Challenges and opportunities in the high-resolution cryo-EM visualization of microtubules and their binding partners.

PubMed

Nogales, Eva; Kellogg, Elizabeth H

2017-10-01

As non-crystallizable polymers, microtubules have been the target of cryo-electron microscopy (cryo-EM) studies since the technique was first established. Over the years, image processing strategies have been developed that take care of the unique, pseudo-helical symmetry of the microtubule. With recent progress in data quality and data processing, cryo-EM reconstructions are now reaching resolutions that allow the generation of atomic models of microtubules and the factors that bind them. These include cellular partners that contribute to microtubule cellular functions, or small ligands that interfere with those functions in the treatment of cancer. The stage is set to generate a family portrait for all identified microtubule interacting proteins and to use cryo-EM as a drug development tool in the targeting of tubulin. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

PubMed Central

Gulati, Srishti; Cao, Vania Y.; Otte, Stephani

2017-01-01

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms. PMID:28654056

Cell Signaling Experiments Driven by Optical Manipulation

PubMed Central

Difato, Francesco; Pinato, Giulietta; Cojoc, Dan

2013-01-01

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

Transparent, Flexible, Low Noise Graphene Electrodes for Simultaneous Electrophysiology and Neuroimaging

PubMed Central

Kuzum, Duygu; Takano, Hajime; Shim, Euijae; Reed, Jason C; Juul, Halvor; Richardson, Andrew G.; de Vries, Julius; Bink, Hank; Dichter, Marc A.; Lucas, Timothy H.; Coulter, Douglas A.; Cubukcu, Ertugrul; Litt, Brian

2014-01-01

Calcium imaging is a versatile experimental approach capable of resolving single neurons with single-cell spatial resolution in the brain. Electrophysiological recordings provide high temporal, but limited spatial resolution, due to the geometrical inaccessibility of the brain. An approach that integrates the advantages of both techniques could provide new insights into functions of neural circuits. Here, we report a transparent, flexible neural electrode technology based on graphene, which enables simultaneous optical imaging and electrophysiological recording. We demonstrate that hippocampal slices can be imaged through transparent graphene electrodes by both confocal and two-photon microscopy without causing any light-induced artifacts in the electrical recordings. Graphene electrodes record high frequency bursting activity and slow synaptic potentials that are hard to resolve by multi-cellular calcium imaging. This transparent electrode technology may pave the way for high spatio-temporal resolution electrooptic mapping of the dynamic neuronal activity. PMID:25327632

Compartmental Genomics in Living Cells Revealed by Single-Cell Nanobiopsy

PubMed Central

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

2014-01-01

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

Single-pulse enhanced coherent diffraction imaging of bacteria with an X-ray free-electron laser

NASA Astrophysics Data System (ADS)

Fan, Jiadong; Sun, Zhibin; Wang, Yaling; Park, Jaehyun; Kim, Sunam; Gallagher-Jones, Marcus; Kim, Yoonhee; Song, Changyong; Yao, Shengkun; Zhang, Jian; Zhang, Jianhua; Duan, Xiulan; Tono, Kensuke; Yabashi, Makina; Ishikawa, Tetsuya; Fan, Chunhai; Zhao, Yuliang; Chai, Zhifang; Gao, Xueyun; Earnest, Thomas; Jiang, Huaidong

2016-09-01

High-resolution imaging offers one of the most promising approaches for exploring and understanding the structure and function of biomaterials and biological systems. X-ray free-electron lasers (XFELs) combined with coherent diffraction imaging can theoretically provide high-resolution spatial information regarding biological materials using a single XFEL pulse. Currently, the application of this method suffers from the low scattering cross-section of biomaterials and X-ray damage to the sample. However, XFELs can provide pulses of such short duration that the data can be collected using the “diffract and destroy” approach before the effects of radiation damage on the data become significant. These experiments combine the use of enhanced coherent diffraction imaging with single-shot XFEL radiation to investigate the cellular architecture of Staphylococcus aureus with and without labeling by gold (Au) nanoclusters. The resolution of the images reconstructed from these diffraction patterns were twice as high or more for gold-labeled samples, demonstrating that this enhancement method provides a promising approach for the high-resolution imaging of biomaterials and biological systems.

Single-pulse enhanced coherent diffraction imaging of bacteria with an X-ray free-electron laser

PubMed Central

Fan, Jiadong; Sun, Zhibin; Wang, Yaling; Park, Jaehyun; Kim, Sunam; Gallagher-Jones, Marcus; Kim, Yoonhee; Song, Changyong; Yao, Shengkun; Zhang, Jian; Zhang, Jianhua; Duan, Xiulan; Tono, Kensuke; Yabashi, Makina; Ishikawa, Tetsuya; Fan, Chunhai; Zhao, Yuliang; Chai, Zhifang; Gao, Xueyun; Earnest, Thomas; Jiang, Huaidong

2016-01-01

High-resolution imaging offers one of the most promising approaches for exploring and understanding the structure and function of biomaterials and biological systems. X-ray free-electron lasers (XFELs) combined with coherent diffraction imaging can theoretically provide high-resolution spatial information regarding biological materials using a single XFEL pulse. Currently, the application of this method suffers from the low scattering cross-section of biomaterials and X-ray damage to the sample. However, XFELs can provide pulses of such short duration that the data can be collected using the “diffract and destroy” approach before the effects of radiation damage on the data become significant. These experiments combine the use of enhanced coherent diffraction imaging with single-shot XFEL radiation to investigate the cellular architecture of Staphylococcus aureus with and without labeling by gold (Au) nanoclusters. The resolution of the images reconstructed from these diffraction patterns were twice as high or more for gold-labeled samples, demonstrating that this enhancement method provides a promising approach for the high-resolution imaging of biomaterials and biological systems. PMID:27659203

Single-pulse enhanced coherent diffraction imaging of bacteria with an X-ray free-electron laser.

PubMed

Fan, Jiadong; Sun, Zhibin; Wang, Yaling; Park, Jaehyun; Kim, Sunam; Gallagher-Jones, Marcus; Kim, Yoonhee; Song, Changyong; Yao, Shengkun; Zhang, Jian; Zhang, Jianhua; Duan, Xiulan; Tono, Kensuke; Yabashi, Makina; Ishikawa, Tetsuya; Fan, Chunhai; Zhao, Yuliang; Chai, Zhifang; Gao, Xueyun; Earnest, Thomas; Jiang, Huaidong

2016-09-23

High-resolution imaging offers one of the most promising approaches for exploring and understanding the structure and function of biomaterials and biological systems. X-ray free-electron lasers (XFELs) combined with coherent diffraction imaging can theoretically provide high-resolution spatial information regarding biological materials using a single XFEL pulse. Currently, the application of this method suffers from the low scattering cross-section of biomaterials and X-ray damage to the sample. However, XFELs can provide pulses of such short duration that the data can be collected using the "diffract and destroy" approach before the effects of radiation damage on the data become significant. These experiments combine the use of enhanced coherent diffraction imaging with single-shot XFEL radiation to investigate the cellular architecture of Staphylococcus aureus with and without labeling by gold (Au) nanoclusters. The resolution of the images reconstructed from these diffraction patterns were twice as high or more for gold-labeled samples, demonstrating that this enhancement method provides a promising approach for the high-resolution imaging of biomaterials and biological systems.

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

NASA Astrophysics Data System (ADS)

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

2016-10-01

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

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

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

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

PubMed Central

Treweek, Jennifer B; Deverman, Benjamin E; Greenbaum, Alon; Lignell, Antti; Xiao, Cheng; Cai, Long; Ladinsky, Mark S; Bjorkman, Pamela J; Fowlkes, Charless C; Gradinaru, Viviana

2016-01-01

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks. PMID:26492141

High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level

PubMed Central

Gong, Hui; Xu, Dongli; Yuan, Jing; Li, Xiangning; Guo, Congdi; Peng, Jie; Li, Yuxin; Schwarz, Lindsay A.; Li, Anan; Hu, Bihe; Xiong, Benyi; Sun, Qingtao; Zhang, Yalun; Liu, Jiepeng; Zhong, Qiuyuan; Xu, Tonghui; Zeng, Shaoqun; Luo, Qingming

2016-01-01

The precise annotation and accurate identification of neural structures are prerequisites for studying mammalian brain function. The orientation of neurons and neural circuits is usually determined by mapping brain images to coarse axial-sampling planar reference atlases. However, individual differences at the cellular level likely lead to position errors and an inability to orient neural projections at single-cell resolution. Here, we present a high-throughput precision imaging method that can acquire a co-localized brain-wide data set of both fluorescent-labelled neurons and counterstained cell bodies at a voxel size of 0.32 × 0.32 × 2.0 μm in 3 days for a single mouse brain. We acquire mouse whole-brain imaging data sets of multiple types of neurons and projections with anatomical annotation at single-neuron resolution. The results show that the simultaneous acquisition of labelled neural structures and cytoarchitecture reference in the same brain greatly facilitates precise tracing of long-range projections and accurate locating of nuclei. PMID:27374071

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

PubMed

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

2016-09-01

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

Design of small confocal endo-microscopic probe working under multiwavelength environment

NASA Astrophysics Data System (ADS)

Kim, Young-Duk; Ahn, MyoungKi; Gweon, Dae-Gab

2010-02-01

Recently, optical imaging system is widely used in medical purpose. By using optical imaging system specific diseases can be easily diagnosed at early stage because optical imaging system has high resolution performance and various imaging method. These methods are used to get high resolution image of human body and can be used to verify whether the cell is infected by virus. Confocal microscope is one of the famous imaging systems which is used for in-vivo imaging. Because most of diseases are accompanied with cellular level changes, doctors can diagnosis at early stage by observing the cellular image of human organ. Current research is focused in the development of endo-microscope that has great advantage in accessibility to human body. In this research, I designed small probe that is connected to confocal microscope through optical fiber bundle and work as endo-microscope. And this small probe is mainly designed to correct chromatic aberration to use various laser sources for both fluorescence type and reflection type confocal images. By using two kinds of laser sources at the same time we demonstrated multi-modality confocal endo-microscope.

Cellular dynamics of bovine aortic smooth muscle cells measured using MEMS force sensors

NASA Astrophysics Data System (ADS)

Tsukagoshi, Takuya; Nguyen, Thanh-Vinh; Hirayama Shoji, Kayoko; Takahashi, Hidetoshi; Matsumoto, Kiyoshi; Shimoyama, Isao

2018-04-01

Adhesive cells perceive the mechanical properties of the substrates to which they adhere, adjusting their cellular mechanical forces according to their biological characteristics. This mechanical interaction subsequently affects the growth, locomotion, and differentiation of the cell. However, little is known about the detailed mechanism that underlies this interaction between adherent cells and substrates because dynamically measuring mechanical phenomena is difficult. Here, we utilize microelectromechamical systems force sensors that can measure cellular traction forces with high temporal resolution (~2.5 µs) over long periods (~3 h). We found that the cellular dynamics reflected physical phenomena with time scales from milliseconds to hours, which contradicts the idea that cellular motion is slow. A single focal adhesion (FA) generates an average force of 7 nN, which disappears in ms via the action of trypsin-ethylenediaminetetraacetic acid. The force-changing rate obtained from our measurements suggests that the time required for an FA to decompose was nearly proportional to the force acting on the FA.

Correlative cellular ptychography with functionalized nanoparticles at the Fe L-edge

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

Gallagher-Jones, Marcus; Dias, Carlos Sato Baraldi; Pryor, Alan

Precise localization of nanoparticles within a cell is crucial to the understanding of cell-particle interactions and has broad applications in nanomedicine. Here in this paper, we report a proof-of-principle experiment for imaging individual functionalized nanoparticles within a mammalian cell by correlative microscopy. Using a chemically-fixed HeLa cell labeled with fluorescent core-shell nanoparticles as a model system, we implemented a graphene-oxide layer as a substrate to significantly reduce background scattering. We identified cellular features of interest by fluorescence microscopy, followed by scanning transmission X-ray tomography to localize the particles in 3D, and ptychographic coherent diffractive imaging of the fine features inmore » the region at high resolution. By tuning the X-ray energy to the Fe L-edge, we demonstrated sensitive detection of nanoparticles composed of a 22 nm magnetic Fe 3O 4 core encased by a 25-nm-thick fluorescent silica (SiO 2) shell. These fluorescent core-shell nanoparticles act as landmarks and offer clarity in a cellular context. Our correlative microscopy results confirmed a subset of particles to be fully internalized, and high-contrast ptychographic images showed two oxidation states of individual nanoparticles with a resolution of ~16.5 nm. The ability to precisely localize individual fluorescent nanoparticles within mammalian cells will expand our understanding of the structure/function relationships for functionalized nanoparticles.« less

Correlative cellular ptychography with functionalized nanoparticles at the Fe L-edge

DOE PAGES

Gallagher-Jones, Marcus; Dias, Carlos Sato Baraldi; Pryor, Alan; ...

2017-07-06

Precise localization of nanoparticles within a cell is crucial to the understanding of cell-particle interactions and has broad applications in nanomedicine. Here in this paper, we report a proof-of-principle experiment for imaging individual functionalized nanoparticles within a mammalian cell by correlative microscopy. Using a chemically-fixed HeLa cell labeled with fluorescent core-shell nanoparticles as a model system, we implemented a graphene-oxide layer as a substrate to significantly reduce background scattering. We identified cellular features of interest by fluorescence microscopy, followed by scanning transmission X-ray tomography to localize the particles in 3D, and ptychographic coherent diffractive imaging of the fine features inmore » the region at high resolution. By tuning the X-ray energy to the Fe L-edge, we demonstrated sensitive detection of nanoparticles composed of a 22 nm magnetic Fe 3O 4 core encased by a 25-nm-thick fluorescent silica (SiO 2) shell. These fluorescent core-shell nanoparticles act as landmarks and offer clarity in a cellular context. Our correlative microscopy results confirmed a subset of particles to be fully internalized, and high-contrast ptychographic images showed two oxidation states of individual nanoparticles with a resolution of ~16.5 nm. The ability to precisely localize individual fluorescent nanoparticles within mammalian cells will expand our understanding of the structure/function relationships for functionalized nanoparticles.« less

Quantitation of Cellular Dynamics in Growing Arabidopsis Roots with Light Sheet Microscopy

PubMed Central

Birnbaum, Kenneth D.; Leibler, Stanislas

2011-01-01

To understand dynamic developmental processes, living tissues have to be imaged frequently and for extended periods of time. Root development is extensively studied at cellular resolution to understand basic mechanisms underlying pattern formation and maintenance in plants. Unfortunately, ensuring continuous specimen access, while preserving physiological conditions and preventing photo-damage, poses major barriers to measurements of cellular dynamics in growing organs such as plant roots. We present a system that integrates optical sectioning through light sheet fluorescence microscopy with hydroponic culture that enables us to image, at cellular resolution, a vertically growing Arabidopsis root every few minutes and for several consecutive days. We describe novel automated routines to track the root tip as it grows, to track cellular nuclei and to identify cell divisions. We demonstrate the system's capabilities by collecting data on divisions and nuclear dynamics. PMID:21731697

3D Printing Variable Stiffness Foams Using Viscous Thread Instability

NASA Astrophysics Data System (ADS)

Lipton, Jeffrey I.; Lipson, Hod

2016-08-01

Additive manufacturing of cellular structures has numerous applications ranging from fabrication of biological scaffolds and medical implants, to mechanical weight reduction and control over mechanical properties. Various additive manufacturing processes have been used to produce open regular cellular structures limited only by the resolution of the printer. These efforts have focused on printing explicitly designed cells or explicitly planning offsets between strands. Here we describe a technique for producing cellular structures implicitly by inducing viscous thread instability when extruding material. This process allows us to produce complex cellular structures at a scale that is finer than the native resolution of the printer. We demonstrate tunable effective elastic modulus and density that span two orders of magnitude. Fine grained cellular structures allow for fabrication of foams for use in a wide range of fields ranging from bioengineering, to robotics to food printing.

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

NASA Astrophysics Data System (ADS)

Lu, Yiming; Liu, Changgeng; Yao, Xincheng

2018-02-01

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

1024-Pixel CMOS Multimodality Joint Cellular Sensor/Stimulator Array for Real-Time Holistic Cellular Characterization and Cell-Based Drug Screening.

PubMed

Park, Jong Seok; Aziz, Moez Karim; Li, Sensen; Chi, Taiyun; Grijalva, Sandra Ivonne; Sung, Jung Hoon; Cho, Hee Cheol; Wang, Hua

2018-02-01

This paper presents a fully integrated CMOS multimodality joint sensor/stimulator array with 1024 pixels for real-time holistic cellular characterization and drug screening. The proposed system consists of four pixel groups and four parallel signal-conditioning blocks. Every pixel group contains 16 × 16 pixels, and each pixel includes one gold-plated electrode, four photodiodes, and in-pixel circuits, within a pixel footprint. Each pixel supports real-time extracellular potential recording, optical detection, charge-balanced biphasic current stimulation, and cellular impedance measurement for the same cellular sample. The proposed system is fabricated in a standard 130-nm CMOS process. Rat cardiomyocytes are successfully cultured on-chip. Measured high-resolution optical opacity images, extracellular potential recordings, biphasic current stimulations, and cellular impedance images demonstrate the unique advantages of the system for holistic cell characterization and drug screening. Furthermore, this paper demonstrates the use of optical detection on the on-chip cultured cardiomyocytes to real-time track their cyclic beating pattern and beating rate.

A nanotube based electron microbeam cellular irradiator for radiobiology research

PubMed Central

Bordelon, David E.; Zhang, Jian; Graboski, Sarah; Cox, Adrienne; Schreiber, Eric; Zhou, Otto Z.; Chang, Sha

2008-01-01

A prototype cellular irradiator utilizing a carbon nanotube (CNT) based field emission electron source has been developed for microscopic image-guided cellular region irradiation. The CNT cellular irradiation system has shown great potential to be a high temporal and spatial resolution research tool to enable researchers to gain a better understanding of the intricate cellular and intercellular microprocesses occurring following radiation deposition, which is essential to improving radiotherapy cancer treatment outcomes. In this paper, initial results of the system development are reported. The relationship between field emission current, the dose rate, and the dose distribution has been investigated. A beam size of 23 μm has been achieved with variable dose rates of 1–100 Gy∕s, and the system dosimetry has been measured using a radiochromic film. Cell irradiation has been demonstrated by the visualization of H2AX phosphorylation at DNA double-strand break sites following irradiation in a rat fibroblast cell monolayer. The prototype single beam cellular irradiator is a preliminary step to a multipixel cell irradiator that is under development. PMID:19123587

Patterning pallet arrays for cell selection based on high-resolution measurements of fluorescent biosensors

PubMed Central

Shadpour, Hamed; Zawistowski, Jon S.; Herman, Annadele; Hahn, Klaus; Allbritton, Nancy L.

2011-01-01

Pallet arrays enable cells to be separated while they remain adherent to a surface and provide a much greater range of cell selection criteria relative to that of current technologies. However there remains a need to further broaden cell selection criteria to include dynamic intracellular signaling events. To demonstrate the feasibility of measuring cellular protein behavior on the arrays using high resolution microscopy, the surfaces of individual pallets were modified to minimize the impact of scattered light at the pallet edges. The surfaces of the three-dimensional pallets on an array were patterned with a coating such as fibronectin using a customized stamping tool. Micropatterns of varying shape and size were printed in designated regions on the pallets in single or multiple steps to demonstrate the reliability and precision of patterning molecules on the pallet surface. Use of a fibronectin matrix stamped at the center of each pallet permitted the localization of H1299 and mouse embryonic fibroblast (MEF) cells to the pallet centers and away from the edges. Compared to pallet arrays with fibronection coating the entire top surface, arrays with a central fibronectin pattern increased the percentage of cells localized to the pallet center by 3-4 fold. Localization of cells to the pallet center also enabled the physical separation of cells from optical artifacts created by the rough pallet side walls. To demonstrate the measurement of dynamic intracellular signaling on the arrays, fluorescence measurements of high spatial resolution were performed using a RhoA GTPase biosensor. This biosensor utilized fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to measure localized RhoA activity in cellular ruffles at the cell periphery. These results demonstrated the ability to perform spatially resolved measurements of fluorescence-based sensors on the pallet arrays. Thus, the patterned pallet arrays should enable novel cell separations in which cell selection is based on complex cellular signaling properties. PMID:21621038

Single particle tracking through highly scattering media with multiplexed two-photon excitation

NASA Astrophysics Data System (ADS)

Perillo, Evan; Liu, Yen-Liang; Liu, Cong; Yeh, Hsin-Chih; Dunn, Andrew K.

2015-03-01

3D single-particle tracking (SPT) has been a pivotal tool to furthering our understanding of dynamic cellular processes in complex biological systems, with a molecular localization accuracy (10-100 nm) often better than the diffraction limit of light. However, current SPT techniques utilize either CCDs or a confocal detection scheme which not only suffer from poor temporal resolution but also limit tracking to a depth less than one scattering mean free path in the sample (typically <15μm). In this report we highlight our novel design for a spatiotemporally multiplexed two-photon microscope which is able to reach sub-diffraction-limit tracking accuracy and sub-millisecond temporal resolution, but with a dramatically extended SPT range of up to 200 μm through dense cell samples. We have validated our microscope by tracking (1) fluorescent nanoparticles in a prescribed motion inside gelatin gel (with 1% intralipid) and (2) labeled single EGFR complexes inside skin cancer spheroids (at least 8 layers of cells thick) for ~10 minutes. Furthermore we discuss future capabilities of our multiplexed two-photon microscope design, specifically to the extension of (1) simultaneous multicolor tracking (i.e. spatiotemporal co-localization analysis) and (2) FRET studies (i.e. lifetime analysis). The high resolution, high depth penetration, and multicolor features of this microscope make it well poised to study a variety of molecular scale dynamics in the cell, especially related to cellular trafficking studies with in vitro tumor models and in vivo.

Two-Dimensional Standing Wave Total Internal Reflection Fluorescence Microscopy: Superresolution Imaging of Single Molecular and Biological Specimens

PubMed Central

Chung, Euiheon; Kim, Daekeun; Cui, Yan; Kim, Yang-Hyo; So, Peter T. C.

2007-01-01

The development of high resolution, high speed imaging techniques allows the study of dynamical processes in biological systems. Lateral resolution improvement of up to a factor of 2 has been achieved using structured illumination. In a total internal reflection fluorescence microscope, an evanescence excitation field is formed as light is total internally reflected at an interface between a high and a low index medium. The <100 nm penetration depth of evanescence field ensures a thin excitation region resulting in low background fluorescence. We present even higher resolution wide-field biological imaging by use of standing wave total internal reflection fluorescence (SW-TIRF). Evanescent standing wave (SW) illumination is used to generate a sinusoidal high spatial frequency fringe pattern on specimen for lateral resolution enhancement. To prevent thermal drift of the SW, novel detection and estimation of the SW phase with real-time feedback control is devised for the stabilization and control of the fringe phase. SW-TIRF is a wide-field superresolution technique with resolution better than a fifth of emission wavelength or ∼100 nm lateral resolution. We demonstrate the performance of the SW-TIRF microscopy using one- and two-directional SW illumination with a biological sample of cellular actin cytoskeleton of mouse fibroblast cells as well as single semiconductor nanocrystal molecules. The results confirm the superior resolution of SW-TIRF in addition to the merit of a high signal/background ratio from TIRF microscopy. PMID:17483188

Non-contact full-field optical coherence tomography: a novel tool for in vivo imaging of the human cornea (Conference Presentation)

NASA Astrophysics Data System (ADS)

Mazlin, Viacheslav; Dalimier, Eugénie; Grieve, Katharine F.; Irsch, Kristina; Sahel, José-Alain; Fink, Mathias; Boccara, A. Claude

2017-02-01

According to the World Health Organization (WHO), corneal diseases alongside with cataract and retinal diseases are major causes of blindness worldwide. For the 95.5% of corneal blindness cases, prevention or rehabilitation could have been possible without negative consequences for vision, provided that disease is diagnosed early. However, diagnostics at the early stage requires cellular-level resolution, which is not achieved with routinely used Slit-lamp and OCT instruments. Confocal microscopy allows examination of the cornea at a resolution approaching histological detail, however requires contact with a patient's eye. The recently developed full-field OCT technique, in which 2D en face tangential optical slices are directly recorded on a camera, was successfully applied for ex vivo eye imaging. However, in vivo human eye imaging has not been demonstrated yet. Here we present a novel non-contact full-field OCT system, which is capable of imaging in air and, therefore, shows potential for in vivo cornea imaging in patients. The first cellular-level resolution ex vivo images of cornea, obtained in a completely non-contact way, were demonstrated. We were able to scan through the entire cornea (400 µm) and resolve epithelium, Bowman's layer, stroma and endothelium. FFOCT images of the human cornea in vivo were obtained for the first time. The epithelium structures and stromal keratocyte cells were distinguishable. Both ex vivo and in vivo images were acquired with a large (1.26 mm x 1.26 mm) field of view. Cellular details in obtained images make this device a promising candidate for realization of high-resolution in vivo cornea imaging.

Adding the third dimension on adaptive optics retina imager thanks to full-field optical coherence tomography

NASA Astrophysics Data System (ADS)

Blavier, Marie; Blanco, Leonardo; Glanc, Marie; Pouplard, Florence; Tick, Sarah; Maksimovic, Ivan; Mugnier, Laurent; Chènegros, Guillaume; Rousset, Gérard; Lacombe, François; Pâques, Michel; Le Gargasson, Jean-François; Sahel, José-Alain

2009-02-01

Retinal pathologies, like ARMD or glaucoma, need to be early detected, requiring imaging instruments with resolution at a cellular scale. However, in vivo retinal cells studies and early diagnoses are severely limited by the lack of resolution on eye-fundus images from classical ophthalmologic instruments. We built a 2D retina imager using Adaptive Optics to improve lateral resolution. This imager is currently used in clinical environment. We are currently developing a time domain full-field optical coherence tomograph. The first step was to conceive the images reconstruction algorithms and validation was realized on non-biological samples. Ex vivo retina are currently being imaged. The final step will consist in coupling both setups to acquire high resolution retina cross-sections.

3D Printing Variable Stiffness Foams Using Viscous Thread Instability

PubMed Central

Lipton, Jeffrey I.; Lipson, Hod

2016-01-01

Additive manufacturing of cellular structures has numerous applications ranging from fabrication of biological scaffolds and medical implants, to mechanical weight reduction and control over mechanical properties. Various additive manufacturing processes have been used to produce open regular cellular structures limited only by the resolution of the printer. These efforts have focused on printing explicitly designed cells or explicitly planning offsets between strands. Here we describe a technique for producing cellular structures implicitly by inducing viscous thread instability when extruding material. This process allows us to produce complex cellular structures at a scale that is finer than the native resolution of the printer. We demonstrate tunable effective elastic modulus and density that span two orders of magnitude. Fine grained cellular structures allow for fabrication of foams for use in a wide range of fields ranging from bioengineering, to robotics to food printing. PMID:27503148

Development of large field-of-view two photon microscopy for imaging mouse cortex (Conference Presentation)

NASA Astrophysics Data System (ADS)

Bumstead, Jonathan; Côté, Daniel C.; Culver, Joseph P.

2017-02-01

Spontaneous neuronal activity has been measured at cellular resolution in mice, zebrafish, and C. elegans using optical sectioning microscopy techniques, such as light sheet microscopy (LSM) and two photon microscopy (TPM). Recent improvements in these modalities and genetically encoded calcium indicators (GECI's) have enabled whole brain imaging of calcium dynamics in zebrafish and C. elegans. However, these whole brain microscopy studies have not been extended to mice due to the limited field of view (FOV) of TPM and the cumbersome geometry of LSM. Conventional TPM is restricted to diffraction limited imaging over this small FOV (around 500 x 500 microns) due to the use of high magnification objectives (e.g. 1.0 NA; 20X) and the aberrations introduced by relay optics used in scanning the beam across the sample. To overcome these limitations, we have redesigned the entire optical path of the two photon microscope (scanning optics and objective lens) to support a field of view of Ø7 mm with relatively high spatial resolution (<10 microns). Using optical engineering software Zemax, we designed our system with commercially available optics that minimize astigmatism, field curvature, chromatic focal shift, and vignetting. Performance of the system was also tested experimentally with fluorescent beads in agarose, fixed samples, and in vivo structural imaging. Our large-FOV TPM provides a modality capable of studying distributed brain networks in mice at cellular resolution.

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.

Nano-Computed Tomography: Technique and Applications.

PubMed

Kampschulte, M; Langheinirch, A C; Sender, J; Litzlbauer, H D; Althöhn, U; Schwab, J D; Alejandre-Lafont, E; Martels, G; Krombach, G A

2016-02-01

Nano-computed tomography (nano-CT) is an emerging, high-resolution cross-sectional imaging technique and represents a technical advancement of the established micro-CT technology. Based on the application of a transmission target X-ray tube, the focal spot size can be decreased down to diameters less than 400 nanometers (nm). Together with specific detectors and examination protocols, a superior spatial resolution up to 400 nm (10 % MTF) can be achieved, thereby exceeding the resolution capacity of typical micro-CT systems. The technical concept of nano-CT imaging as well as the basics of specimen preparation are demonstrated exemplarily. Characteristics of atherosclerotic plaques (intraplaque hemorrhage and calcifications) in a murine model of atherosclerosis (ApoE (-/-)/LDLR(-/-) double knockout mouse) are demonstrated in the context of superior spatial resolution in comparison to micro-CT. Furthermore, this article presents the application of nano-CT for imaging cerebral microcirculation (murine), lung structures (porcine), and trabecular microstructure (ovine) in contrast to micro-CT imaging. This review shows the potential of nano-CT as a radiological method in biomedical basic research and discusses the application of experimental, high resolution CT techniques in consideration of other high resolution cross-sectional imaging techniques. Nano-computed tomography is a high resolution CT-technology for 3D imaging at sub-micrometer resolution. The technical concept bases on a further development of the established ex-vivo-micro-CT technology. By improvement of the spatial resolution, structures at a cellular level become visible (e.g. osteocyte lacunae). © Georg Thieme Verlag KG Stuttgart · New York.

Dual-wavelength optical-resolution photoacoustic microscopy for cells with gold nanoparticle bioconjugates in three-dimensional cultures

NASA Astrophysics Data System (ADS)

Lee, Po-Yi; Liu, Wei-Wen; Chen, Shu-Ching; Li, Pai-Chi

2016-03-01

Three-dimensional (3D) in vitro models bridge the gap between typical two-dimensional cultures and in vivo conditions. However, conventional optical imaging methods such as confocal microscopy and two-photon microscopy cannot accurately depict cellular processing in 3D models due to limited penetration of photons. We developed a dualwavelength optical-resolution photoacoustic microscopy (OR-PAM), which provides sufficient penetration depth and spatial resolution, for studying CD8+ cytotoxic T lymphocytes (CTLs) trafficking in an in vitro 3D tumor microenvironment. CTLs play a cardinal role in host defense against tumor. Efficient trafficking of CTLs to the tumor microenvironment is a critical step for cancer immunotherapy. For the proposed system, gold nanospheres and indocyanine green (ICG) have been remarkable choices for contrast agents for photoacoustic signals due to their excellent biocompatibility and high optical absorption. With distinct absorption spectrums, targeted cells with gold nanospheres and ICG respectively can be identified by switching 523-nm and 800-nm laser irradiation. Moreover, we use an x-y galvanometer scanner to obtain high scanning rate. In the developed system, lateral and axial resolutions were designed at 1.6 μm and 5 μm, respectively. We successfully showed that dual-spectral OR-PAM can map either the distribution of CTLs with gold nanospheres at a visible wavelength of 523 nm or the 3D structure of tumor spheres with ICG in an in vitro 3D microenvironment. Our OR-PAM can provide better biological relevant information in cellular interaction and is potential for preclinical screening of anti-cancer drugs.

High resolution simulations of energy absorption in dynamically loaded cellular structures

NASA Astrophysics Data System (ADS)

Winter, R. E.; Cotton, M.; Harris, E. J.; Eakins, D. E.; McShane, G.

2017-03-01

Cellular materials have potential application as absorbers of energy generated by high velocity impact. CTH, a Sandia National Laboratories Code which allows very severe strains to be simulated, has been used to perform very high resolution simulations showing the dynamic crushing of a series of two-dimensional, stainless steel metal structures with varying architectures. The structures are positioned to provide a cushion between a solid stainless steel flyer plate with velocities ranging from 300 to 900 m/s, and an initially stationary stainless steel target. Each of the alternative architectures under consideration was formed by an array of identical cells each of which had a constant volume and a constant density. The resolution of the simulations was maximised by choosing a configuration in which one-dimensional conditions persisted for the full period over which the specimen densified, a condition which is most readily met by impacting high density specimens at high velocity. It was found that the total plastic flow and, therefore, the irreversible energy dissipated in the fully densified energy absorbing cell, increase (a) as the structure becomes more rodlike and less platelike and (b) as the impact velocity increases. Sequential CTH images of the deformation processes show that the flow of the cell material may be broadly divided into macroscopic flow perpendicular to the compression direction and jetting-type processes (microkinetic flow) which tend to predominate in rod and rodlike configurations and also tend to play an increasing role at increased strain rates. A very simple analysis of a configuration in which a solid flyer impacts a solid target provides a baseline against which to compare and explain features seen in the simulations. The work provides a basis for the development of energy absorbing structures for application in the 200-1000 m/s impact regime.

Three-dimensional super-resolved live cell imaging through polarized multi-angle TIRF.

PubMed

Zheng, Cheng; Zhao, Guangyuan; Liu, Wenjie; Chen, Youhua; Zhang, Zhimin; Jin, Luhong; Xu, Yingke; Kuang, Cuifang; Liu, Xu

2018-04-01

Measuring three-dimensional nanoscale cellular structures is challenging, especially when the structure is dynamic. Owing to the informative total internal reflection fluorescence (TIRF) imaging under varied illumination angles, multi-angle (MA) TIRF has been examined to offer a nanoscale axial and a subsecond temporal resolution. However, conventional MA-TIRF still performs badly in lateral resolution and fails to characterize the depth image in densely distributed regions. Here, we emphasize the lateral super-resolution in the MA-TIRF, exampled by simply introducing polarization modulation into the illumination procedure. Equipped with a sparsity and accelerated proximal algorithm, we examine a more precise 3D sample structure compared with previous methods, enabling live cell imaging with a temporal resolution of 2 s and recovering high-resolution mitochondria fission and fusion processes. We also shared the recovery program, which is the first open-source recovery code for MA-TIRF, to the best of our knowledge.

Structural studies of RNA-protein complexes: A hybrid approach involving hydrodynamics, scattering, and computational methods.

PubMed

Patel, Trushar R; Chojnowski, Grzegorz; Astha; Koul, Amit; McKenna, Sean A; Bujnicki, Janusz M

2017-04-15

The diverse functional cellular roles played by ribonucleic acids (RNA) have emphasized the need to develop rapid and accurate methodologies to elucidate the relationship between the structure and function of RNA. Structural biology tools such as X-ray crystallography and Nuclear Magnetic Resonance are highly useful methods to obtain atomic-level resolution models of macromolecules. However, both methods have sample, time, and technical limitations that prevent their application to a number of macromolecules of interest. An emerging alternative to high-resolution structural techniques is to employ a hybrid approach that combines low-resolution shape information about macromolecules and their complexes from experimental hydrodynamic (e.g. analytical ultracentrifugation) and solution scattering measurements (e.g., solution X-ray or neutron scattering), with computational modeling to obtain atomic-level models. While promising, scattering methods rely on aggregation-free, monodispersed preparations and therefore the careful development of a quality control pipeline is fundamental to an unbiased and reliable structural determination. This review article describes hydrodynamic techniques that are highly valuable for homogeneity studies, scattering techniques useful to study the low-resolution shape, and strategies for computational modeling to obtain high-resolution 3D structural models of RNAs, proteins, and RNA-protein complexes. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

All-passive pixel super-resolution of time-stretch imaging

PubMed Central

Chan, Antony C. S.; Ng, Ho-Cheung; Bogaraju, Sharat C. V.; So, Hayden K. H.; Lam, Edmund Y.; Tsia, Kevin K.

2017-01-01

Based on image encoding in a serial-temporal format, optical time-stretch imaging entails a stringent requirement of state-of-the-art fast data acquisition unit in order to preserve high image resolution at an ultrahigh frame rate — hampering the widespread utilities of such technology. Here, we propose a pixel super-resolution (pixel-SR) technique tailored for time-stretch imaging that preserves pixel resolution at a relaxed sampling rate. It harnesses the subpixel shifts between image frames inherently introduced by asynchronous digital sampling of the continuous time-stretch imaging process. Precise pixel registration is thus accomplished without any active opto-mechanical subpixel-shift control or other additional hardware. Here, we present the experimental pixel-SR image reconstruction pipeline that restores high-resolution time-stretch images of microparticles and biological cells (phytoplankton) at a relaxed sampling rate (≈2–5 GSa/s)—more than four times lower than the originally required readout rate (20 GSa/s) — is thus effective for high-throughput label-free, morphology-based cellular classification down to single-cell precision. Upon integration with the high-throughput image processing technology, this pixel-SR time-stretch imaging technique represents a cost-effective and practical solution for large scale cell-based phenotypic screening in biomedical diagnosis and machine vision for quality control in manufacturing. PMID:28303936

High-Resolution Intravital Microscopy

PubMed Central

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

2012-01-01

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

The sweet spot: FDG and other 2-carbon glucose analogs for multi-modal metabolic imaging of tumor metabolism

PubMed Central

Cox, Benjamin L; Mackie, Thomas R; Eliceiri, Kevin W

2015-01-01

Multi-modal imaging approaches of tumor metabolism that provide improved specificity, physiological relevance and spatial resolution would improve diagnosing of tumors and evaluation of tumor progression. Currently, the molecular probe FDG, glucose fluorinated with 18F at the 2-carbon, is the primary metabolic approach for clinical diagnostics with PET imaging. However, PET lacks the resolution necessary to yield intratumoral distributions of deoxyglucose, on the cellular level. Multi-modal imaging could elucidate this problem, but requires the development of new glucose analogs that are better suited for other imaging modalities. Several such analogs have been created and are reviewed here. Also reviewed are several multi-modal imaging studies that have been performed that attempt to shed light on the cellular distribution of glucose analogs within tumors. Some of these studies are performed in vitro, while others are performed in vivo, in an animal model. The results from these studies introduce a visualization gap between the in vitro and in vivo studies that, if solved, could enable the early detection of tumors, the high resolution monitoring of tumors during treatment, and the greater accuracy in assessment of different imaging agents. PMID:25625022

High-resolution proton NMR studies of intracellular metabolites in yeast using 13C decoupling

NASA Astrophysics Data System (ADS)

Sillerud, Laurel O.; Alger, Jeffry R.; Shulman, Robert G.

The resolution and specificity of 1H NMR in studies of yeast cellular metabolism were increased by feeding a 13C-labeled substrate and observing 1H difference spectra in the presence and absence of 13C decoupling fields. [2- 13C]Acetate was utilized as a respiratory substrate in an aerobic suspension of Saccharomyces cerevisiae. The broad cellular background proton resonances are removed by the technique, leaving only signals from the protons of the substrate, or its metabolites, that are coupled to 13C. Spectra of the yeast suspension after acetate feeding show the disappearance of label from the acetate pool and the subsequent appearance of 13C in glutamate C 3 and C 4 and in aspartate C 3. These results are in accord with the known fluxes of metabolites. Selective single-frequency 13C decoupling was used to provide assignments for the difference signals. The limitations on single-frequency decoupling coming from finite decoupling fields are investigated. The technique shows a potential for application in a wide variety of systems where the resolution of the 13C spectrum may be combined with the sensitivity for proton detection to observe metabolites that have been previously unobservable.

High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

NASA Astrophysics Data System (ADS)

Regmi, Raju; Mohan, Kavya; Mondal, Partha Pratim

2014-09-01

Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro- to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine.

Hydrogel-Tissue Chemistry: Principles and Applications.

PubMed

Gradinaru, Viviana; Treweek, Jennifer; Overton, Kristin; Deisseroth, Karl

2018-05-20

Over the past five years, a rapidly developing experimental approach has enabled high-resolution and high-content information retrieval from intact multicellular animal (metazoan) systems. New chemical and physical forms are created in the hydrogel-tissue chemistry process, and the retention and retrieval of crucial phenotypic information regarding constituent cells and molecules (and their joint interrelationships) are thereby enabled. For example, rich data sets defining both single-cell-resolution gene expression and single-cell-resolution activity during behavior can now be collected while still preserving information on three-dimensional positioning and/or brain-wide wiring of those very same neurons-even within vertebrate brains. This new approach and its variants, as applied to neuroscience, are beginning to illuminate the fundamental cellular and chemical representations of sensation, cognition, and action. More generally, reimagining metazoans as metareactants-or positionally defined three-dimensional graphs of constituent chemicals made available for ongoing functionalization, transformation, and readout-is stimulating innovation across biology and medicine.

Comparing high-resolution microscopy techniques for potential intraoperative use in guiding low-grade glioma resections.

PubMed

Meza, Daphne; Wang, Danni; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T C

2015-04-01

Fluorescence image-guided surgery (FIGS), with contrast provided by 5-ALA-induced PpIX, has been shown to enable a higher extent of resection of high-grade gliomas. However, conventional FIGS with low-power microscopy lacks the sensitivity to aid in low-grade glioma (LGG) resection because PpIX signal is weak and sparse in such tissues. Intraoperative high-resolution microscopy of PpIX fluorescence has been proposed as a method to guide LGG resection, where sub-cellular resolution allows for the visualization of sparse and punctate mitochondrial PpIX production in tumor cells. Here, we assess the performance of three potentially portable high-resolution microscopy techniques that may be used for the intraoperative imaging of human LGG tissue samples with PpIX contrast: high-resolution fiber-optic microscopy (HRFM), high-resolution wide-field microscopy (WFM), and dual-axis confocal (DAC) microscopy. Thick unsectioned human LGG tissue samples (n = 7) with 5-ALA-induced PpIX contrast were imaged using three imaging techniques (HRFM, WFM, DAC). The average signal-to-background ratio (SBR) was then calculated for each imaging modality (5 images per tissue, per modality). HRFM provides the ease of use and portability of a flexible fiber bundle, and is simple and inexpensive to build. However, in most cases (6/7), HRFM is not capable of detecting PpIX signal from LGGs due to high autofluorescence, generated by the fiber bundle under laser illumination at 405 nm, which overwhelms the PpIX signal and impedes its visualization. WFM is a camera-based method possessing high lateral resolution but poor axial resolution, resulting in sub-optimal image contrast. Consistent successful detection of PpIX signal throughout our human LGG tissue samples (n = 7), with an acceptable image contrast (SBR >2), was only achieved using DAC microscopy, which offers superior image resolution and contrast that is comparable to histology, but requires a laser-scanning mechanism to achieve optical sectioning. © 2015 Wiley Periodicals, Inc.

Nanowire-based single-cell endoscopy

NASA Astrophysics Data System (ADS)

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

2012-03-01

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

Novel lipid mediators promote resolution of acute inflammation: impact of aspirin and statins

PubMed Central

Spite, Matthew; Serhan, Charles N.

2010-01-01

The resolution of acute inflammation is a process that allows for inflamed tissues to return to homeostasis. Resolution was held to be a passive process, a concept now overturned with new evidence demonstrating that resolution is actively orchestrated by distinct cellular events and endogenous chemical mediators. Among these, lipid mediators, such as the lipoxins, resolvins, protectins and newly identified maresins, have emerged as a novel genus of potent and stereoselective players that counter-regulate excessive acute inflammation and stimulate molecular and cellular events that define resolution. Given that uncontrolled, chronic inflammation is associated with many cardiovascular pathologies, an appreciation of the endogenous pathways and mediators that control timely resolution can open new terrain for therapeutic approaches targeted at stimulating resolution of local inflammation, as well as correcting the impact of chronic inflammation in cardiovascular disorders. Here, we overview and update the biosynthesis and actions of pro-resolving lipid mediators, highlighting their diverse protective roles relevant to vascular systems and their relation to aspirin and statin therapies. PMID:21071715

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.

Label free cell tracking in 3D tissue engineering constructs with high resolution imaging

NASA Astrophysics Data System (ADS)

Smith, W. A.; Lam, K.-P.; Dempsey, K. P.; Mazzocchi-Jones, D.; Richardson, J. B.; Yang, Y.

2014-02-01

Within the field of tissue engineering there is an emphasis on studying 3-D live tissue structures. Consequently, to investigate and identify cellular activities and phenotypes in a 3-D environment for all in vitro experiments, including shape, migration/proliferation and axon projection, it is necessary to adopt an optical imaging system that enables monitoring 3-D cellular activities and morphology through the thickness of the construct for an extended culture period without cell labeling. This paper describes a new 3-D tracking algorithm developed for Cell-IQ®, an automated cell imaging platform, which has been equipped with an environmental chamber optimized to enable capturing time-lapse sequences of live cell images over a long-term period without cell labeling. As an integral part of the algorithm, a novel auto-focusing procedure was developed for phase contrast microscopy equipped with 20x and 40x objectives, to provide a more accurate estimation of cell growth/trajectories by allowing 3-D voxels to be computed at high spatiotemporal resolution and cell density. A pilot study was carried out in a phantom system consisting of horizontally aligned nanofiber layers (with precise spacing between them), to mimic features well exemplified in cellular activities of neuronal growth in a 3-D environment. This was followed by detailed investigations concerning axonal projections and dendritic circuitry formation in a 3-D tissue engineering construct. Preliminary work on primary animal neuronal cells in response to chemoattractant and topographic cue within the scaffolds has produced encouraging results.

Towards a magnetoresistive platform for neural signal recording

NASA Astrophysics Data System (ADS)

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

2017-05-01

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

Automated imaging of cellular spheroids with selective plane illumination microscopy on a chip (Conference Presentation)

NASA Astrophysics Data System (ADS)

Paiè, Petra; Bassi, Andrea; Bragheri, Francesca; Osellame, Roberto

2017-02-01

Selective plane illumination microscopy (SPIM) is an optical sectioning technique that allows imaging of biological samples at high spatio-temporal resolution. Standard SPIM devices require dedicated set-ups, complex sample preparation and accurate system alignment, thus limiting the automation of the technique, its accessibility and throughput. We present a millimeter-scaled optofluidic device that incorporates selective plane illumination and fully automatic sample delivery and scanning. To this end an integrated cylindrical lens and a three-dimensional fluidic network were fabricated by femtosecond laser micromachining into a single glass chip. This device can upgrade any standard fluorescence microscope to a SPIM system. We used SPIM on a CHIP to automatically scan biological samples under a conventional microscope, without the need of any motorized stage: tissue spheroids expressing fluorescent proteins were flowed in the microchannel at constant speed and their sections were acquired while passing through the light sheet. We demonstrate high-throughput imaging of the entire sample volume (with a rate of 30 samples/min), segmentation and quantification in thick (100-300 μm diameter) cellular spheroids. This optofluidic device gives access to SPIM analyses to non-expert end-users, opening the way to automatic and fast screening of a high number of samples at subcellular resolution.

In vivo imaging through the entire thickness of human cornea by full-field optical coherence tomography

NASA Astrophysics Data System (ADS)

Mazlin, Viacheslav; Xiao, Peng; Dalimier, Eugénie; Grieve, Kate; Irsch, Kristina; Sahel, José; Fink, Mathias; Boccara, Claude

2018-02-01

Despite obvious improvements in visualization of the in vivo cornea through the faster imaging speeds and higher axial resolutions, cellular imaging stays unresolvable task for OCT, as en face viewing with a high lateral resolution is required. The latter is possible with FFOCT, a method that relies on a camera, moderate numerical aperture (NA) objectives and an incoherent light source to provide en face images with a micrometer-level resolution. Recently, we for the first time demonstrated the ability of FFOCT to capture images from the in vivo human cornea1. In the current paper we present an extensive study of appearance of healthy in vivo human corneas under FFOCT examination. En face corneal images with a micrometer-level resolution were obtained from the three healthy subjects. For each subject it was possible to acquire images through the entire corneal depth and visualize the epithelium structures, Bowman's layer, sub-basal nerve plexus (SNP) fibers, anterior, middle and posterior stroma, endothelial cells with nuclei. Dimensions and densities of the structures visible with FFOCT, are in agreement with those seen by other cornea imaging methods. Cellular-level details in the images obtained together with the relatively large field-of-view (FOV) and contactless way of imaging make this device a promising candidate for becoming a new tool in ophthalmological diagnostics.

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

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

Deng, Junjing; Vine, David J.; Chen, Si

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ~90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. Finally, this combined approach offers a way to study the role of trace elements in their structural context.« less

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

DOE PAGES

Deng, Junjing; Vine, David J.; Chen, Si; ...

2015-02-24

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ~90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. Finally, this combined approach offers a way to study the role of trace elements in their structural context.« less

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

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

Deng, Junjing; Vine, David J.; Chen, Si

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and similar to 90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. This combined approach offers a way to study the role of trace elements in their structural context.« less

X-ray micro-modulated luminescence tomography (XMLT)

PubMed Central

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

2014-01-01

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

Learning from Heterogeneous Data Sources: An Application in Spatial Proteomics

PubMed Central

Breckels, Lisa M.; Holden, Sean B.; Wojnar, David; Mulvey, Claire M.; Christoforou, Andy; Groen, Arnoud; Trotter, Matthew W. B.; Kohlbacher, Oliver; Lilley, Kathryn S.; Gatto, Laurent

2016-01-01

Sub-cellular localisation of proteins is an essential post-translational regulatory mechanism that can be assayed using high-throughput mass spectrometry (MS). These MS-based spatial proteomics experiments enable us to pinpoint the sub-cellular distribution of thousands of proteins in a specific system under controlled conditions. Recent advances in high-throughput MS methods have yielded a plethora of experimental spatial proteomics data for the cell biology community. Yet, there are many third-party data sources, such as immunofluorescence microscopy or protein annotations and sequences, which represent a rich and vast source of complementary information. We present a unique transfer learning classification framework that utilises a nearest-neighbour or support vector machine system, to integrate heterogeneous data sources to considerably improve on the quantity and quality of sub-cellular protein assignment. We demonstrate the utility of our algorithms through evaluation of five experimental datasets, from four different species in conjunction with four different auxiliary data sources to classify proteins to tens of sub-cellular compartments with high generalisation accuracy. We further apply the method to an experiment on pluripotent mouse embryonic stem cells to classify a set of previously unknown proteins, and validate our findings against a recent high resolution map of the mouse stem cell proteome. The methodology is distributed as part of the open-source Bioconductor pRoloc suite for spatial proteomics data analysis. PMID:27175778

Combined system for high-time-resolution dual-excitation fluorescence photometry and fluorescence imaging of calcium transients in single normal and diseased skeletal muscle fibers

NASA Astrophysics Data System (ADS)

Uttenweiler, Dietmar; Wojciechowski, Reinhold; Makabe, Makoto; Veigel, Claudia; Fink, Rainer H.

1994-12-01

Fast photometric measurements and video-imaging of fluorescent indicators both are powerful tools in measuring the intracellular free calcium concentration of muscle and many other cells. as photometric systems yield a high temporal resolution, calcium imaging systems have high spatial but significantly reduced temporal resolution. Therefore we have developed an integrated system combining both methods and based mostly on standard components. As a common, sensitive Ca2+- indicator we used the fluorescent probe Fura-2, which is alternatingly excited for ratio measurements at 340/380 nm. We used a commercially available dual excitation photometric system (OSP-3; Olympus) for attaching a CCD-camera and a frame grabber board. To achieve the synchronization we had to design circuitries for external triggering, synchronization and accurate control of the filter changer, which we added to the system. Additionally, the software for a triggered image acquisition was developed. With this integrated setup one can easily switch between the fast photometric mode (ratio frequency 100 Hz) and the imaging mode (ratio frequency 4.17 Hz). The calcium images are correlated with the 25 times faster spot measurements and are analyzed by means of image processing. With this combined system we study release and uptake of calcium ions of normal and diseased skeletal muscle from mdx mice. Such a system will also be important for other cellular studies in which fluorescence indicators are used to monitor similar time dependent alterations as well as changes in cellular distributions of calcium.

Using Imaging Methods to Interrogate Radiation-Induced Cell Signaling

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

Shankaran, Harish; Weber, Thomas J.; Freiin von Neubeck, Claere H.

2012-04-01

There is increasing emphasis on the use of systems biology approaches to define radiation induced responses in cells and tissues. Such approaches frequently rely on global screening using various high throughput 'omics' platforms. Although these methods are ideal for obtaining an unbiased overview of cellular responses, they often cannot reflect the inherent heterogeneity of the system or provide detailed spatial information. Additionally, performing such studies with multiple sampling time points can be prohibitively expensive. Imaging provides a complementary method with high spatial and temporal resolution capable of following the dynamics of signaling processes. In this review, we utilize specific examplesmore » to illustrate how imaging approaches have furthered our understanding of radiation induced cellular signaling. Particular emphasis is placed on protein co-localization, and oscillatory and transient signaling dynamics.« less

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2015-07-07

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

Study of cellular oncometabolism via multidimensional protein identification technology.

PubMed

Aukim-Hastie, Claire; Garbis, Spiros D

2014-01-01

Cellular proteomics is becoming a widespread clinical application, matching the definition of bench-to-bedside translation. Among various fields of investigation, this approach can be applied to the study of the metabolic alterations that accompany oncogenesis and tumor progression, which are globally referred to as oncometabolism. Here, we describe a multidimensional protein identification technology (MuDPIT)-based strategy that can be employed to study the cellular proteome of malignant cells and tissues. This method has previously been shown to be compatible with the reproducible, in-depth analysis of up to a thousand proteins in clinical samples. The possibility to employ this technique to study clinical specimens demonstrates its robustness. MuDPIT is advantageous as compared to other approaches because it is direct, highly sensitive, and reproducible, it provides high resolution with ultra-high mass accuracy, it allows for relative quantifications, and it is compatible with multiplexing (thus limiting costs).This method enables the direct assessment of the proteomic profile of neoplastic cells and tissues and could be employed in the near future as a high-throughput, rapid, quantitative, and cost-effective screening platform for clinical samples. © 2014 Elsevier Inc. All rights reserved.

Far-field photostable optical nanoscopy (PHOTON) for real-time super-resolution single-molecular imaging of signaling pathways of single live cells

NASA Astrophysics Data System (ADS)

Huang, Tao; Browning, Lauren M.; Xu, Xiao-Hong Nancy

2012-04-01

Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions.Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr11739h

A comparison of high-throughput techniques for assaying circadian rhythms in plants.

PubMed

Tindall, Andrew J; Waller, Jade; Greenwood, Mark; Gould, Peter D; Hartwell, James; Hall, Anthony

2015-01-01

Over the last two decades, the development of high-throughput techniques has enabled us to probe the plant circadian clock, a key coordinator of vital biological processes, in ways previously impossible. With the circadian clock increasingly implicated in key fitness and signalling pathways, this has opened up new avenues for understanding plant development and signalling. Our tool-kit has been constantly improving through continual development and novel techniques that increase throughput, reduce costs and allow higher resolution on the cellular and subcellular levels. With circadian assays becoming more accessible and relevant than ever to researchers, in this paper we offer a review of the techniques currently available before considering the horizons in circadian investigation at ever higher throughputs and resolutions.

Multi-scale Functional and Molecular Photoacoustic Tomography

PubMed Central

Yao, Junjie; Xia, Jun; Wang, Lihong V.

2015-01-01

Photoacoustic tomography (PAT) combines rich optical absorption contrast with the high spatial resolution of ultrasound at depths in tissue. The high scalability of PAT has enabled anatomical imaging of biological structures ranging from organelles to organs. The inherent functional and molecular imaging capabilities of PAT have further allowed it to measure important physiological parameters and track critical cellular activities. Integration of PAT with other imaging technologies provides complementary capabilities and can potentially accelerate the clinical translation of PAT. PMID:25933617

Rare-earth-doped nanophosphors for multicolor cathodoluminescence nanobioimaging using scanning transmission electron microscopy.

PubMed

Furukawa, Taichi; Fukushima, Shoichiro; Niioka, Hirohiko; Yamamoto, Naoki; Miyake, Jun; Araki, Tsutomu; Hashimoto, Mamoru

2015-05-01

We describe rare-earth-doped nanophosphors (RE-NPs) for biological imaging using cathodoluminescence(CL) microscopy based on scanning transmission electron microscopy (STEM). We report the first demonstration of multicolor CL nanobioimaging using STEM with nanophosphors. The CL spectra of the synthesized nanophosphors (Y2O3∶Eu, Y2O3∶Tb) were sufficiently narrow to be distinguished. From CL images of RE-NPs on an elastic carbon-coated copper grid, the spatial resolution was beyond the diffraction limit of light.Y2O3∶Tb and Y2O3∶Eu RE-NPs showed a remarkable resistance against electron beam exposure even at high acceleration voltage (80 kV) and retained a CL intensity of more than 97% compared with the initial intensity for 1 min. In biological CL imaging with STEM, heavy-metal-stained cell sections containing the RE-NPs were prepared,and both the CL images of RE-NPs and cellular structures, such as mitochondria, were clearly observed from STEM images with high contrast. The cellular CL imaging using RE-NPs also had high spatial resolution even though heavy-metal-stained cells are normally regarded as highly scattering media. Moreover, since theRE-NPs exhibit photoluminescence (PL) excited by UV light, they are useful for multimodal correlative imaging using CL and PL.

Metallic powder-bed based 3D printing of cellular scaffolds for orthopaedic implants: A state-of-the-art review on manufacturing, topological design, mechanical properties and biocompatibility.

PubMed

Tan, X P; Tan, Y J; Chow, C S L; Tor, S B; Yeong, W Y

2017-07-01

Metallic cellular scaffold is one of the best choices for orthopaedic implants as a replacement of human body parts, which could improve life quality and increase longevity for the people needed. Unlike conventional methods of making cellular scaffolds, three-dimensional (3D) printing or additive manufacturing opens up new possibilities to fabricate those customisable intricate designs with highly interconnected pores. In the past decade, metallic powder-bed based 3D printing methods emerged and the techniques are becoming increasingly mature recently, where selective laser melting (SLM) and selective electron beam melting (SEBM) are the two representatives. Due to the advantages of good dimensional accuracy, high build resolution, clean build environment, saving materials, high customisability, etc., SLM and SEBM show huge potential in direct customisable manufacturing of metallic cellular scaffolds for orthopaedic implants. Ti-6Al-4V to date is still considered to be the optimal materials for producing orthopaedic implants due to its best combination of biocompatibility, corrosion resistance and mechanical properties. This paper presents a state-of-the-art overview mainly on manufacturing, topological design, mechanical properties and biocompatibility of cellular Ti-6Al-4V scaffolds via SLM and SEBM methods. Current manufacturing limitations, topological shortcomings, uncertainty of biocompatible test were sufficiently discussed herein. Future perspectives and recommendations were given at the end. Copyright © 2017 Elsevier B.V. All rights reserved.

Hard X-ray Ptychography: Making It Cool, Colorful and Fast

NASA Astrophysics Data System (ADS)

Deng, Junjing

Ptychography is a recently developed coherent imaging technique for extended objects, with a resolution not limited by the lens. Because X-rays have short wavelengths and high penetration ability, X-ray ptychography provides a powerful and unique tool for studying thick samples at high spatial resolution. We have advanced X-ray ptychography by making it cool, colorful, and fast. We make it cool by carrying out ptychography experiments at cryogenic conditions to image frozen-hydrated specimens. This largely removes the limitations of radiation damage on the achievable resolution, and allows one to obtain excellent preservation of structure and chemistry in biological specimens. We make it colorful by combining it with X-ray fluorescence measurements of chemical element distributions. In studies of biological specimens, this means that ptychography can reveal cellular ultrastructure at high contrast and at a resolution well beyond that of X-ray focusing optics, while X-ray fluorescence is used to simultaneously image the distribution of trace elements in cells (such as metals that play key roles in cell functions and which can be used in various disease therapeutic agents). Because X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular materials, this combined approach provides the unique tool to obtain simultaneous views of ultrastructure and elemental compositions of specimens. We make it fast by using continuous-scan (or "fly-scan") methods. Conventional ptychography is implemented in a move-settle-measure approach, which is slow due to the positioning overheads. To overcome this bottleneck, we have developed fly-scan ptychography that is able to speed up the data collection, and real time on-site data analysis can be achieved by using a parallelized reconstruction code. With these advances, we conducted combined cryo X-ray ptychography and fluorescence imaging at 5.2 keV in a more practical way using fly scan, well-preserved cryogenic samples and rapid reconstructions, and obtained images of a whole frozen-hydrated eukaryotic cell at 18 nm resolution which we believe to be the highest spatial resolution obtained in X-ray imaging of frozen-hydrated biological samples to date. After a successful demonstration of fly-scan 3D ptychography on a gold test sample, we also obtained fly-scan 3D ptychography and fluorescence data on frozen-hydrated cells with an imaging speedup of factor more than 7. Finally, we applied fly-scan X-ray ptychography on un-thinned integrated circuits (ICs) using 10 keV X-rays, and were able to see the circuit details within the thick IC chips with a high resolution of 11.6 nm. All of these achievements point the way toward high-speed X-ray imaging without lens-imposed resolution limit.

Introduction to the Minireview Series on Modern Technologies for In-cell Biochemistry.

PubMed

Lutsenko, Svetlana

2016-02-19

The last decade has seen enormous progress in the exploration and understanding of the behavior of molecules in their natural cellular environments at increasingly high spatial and temporal resolution. Advances in microscopy and the development of new fluorescent reagents as well as genetic editing techniques have enabled quantitative analysis of protein interactions, intracellular trafficking, metabolic changes, and signaling. Modern biochemistry now faces new and exciting challenges. Can traditionally "in vitro" experiments, e.g. analysis of protein folding and conformational transitions, be done in cells? Can the structure and behavior of endogenous and/or non-tagged recombinant proteins be analyzed and altered within the cell or in cellular compartments? How can molecules and their actions be studied mechanistically in tissues and organs? Is personalized cellular biochemistry a reality? This thematic series summarizes recent studies that illustrate some first steps toward successfully answering these modern biochemical questions. The first minireview focuses on utilization of three-dimensional primary enteroids and organoids for mechanistic studies of intestinal biology with molecular resolution. The second minireview describes application of single chain antibodies (nanobodies) for monitoring and regulating protein dynamics in vitro and in cells. The third minireview highlights advances in using NMR spectroscopy for analysis of protein folding and assembly in cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

FRET-based genetically-encoded sensors for quantitative monitoring of metabolites.

PubMed

Mohsin, Mohd; Ahmad, Altaf; Iqbal, Muhammad

2015-10-01

Neighboring cells in the same tissue can exist in different states of dynamic activities. After genomics, proteomics and metabolomics, fluxomics is now equally important for generating accurate quantitative information on the cellular and sub-cellular dynamics of ions and metabolite, which is critical for functional understanding of organisms. Various spectrometry techniques are used for monitoring ions and metabolites, although their temporal and spatial resolutions are limited. Discovery of the fluorescent proteins and their variants has revolutionized cell biology. Therefore, novel tools and methods targeting sub-cellular compartments need to be deployed in specific cells and targeted to sub-cellular compartments in order to quantify the target-molecule dynamics directly. We require tools that can measure cellular activities and protein dynamics with sub-cellular resolution. Biosensors based on fluorescence resonance energy transfer (FRET) are genetically encoded and hence can specifically target sub-cellular organelles by fusion to proteins or targetted sequences. Since last decade, FRET-based genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of cellular physiology. This review, describing the design and principles of sensors, presents a database of sensors for different analytes/processes, and illustrate examples of application in quantitative live cell imaging.

Whole mouse cryo-imaging

NASA Astrophysics Data System (ADS)

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

2008-03-01

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

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

PubMed Central

Kim, Doory; Deerinck, Thomas J.; Sigal, Yaron M.; Babcock, Hazen P.; Ellisman, Mark H.; Zhuang, Xiaowei

2015-01-01

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets. PMID:25874453

Photoacoustic imaging of single circulating melanoma cells in vivo

NASA Astrophysics Data System (ADS)

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

2015-03-01

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

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology.

PubMed

Rajagopal, Vijay; Bass, Gregory; Ghosh, Shouryadipta; Hunt, Hilary; Walker, Cameron; Hanssen, Eric; Crampin, Edmund; Soeller, Christian

2018-04-18

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.

A Conserved Developmental Patterning Network Produces Quantitatively Different Output in Multiple Species of Drosophila

PubMed Central

Meyer, Miriah; Wunderlich, Zeba; Simirenko, Lisa; Luengo Hendriks, Cris L.; Keränen, Soile V. E.; Henriquez, Clara; Knowles, David W.; Biggin, Mark D.; Eisen, Michael B.; DePace, Angela H.

2011-01-01

Differences in the level, timing, or location of gene expression can contribute to alternative phenotypes at the molecular and organismal level. Understanding the origins of expression differences is complicated by the fact that organismal morphology and gene regulatory networks could potentially vary even between closely related species. To assess the scope of such changes, we used high-resolution imaging methods to measure mRNA expression in blastoderm embryos of Drosophila yakuba and Drosophila pseudoobscura and assembled these data into cellular resolution atlases, where expression levels for 13 genes in the segmentation network are averaged into species-specific, cellular resolution morphological frameworks. We demonstrate that the blastoderm embryos of these species differ in their morphology in terms of size, shape, and number of nuclei. We present an approach to compare cellular gene expression patterns between species, while accounting for varying embryo morphology, and apply it to our data and an equivalent dataset for Drosophila melanogaster. Our analysis reveals that all individual genes differ quantitatively in their spatio-temporal expression patterns between these species, primarily in terms of their relative position and dynamics. Despite many small quantitative differences, cellular gene expression profiles for the whole set of genes examined are largely similar. This suggests that cell types at this stage of development are conserved, though they can differ in their relative position by up to 3–4 cell widths and in their relative proportion between species by as much as 5-fold. Quantitative differences in the dynamics and relative level of a subset of genes between corresponding cell types may reflect altered regulatory functions between species. Our results emphasize that transcriptional networks can diverge over short evolutionary timescales and that even small changes can lead to distinct output in terms of the placement and number of equivalent cells. PMID:22046143

STED Imaging of Golgi Dynamics with Cer-SiR: A Two-Component, Photostable, High-Density Lipid Probe for Live Cells.

PubMed

Erdmann, Roman S; Toomre, Derek; Schepartz, Alanna

2017-01-01

Long time-lapse super-resolution imaging in live cells requires a labeling strategy that combines a bright, photostable fluorophore with a high-density localization probe. Lipids are ideal high-density localization probes, as they are >100 times more abundant than most membrane-bound proteins and simultaneously demark the boundaries of cellular organelles. Here, we describe Cer-SiR, a two-component, high-density lipid probe that is exceptionally photostable. Cer-SiR is generated in cells via a bioorthogonal reaction of two components: a ceramide lipid tagged with trans-cyclooctene (Cer-TCO) and a reactive, photostable Si-rhodamine dye (SiR-Tz). These components assemble within the Golgi apparatus of live cells to form Cer-SiR. Cer-SiR is benign to cellular function, localizes within the Golgi at a high density, and is sufficiently photostable to enable visualization of Golgi structure and dynamics by 3D confocal or long time-lapse STED microscopy.

Advanced spinning disk-TIRF microscopy for faster imaging of the cell interior and the plasma membrane.

PubMed

Zobiak, Bernd; Failla, Antonio Virgilio

2018-03-01

Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high-resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high-resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD-TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD-TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup. © 2017 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Super-resolution photon-efficient imaging by nanometric double-helix point spread function localization of emitters (SPINDLE)

PubMed Central

Grover, Ginni; DeLuca, Keith; Quirin, Sean; DeLuca, Jennifer; Piestun, Rafael

2012-01-01

Super-resolution imaging with photo-activatable or photo-switchable probes is a promising tool in biological applications to reveal previously unresolved intra-cellular details with visible light. This field benefits from developments in the areas of molecular probes, optical systems, and computational post-processing of the data. The joint design of optics and reconstruction processes using double-helix point spread functions (DH-PSF) provides high resolution three-dimensional (3D) imaging over a long depth-of-field. We demonstrate for the first time a method integrating a Fisher information efficient DH-PSF design, a surface relief optical phase mask, and an optimal 3D localization estimator. 3D super-resolution imaging using photo-switchable dyes reveals the 3D microtubule network in mammalian cells with localization precision approaching the information theoretical limit over a depth of 1.2 µm. PMID:23187521

High-resolution confocal imaging of wall ingrowth deposition in plant transfer cells: Semi-quantitative analysis of phloem parenchyma transfer cell development in leaf minor veins of Arabidopsis.

PubMed

Nguyen, Suong T T; McCurdy, David W

2015-04-23

Transfer cells (TCs) are trans-differentiated versions of existing cell types designed to facilitate enhanced membrane transport of nutrients at symplasmic/apoplasmic interfaces. This transport capacity is conferred by intricate wall ingrowths deposited secondarily on the inner face of the primary cell wall, hence promoting the potential trans-membrane flux of solutes and consequently assigning TCs as having key roles in plant growth and productivity. However, TCs are typically positioned deep within tissues and have been studied mostly by electron microscopy. Recent advances in fluorophore labelling of plant cell walls using a modified pseudo-Schiff-propidium iodide (mPS-PI) staining procedure in combination with high-resolution confocal microscopy have allowed visualization of cellular details of individual tissue layers in whole mounts, hence enabling study of tissue and cellular architecture without the need for tissue sectioning. Here we apply a simplified version of the mPS-PI procedure for confocal imaging of cellulose-enriched wall ingrowths in vascular TCs at the whole tissue level. The simplified mPS-PI staining procedure produced high-resolution three-dimensional images of individual cell types in vascular bundles and, importantly, wall ingrowths in phloem parenchyma (PP) TCs in minor veins of Arabidopsis leaves and companion cell TCs in pea. More efficient staining of tissues was obtained by replacing complex clearing procedures with a simple post-fixation bleaching step. We used this modified procedure to survey the presence of PP TCs in other tissues of Arabidopsis including cotyledons, cauline leaves and sepals. This high-resolution imaging enabled us to classify different stages of wall ingrowth development in Arabidopsis leaves, hence enabling semi-quantitative assessment of the extent of wall ingrowth deposition in PP TCs at the whole leaf level. Finally, we conducted a defoliation experiment as an example of using this approach to statistically analyze responses of PP TC development to leaf ablation. Use of a modified mPS-PI staining technique resulted in high-resolution confocal imaging of polarized wall ingrowth deposition in TCs. This technique can be used in place of conventional electron microscopy and opens new possibilities to study mechanisms determining polarized deposition of wall ingrowths and use reverse genetics to identify regulatory genes controlling TC trans-differentiation.

Label-free high-throughput imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mahjoubfar, A.; Chen, C.; Niazi, K. R.; Rabizadeh, S.; Jalali, B.

2014-03-01

Flow cytometry is an optical method for studying cells based on their individual physical and chemical characteristics. It is widely used in clinical diagnosis, medical research, and biotechnology for analysis of blood cells and other cells in suspension. Conventional flow cytometers aim a laser beam at a stream of cells and measure the elastic scattering of light at forward and side angles. They also perform single-point measurements of fluorescent emissions from labeled cells. However, many reagents used in cell labeling reduce cellular viability or change the behavior of the target cells through the activation of undesired cellular processes or inhibition of normal cellular activity. Therefore, labeled cells are not completely representative of their unaltered form nor are they fully reliable for downstream studies. To remove the requirement of cell labeling in flow cytometry, while still meeting the classification sensitivity and specificity goals, measurement of additional biophysical parameters is essential. Here, we introduce an interferometric imaging flow cytometer based on the world's fastest continuous-time camera. Our system simultaneously measures cellular size, scattering, and protein concentration as supplementary biophysical parameters for label-free cell classification. It exploits the wide bandwidth of ultrafast laser pulses to perform blur-free quantitative phase and intensity imaging at flow speeds as high as 10 meters per second and achieves nanometer-scale optical path length resolution for precise measurements of cellular protein concentration.

Optical Diagnostics in Medicine

NASA Astrophysics Data System (ADS)

Iftimia, Nicusor

2003-03-01

Light has a unique potential for non-invasive tissue diagnosis. The relatively short wavelength of light allows imaging of tissue at the resolution of histopathology. While strong multiple scattering of light in tissue makes attainment of this resolution difficult for thick tissues, most pathology emanates from epithelial surfaces. Therefore, high-resolution diagnosis of many important diseases may be achieved by transmitting light to the surface of interest. The recent fiber-optic implementation of technologies that reject multiple scattering, such as confocal microscopy and optical low coherence interferometry, have brought us one step closer to realizing non-invasive imaging of architectural and cellular features of tissue. Optical coherence tomography (OCT) can produce high-resolution cross-sectional images of biological structures. Clinical OCT studies conducted in the gastrointestinal tract and cardiovascular system have shown that OCT is capable of providing images of the architectural (> 20 µm) microanatomy of a variety of epithelial tissues, including the layered structure of squamous epithelium and arterial vessels. Fine Needle Aspiration- Low Coherence Interferometry (FNA-LCI) is another optical diagnostics technique, which is a suitable solution to increase the effectiveness of the FNA procedures. LCI is capable of measuring depth resolved (axial, z) tissue structure, birefringence, flow (Doppler shift), and spectra at a resolution of several microns. Since LCI systems are fiber-optic based, LCI probes may easily fit within the bore of a fine gauge needle, allowing diagnostic information to be obtained directly from the FNA biopsy site. Fiber optic spectrally encoded confocal microscopy (SECM) is a new confocal microscopy method, which eliminates the need for rapid beam scanning within the optical probe. This advance enables confocal microscopy to be performed through small diameter probes and will allow assessment of internal human tissues in vivo at the cellular level. A detailed description of several fiber optics based systems for early diseases diagnosis, as well as preliminary clinic results, will be presented.

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.

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

DOE PAGES

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

2017-01-03

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

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

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

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

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

Confocal Endomicroscopy: Instrumentation and Medical Applications

PubMed Central

Jabbour, Joey M.; Saldua, Meagan A.; Bixler, Joel N.; Maitland, Kristen C.

2013-01-01

Advances in fiber optic technology and miniaturized optics and mechanics have propelled confocal endomicroscopy into the clinical realm. This high resolution, non-invasive imaging technology provides the ability to microscopically evaluate cellular and sub-cellular features in tissue in vivo by optical sectioning. Because many cancers originate in epithelial tissues accessible by endoscopes, confocal endomicroscopy has been explored to detect regions of possible neoplasia at an earlier stage by imaging morphological features in vivo that are significant in histopathologic evaluation. This technique allows real-time assessment of tissue which may improve diagnostic yield by guiding biopsy. Research and development continues to reduce the overall size of the imaging probe, increase the image acquisition speed, and improve resolution and field of view of confocal endomicroscopes. Technical advances will continue to enable application to less accessible organs and more complex systems in the body. Lateral and axial resolutions down to 0.5 μm and 3 μm, respectively, field of view as large as 800×450 μm, and objective lens and total probe outer diameters down to 350 μm and 1.25 mm, respectively, have been achieved. We provide a review of the historical developments of confocal imaging in vivo, the evolution of endomicroscope instrumentation, and the medical applications of confocal endomicroscopy. PMID:21994069

A Fast Microfluidic Temperature Control Device for Studying Microtubule Dynamics in Fission Yeast

PubMed Central

Velve-Casquillas, Guilhem; Costa, Judite; Carlier-Grynkorn, Frédérique; Mayeux, Adeline; Tran, Phong T.

2010-01-01

Recent development in soft lithography and microfluidics enables biologists to create tools to control the cellular microenvironment. One such control is the ability to quickly change the temperature of the cells. Genetic model organism such as fission yeast has been useful for studies of the cell cytoskeleton. In particular, the dynamic microtubule cytoskeleton responds to changes in temperature. In addition, there are temperature-sensitive mutations of cytoskeletal proteins. We describe here the fabrication and use of a microfluidic device to quickly and reversibly change cellular temperature between 2°C and 50°C. We demonstrate the use of this device while imaging at high-resolution microtubule dynamics in fission yeast. PMID:20719272

Looking inside the heart: a see-through view of the vascular tree

PubMed Central

Nehrhoff, Imke; Ripoll, Jorge; Samaniego, Rafael; Desco, Manuel; Gómez-Gaviro, Maria Victoria

2017-01-01

The ability to acquire 3D images of the heart and its vasculature at cellular resolution facilitates a more detailed study of many heart diseases. Here, we describe a novel technique to image in 3D the heart vasculature by combining the CUBIC clearing protocol combined with in vivo administration of fluorescent-labeled lectin. The use of these techniques in combination with Selective Plane Illumination Microscopy (SPIM) made it possible to obtain high resolution 3D images of the cardiac vascular tree. This methodological approach may enhance the visualization of 3D images of the cardiac vasculature remodeling associated with coronary disease. PMID:28663930

Optical coherence elastography for cellular-scale stiffness imaging of mouse aorta

NASA Astrophysics Data System (ADS)

Wijesinghe, Philip; Johansen, Niloufer J.; Curatolo, Andrea; Sampson, David D.; Ganss, Ruth; Kennedy, Brendan F.

2017-04-01

We have developed a high-resolution optical coherence elastography system capable of estimating Young's modulus in tissue volumes with an isotropic resolution of 15 μm over a 1 mm lateral field of view and a 100 μm axial depth of field. We demonstrate our technique on healthy and hypertensive, freshly excised and intact mouse aortas. Our technique has the capacity to delineate the individual mechanics of elastic lamellae and vascular smooth muscle. Further, we observe global and regional vascular stiffening in hypertensive aortas, and note the presence of local micro-mechanical signatures, characteristic of fibrous and lipid-rich regions.

High-resolution simultaneous voltage and Ca2+ imaging

PubMed Central

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

2011-01-01

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

Organization of the ER–Golgi interface for membrane traffic control

PubMed Central

Brandizzi, Federica; Barlowe, Charles

2014-01-01

Coat protein complex I (COPI) and COPII are required for bidirectional membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. While these core coat machineries and other transport factors are highly conserved across species, high-resolution imaging studies indicate that the organization of the ER–Golgi interface is varied in eukaryotic cells. Regulation of COPII assembly, in some cases to manage distinct cellular cargo, is emerging as one important component in determining this structure. Comparison of the ER–Golgi interface across different systems, particularly mammalian and plant cells, reveals fundamental elements and distinct organization of this interface. A better understanding of how these interfaces are regulated to meet varying cellular secretory demands should provide key insights into the mechanisms that control efficient trafficking of proteins and lipids through the secretory pathway. PMID:23698585

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

NASA Astrophysics Data System (ADS)

Dmitriev, Ruslan I.; Papkovsky, Dmitri B.

2015-09-01

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

Novel method for water vapour monitoring using wireless communication networks measurements

NASA Astrophysics Data System (ADS)

David, N.; Alpert, P.; Messer, H.

2010-09-01

We propose a new technique for monitoring near-surface water vapour, by estimating humidity from data collected through existing wireless communication networks. Weather conditions and atmospheric phenomena affect the electromagnetic channel, causing attenuations to the radio signals. Thus, wireless communication networks are in effect built-in environmental monitoring facilities. The wireless microwave links, used in these networks, are widely deployed by cellular providers for backhaul communication between base stations, a few tens of meters above ground level. As a result, if all available measurements are used, the proposed method can provide moisture observations with high spatial resolution and potentially high temporal resolution. Further, the implementation cost is minimal, since the data used are already collected and saved by the cellular operators. In addition - many of these links are installed in areas where access is difficult such as orographic terrain and complex topography. As such, our method enables measurements in places that have been hard to measure in the past, or have never been measured before. The technique is restricted to weather conditions which exclude rain, fog or clouds along the propagation path. Strong winds that may cause movement of the link transmitter or receiver (or both) may also interfere with the ability to conduct accurate measurements. We present results from real-data measurements taken from microwave links used in a backhaul cellular network that show very good correlation with surface station humidity measurements (comparisons were performed for several links, found at different locations, during different time periods, showing correlations in the range of 0.5-0.9).

Light, heat, action: neural control of fruit fly behaviour.

PubMed

Owald, David; Lin, Suewei; Waddell, Scott

2015-09-19

The fruit fly Drosophila melanogaster has emerged as a popular model to investigate fundamental principles of neural circuit operation. The sophisticated genetics and small brain permit a cellular resolution understanding of innate and learned behavioural processes. Relatively recent genetic and technical advances provide the means to specifically and reproducibly manipulate the function of many fly neurons with temporal resolution. The same cellular precision can also be exploited to express genetically encoded reporters of neural activity and cell-signalling pathways. Combining these approaches in living behaving animals has great potential to generate a holistic view of behavioural control that transcends the usual molecular, cellular and systems boundaries. In this review, we discuss these approaches with particular emphasis on the pioneering studies and those involving learning and memory.

Ultrahigh resolution optical coherence elastography combined with a rigid micro-endoscope (Conference Presentation)

NASA Astrophysics Data System (ADS)

Fang, Qi; Curatolo, Andrea; Wijesinghe, Philip; Hamzah, Juliana; Ganss, Ruth; Noble, Peter B.; Karnowski, Karol; Sampson, David D.; Kim, Jun Ki; Lee, Wei M.; Kennedy, Brendan F.

2017-02-01

The mechanical forces that living cells experience represent an important framework in the determination of a range of intricate cellular functions and processes. Current insight into cell mechanics is typically provided by in vitro measurement systems; for example, atomic force microscopy (AFM) measurements are performed on cells in culture or, at best, on freshly excised tissue. Optical techniques, such as Brillouin microscopy and optical elastography, have been used for ex vivo and in situ imaging, recently achieving cellular-scale resolution. The utility of these techniques in cell mechanics lies in quick, three-dimensional and label-free mechanical imaging. Translation of these techniques toward minimally invasive in vivo imaging would provide unprecedented capabilities in tissue characterization. Here, we take the first steps along this path by incorporating a gradient-index micro-endoscope into an ultrahigh resolution optical elastography system. Using this endoscope, a lateral resolution of 2 µm is preserved over an extended depth-of-field of 80 µm, achieved by Bessel beam illumination. We demonstrate this combined system by imaging stiffness of a silicone phantom containing stiff inclusions and a freshly excised murine liver tissue. Additionally, we test this system on murine ribs in situ. We show that our approach can provide high quality extended depth-of-field images through an endoscope and has the potential to measure cell mechanics deep in tissue. Eventually, we believe this tool will be capable of studying biological processes and disease progression in vivo.

Pollen structure visualization using high-resolution laboratory-based hard X-ray tomography.

PubMed

Li, Qiong; Gluch, Jürgen; Krüger, Peter; Gall, Martin; Neinhuis, Christoph; Zschech, Ehrenfried

2016-10-14

A laboratory-based X-ray microscope is used to investigate the 3D structure of unstained whole pollen grains. For the first time, high-resolution laboratory-based hard X-ray microscopy is applied to study pollen grains. Based on the efficient acquisition of statistically relevant information-rich images using Zernike phase contrast, both surface- and internal structures of pine pollen - including exine, intine and cellular structures - are clearly visualized. The specific volumes of these structures are calculated from the tomographic data. The systematic three-dimensional study of pollen grains provides morphological and structural information about taxonomic characters that are essential in palynology. Such studies have a direct impact on disciplines such as forestry, agriculture, horticulture, plant breeding and biodiversity. Copyright © 2016 Elsevier Inc. All rights reserved.

Hybrid-coded 3D structured illumination imaging with Bayesian estimation (Conference Presentation)

NASA Astrophysics Data System (ADS)

Chen, Hsi-Hsun; Luo, Yuan; Singh, Vijay R.

2016-03-01

Light induced fluorescent microscopy has long been developed to observe and understand the object at microscale, such as cellular sample. However, the transfer function of lense-based imaging system limits the resolution so that the fine and detailed structure of sample cannot be identified clearly. The techniques of resolution enhancement are fascinated to break the limit of resolution for objective given. In the past decades, the resolution enhancement imaging has been investigated through variety of strategies, including photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), stimulated emission depletion (STED), and structure illuminated microscopy (SIM). In those methods, only SIM can intrinsically improve the resolution limit for a system without taking the structure properties of object into account. In this paper, we develop a SIM associated with Bayesian estimation, furthermore, with optical sectioning capability rendered from HiLo processing, resulting the high resolution through 3D volume. This 3D SIM can provide the optical sectioning and resolution enhancement performance, and be robust to noise owing to the Data driven Bayesian estimation reconstruction proposed. For validating the 3D SIM, we show our simulation result of algorithm, and the experimental result demonstrating the 3D resolution enhancement.

Quantification of silver nanoparticle uptake and distribution within individual human macrophages by FIB/SEM slice and view.

PubMed

Guehrs, Erik; Schneider, Michael; Günther, Christian M; Hessing, Piet; Heitz, Karen; Wittke, Doreen; López-Serrano Oliver, Ana; Jakubowski, Norbert; Plendl, Johanna; Eisebitt, Stefan; Haase, Andrea

2017-03-21

Quantification of nanoparticle (NP) uptake in cells or tissues is very important for safety assessment. Often, electron microscopy based approaches are used for this purpose, which allow imaging at very high resolution. However, precise quantification of NP numbers in cells and tissues remains challenging. The aim of this study was to present a novel approach, that combines precise quantification of NPs in individual cells together with high resolution imaging of their intracellular distribution based on focused ion beam/ scanning electron microscopy (FIB/SEM) slice and view approaches. We quantified cellular uptake of 75 nm diameter citrate stabilized silver NPs (Ag 75 Cit) into an individual human macrophage derived from monocytic THP-1 cells using a FIB/SEM slice and view approach. Cells were treated with 10 μg/ml for 24 h. We investigated a single cell and found in total 3138 ± 722 silver NPs inside this cell. Most of the silver NPs were located in large agglomerates, only a few were found in clusters of fewer than five NPs. Furthermore, we cross-checked our results by using inductively coupled plasma mass spectrometry and could confirm the FIB/SEM results. Our approach based on FIB/SEM slice and view is currently the only one that allows the quantification of the absolute dose of silver NPs in individual cells and at the same time to assess their intracellular distribution at high resolution. We therefore propose to use FIB/SEM slice and view to systematically analyse the cellular uptake of various NPs as a function of size, concentration and incubation time.

Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2-deoxyglucose using pulsed stable isotope-resolved metabolomics.

PubMed

Pietzke, Matthias; Zasada, Christin; Mudrich, Susann; Kempa, Stefan

2014-01-01

Cellular metabolism is highly dynamic and continuously adjusts to the physiological program of the cell. The regulation of metabolism appears at all biological levels: (post-) transcriptional, (post-) translational, and allosteric. This regulatory information is expressed in the metabolome, but in a complex manner. To decode such complex information, new methods are needed in order to facilitate dynamic metabolic characterization at high resolution. Here, we describe pulsed stable isotope-resolved metabolomics (pSIRM) as a tool for the dynamic metabolic characterization of cellular metabolism. We have adapted gas chromatography-coupled mass spectrometric methods for metabolomic profiling and stable isotope-resolved metabolomics. In addition, we have improved robustness and reproducibility and implemented a strategy for the absolute quantification of metabolites. By way of examples, we have applied this methodology to characterize central carbon metabolism of a panel of cancer cell lines and to determine the mode of metabolic inhibition of glycolytic inhibitors in times ranging from minutes to hours. Using pSIRM, we observed that 2-deoxyglucose is a metabolic inhibitor, but does not directly act on the glycolytic cascade.

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

PubMed Central

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

2012-01-01

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

Multi-MHz laser-scanning single-cell fluorescence microscopy by spatiotemporally encoded virtual source array

PubMed Central

Wu, Jianglai; Tang, Anson H. L.; Mok, Aaron T. Y.; Yan, Wenwei; Chan, Godfrey C. F.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2017-01-01

Apart from the spatial resolution enhancement, scaling of temporal resolution, equivalently the imaging throughput, of fluorescence microscopy is of equal importance in advancing cell biology and clinical diagnostics. Yet, this attribute has mostly been overlooked because of the inherent speed limitation of existing imaging strategies. To address the challenge, we employ an all-optical laser-scanning mechanism, enabled by an array of reconfigurable spatiotemporally-encoded virtual sources, to demonstrate ultrafast fluorescence microscopy at line-scan rate as high as 8 MHz. We show that this technique enables high-throughput single-cell microfluidic fluorescence imaging at 75,000 cells/second and high-speed cellular 2D dynamical imaging at 3,000 frames per second, outperforming the state-of-the-art high-speed cameras and the gold-standard laser scanning strategies. Together with its wide compatibility to the existing imaging modalities, this technology could empower new forms of high-throughput and high-speed biological fluorescence microscopy that was once challenged. PMID:28966855

Coordination of Cellular Dynamics Contributes to Tooth Epithelium Deformations

PubMed Central

Morita, Ritsuko; Kihira, Miho; Nakatsu, Yousuke; Nomoto, Yohei; Ogawa, Miho; Ohashi, Kazumasa; Mizuno, Kensaku; Tachikawa, Tetsuhiko; Ishimoto, Yukitaka; Morishita, Yoshihiro; Tsuji, Takashi

2016-01-01

The morphologies of ectodermal organs are shaped by appropriate combinations of several deformation modes, such as invagination and anisotropic tissue elongation. However, how multicellular dynamics are coordinated during deformation processes remains to be elucidated. Here, we developed a four-dimensional (4D) analysis system for tracking cell movement and division at a single-cell resolution in developing tooth epithelium. The expression patterns of a Fucci probe clarified the region- and stage-specific cell cycle patterns within the tooth germ, which were in good agreement with the pattern of the volume growth rate estimated from tissue-level deformation analysis. Cellular motility was higher in the regions with higher growth rates, while the mitotic orientation was significantly biased along the direction of tissue elongation in the epithelium. Further, these spatio-temporal patterns of cellular dynamics and tissue-level deformation were highly correlated with that of the activity of cofilin, which is an actin depolymerization factor, suggesting that the coordination of cellular dynamics via actin remodeling plays an important role in tooth epithelial morphogenesis. Our system enhances the understanding of how cellular behaviors are coordinated during ectodermal organogenesis, which cannot be observed from histological analyses. PMID:27588418

An insight into morphometric descriptors of cell shape that pertain to regenerative medicine.

PubMed

Lobo, Joana; See, Eugene Yong-Shun; Biggs, Manus; Pandit, Abhay

2016-07-01

Cellular morphology has recently been indicated as a powerful indicator of cellular function. The analysis of cell shape has evolved from rudimentary forms of microscopic visual inspection to more advanced methodologies that utilize high-resolution microscopy coupled with sophisticated computer hardware and software for data analysis. Despite this progress, there is still a lack of standardization in quantification of morphometric parameters. In addition, uncertainty remains as to which methodologies and parameters of cell morphology will yield meaningful data, which methods should be utilized to categorize cell shape, and the extent of reliability of measurements and the interpretation of the resulting analysis. A large range of descriptors has been employed to objectively assess the cellular morphology in two-dimensional and three-dimensional domains. Intuitively, simple and applicable morphometric descriptors are preferable and standardized protocols for cell shape analysis can be achieved with the help of computerized tools. In this review, cellular morphology is discussed as a descriptor of cellular function and the current morphometric parameters that are used quantitatively in two- and three-dimensional environments are described. Furthermore, the current problems associated with these morphometric measurements are addressed. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

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

X-ray phase contrast tomography from whole organ down to single cells

NASA Astrophysics Data System (ADS)

Krenkel, Martin; Töpperwien, Mareike; Bartels, Matthias; Lingor, Paul; Schild, Detlev; Salditt, Tim

2014-09-01

We use propagation based hard x-ray phase contrast tomography to explore the three dimensional structure of neuronal tissues from the organ down to sub-cellular level, based on combinations of synchrotron radiation and laboratory sources. To this end a laboratory based microfocus tomography setup has been built in which the geometry was optimized for phase contrast imaging and tomography. By utilizing phase retrieval algorithms, quantitative reconstructions can be obtained that enable automatic renderings without edge artifacts. A high brightness liquid metal microfocus x-ray source in combination with a high resolution detector yielding a resolution down to 1.5 μm. To extend the method to nanoscale resolution we use a divergent x-ray waveguide beam geometry at the synchrotron. Thus, the magnification can be easily tuned by placing the sample at different defocus distances. Due to the small Fresnel numbers in this geometry the measured images are of holographic nature which poses a challenge in phase retrieval.

Three-dimensional cellular deformation analysis with a two-photon magnetic manipulator workstation.

PubMed

Huang, Hayden; Dong, Chen Y; Kwon, Hyuk-Sang; Sutin, Jason D; Kamm, Roger D; So, Peter T C

2002-04-01

The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses noninvasively. This paper describes the development of a micromanipulation workstation integrating two-photon, three-dimensional imaging with a high-force, uniform-gradient magnetic manipulator. The uniform-gradient magnetic field applies nearly uniform forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5-microm-diameter paramagnetic particles and over 800 pN on 5.0-microm ferromagnetic particles. These forces vary within +/-10% over an area 500 x 500 microm2. The compatibility with the use of high numerical aperture (approximately 1.0) objectives is an integral part of the workstation design allowing submicron-resolution, three-dimensional, two-photon imaging. Three-dimensional analyses of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses may contain three-dimensional global deformations and show the suitability of this workstation to further studying cellular response to mechanical stresses.

Evaluation of variability in high-resolution protein structures by global distance scoring.

PubMed

Anzai, Risa; Asami, Yoshiki; Inoue, Waka; Ueno, Hina; Yamada, Koya; Okada, Tetsuji

2018-01-01

Systematic analysis of the statistical and dynamical properties of proteins is critical to understanding cellular events. Extraction of biologically relevant information from a set of high-resolution structures is important because it can provide mechanistic details behind the functional properties of protein families, enabling rational comparison between families. Most of the current structural comparisons are pairwise-based, which hampers the global analysis of increasing contents in the Protein Data Bank. Additionally, pairing of protein structures introduces uncertainty with respect to reproducibility because it frequently accompanies other settings for superimposition. This study introduces intramolecular distance scoring for the global analysis of proteins, for each of which at least several high-resolution structures are available. As a pilot study, we have tested 300 human proteins and showed that the method is comprehensively used to overview advances in each protein and protein family at the atomic level. This method, together with the interpretation of the model calculations, provide new criteria for understanding specific structural variation in a protein, enabling global comparison of the variability in proteins from different species.

High-speed adaptive optics line scan confocal retinal imaging for human eye

PubMed Central

Wang, Xiaolin; Zhang, Yuhua

2017-01-01

Purpose Continuous and rapid eye movement causes significant intraframe distortion in adaptive optics high resolution retinal imaging. To minimize this artifact, we developed a high speed adaptive optics line scan confocal retinal imaging system. Methods A high speed line camera was employed to acquire retinal image and custom adaptive optics was developed to compensate the wave aberration of the human eye’s optics. The spatial resolution and signal to noise ratio were assessed in model eye and in living human eye. The improvement of imaging fidelity was estimated by reduction of intra-frame distortion of retinal images acquired in the living human eyes with frame rates at 30 frames/second (FPS), 100 FPS, and 200 FPS. Results The device produced retinal image with cellular level resolution at 200 FPS with a digitization of 512×512 pixels/frame in the living human eye. Cone photoreceptors in the central fovea and rod photoreceptors near the fovea were resolved in three human subjects in normal chorioretinal health. Compared with retinal images acquired at 30 FPS, the intra-frame distortion in images taken at 200 FPS was reduced by 50.9% to 79.7%. Conclusions We demonstrated the feasibility of acquiring high resolution retinal images in the living human eye at a speed that minimizes retinal motion artifact. This device may facilitate research involving subjects with nystagmus or unsteady fixation due to central vision loss. PMID:28257458

High-speed adaptive optics line scan confocal retinal imaging for human eye.

PubMed

Lu, Jing; Gu, Boyu; Wang, Xiaolin; Zhang, Yuhua

2017-01-01

Continuous and rapid eye movement causes significant intraframe distortion in adaptive optics high resolution retinal imaging. To minimize this artifact, we developed a high speed adaptive optics line scan confocal retinal imaging system. A high speed line camera was employed to acquire retinal image and custom adaptive optics was developed to compensate the wave aberration of the human eye's optics. The spatial resolution and signal to noise ratio were assessed in model eye and in living human eye. The improvement of imaging fidelity was estimated by reduction of intra-frame distortion of retinal images acquired in the living human eyes with frame rates at 30 frames/second (FPS), 100 FPS, and 200 FPS. The device produced retinal image with cellular level resolution at 200 FPS with a digitization of 512×512 pixels/frame in the living human eye. Cone photoreceptors in the central fovea and rod photoreceptors near the fovea were resolved in three human subjects in normal chorioretinal health. Compared with retinal images acquired at 30 FPS, the intra-frame distortion in images taken at 200 FPS was reduced by 50.9% to 79.7%. We demonstrated the feasibility of acquiring high resolution retinal images in the living human eye at a speed that minimizes retinal motion artifact. This device may facilitate research involving subjects with nystagmus or unsteady fixation due to central vision loss.

Quantification of the fluorine containing drug 5-fluorouracil in cancer cells by GaF molecular absorption via high-resolution continuum source molecular absorption spectrometry

NASA Astrophysics Data System (ADS)

Krüger, Magnus; Huang, Mao-Dong; Becker-Roß, Helmut; Florek, Stefan; Ott, Ingo; Gust, Ronald

The development of high-resolution continuum source molecular absorption spectrometry made the quantification of fluorine feasible by measuring the molecular absorption as gallium monofluoride (GaF). Using this new technique, we developed on the example of 5-fluorouracil (5-FU) a graphite furnace method to quantify fluorine in organic molecules. The effect of 5-FU on the generation of the diatomic GaF molecule was investigated. The experimental conditions such as gallium nitrate amount, temperature program, interfering anions (represented as corresponding acids) and calibration for the determination of 5-FU in standard solution and in cellular matrix samples were investigated and optimized. The sample matrix showed no effect on the sensitivity of GaF molecular absorption. A simple calibration curve using an inorganic sodium fluoride solution can conveniently be used for the calibration. The described method is sensitive and the achievable limit of detection is 0.23 ng of 5-FU. In order to establish the concept of "fluorine as a probe in medicinal chemistry" an exemplary application was selected, in which the developed method was successfully demonstrated by performing cellular uptake studies of the 5-FU in human colon carcinoma cells.

Linking environmental processes to the in situ functioning of microorganisms by high-resolution secondary ion mass spectrometry (NanoSIMS) and scanning transmission X-ray microscopy (STXM).

PubMed

Behrens, Sebastian; Kappler, Andreas; Obst, Martin

2012-11-01

Environmental microbiology research increasingly focuses on the single microbial cell as the defining entity that drives environmental processes. The interactions of individual microbial cells with each other, the environment and with higher organisms shape microbial communities and control the functioning of whole ecosystems. A single-cell view of microorganisms in their natural environment requires analytical tools that measure both cell function and chemical speciation at the submicrometre scale. Here we review the technical capabilities and limitations of high-resolution secondary ion mass spectrometry (NanoSIMS) and scanning transmission (soft) X-ray microscopy (STXM) and give examples of their applications. Whereas NanoSIMS can be combined with isotope-labelling, thereby localizing the distribution of cellular activities (e.g. carbon/nitrogen fixation/turnover), STXM provides information on the location and chemical speciation of metabolites and products of redox reactions. We propose the combined use of both techniques and discuss the technical challenges of their joint application. Both techniques have the potential to enhance our understanding of cellular mechanisms and activities that contribute to microbially mediated processes, such as the biogeochemical cycling of elements, the transformation of contaminants and the precipitation of mineral phases. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

High-resolution imaging of living mammalian cells bound by nanobeads-connected antibodies in a medium using scanning electron-assisted dielectric microscopy

NASA Astrophysics Data System (ADS)

Okada, Tomoko; Ogura, Toshihiko

2017-02-01

Nanometre-scale-resolution imaging technologies for liquid-phase specimens are indispensable tools in various scientific fields. In biology, observing untreated living cells in a medium is essential for analysing cellular functions. However, nanoparticles that bind living cells in a medium are hard to detect directly using traditional optical or electron microscopy. Therefore, we previously developed a novel scanning electron-assisted dielectric microscope (SE-ADM) capable of nanoscale observations. This method enables observation of intact cells in aqueous conditions. Here, we use this SE-ADM system to clearly observe antibody-binding nanobeads in liquid-phase. We also report the successful direct detection of streptavidin-conjugated nanobeads binding to untreated cells in a medium via a biotin-conjugated anti-CD44 antibody. Our system is capable of obtaining clear images of cellular organelles and beads on the cells at the same time. The direct observation of living cells with nanoparticles in a medium allowed by our system may contribute the development of carriers for drug delivery systems (DDS).

Multi-photon microfabrication of three-dimensional capillary-scale vascular networks

NASA Astrophysics Data System (ADS)

Skylar-Scott, Mark A.; Liu, Man-Chi; Wu, Yuelong; Yanik, Mehmet Fatih

2017-02-01

Biomimetic models of microvasculature could enable assays of complex cellular behavior at the capillary-level, and enable efficient nutrient perfusion for the maintenance of tissues. However, existing three-dimensional printing methods for generating perfusable microvasculature with have insufficient resolution to recapitulate the microscale geometry of capillaries. Here, we present a collection of multiphoton microfabrication methods that enable the production of precise, three-dimensional, branched microvascular networks in collagen. When endothelial cells are added to the channels, they form perfusable lumens with diameters as small as 10 μm. Using a similar photochemistry, we also demonstrate the micropatterning of proteins embedded in microfabricated collagen scaffolds, producing hybrid scaffolds with both defined microarchitecture with integrated gradients of chemical cues. We provide examples for how these hybrid microfabricated scaffolds could be used in angiogenesis and cell homing assays. Finally, we describe a new method for increasing the micropatterning speed by synchronous laser and stage scanning. Using these technologies, we are working towards large-scale (>1 cm), high resolution ( 1 μm) scaffolds with both microarchitecture and embedded protein cues, with applications in three-dimensional assays of cellular behavior.

Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid.

PubMed

Dukes, Madeline J; Peckys, Diana B; de Jonge, Niels

2010-07-27

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.

Correlative Fluorescence Microscopy and Scanning Transmission Electron Microscopy of Quantum Dot Labeled Proteins in Whole Cells in Liquid

PubMed Central

Dukes, Madeline J.; Peckys, Diana B.; de Jonge, Niels

2010-01-01

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7 × 12 nm were visible in a 5 μm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs. PMID:20550177

Multi-view light-sheet imaging and tracking with the MaMuT software reveals the cell lineage of a direct developing arthropod limb

PubMed Central

Stamataki, Evangelia; Harich, Benjamin; Guignard, Léo; Preibisch, Stephan; Shorte, Spencer; Keller, Philipp J

2018-01-01

During development, coordinated cell behaviors orchestrate tissue and organ morphogenesis. Detailed descriptions of cell lineages and behaviors provide a powerful framework to elucidate the mechanisms of morphogenesis. To study the cellular basis of limb development, we imaged transgenic fluorescently-labeled embryos from the crustacean Parhyale hawaiensis with multi-view light-sheet microscopy at high spatiotemporal resolution over several days of embryogenesis. The cell lineage of outgrowing thoracic limbs was reconstructed at single-cell resolution with new software called Massive Multi-view Tracker (MaMuT). In silico clonal analyses suggested that the early limb primordium becomes subdivided into anterior-posterior and dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb-bud formation is associated with spatial modulation of cell proliferation, while limb elongation is also driven by preferential orientation of cell divisions along the proximal-distal growth axis. Cellular reconstructions were predictive of the expression patterns of limb development genes including the BMP morphogen Decapentaplegic. PMID:29595475

High resolution multiplexed functional imaging in live embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Zhou, Weibin; Peng, Leilei

2017-02-01

Fourier multiplexed fluorescence lifetime imaging (FmFLIM) scanning laser optical tomography (FmFLIM-SLOT) combines FmFLIM and Scanning laser optical tomography (SLOT) to perform multiplexed 3D FLIM imaging of live embryos. The system had demonstrate multiplexed functional imaging of zebrafish embryos genetically express Foster Resonant Energy Transfer (FRET) sensors. However, previous system has a 20 micron resolution because the focused Gaussian beam diverges quickly from the focused plane, makes it difficult to achieve high resolution imaging over a long projection depth. Here, we present a high-resolution FmFLIM-SLOT system with achromatic Bessel beam, which achieves 3 micron resolution in 3D deep tissue imaging. In Bessel-FmFLIM-SLOT, multiple laser excitation lines are firstly intensity modulated by a Michelson interferometer with a spinning polygon mirror optical delay line, which enables Fourier multiplexed multi-channel lifetime measurements. Then, a spatial light modulator and a prism are used to transform the modulated Gaussian laser beam to an achromatic Bessel beam. The achromatic Bessel beam scans across the whole specimen with equal angular intervals as sample rotated. After tomography reconstruction and the frequency domain lifetime analysis method, both the 3D intensity and lifetime image of multiple excitation-emission can be obtained. Using Bessel-FmFLIM-SLOT system, we performed cellular-resolution FLIM tomography imaging of live zebrafish embryo. Genetically expressed FRET sensors in these embryo will allow non-invasive observation of multiple biochemical processes in vivo.

Cell signaling, post-translational protein modifications and NMR spectroscopy

PubMed Central

Theillet, Francois-Xavier; Smet-Nocca, Caroline; Liokatis, Stamatios; Thongwichian, Rossukon; Kosten, Jonas; Yoon, Mi-Kyung; Kriwacki, Richard W.; Landrieu, Isabelle; Lippens, Guy

2016-01-01

Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy. PMID:23011410

Force-activatable biosensor enables single platelet force mapping directly by fluorescence imaging.

PubMed

Wang, Yongliang; LeVine, Dana N; Gannon, Margaret; Zhao, Yuanchang; Sarkar, Anwesha; Hoch, Bailey; Wang, Xuefeng

2018-02-15

Integrin-transmitted cellular forces are critical for platelet adhesion, activation, aggregation and contraction during hemostasis and thrombosis. Measuring and mapping single platelet forces are desired in both research and clinical applications. Conventional force-to-strain based cell traction force microscopies have low resolution which is not ideal for cellular force mapping in small platelets. To enable platelet force mapping with submicron resolution, we developed a force-activatable biosensor named integrative tension sensor (ITS) which directly converts molecular tensions to fluorescent signals, therefore enabling cellular force mapping directly by fluorescence imaging. With ITS, we mapped cellular forces in single platelets at 0.4µm resolution. We found that platelet force distribution has strong polarization which is sensitive to treatment with the anti-platelet drug tirofiban, suggesting that the ITS force map can report anti-platelet drug efficacy. The ITS also calibrated integrin molecular tensions in platelets and revealed two distinct tension levels: 12-54 piconewton (nominal values) tensions generated during platelet adhesion and tensions above 54 piconewton generated during platelet contraction. Overall, the ITS is a powerful biosensor for the study of platelet mechanobiology, and holds great potential in antithrombotic drug development and assessing platelet activity in health and disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Full-field OCT: applications in ophthalmology

NASA Astrophysics Data System (ADS)

Grieve, Kate; Dubois, Arnaud; Paques, Michel; Le Gargasson, Jean-Francois; Boccara, Albert C.

2005-04-01

We present images of ocular tissues obtained using ultrahigh resolution full-field OCT. The experimental setup is based on the Linnik interferometer, illuminated by a tungsten halogen lamp. En face tomographic images are obtained in real-time without scanning by computing the difference of two phase-opposed interferometric images recorded by a high-resolution CCD camera. A spatial resolution of 0.7 μm × 0.9 μm (axial × transverse) is achieved thanks to the short source coherence length and the use of high numerical aperture microscope objectives. A detection sensitivity of 90 dB is obtained by means of image averaging and pixel binning. Whole unfixed eyes and unstained tissue samples (cornea, lens, retina, choroid and sclera) of ex vivo rat, mouse, rabbit and porcine ocular tissues were examined. The unprecedented resolution of our instrument allows cellular-level resolution in the cornea and retina, and visualization of individual fibers in the lens. Transcorneal lens imaging was possible in all animals, and in albino animals, transscleral retinal imaging was achieved. We also introduce our rapid acquisition full-field optical coherence tomography system designed to accommodate in vivo ophthalmologic imaging. The variations on the original system technology include the introduction of a xenon arc lamp as source, and rapid image acquisition performed by a high-speed CMOS camera, reducing acquisition time to 5 ms per frame.

Specialized Proresolving Mediators in Innate and Adaptive Immune Responses in Airway Diseases.

PubMed

Krishnamoorthy, Nandini; Abdulnour, Raja-Elie E; Walker, Katherine H; Engstrom, Braden D; Levy, Bruce D

2018-07-01

Airborne pathogens and environmental stimuli evoke immune responses in the lung. It is critical to health that these responses be controlled to prevent tissue damage and the compromise of organ function. Resolution of inflammation is a dynamic process that is coordinated by biochemical and cellular mechanisms. Recently, specialized proresolving mediators (SPMs) have been identified in resolution exudates. These molecules orchestrate anti-inflammatory and proresolving actions that are cell type specific. In this review, we highlight SPM biosynthesis, the influence of SPMs on the innate and adaptive immune responses in the lung, as well as recent insights from SPMs on inflammatory disease pathophysiology. Uncovering these mediators and cellular mechanisms for resolution is providing new windows into physiology and disease pathogenesis.

Anatomically accurate high resolution modeling of human whole heart electromechanics: A strongly scalable algebraic multigrid solver method for nonlinear deformation

NASA Astrophysics Data System (ADS)

Augustin, Christoph M.; Neic, Aurel; Liebmann, Manfred; Prassl, Anton J.; Niederer, Steven A.; Haase, Gundolf; Plank, Gernot

2016-01-01

Electromechanical (EM) models of the heart have been used successfully to study fundamental mechanisms underlying a heart beat in health and disease. However, in all modeling studies reported so far numerous simplifications were made in terms of representing biophysical details of cellular function and its heterogeneity, gross anatomy and tissue microstructure, as well as the bidirectional coupling between electrophysiology (EP) and tissue distension. One limiting factor is the employed spatial discretization methods which are not sufficiently flexible to accommodate complex geometries or resolve heterogeneities, but, even more importantly, the limited efficiency of the prevailing solver techniques which is not sufficiently scalable to deal with the incurring increase in degrees of freedom (DOF) when modeling cardiac electromechanics at high spatio-temporal resolution. This study reports on the development of a novel methodology for solving the nonlinear equation of finite elasticity using human whole organ models of cardiac electromechanics, discretized at a high para-cellular resolution. Three patient-specific, anatomically accurate, whole heart EM models were reconstructed from magnetic resonance (MR) scans at resolutions of 220 μm, 440 μm and 880 μm, yielding meshes of approximately 184.6, 24.4 and 3.7 million tetrahedral elements and 95.9, 13.2 and 2.1 million displacement DOF, respectively. The same mesh was used for discretizing the governing equations of both electrophysiology (EP) and nonlinear elasticity. A novel algebraic multigrid (AMG) preconditioner for an iterative Krylov solver was developed to deal with the resulting computational load. The AMG preconditioner was designed under the primary objective of achieving favorable strong scaling characteristics for both setup and solution runtimes, as this is key for exploiting current high performance computing hardware. Benchmark results using the 220 μm, 440 μm and 880 μm meshes demonstrate efficient scaling up to 1024, 4096 and 8192 compute cores which allowed the simulation of a single heart beat in 44.3, 87.8 and 235.3 minutes, respectively. The efficiency of the method allows fast simulation cycles without compromising anatomical or biophysical detail.

Anatomically accurate high resolution modeling of human whole heart electromechanics: A strongly scalable algebraic multigrid solver method for nonlinear deformation

PubMed Central

Augustin, Christoph M.; Neic, Aurel; Liebmann, Manfred; Prassl, Anton J.; Niederer, Steven A.; Haase, Gundolf; Plank, Gernot

2016-01-01

Electromechanical (EM) models of the heart have been used successfully to study fundamental mechanisms underlying a heart beat in health and disease. However, in all modeling studies reported so far numerous simplifications were made in terms of representing biophysical details of cellular function and its heterogeneity, gross anatomy and tissue microstructure, as well as the bidirectional coupling between electrophysiology (EP) and tissue distension. One limiting factor is the employed spatial discretization methods which are not sufficiently flexible to accommodate complex geometries or resolve heterogeneities, but, even more importantly, the limited efficiency of the prevailing solver techniques which are not sufficiently scalable to deal with the incurring increase in degrees of freedom (DOF) when modeling cardiac electromechanics at high spatio-temporal resolution. This study reports on the development of a novel methodology for solving the nonlinear equation of finite elasticity using human whole organ models of cardiac electromechanics, discretized at a high para-cellular resolution. Three patient-specific, anatomically accurate, whole heart EM models were reconstructed from magnetic resonance (MR) scans at resolutions of 220 μm, 440 μm and 880 μm, yielding meshes of approximately 184.6, 24.4 and 3.7 million tetrahedral elements and 95.9, 13.2 and 2.1 million displacement DOF, respectively. The same mesh was used for discretizing the governing equations of both electrophysiology (EP) and nonlinear elasticity. A novel algebraic multigrid (AMG) preconditioner for an iterative Krylov solver was developed to deal with the resulting computational load. The AMG preconditioner was designed under the primary objective of achieving favorable strong scaling characteristics for both setup and solution runtimes, as this is key for exploiting current high performance computing hardware. Benchmark results using the 220 μm, 440 μm and 880 μm meshes demonstrate efficient scaling up to 1024, 4096 and 8192 compute cores which allowed the simulation of a single heart beat in 44.3, 87.8 and 235.3 minutes, respectively. The efficiency of the method allows fast simulation cycles without compromising anatomical or biophysical detail. PMID:26819483

Systematic, spatial imaging of large multimolecular assemblies and the emerging principles of supramolecular order in biological systems

PubMed Central

Schubert, Walter

2013-01-01

Understanding biological systems at the level of their relational (emergent) molecular properties in functional protein networks relies on imaging methods, able to spatially resolve a tissue or a cell as a giant, non-random, topologically defined collection of interacting supermolecules executing myriads of subcellular mechanisms. Here, the development and findings of parameter-unlimited functional super-resolution microscopy are described—a technology based on the fluorescence imaging cycler (IC) principle capable of co-mapping thousands of distinct biomolecular assemblies at high spatial resolution and differentiation (<40 nm distances). It is shown that the subcellular and transcellular features of such supermolecules can be described at the compositional and constitutional levels; that the spatial connection, relational stoichiometry, and topology of supermolecules generate hitherto unrecognized functional self-segmentation of biological tissues; that hierarchical features, common to thousands of simultaneously imaged supermolecules, can be identified; and how the resulting supramolecular order relates to spatial coding of cellular functionalities in biological systems. A large body of observations with IC molecular systems microscopy collected over 20 years have disclosed principles governed by a law of supramolecular segregation of cellular functionalities. This pervades phenomena, such as exceptional orderliness, functional selectivity, combinatorial and spatial periodicity, and hierarchical organization of large molecular systems, across all species investigated so far. This insight is based on the high degree of specificity, selectivity, and sensitivity of molecular recognition processes for fluorescence imaging beyond the spectral resolution limit, using probe libraries controlled by ICs. © 2013 The Authors. Journal of Molecular Recognition published by John Wiley & Sons, Ltd. PMID:24375580

Light, heat, action: neural control of fruit fly behaviour

PubMed Central

Owald, David; Lin, Suewei; Waddell, Scott

2015-01-01

The fruit fly Drosophila melanogaster has emerged as a popular model to investigate fundamental principles of neural circuit operation. The sophisticated genetics and small brain permit a cellular resolution understanding of innate and learned behavioural processes. Relatively recent genetic and technical advances provide the means to specifically and reproducibly manipulate the function of many fly neurons with temporal resolution. The same cellular precision can also be exploited to express genetically encoded reporters of neural activity and cell-signalling pathways. Combining these approaches in living behaving animals has great potential to generate a holistic view of behavioural control that transcends the usual molecular, cellular and systems boundaries. In this review, we discuss these approaches with particular emphasis on the pioneering studies and those involving learning and memory. PMID:26240426

Resonance Raman Probes for Organelle-Specific Labeling in Live Cells

NASA Astrophysics Data System (ADS)

Kuzmin, Andrey N.; Pliss, Artem; Lim, Chang-Keun; Heo, Jeongyun; Kim, Sehoon; Rzhevskii, Alexander; Gu, Bobo; Yong, Ken-Tye; Wen, Shangchun; Prasad, Paras N.

2016-06-01

Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging.

Full-field OCT: ex vivo and in vivo biological imaging applications

NASA Astrophysics Data System (ADS)

Grieve, Katharine; Dubois, Arnaud; Moneron, Gael; Guyot, Elvire; Boccara, Albert C.

2005-04-01

We present results of studies in embryology and ophthalmology performed using our ultrahigh-resolution full-field OCT system. We also discuss recent developments to our ultrashort acquisition time full-field optical coherence tomography system designed to allow in vivo biological imaging. Preliminary results of high-speed imaging in biological samples are presented. The core of the experimental setup is the Linnik interferometer, illuminated by a white light source. En face tomographic images are obtained in real-time without scanning by computing the difference of two phase-opposed interferometric images recorded by high-resolution CCD cameras. An isotropic spatial resolution of ~1 μm is achieved thanks to the short source coherence length and the use of high numerical aperture microscope objectives. A detection sensitivity of ~90 dB is obtained by means of image averaging and pixel binning. In ophthalmology, reconstructed xz images from rat ocular tissue are presented, where cellular-level structures in the retina are revealed, demonstrating the unprecedented resolution of our instrument. Three-dimensional reconstructions of the mouse embryo allowing the study of the establishment of the anterior-posterior axis are shown. Finally we present the first results of embryonic imaging using the new rapid acquisition full-field OCT system, which offers an acquisition time of 10 μs per frame.

Cell electrophysiology with carbon nanopipettes.

PubMed

Schrlau, Michael G; Dun, Nae J; Bau, Haim H

2009-03-24

The ability to monitor living cell behavior in real time and with high spatial resolution is vital for advancing our knowledge of cellular machinery and evaluating cellular response to various drugs. Here, we report the development and utilization of carbon-based nanoelectrodes for cell electrophysiology. We employ carbon nanopipettes (CNPs), novel carbon-based nanoprobes which integrate carbon nanopipes into the tips of pulled glass capillaries, to measure electrical signals in the mouse hippocampal cell line HT-22. Using a standard electrophysiology amplifier in current-clamp mode, we measured the resting membrane potential of cells and their transient membrane response to extracellular pharmacological agents. In addition to their superior injection capabilities reported previously, CNPs are capable of multifunctionality, enabling, for example, concurrent intracellular injection and electrical measurements without damaging cells.

Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

PubMed Central

González-Vera, Juan A.; Morris, May C.

2015-01-01

Probing the dynamic activities of protein kinases in real-time in living cells constitutes a major challenge that requires specific and sensitive tools tailored to meet the particular demands associated with cellular imaging. The development of genetically-encoded and synthetic fluorescent biosensors has provided means of monitoring protein kinase activities in a non-invasive fashion in their native cellular environment with high spatial and temporal resolution. Here, we review existing technologies to probe different dynamic features of protein kinases and discuss limitations where new developments are required to implement more performant tools, in particular with respect to infrared and near-infrared fluorescent probes and strategies which enable improved signal-to-noise ratio and controlled activation of probes. PMID:28248276

Motion compensation using a suctioning stabilizer for intravital microscopy

PubMed Central

Vinegoni, Claudio; Lee, Sungon; Gorbatov, Rostic; Weissleder, Ralph

2013-01-01

Motion artifacts continue to present a major challenge to single cell imaging in cardiothoracic organs such as the beating heart, blood vessels, or lung. In this study, we present a new water-immersion suctioning stabilizer that enables minimally invasive intravital fluorescence microscopy using water-based stick objectives. The stabilizer works by reducing major motion excursions and can be used in conjunction with both prospective or retrospective gating approaches. We show that the new approach offers cellular resolution in the beating murine heart without perturbing normal physiology. In addition, because this technique allows multiple areas to be easily probed, it offers the opportunity for wide area coverage at high resolution. PMID:24086796

High resolution IVEM tomography of biological specimens

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

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

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

Cardiovascular Imaging Using Two-Photon Microscopy

PubMed Central

Scherschel, John A.; Rubart, Michael

2008-01-01

Two-photon excitation microscopy has become the standard technique for high resolution deep tissue and intravital imaging. It provides intrinsic three-dimensional resolution in combination with increased penetration depth compared to single-photon confocal microscopy. This article will describe the basic physical principles of two-photon excitation and will review its multiple applications to cardiovascular imaging, including second harmonic generation and fluorescence laser scanning microscopy. In particular, the capability and limitations of multiphoton microscopy to assess functional heterogeneity on a cellular scale deep within intact, Langendorff-perfused hearts are demonstrated. It will also discuss the use of two-photon excitation-induced release of caged compounds for the study of intracellular calcium signaling and intercellular dye transfer. PMID:18986603

Robust-mode analysis of hydrodynamic flows

NASA Astrophysics Data System (ADS)

Roy, Sukesh; Gord, James R.; Hua, Jia-Chen; Gunaratne, Gemunu H.

2017-04-01

The emergence of techniques to extract high-frequency high-resolution data introduces a new avenue for modal decomposition to assess the underlying dynamics, especially of complex flows. However, this task requires the differentiation of robust, repeatable flow constituents from noise and other irregular features of a flow. Traditional approaches involving low-pass filtering and principle components analysis have shortcomings. The approach outlined here, referred to as robust-mode analysis, is based on Koopman decomposition. Three applications to (a) a counter-rotating cellular flame state, (b) variations in financial markets, and (c) turbulent injector flows are provided.

Serial Femtosecond Crystallography of G Protein-Coupled Receptors

PubMed Central

Liu, Wei; Wacker, Daniel; Gati, Cornelius; Han, Gye Won; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; Katritch, Vsevolod; Barty, Anton; Zatsepin, Nadia A.; Li, Dianfan; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Koglin, Jason E.; Seibert, M. Marvin; Wang, Chong; Shah, Syed T.A.; Basu, Shibom; Fromme, Raimund; Kupitz, Christopher; Rendek, Kimberley N.; Grotjohann, Ingo; Fromme, Petra; Kirian, Richard A.; Beyerlein, Kenneth R.; White, Thomas A.; Chapman, Henry N.; Caffrey, Martin; Spence, John C.H.; Stevens, Raymond C.; Cherezov, Vadim

2014-01-01

X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. Here we used an x-ray free-electron laser (XFEL) with individual 50-fs duration x-ray pulses to minimize radiation damage and obtained a high-resolution room temperature structure of a human serotonin receptor using sub-10 µm microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared to the structure solved by traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment. PMID:24357322

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

NASA Astrophysics Data System (ADS)

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

2014-01-01

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

Toward correlating structure and mechanics of platelets.

PubMed

Sorrentino, Simona; Studt, Jan-Dirk; Horev, Melanie Bokstad; Medalia, Ohad; Sapra, K Tanuj

2016-09-02

The primary physiological function of blood platelets is to seal vascular lesions after injury and form hemostatic thrombi in order to prevent blood loss. This task relies on the formation of strong cellular-extracellular matrix interactions in the subendothelial lesions. The cytoskeleton of a platelet is key to all of its functions: its ability to spread, adhere and contract. Despite the medical significance of platelets, there is still no high-resolution structural information of their cytoskeleton. Here, we discuss and present 3-dimensional (3D) structural analysis of intact platelets by using cryo-electron tomography (cryo-ET) and atomic force microscopy (AFM). Cryo-ET provides in situ structural analysis and AFM gives stiffness maps of the platelets. In the future, combining high-resolution structural and mechanical techniques will bring new understanding of how structural changes modulate platelet stiffness during activation and adhesion.

High-resolution structure of the Escherichia coli ribosome

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

Noeske, Jonas; Wasserman, Michael R.; Terry, Daniel S.

Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation remain poorly understood. Moreover, the function of modifications to ribosomal RNA and ribosomal proteins are unclear. Here we present the structure of the Escherichia coli 70S ribosome to 2.4 Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and suggest how solvation contributes to ribosome integrity and function. The structure also suggests how the conformation of ribosomal protein uS12 likely impacts its contribution to messenger RNA decoding. Inmore » conclusion, this structure helps to explain the phylogenetic conservation of key elements of the ribosome, including posttranscriptional and posttranslational modifications and should serve as a basis for future antibiotic development.« less

High-resolution structure of the Escherichia coli ribosome

DOE PAGES

Noeske, Jonas; Wasserman, Michael R.; Terry, Daniel S.; ...

2015-03-16

Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation remain poorly understood. Moreover, the function of modifications to ribosomal RNA and ribosomal proteins are unclear. Here we present the structure of the Escherichia coli 70S ribosome to 2.4 Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and suggest how solvation contributes to ribosome integrity and function. The structure also suggests how the conformation of ribosomal protein uS12 likely impacts its contribution to messenger RNA decoding. Inmore » conclusion, this structure helps to explain the phylogenetic conservation of key elements of the ribosome, including posttranscriptional and posttranslational modifications and should serve as a basis for future antibiotic development.« less

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

PubMed Central

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

2013-01-01

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

Macrophage and T cell dynamics during the development and disintegration of mycobacterial granulomas.

PubMed

Egen, Jackson G; Rothfuchs, Antonio Gigliotti; Feng, Carl G; Winter, Nathalie; Sher, Alan; Germain, Ronald N

2008-02-01

Granulomas play a key role in host protection against mycobacterial pathogens, with their breakdown contributing to exacerbated disease. To better understand the initiation and maintenance of these structures, we employed both high-resolution multiplex static imaging and intravital multiphoton microscopy of Mycobacterium bovis BCG-induced liver granulomas. We found that Kupffer cells directly capture blood-borne bacteria and subsequently nucleate formation of a nascent granuloma by recruiting both uninfected liver-resident macrophages and blood-derived monocytes. Within the mature granuloma, these myeloid cell populations formed a relatively immobile cellular matrix that interacted with a highly dynamic effector T cell population. The efficient recruitment of these T cells was highly dependent on TNF-alpha-derived signals, which also maintained the granuloma structure through preferential effects on uninfected macrophage populations. By characterizing the migration of both innate and adaptive immune cells throughout the process of granuloma development, these studies provide a new perspective on the cellular events involved in mycobacterial containment and escape.

Label-free chemical imaging of live Euglena gracilis by high-speed SRS spectral microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Wakisaka, Yoshifumi; Suzuki, Yuta; Tokunaga, Kyoya; Hirose, Misa; Domon, Ryota; Akaho, Rina; Kuroshima, Mai; Tsumura, Norimichi; Shimobaba, Tomoyoshi; Iwata, Osamu; Suzuki, Kengo; Nakashima, Ayaka; Goda, Keisuke; Ozeki, Yasuyuki

2016-03-01

Microbes, especially microalgae, have recently been of great interest for developing novel biofuels, drugs, and biomaterials. Imaging-based screening of live cells can provide high selectivity and is attractive for efficient bio-production from microalgae. Although conventional cellular screening techniques use cell labeling, labeling of microbes is still under development and can interfere with their cellular functions. Furthermore, since live microbes move and change their shapes rapidly, a high-speed imaging technique is required to suppress motion artifacts. Stimulated Raman scattering (SRS) microscopy allows for label-free and high-speed spectral imaging, which helps us visualize chemical components inside biological cells and tissues. Here we demonstrate high-speed SRS imaging, with temporal resolution of 0.14 seconds, of intracellular distributions of lipid, polysaccharide, and chlorophyll concentrations in rapidly moving Euglena gracilis, a unicellular phytoflagellate. Furthermore, we show that our method allows us to analyze the amount of chemical components inside each living cell. Our results indicate that SRS imaging may be applied to label-free screening of living microbes based on chemical information.

LABORATORY EVALUATION OF A MICROFLUIDIC ELECTROCHEMICAL SENSOR FOR AEROSOL OXIDATIVE LOAD.

PubMed

Koehler, Kirsten; Shapiro, Jeffrey; Sameenoi, Yupaporn; Henry, Charles; Volckens, John

2014-05-01

Human exposure to particulate matter (PM) air pollution is associated with human morbidity and mortality. The mechanisms by which PM impacts human health are unresolved, but evidence suggests that PM intake leads to cellular oxidative stress through the generation of reactive oxygen species (ROS). Therefore, reliable tools are needed for estimating the oxidant generating capacity, or oxidative load, of PM at high temporal resolution (minutes to hours). One of the most widely reported methods for assessing PM oxidative load is the dithiothreitol (DTT) assay. The traditional DTT assay utilizes filter-based PM collection in conjunction with chemical analysis to determine the oxidation rate of reduced DTT in solution with PM. However, the traditional DTT assay suffers from poor time resolution, loss of reactive species during sampling, and high limit of detection. Recently, a new DTT assay was developed that couples a Particle-Into-Liquid-Sampler with microfluidic-electrochemical detection. This 'on-line' system allows high temporal resolution monitoring of PM reactivity with improved detection limits. This study reports on a laboratory comparison of the traditional and on-line DTT approaches. An urban dust sample was aerosolized in a laboratory test chamber at three atmospherically-relevant concentrations. The on-line system gave a stronger correlation between DTT consumption rate and PM mass (R 2 = 0.69) than the traditional method (R 2 = 0.40) and increased precision at high temporal resolution, compared to the traditional method.

Phenotypic diversity in autosomal-dominant cone-rod dystrophy elucidated by adaptive optics retinal imaging

PubMed Central

Song, Hongxin; Rossi, Ethan A; Stone, Edwin; Latchney, Lisa; Williams, David; Dubra, Alfredo; Chung, Mina

2018-01-01

Purpose Several genes causing autosomal-dominant cone-rod dystrophy (AD-CRD) have been identified. However, the mechanisms by which genetic mutations lead to cellular loss in human disease remain poorly understood. Here we combine genotyping with high-resolution adaptive optics retinal imaging to elucidate the retinal phenotype at a cellular level in patients with AD-CRD harbouring a defect in the GUCA1A gene. Methods Nine affected members of a four-generation AD-CRD pedigree and three unaffected first-degree relatives underwent clinical examinations including visual acuity, fundus examination, Goldmann perimetry, spectral domain optical coherence tomography and electroretinography. Genome-wide scan followed by bidirectional sequencing was performed on all affected participants. High-resolution imaging using a custom adaptive optics scanning light ophthalmoscope (AOSLO) was performed for selected participants. Results Clinical evaluations showed a range of disease severity from normal fundus appearance in teenaged patients to pronounced macular atrophy in older patients. Molecular genetic testing showed a mutation in in GUCA1A segregating with disease. AOSLO imaging revealed that of the two teenage patients with mild disease, one had severe disruption of the photoreceptor mosaic while the other had a normal cone mosaic. Conclusions AOSLO imaging demonstrated variability in the pattern of cone and rod cell loss between two teenage cousins with early AD-CRD, who had similar clinical features and had the identical disease-causing mutation in GUCA1A. This finding suggests that a mutation in GUCA1A does not lead to the same degree of AD-CRD in all patients. Modifying factors may mitigate or augment disease severity, leading to different retinal cellular phenotypes. PMID:29074494

Novel method for water vapour monitoring using wireless communication networks measurements

NASA Astrophysics Data System (ADS)

David, N.; Alpert, P.; Messer, H.

2009-04-01

We propose a new technique for monitoring near-surface water vapour, by estimating humidity from data collected through existing wireless communication networks. Water vapour plays a crucial part in a variety of atmospheric processes. As the most influential of greenhouse gases, it absorbs long-wave terrestrial radiation. The water vapour cycle of evaporation and recondensation is a major energy redistributing mechanism transferring heat energy from the Earth's surface to the atmosphere. Additionally, humidity has an important role in weather forecasting as a key variable required for initialization of atmospheric models and hazard warning techniques. However, current methods of monitoring humidity suffer from low spatial resolution, high cost or a lack of precision when measuring near ground levels. Weather conditions and atmospheric phenomena affect the electromagnetic channel, causing attenuations to the radio signals. Thus, wireless communication networks are in effect built-in environmental monitoring facilities. The wireless microwave links, used in these networks, are widely deployed by cellular providers for backhaul communication between base stations, a few tens of meters above ground level. As a result, the proposed method can provide moisture observations at high temporal and spatial resolution. Further, the implementation cost is minimal, since the data used is already collected and saved by the cellular operators. In addition - many of these links are installed in areas where access is difficult such as orographic terrain and complex topography. As such, our method enables measurements in places that have been hard to measure in the past, or have never been measured before. The technique is restricted to weather conditions which include absence of rain, fog or clouds along the propagation path. We present results from real-data measurements taken from microwave links used in a backhaul cellular network that show very good agreement with surface station humidity measurements.

SECM half-inch tethered endoscopic capsule (HITEC) for esophageal imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kang, DongKyun; Kim, Minkyu; Carruth, Robert W.; Lu, Weina; Wu, Tao; Alali, Sanaz; Do, Dukho; Soomro, Amna R.; Grant, Catriona N.; Tiernan, Aubrey R.; Rosenberg, Mireille; Nishioka, Norman S.; Tearney, Guillermo J.

2016-03-01

Spectrally encoded confocal microscopy (SECM) is a high-speed confocal endomicroscopy technology that can image extremely large regions of human tissue at cellular resolution within a short imaging time. Previously, we have developed a 7-mm-diameter SECM endoscopic capsule and successfully demonstrated imaging of human esophagus in vivo. Even though we were able to successfully capture images with the previous capsule, it suffered from two limitations: (1) the capsule had a small diameter, which provided a limited contact between SECM capsule and esophagus; and (2) speckle noise in SECM images made it challenging to appreciate cellular features. In this paper, we present a new SECM capsule, termed SECM half-inch tethered endoscopic capsule (HITEC), which addresses the two aforementioned technical challenges. With the SECM HITEC, a dual-clad fiber was used to reduce the speckle noise. Miniature GRIN optics was used to increase the NA of the fiber from 0.09 to 0.25, which made it possible to build a SECM capsule with large diameter (12.7 mm) while maintaining a short rigid length (22 mm). A water-immersion objective lens was custom designed and manufactured to provide high NA of 0.7. We have manufactured the SECM HITEC catheter and tested its optical and mechanical performance. Lateral and axial resolution was measured as 1.2 µm and 13 µm, respectively. We have imaged swine esophageal tissues ex vivo, and SECM images clearly visualized cell nuclei. Non-uniform rotational distortion (NURD) was small, less than 5%. Preliminary results suggest that SECM HITEC provides sufficient optical and mechanical performance for tissue imaging. In a future clinical study, we will test the feasibility of utilizing SECM HITEC for improved cellular imaging human of the human esophagus in vivo.

Phenotypic diversity in autosomal-dominant cone-rod dystrophy elucidated by adaptive optics retinal imaging.

PubMed

Song, Hongxin; Rossi, Ethan A; Stone, Edwin; Latchney, Lisa; Williams, David; Dubra, Alfredo; Chung, Mina

2018-01-01

Several genes causing autosomal-dominant cone-rod dystrophy (AD-CRD) have been identified. However, the mechanisms by which genetic mutations lead to cellular loss in human disease remain poorly understood. Here we combine genotyping with high-resolution adaptive optics retinal imaging to elucidate the retinal phenotype at a cellular level in patients with AD-CRD harbouring a defect in the GUCA1A gene. Nine affected members of a four-generation AD-CRD pedigree and three unaffected first-degree relatives underwent clinical examinations including visual acuity, fundus examination, Goldmann perimetry, spectral domain optical coherence tomography and electroretinography. Genome-wide scan followed by bidirectional sequencing was performed on all affected participants. High-resolution imaging using a custom adaptive optics scanning light ophthalmoscope (AOSLO) was performed for selected participants. Clinical evaluations showed a range of disease severity from normal fundus appearance in teenaged patients to pronounced macular atrophy in older patients. Molecular genetic testing showed a mutation in in GUCA1A segregating with disease. AOSLO imaging revealed that of the two teenage patients with mild disease, one had severe disruption of the photoreceptor mosaic while the other had a normal cone mosaic. AOSLO imaging demonstrated variability in the pattern of cone and rod cell loss between two teenage cousins with early AD-CRD, who had similar clinical features and had the identical disease-causing mutation in GUCA1A . This finding suggests that a mutation in GUCA1A does not lead to the same degree of AD-CRD in all patients. Modifying factors may mitigate or augment disease severity, leading to different retinal cellular phenotypes. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

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.

X-ray micro-tomography for investigations of brain tissues on cellular level

NASA Astrophysics Data System (ADS)

Khimchenko, Anna; Schulz, Georg; Deyhle, Hans; Thalmann, Peter; Zanette, Irene; Zdora, Marie-Christine; Bikis, Christos; Hipp, Alexander; Hieber, Simone E.; Schweighauser, Gabriel; Hench, Jürgen; Müller, Bert

2016-10-01

X-ray imaging in absorption contrast mode is well established for hard tissue visualization. However, performance for lower density materials is limited due to a reduced contrast. Our aim is three-dimensional (3D) characterization of micro-morphology of human brain tissues down to (sub-)cellular resolution within a laboratory environment. Using the laboratory-based microtomography (μCT) system nanotom m (GE Sensing and Inspection Technologies GmbH, Wunstorf, Germany) and synchrotron radiation at the Diamond-Manchester Imaging Branchline I13-2 (Diamond Light Source, Didcot, UK), we have acquired 3D data with a resolution down to 0.45 μm for visualization of a human cerebellum specimen down to cellular level. We have shown that all selected modalities, namely laboratory-based absorption contrast micro-tomography (LBμCT), synchrotron radiation based in-line single distance phase contrast tomography (SDPR) and synchrotron radiation based single-grating interferometry (GI), can reach cellular resolution for tissue samples with a size in the mm-range. The results are discussed qualitatively in comparison to optical microscopy of haematoxylin and eosin (HE) stained sections. As phase contrast yields to a better data quality for soft tissues and in order to overcome restrictions of limited beamline access for phase contrast measurements, we have equipped the μCT system nanotom m with a double-grating phase contrast set-up. Preliminary experimental results of a knee sample consisting of a bony part and a cartilage demonstrate that phase contrast data exhibits better quality compared to absorption contrast. Currently, the set-up is under adjustment. It is expected that cellular resolution would also be achieved. The questions arise (1) what would be the quality gain of laboratory-based phase contrast in comparison to laboratory-based absorption contrast tomography and (2) could laboratory-based phase contrast data provide comparable results to synchrotron radiation based phase contrast data.

Visual analysis of mass cytometry data by hierarchical stochastic neighbour embedding reveals rare cell types.

PubMed

van Unen, Vincent; Höllt, Thomas; Pezzotti, Nicola; Li, Na; Reinders, Marcel J T; Eisemann, Elmar; Koning, Frits; Vilanova, Anna; Lelieveldt, Boudewijn P F

2017-11-23

Mass cytometry allows high-resolution dissection of the cellular composition of the immune system. However, the high-dimensionality, large size, and non-linear structure of the data poses considerable challenges for the data analysis. In particular, dimensionality reduction-based techniques like t-SNE offer single-cell resolution but are limited in the number of cells that can be analyzed. Here we introduce Hierarchical Stochastic Neighbor Embedding (HSNE) for the analysis of mass cytometry data sets. HSNE constructs a hierarchy of non-linear similarities that can be interactively explored with a stepwise increase in detail up to the single-cell level. We apply HSNE to a study on gastrointestinal disorders and three other available mass cytometry data sets. We find that HSNE efficiently replicates previous observations and identifies rare cell populations that were previously missed due to downsampling. Thus, HSNE removes the scalability limit of conventional t-SNE analysis, a feature that makes it highly suitable for the analysis of massive high-dimensional data sets.

Wood Cellular Dendroclimatology: A Pilot Study on Bristlecone Pine in the Southwest US

NASA Astrophysics Data System (ADS)

Ziaco, E.; Biondi, F.; Heinrich, I.

2015-12-01

Tree-rings provide paleoclimatic records at annual to seasonal resolution for regions or periods with no instrumental climatic data. Relationships between climatic variability and wood cellular features allow for a more complete understanding of the physiological mechanisms that control the climatic response of trees. Given the increasing importance of wood anatomy as a source of dendroecological information, such studies are now starting in the US. We analyzed 10 cores of bristlecone pine (Pinus longaeva D.K. Bailey) from a high-elevation site included in the Nevada Climate-ecohydrological Assessment Network (NevCAN). Century-long chronologies (1870-2013) of wood anatomical parameters (lumen area, cell diameter, cell wall thickness) can be developed by capturing strongly contrasted microscopic images using a Confocal Laser Scanning Microscope, and then measuring cellular parameters with task-specific software. Measures of empirical signal strength were used to test the strength of the environmental information embedded in wood anatomy. Correlation functions between ring-width, cellular features, and PRISM climatic variables were produced for the period 1926-2013. Time series of anatomical features present lower autocorrelation compared to ring widths, highlighting the role of environmental conditions occurring at the time of cell formation. Mean chronologies of radial lumen length and cell diameter carry a stronger climatic signal compared to cell wall thickness, and are significantly correlated with climatic variables (maximum temperature and total precipitation) in spring (Mar-Apr) and during the growing season (Jun-Sep), whereas ring widths show weaker or no correlation. Wood anatomy holds great potential to refine dendroclimatic reconstructions at higher temporal resolution, providing better estimates of hydroclimatic variability and plant physiological adaptations in the southwest US.

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

PubMed

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

2010-11-19

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

Optical properties of photoreceptor and retinal pigment epithelium cells investigated with adaptive optics optical coherence tomography

NASA Astrophysics Data System (ADS)

Liu, Zhuolin

Human vision starts when photoreceptors collect and respond to light. Photoreceptors do not function in isolation though, but share close interdependence with neighboring photoreceptors and underlying retinal pigment epithelium (RPE) cells. These cellular interactions are essential for normal function of the photoreceptor-RPE complex, but methods to assess these in the living human eye are limited. One approach that has gained increased promise is high-resolution retinal imaging that has undergone tremendous technological advances over the last two decades to probe the living retina at the cellular level. Pivotal in these advances has been adaptive optics (AO) and optical coherence tomography (OCT) that together allow unprecedented spatial resolution of retinal structures in all three dimensions. Using these high-resolution systems, cone photoreceptor are now routinely imaged in healthy and diseased retina enabling fundamental structural properties of cones to be studied such as cell spacing, packing arrangement, and alignment. Other important cell properties, however, have remained elusive to investigation as even better imaging performance is required and thus has resulted in an incomplete understanding of how cells in the photoreceptor-RPE complex interact with light. To address this technical bottleneck, we expanded the imaging capability of AO-OCT to detect and quantify more accurately and completely the optical properties of cone photoreceptor and RPE cells at the cellular level in the living human retina. The first objective of this thesis was development of a new AO-OCT method that is more precise and sensitive, thus enabling a more detailed view of the 3D optical signature of the photoreceptor-RPE complex than was previously possible (Chapter 2). Using this new system, the second objective was quantifying the waveguide properties of individual cone photoreceptor inner and outer segments across the macula (Chapter 3). The third objective extended the AO-OCT method to RPE cell imaging. This entailed using AO-OCT in conjunction with organelle motility as a novel contrast mechanism to visualize RPE cells and to characterize their 3D reflectance profile (Chapter 4).

Metabolomics for undergraduates: Identification and pathway assignment of mitochondrial metabolites.

PubMed

Marques, Ana Patrícia; Serralheiro, Maria Luisa; Ferreira, António E N; Freire, Ana Ponces; Cordeiro, Carlos; Silva, Marta Sousa

2016-01-01

Metabolomics is a key discipline in systems biology, together with genomics, transcriptomics, and proteomics. In this omics cascade, the metabolome represents the biochemical products that arise from cellular processes and is often regarded as the final response of a biological system to environmental or genetic changes. The overall screening approach to identify all the metabolites in a given biological system is called metabolic fingerprinting. Using high-resolution and high-mass accuracy mass spectrometry, large metabolome coverage, sensitivity, and specificity can be attained. Although the theoretical concepts of this methodology are usually provided in life-science programs, hands-on laboratory experiments are not usually accessible to undergraduate students. Even if the instruments are available, there are not simple laboratory protocols created specifically for teaching metabolomics. We designed a straightforward hands-on laboratory experiment to introduce students to this methodology, relating it to biochemical knowledge through metabolic pathway mapping of the identified metabolites. This study focuses on mitochondrial metabolomics since mitochondria have a well-known, medium-sized cellular sub-metabolome. These features facilitate both data processing and pathway mapping. In this experiment, students isolate mitochondria from potatoes, extract the metabolites, and analyze them by high-resolution mass spectrometry (using an FT-ICR mass spectrometer). The resulting mass list is submitted to an online program for metabolite identification, and compounds associated with mitochondrial pathways can be highlighted in a metabolic network map. © 2015 The International Union of Biochemistry and Molecular Biology.

Multicellular tumor spheroids as an in vivo-like tumor model for three-dimensional imaging of chemotherapeutic and nano material cellular penetration.

PubMed

Ma, Hui-li; Jiang, Qiao; Han, Siyuan; Wu, Yan; Cui Tomshine, Jin; Wang, Dongliang; Gan, Yaling; Zou, Guozhang; Liang, Xing-Jie

2012-01-01

We present a flexible and highly reproducible method using three-dimensional (3D) multicellular tumor spheroids to quantify chemotherapeutic and nanoparticle penetration properties in vitro. We generated HeLa cell-derived spheroids using the liquid overlay method. To properly characterize HeLa spheroids, scanning electron microscopy, transmission electron microscopy, and multiphoton microscopy were used to obtain high-resolution 3D images of HeLa spheroids. Next, pairing high-resolution optical characterization techniques with flow cytometry, we quantitatively compared the penetration of doxorubicin, quantum dots, and synthetic micelles into 3D HeLa spheroid versus HeLa cells grown in a traditional two-dimensional culturing system. Our data revealed that 3D cultured HeLa cells acquired several clinically relevant morphologic and cellular characteristics (such as resistance to chemotherapeutics) often found in human solid tumors. These characteristic, however, could not be captured using conventional two-dimensional cell culture techniques. This study demonstrated the remarkable versatility of HeLa spheroid 3D imaging. In addition, our results revealed the capability of HeLa spheroids to function as a screening tool for nanoparticles or synthetic micelles that, due to their inherent size, charge, and hydrophobicity, can penetrate into solid tumors and act as delivery vehicles for chemotherapeutics. The development of this image-based, reproducible, and quantifiable in vitro HeLa spheroid screening tool will greatly aid future exploration of chemotherapeutics and nanoparticle delivery into solid tumors.

Measuring spatial and temporal Ca2+ signals in Arabidopsis plants.

PubMed

Zhu, Xiaohong; Taylor, Aaron; Zhang, Shenyu; Zhang, Dayong; Feng, Ying; Liang, Gaimei; Zhu, Jian-Kang

2014-09-02

Developmental and environmental cues induce Ca(2+) fluctuations in plant cells. Stimulus-specific spatial-temporal Ca(2+) patterns are sensed by cellular Ca(2+) binding proteins that initiate Ca(2+) signaling cascades. However, we still know little about how stimulus specific Ca(2+) signals are generated. The specificity of a Ca(2+) signal may be attributed to the sophisticated regulation of the activities of Ca(2+) channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca(2+) signals at both the tissue and cellular levels. Genetically encoded Ca(2+) indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca(2+) signals. Here we describe instructions for the use of two Ca(2+) detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca(2+) imaging and case12 based live cell confocal fluorescence Ca(2+) imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca(2+) signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca(2+) signals at a high resolution.

Optical Imaging with a High Resolution Microendoscope to Identify Cholesteatoma of the Middle Ear

PubMed Central

Levy, Lauren L.; Jiang, Nancy; Smouha, Eric; Richards-Kortum, Rebecca; Sikora, Andrew G.

2013-01-01

Objective High resolution optical imaging is an imaging modality which allows visualization of structural changes in epithelial tissue in real time. Our prior studies using contrast-enhanced microendoscopy to image squamous cell carcinoma in the head and neck demonstrated that the contrast agent, proflavine, has high affinity for keratinized tissue. Thus, high-resolution microendoscopy with proflavine provides a potential mechanism to identify ectopic keratin production, such as that associated with cholesteatoma formation and distinguish between uninvolved mucosa and residual keratin at the time of surgery. Study Design Ex vivo imaging of histopathologically-confirmed samples of cholesteatoma and uninvolved middle-ear epithelium. Methods Seven separate specimens collected from patients who underwent surgical treatment for cholesteatoma were imaged ex vivo with the fiberoptic endoscope after surface staining with proflavine. Following imaging, the specimens were submitted for hematoxylin &eosin staining to allow histopathological correlation. Results Cholesteatoma and surrounding middle ear epithelium have distinct imaging characteristics. Keratin-bearing areas of cholesteatoma lack nuclei and appear as confluent hyperfluorescence, while nuclei are easily visualized in specimens containing normal middle ear epithelium. Hyperfluorescence and loss of cellular detail is the imaging hallmark of keratin allowing for discrimination of cholesteatoma from normal middle ear epithelium. Conclusions This study demonstrates the feasibility of high-resolution optical imaging to discriminate cholesteatoma from uninvolved middle ear mucosa, based on the unique staining properties of keratin. Use of real-time imaging may facilitate more complete extirpation of cholesteatoma by identifying areas of residual disease. PMID:23299781

Length of intact plasma membrane determines the diffusion properties of cellular water.

PubMed

Eida, Sato; Van Cauteren, Marc; Hotokezaka, Yuka; Katayama, Ikuo; Sasaki, Miho; Obara, Makoto; Okuaki, Tomoyuki; Sumi, Misa; Nakamura, Takashi

2016-01-11

Molecular diffusion in a boundary-free medium depends only on the molecular size, the temperature, and medium viscosity. However, the critical determinant of the molecular diffusion property in inhomogeneous biological tissues has not been identified. Here, using an in vitro system and a high-resolution MR imaging technique, we show that the length of the intact plasma membrane is a major determinant of water diffusion in a controlled cellular environment and that the cell perimeter length (CPL) is sufficient to estimate the apparent diffusion coefficient (ADC) of water in any cellular environment in our experimental system (ADC = -0.21 × CPL + 1.10). We used this finding to further explain the different diffusion kinetics of cells that are dying via apoptotic or non-apoptotic cell death pathways exhibiting characteristic changes in size, nuclear and cytoplasmic architectures, and membrane integrity. These results suggest that the ADC value can be used as a potential biomarker for cell death.

Length of intact plasma membrane determines the diffusion properties of cellular water

PubMed Central

Eida, Sato; Van Cauteren, Marc; Hotokezaka, Yuka; Katayama, Ikuo; Sasaki, Miho; Obara, Makoto; Okuaki, Tomoyuki; Sumi, Misa; Nakamura, Takashi

2016-01-01

Molecular diffusion in a boundary-free medium depends only on the molecular size, the temperature, and medium viscosity. However, the critical determinant of the molecular diffusion property in inhomogeneous biological tissues has not been identified. Here, using an in vitro system and a high-resolution MR imaging technique, we show that the length of the intact plasma membrane is a major determinant of water diffusion in a controlled cellular environment and that the cell perimeter length (CPL) is sufficient to estimate the apparent diffusion coefficient (ADC) of water in any cellular environment in our experimental system (ADC = −0.21 × CPL + 1.10). We used this finding to further explain the different diffusion kinetics of cells that are dying via apoptotic or non-apoptotic cell death pathways exhibiting characteristic changes in size, nuclear and cytoplasmic architectures, and membrane integrity. These results suggest that the ADC value can be used as a potential biomarker for cell death. PMID:26750342

Label-free imaging of gold nanoparticles in single live cells by photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

2016-03-01

Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

Geomorphological Mapping of Sputnik Planum on Pluto: Convection, Glacial Flow, Sublimation and Re-deposition of Nitrogen Ice

NASA Astrophysics Data System (ADS)

White, O. L.; Moore, J. M.; Stern, S. A.; Weaver, H. A., Jr.; Olkin, C.; Ennico Smith, K.; Young, L. A.; Cheng, A. F.

2016-12-01

The New Horizons flyby of Pluto provided extensive high-resolution coverage of its encounter hemisphere. The most prominent surface feature in this hemisphere is the high albedo region informally named Tombaugh Regio, the western portion of which is represented by the expansive nitrogen ice plains informally named Sputnik Planum. A large fraction of Sputnik Planum displays a distinct cellular pattern, with individual cells typically displaying ovoid planforms and shallow pitting on a scale of a few hundred meters. Troughs with medial ridges define the boundaries between cells. Prior studies have argued that this pattern is indicative of solid-state convection occurring within the nitrogen ice. The southern non-cellular plains are either featureless or display dense fields of often elongate and aligned pits typically reaching a few km across, interpreted to have formed via sublimation. The mapping that will be presented at AGU focuses on identifying the different plains units that compose Sputnik Planum and defining the boundaries between them, which aids in assessing their time sequencing and correlation to one another. The cellular plains are divided into bright and dark units, with the bright unit forming a continuous high albedo zone with the bright uplands of east Tombaugh Regio. We interpret the dark plains to represent the main body of convecting N2 ice that forms the cellular plains of Sputnik Planum, with the low albedo caused by a high concentration of entrained dark material (likely tholins). Preferential sublimation of N2 ice from these plains would leave the dark ice exposed, and re-deposition of the N2 ice on the eastern cellular plains and uplands of east Tombaugh Regio would create a thin veneer of pure, bright N2 ice covering these landscapes. The non-cellular plains are universally bright and display evidence for southwards flow of the N2 ice, based on the orientations of fields of elongate sublimation pits as well as the presence of `extinct cells' that appear to have migrated away from the zone of active convection. The larger pits that occur within the non-cellular plains imply that these plains are older than the cellular plains, where resurfacing via convection limits the maximum size attainable by sublimation pits.

Identification and High-Resolution Imaging of α-Tocopherol from Human Cells to Whole Animals by TOF-SIMS Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Bruinen, Anne L.; Fisher, Gregory L.; Balez, Rachelle; van der Sar, Astrid M.; Ooi, Lezanne; Heeren, Ron M. A.

2018-06-01

A unique method for identification of biomolecular components in different biological specimens, while preserving the capability for high speed 2D and 3D molecular imaging, is employed to investigate cellular response to oxidative stress. The employed method enables observing the distribution of the antioxidant α-tocopherol and other molecules in cellular structures via time-of-flight secondary ion mass spectrometry (TOF-SIMS (MS1)) imaging in parallel with tandem mass spectrometry (MS2) imaging, collected simultaneously. The described method is employed to examine a network formed by neuronal cells differentiated from human induced pluripotent stem cells (iPSCs), a model for investigating human neurons in vitro. The antioxidant α-tocopherol is identified in situ within different cellular layers utilizing a 3D TOF-SIMS tandem MS imaging analysis. As oxidative stress also plays an important role in mediating inflammation, the study was expanded to whole body tissue sections of M. marinum-infected zebrafish, a model organism for tuberculosis. The TOF-SIMS tandem MS imaging results reveal an increased presence of α-tocopherol in response to the pathogen. [Figure not available: see fulltext.

A high-content image-based method for quantitatively studying context-dependent cell population dynamics

PubMed Central

Garvey, Colleen M.; Spiller, Erin; Lindsay, Danika; Chiang, Chun-Te; Choi, Nathan C.; Agus, David B.; Mallick, Parag; Foo, Jasmine; Mumenthaler, Shannon M.

2016-01-01

Tumor progression results from a complex interplay between cellular heterogeneity, treatment response, microenvironment and heterocellular interactions. Existing approaches to characterize this interplay suffer from an inability to distinguish between multiple cell types, often lack environmental context, and are unable to perform multiplex phenotypic profiling of cell populations. Here we present a high-throughput platform for characterizing, with single-cell resolution, the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations both during standard growth and in response to multiple, co-occurring selective pressures. The speed of this platform enables a thorough investigation of the impacts of diverse selective pressures including genetic alterations, therapeutic interventions, heterocellular components and microenvironmental factors. The platform has been applied to both 2D and 3D culture systems and readily distinguishes between (1) cytotoxic versus cytostatic cellular responses; and (2) changes in morphological features over time and in response to perturbation. These important features can directly influence tumor evolution and clinical outcome. Our image-based approach provides a deeper insight into the cellular dynamics and heterogeneity of tumors (or other complex systems), with reduced reagents and time, offering advantages over traditional biological assays. PMID:27452732

A high-content image-based method for quantitatively studying context-dependent cell population dynamics

NASA Astrophysics Data System (ADS)

Garvey, Colleen M.; Spiller, Erin; Lindsay, Danika; Chiang, Chun-Te; Choi, Nathan C.; Agus, David B.; Mallick, Parag; Foo, Jasmine; Mumenthaler, Shannon M.

2016-07-01

Tumor progression results from a complex interplay between cellular heterogeneity, treatment response, microenvironment and heterocellular interactions. Existing approaches to characterize this interplay suffer from an inability to distinguish between multiple cell types, often lack environmental context, and are unable to perform multiplex phenotypic profiling of cell populations. Here we present a high-throughput platform for characterizing, with single-cell resolution, the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations both during standard growth and in response to multiple, co-occurring selective pressures. The speed of this platform enables a thorough investigation of the impacts of diverse selective pressures including genetic alterations, therapeutic interventions, heterocellular components and microenvironmental factors. The platform has been applied to both 2D and 3D culture systems and readily distinguishes between (1) cytotoxic versus cytostatic cellular responses; and (2) changes in morphological features over time and in response to perturbation. These important features can directly influence tumor evolution and clinical outcome. Our image-based approach provides a deeper insight into the cellular dynamics and heterogeneity of tumors (or other complex systems), with reduced reagents and time, offering advantages over traditional biological assays.

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

PubMed Central

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

2010-01-01

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

Chemotaxis of Cell Populations through Confined Spaces at Single-Cell Resolution

PubMed Central

Tong, ZiQiu; Balzer, Eric M.; Dallas, Matthew R.; Hung, Wei-Chien; Stebe, Kathleen J.; Konstantopoulos, Konstantinos

2012-01-01

Cell migration is crucial for both physiological and pathological processes. Current in vitro cell motility assays suffer from various drawbacks, including insufficient temporal and/or optical resolution, or the failure to include a controlled chemotactic stimulus. Here, we address these limitations with a migration chamber that utilizes a self-sustaining chemotactic gradient to induce locomotion through confined environments that emulate physiological settings. Dynamic real-time analysis of both population-scale and single-cell movement are achieved at high resolution. Interior surfaces can be functionalized through adsorption of extracellular matrix components, and pharmacological agents can be administered to cells directly, or indirectly through the chemotactic reservoir. Direct comparison of multiple cell types can be achieved in a single enclosed system to compare inherent migratory potentials. Our novel microfluidic design is therefore a powerful tool for the study of cellular chemotaxis, and is suitable for a wide range of biological and biomedical applications. PMID:22279529

Multi-scale approaches for high-speed imaging and analysis of large neural populations

PubMed Central

Ahrens, Misha B.; Yuste, Rafael; Peterka, Darcy S.; Paninski, Liam

2017-01-01

Progress in modern neuroscience critically depends on our ability to observe the activity of large neuronal populations with cellular spatial and high temporal resolution. However, two bottlenecks constrain efforts towards fast imaging of large populations. First, the resulting large video data is challenging to analyze. Second, there is an explicit tradeoff between imaging speed, signal-to-noise, and field of view: with current recording technology we cannot image very large neuronal populations with simultaneously high spatial and temporal resolution. Here we describe multi-scale approaches for alleviating both of these bottlenecks. First, we show that spatial and temporal decimation techniques based on simple local averaging provide order-of-magnitude speedups in spatiotemporally demixing calcium video data into estimates of single-cell neural activity. Second, once the shapes of individual neurons have been identified at fine scale (e.g., after an initial phase of conventional imaging with standard temporal and spatial resolution), we find that the spatial/temporal resolution tradeoff shifts dramatically: after demixing we can accurately recover denoised fluorescence traces and deconvolved neural activity of each individual neuron from coarse scale data that has been spatially decimated by an order of magnitude. This offers a cheap method for compressing this large video data, and also implies that it is possible to either speed up imaging significantly, or to “zoom out” by a corresponding factor to image order-of-magnitude larger neuronal populations with minimal loss in accuracy or temporal resolution. PMID:28771570

Fluorescence Molecular Tomography: Principles and Potential for Pharmaceutical Research

PubMed Central

Stuker, Florian; Ripoll, Jorge; Rudin, Markus

2011-01-01

Fluorescence microscopic imaging is widely used in biomedical research to study molecular and cellular processes in cell culture or tissue samples. This is motivated by the high inherent sensitivity of fluorescence techniques, the spatial resolution that compares favorably with cellular dimensions, the stability of the fluorescent labels used and the sophisticated labeling strategies that have been developed for selectively labeling target molecules. More recently, two and three-dimensional optical imaging methods have also been applied to monitor biological processes in intact biological organisms such as animals or even humans. These whole body optical imaging approaches have to cope with the fact that biological tissue is a highly scattering and absorbing medium. As a consequence, light propagation in tissue is well described by a diffusion approximation and accurate reconstruction of spatial information is demanding. While in vivo optical imaging is a highly sensitive method, the signal is strongly surface weighted, i.e., the signal detected from the same light source will become weaker the deeper it is embedded in tissue, and strongly depends on the optical properties of the surrounding tissue. Derivation of quantitative information, therefore, requires tomographic techniques such as fluorescence molecular tomography (FMT), which maps the three-dimensional distribution of a fluorescent probe or protein concentration. The combination of FMT with a structural imaging method such as X-ray computed tomography (CT) or Magnetic Resonance Imaging (MRI) will allow mapping molecular information on a high definition anatomical reference and enable the use of prior information on tissue's optical properties to enhance both resolution and sensitivity. Today many of the fluorescent assays originally developed for studies in cellular systems have been successfully translated for experimental studies in animals. The opportunity of monitoring molecular processes non-invasively in the intact organism is highly attractive from a diagnostic point of view but even more so for the drug developer, who can use the techniques for proof-of-mechanism and proof-of-efficacy studies. This review shall elucidate the current status and potential of fluorescence tomography including recent advances in multimodality imaging approaches for preclinical and clinical drug development. PMID:24310495

X-ray fluorescence microscopy reveals the role of selenium in spermatogenesis

PubMed Central

Kehr, Sebastian; Malinouski, Mikalai; Finney, Lydia; Vogt, Stefan; Labunskyy, Vyacheslav M.; Kasaikina, Marina V.; Carlson, Bradley A.; Zhou, You; Hatfield, Dolph L.; Gladyshev, Vadim N.

2009-01-01

Selenium (Se) is a trace element with important roles in human health. Several selenoproteins have essential functions in development. However, the cellular and tissue distribution of Se remains largely unknown because of the lack of analytical techniques that image this element with sufficient sensitivity and resolution. Herein, we report that X-ray fluorescence microscopy (XFM) can be used to visualize and quantify the tissue, cellular and subcellular topography of Se. We applied this technique to characterize the role of Se in spermatogenesis and identified a dramatic Se enrichment specifically in late spermatids, a pattern that was not seen in any other elemental maps. This enrichment was due to elevated levels of the mitochondrial form of glutathione peroxidase 4 and was fully dependent on the supplies of Se by Selenoprotein P. High-resolution scans revealed that Se concentrated near the lumen side of elongating spermatids, where structural components of sperm are formed. During spermatogenesis, maximal Se associated with decreased phosphorus, whereas Zn did not change. In sperm, Se was primarily in the midpiece and co-localized with Cu and Fe. XFM allowed quantification of Se in the midpiece (0.8 fg) and head (0.14 fg) of individual sperm cells, revealing the ability of sperm cells to handle the amounts of this element well above its toxic levels. Overall, the use of XFM allowed visualization of tissue and cellular Se and provided important insights in the role of this and other trace elements in spermatogenesis. PMID:19379757

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

NASA Astrophysics Data System (ADS)

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

2012-11-01

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

Mapping the Complex Morphology of Cell Interactions with Nanowire Substrates Using FIB-SEM

PubMed Central

Jensen, Mikkel R. B.; Łopacińska, Joanna; Schmidt, Michael S.; Skolimowski, Maciej; Abeille, Fabien; Qvortrup, Klaus; Mølhave, Kristian

2013-01-01

Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition and image post-processing were specifically optimised for cellular monolayers cultured on nanostructured substrates. Cells display a wide range of interactions with the nanostructures depending on the surface morphology, but also greatly varying from one cell to another on the same substrate, illustrating a wide phenotypic variability. Depending on the substrate and cell, we observe that cells could for instance: break the nanowires and engulf them, flatten the nanowires or simply reside on top of them. Given the complexity of interactions, we have categorised our observations and created an overview map. The results demonstrate that detailed nanoscale resolution images are required to begin understanding the wide variety of individual cells’ interactions with a structured substrate. The map will provide a framework for light microscopy studies of such interactions indicating what modes of interactions must be considered. PMID:23326412

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.

A detailed study of gold-nanoparticle loaded cells using X-ray based techniques for cell-tracking applications with single-cell sensitivity

NASA Astrophysics Data System (ADS)

Astolfo, Alberto; Arfelli, Fulvia; Schültke, Elisabeth; James, Simon; Mancini, Lucia; Menk, Ralf-Hendrik

2013-03-01

In the present study complementary high-resolution imaging techniques on different length scales are applied to elucidate a cellular loading protocol of gold nanoparticles and subsequently its impact on long term and high-resolution cell-tracking utilizing X-ray technology. Although demonstrated for malignant cell lines the results can be applied to non-malignant cell lines as well. In particular the accumulation of the gold marker per cell has been assessed quantitatively by virtue of electron microscopy, two-dimensional X-ray fluorescence imaging techniques and X-ray CT with micrometric and sub-micrometric resolution. Moreover, utilizing these techniques the three dimensional distribution of the incorporated nanoparticles, which are sequestered in lysosomes as a permanent marker, could be determined. The latter allowed elucidation of the gold partition during mitosis and the cell size, which subsequently enabled us to define the optimal instrument settings of a compact microCT system to visualize gold loaded cells. The results obtained demonstrate the feasibility of cell-tracking using X-ray CT with compact sources.

Creation of a virtual cutaneous tissue bank

NASA Astrophysics Data System (ADS)

LaFramboise, William A.; Shah, Sujal; Hoy, R. W.; Letbetter, D.; Petrosko, P.; Vennare, R.; Johnson, Peter C.

2000-04-01

Cellular and non-cellular constituents of skin contain fundamental morphometric features and structural patterns that correlate with tissue function. High resolution digital image acquisitions performed using an automated system and proprietary software to assemble adjacent images and create a contiguous, lossless, digital representation of individual microscope slide specimens. Serial extraction, evaluation and statistical analysis of cutaneous feature is performed utilizing an automated analysis system, to derive normal cutaneous parameters comprising essential structural skin components. Automated digital cutaneous analysis allows for fast extraction of microanatomic dat with accuracy approximating manual measurement. The process provides rapid assessment of feature both within individual specimens and across sample populations. The images, component data, and statistical analysis comprise a bioinformatics database to serve as an architectural blueprint for skin tissue engineering and as a diagnostic standard of comparison for pathologic specimens.

Towards high resolution analysis of metabolic flux in cells and tissues.

PubMed

Sims, James K; Manteiga, Sara; Lee, Kyongbum

2013-10-01

Metabolism extracts chemical energy from nutrients, uses this energy to form building blocks for biosynthesis, and interconverts between various small molecules that coordinate the activities of cellular pathways. The metabolic state of a cell is increasingly recognized to determine the phenotype of not only metabolically active cell types such as liver, muscle, and adipose, but also other specialized cell types such as neurons and immune cells. This review focuses on methods to quantify intracellular reaction flux as a measure of cellular metabolic activity, with emphasis on studies involving cells of mammalian tissue. Two key areas are highlighted for future development, single cell metabolomics and noninvasive imaging, which could enable spatiotemporally resolved analysis and thereby overcome issues of heterogeneity, a distinctive feature of tissue metabolism. Copyright © 2013 Elsevier Ltd. All rights reserved.

Towards High Resolution Analysis of Metabolic Flux in Cells and Tissues

PubMed Central

Sims, James K; Manteiga, Sara; Lee, Kyongbum

2013-01-01

Metabolism extracts chemical energy from nutrients, uses this energy to form building blocks for biosynthesis, and interconverts between various small molecules that coordinate the activities of cellular pathways. The metabolic state of a cell is increasingly recognized to determine the phenotype of not only metabolically active cell types such as liver, muscle, and adipose, but also other specialized cell types such as neurons and immune cells. This review focuses on methods to quantify intracellular reaction flux as a measure of cellular metabolic activity, with emphasis on studies involving cells of mammalian tissue. Two key areas are highlighted for future development, single cell metabolomics and noninvasive imaging, which could enable spatiotemporally resolved analysis and thereby overcome issues of heterogeneity, a distinctive feature of tissue metabolism. PMID:23906926

Fast and accurate automated cell boundary determination for fluorescence microscopy

NASA Astrophysics Data System (ADS)

Arce, Stephen Hugo; Wu, Pei-Hsun; Tseng, Yiider

2013-07-01

Detailed measurement of cell phenotype information from digital fluorescence images has the potential to greatly advance biomedicine in various disciplines such as patient diagnostics or drug screening. Yet, the complexity of cell conformations presents a major barrier preventing effective determination of cell boundaries, and introduces measurement error that propagates throughout subsequent assessment of cellular parameters and statistical analysis. State-of-the-art image segmentation techniques that require user-interaction, prolonged computation time and specialized training cannot adequately provide the support for high content platforms, which often sacrifice resolution to foster the speedy collection of massive amounts of cellular data. This work introduces a strategy that allows us to rapidly obtain accurate cell boundaries from digital fluorescent images in an automated format. Hence, this new method has broad applicability to promote biotechnology.

Update of the FANTOM web resource: high resolution transcriptome of diverse cell types in mammals

PubMed Central

Lizio, Marina; Harshbarger, Jayson; Abugessaisa, Imad; Noguchi, Shuei; Kondo, Atsushi; Severin, Jessica; Mungall, Chris; Arenillas, David; Mathelier, Anthony; Medvedeva, Yulia A.; Lennartsson, Andreas; Drabløs, Finn; Ramilowski, Jordan A.; Rackham, Owen; Gough, Julian; Andersson, Robin; Sandelin, Albin; Ienasescu, Hans; Ono, Hiromasa; Bono, Hidemasa; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R.R.; Kasukawa, Takeya; Kawaji, Hideya

2017-01-01

Upon the first publication of the fifth iteration of the Functional Annotation of Mammalian Genomes collaborative project, FANTOM5, we gathered a series of primary data and database systems into the FANTOM web resource (http://fantom.gsc.riken.jp) to facilitate researchers to explore transcriptional regulation and cellular states. In the course of the collaboration, primary data and analysis results have been expanded, and functionalities of the database systems enhanced. We believe that our data and web systems are invaluable resources, and we think the scientific community will benefit for this recent update to deepen their understanding of mammalian cellular organization. We introduce the contents of FANTOM5 here, report recent updates in the web resource and provide future perspectives. PMID:27794045

Metastable and unstable cellular solidification of colloidal suspensions

NASA Astrophysics Data System (ADS)

Deville, Sylvain; Maire, Eric; Bernard-Granger, Guillaume; Lasalle, Audrey; Bogner, Agnès; Gauthier, Catherine; Leloup, Jérôme; Guizard, Christian

2009-12-01

Colloidal particles are often seen as big atoms that can be directly observed in real space. They are therefore becoming increasingly important as model systems to study processes of interest in condensed-matter physics such as melting, freezing and glass transitions. The solidification of colloidal suspensions has long been a puzzling phenomenon with many unexplained features. Here, we demonstrate and rationalize the existence of instability and metastability domains in cellular solidification of colloidal suspensions, by direct in situ high-resolution X-ray radiography and tomography observations. We explain such interface instabilities by a partial Brownian diffusion of the particles leading to constitutional supercooling situations. Processing under unstable conditions leads to localized and global kinetic instabilities of the solid/liquid interface, affecting the crystal morphology and particle redistribution behaviour.

Long-term C. elegans immobilization enables high resolution developmental studies in vivo.

PubMed

Berger, Simon; Lattmann, Evelyn; Aegerter-Wilmsen, Tinri; Hengartner, Michael; Hajnal, Alex; deMello, Andrew; Casadevall I Solvas, Xavier

2018-05-01

Live-imaging of C. elegans is essential for the study of conserved cellular pathways (e.g. EGFR/Wnt signaling) and morphogenesis in vivo. However, the usefulness of live imaging as a research tool has been severely limited by the need to immobilize worms prior to and during imaging. Conventionally, immobilization is achieved by employing both physical and chemical interventions. These are known to significantly affect many physiological processes, and thus limit our understanding of dynamic developmental processes. Herein we present a novel, easy-to-use microfluidic platform for the long-term immobilization of viable, normally developing C. elegans, compatible with image acquisition at high resolution, thereby overcoming the limitations associated with conventional worm immobilization. The capabilities of the platform are demonstrated through the continuous assessment of anchor cell (AC) invasion and distal tip cell (DTC) migration in larval C. elegans and germ cell apoptosis in adult C. elegans in vivo for the first time.

Imaging plasmodesmata with high-resolution scanning electron microscopy.

PubMed

Barton, Deborah A; Overall, Robyn L

2015-01-01

High-resolution scanning electron microscopy (HRSEM) is an effective tool to investigate the distribution of plasmodesmata within plant cell walls as well as to probe their complex, three-dimensional architecture. It is a useful alternative to traditional transmission electron microscopy (TEM) in which plasmodesmata are sectioned to reveal their internal substructures. Benefits of adopting an HRSEM approach to studies of plasmodesmata are that the specimen preparation methods are less complex and time consuming than for TEM, many plasmodesmata within a large region of tissue can be imaged in a single session, and three-dimensional information is readily available without the need for reconstructing TEM serial sections or employing transmission electron tomography, both of which are lengthy processes. Here we describe methods to prepare plant samples for HRSEM using pre- or postfixation extraction of cellular material in order to visualize plasmodesmata embedded within plant cell walls.

High temporal-resolution view of transcription and chromatin states across distinct metabolic states in budding yeast

PubMed Central

Kuang, Zheng; Cai, Ling; Zhang, Xuekui; Ji, Hongkai; Tu, Benjamin P.; Boeke, Jef D.

2014-01-01

Under continuous, glucose-limited conditions, budding yeast exhibit robust metabolic cycles associated with major oscillations of gene expression. How such fluctuations are linked to changes in chromatin status is not well understood. Here we examine the correlated genome-wide transcription and chromatin states across the yeast metabolic cycle at unprecedented temporal resolution, revealing a “just-in-time supply chain” by which components from specific cellular processes such as ribosome biogenesis become available in a highly coordinated manner. We identify distinct chromatin and splicing patterns associated with different gene categories and determine the relative timing of chromatin modifications to maximal transcription. There is unexpected variation in the chromatin modification and expression relationship, with histone acetylation peaks occurring with varying timing and “sharpness” relative to RNA expression both within and between cycle phases. Chromatin modifier occupancy reveals subtly distinct spatial and temporal patterns compared to the modifications themselves. PMID:25173176

Polarization Sensitive Coherent Anti-Stokes Raman Spectroscopy of DCVJ in Doped Polymer

NASA Astrophysics Data System (ADS)

Ujj, Laszlo

2014-05-01

Coherent Raman Microscopy is an emerging technic and method to image biological samples such as living cells by recording vibrational fingerprints of molecules with high spatial resolution. The race is on to record the entire image during the shortest time possible in order to increase the time resolution of the recorded cellular events. The electronically enhanced polarization sensitive version of Coherent anti-Stokes Raman scattering is one of the method which can shorten the recording time and increase the sharpness of an image by enhancing the signal level of special molecular vibrational modes. In order to show the effectiveness of the method a model system, a highly fluorescence sample, DCVJ in a polymer matrix is investigated. Polarization sensitive resonance CARS spectra are recorded and analyzed. Vibrational signatures are extracted with model independent methods. Details of the measurements and data analysis will be presented. The author gratefully acknowledge the UWF for financial support.

Confocal fluorescence microscope with dual-axis architecture and biaxial postobjective scanning

PubMed Central

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

2007-01-01

We present a novel confocal microscope that has dual-axis architecture and biaxial postobjective scanning for the collection of fluorescence images from biological specimens. This design uses two low-numerical-aperture lenses to achieve high axial resolution and long working distance, and the scanning mirror located distal to the lenses rotates along the orthogonal axes to produce arc-surface images over a large field of view (FOV). With fiber optic coupling, this microscope can potentially be scaled down to millimeter dimensions via microelectromechanical systems (MEMS) technology. We demonstrate a benchtop prototype with a spatial resolution ≤4.4 μm that collects fluorescence images with a high SNR and a good contrast ratio from specimens expressing GFP. Furthermore, the scanning mechanism produces only small differences in aberrations over the image FOV. These results demonstrate proof of concept of the dual-axis confocal architecture for in vivo molecular and cellular imaging. PMID:15250760

Objective-lens-free Fiber-based Position Detection with Nanometer Resolution in a Fiber Optical Trapping System.

PubMed

Ti, Chaoyang; Ho-Thanh, Minh-Tri; Wen, Qi; Liu, Yuxiang

2017-10-13

Position detection with high accuracy is crucial for force calibration of optical trapping systems. Most existing position detection methods require high-numerical-aperture objective lenses, which are bulky, expensive, and difficult to miniaturize. Here, we report an affordable objective-lens-free, fiber-based position detection scheme with 2 nm spatial resolution and 150 MHz bandwidth. This fiber based detection mechanism enables simultaneous trapping and force measurements in a compact fiber optical tweezers system. In addition, we achieved more reliable signal acquisition with less distortion compared with objective based position detection methods, thanks to the light guiding in optical fibers and small distance between the fiber tips and trapped particle. As a demonstration of the fiber based detection, we used the fiber optical tweezers to apply a force on a cell membrane and simultaneously measure the cellular response.

Highly multiplexed subcellular RNA sequencing in situ

PubMed Central

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

2014-01-01

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

Applicability of confocal laser scanning microscopy for evaluation and monitoring of cutaneous wound healing

NASA Astrophysics Data System (ADS)

Lange-Asschenfeldt, Susanne; Bob, Adrienne; Terhorst, Dorothea; Ulrich, Martina; Fluhr, Joachim; Mendez, Gil; Roewert-Huber, Hans-Joachim; Stockfleth, Eggert; Lange-Asschenfeldt, Bernhard

2012-07-01

There is a high demand for noninvasive imaging techniques for wound assessment. In vivo reflectance confocal laser scanning microscopy (CLSM) represents an innovative optical technique for noninvasive evaluation of normal and diseased skin in vivo at near cellular resolution. This study was designed to test the feasibility of CLSM for noninvasive analysis of cutaneous wound healing in 15 patients (7 male/8 female), including acute and chronic, superficial and deep dermal skin wounds. A commercially available CLSM system was used for the assessment of wound bed and wound margins in order to obtain descriptive cellular and morphological parameters of cutaneous wound repair noninvasively and over time. CLSM was able to visualize features of cutaneous wound repair in epidermal and superficial dermal wounds, including aspects of inflammation, neovascularisation, and tissue remodelling in vivo. Limitations include the lack of mechanic fixation of the optical system on moist surfaces restricting the analysis of chronic skin wounds to the wound margins, as well as a limited optical resolution in areas of significant slough formation. By describing CLSM features of cutaneous inflammation, vascularisation, and epithelialisation, the findings of this study support the role of CLSM in modern wound research and management.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications.

PubMed

Fridman, Yulia; Holland, Neta; Elbaum, Rivka; Savaldi-Goldstein, Sigal

2016-05-10

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

Nanoscale cellular imaging with scanning angle interference microscopy.

PubMed

DuFort, Christopher; Paszek, Matthew

2014-01-01

Fluorescence microscopy is among the most widely utilized tools in cell and molecular biology due to its ability to noninvasively obtain time-resolved images of live cells with molecule-specific contrast. In this chapter, we describe a simple high-resolution technique, scanning angle interference microscopy (SAIM), for the imaging and localization of fluorescent molecules with nanometer precision along the optical axis. In SAIM, samples above a reflective surface are sequentially scanned with an excitation laser at varying angles of incidence. Interference patterns generated between the incident and reflected lights result in an emission intensity that depends on the height of a fluorophore above the silicon surface and the angle of the incident radiation. The measured fluorescence intensities are then fit to an optical model to localize the labeled molecules along the z-axis with 5-10 nm precision and diffraction-limited lateral resolution. SAIM is easily implemented on widely available commercial total internal reflection fluorescence microscopes, offering potential for widespread use in cell biology. Here, we describe the setup of SAIM and its application for imaging cellular structures near (<1 μm) the sample substrate. © 2014 Elsevier Inc. All rights reserved.

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.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

PubMed Central

Fridman, Yulia; Holland, Neta; Elbaum, Rivka; Savaldi-Goldstein, Sigal

2016-01-01

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth. PMID:27214583

Metabolic profiling of Arabidopsis thaliana epidermal cells

PubMed Central

Ebert, Berit; Zöller, Daniela; Erban, Alexander; Fehrle, Ines; Hartmann, Jürgen; Niehl, Annette; Kopka, Joachim; Fisahn, Joachim

2010-01-01

Metabolic phenotyping at cellular resolution may be considered one of the challenges in current plant physiology. A method is described which enables the cell type-specific metabolic analysis of epidermal cell types in Arabidopsis thaliana pavement, basal, and trichome cells. To achieve the required high spatial resolution, single cell sampling using microcapillaries was combined with routine gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) based metabolite profiling. The identification and relative quantification of 117 mostly primary metabolites has been demonstrated. The majority, namely 90 compounds, were accessible without analytical background correction. Analyses were performed using cell type-specific pools of 200 microsampled individual cells. Moreover, among these identified metabolites, 38 exhibited differential pool sizes in trichomes, basal or pavement cells. The application of an independent component analysis confirmed the cell type-specific metabolic phenotypes. Significant pool size changes between individual cells were detectable within several classes of metabolites, namely amino acids, fatty acids and alcohols, alkanes, lipids, N-compounds, organic acids and polyhydroxy acids, polyols, sugars, sugar conjugates and phenylpropanoids. It is demonstrated here that the combination of microsampling and GC-MS based metabolite profiling provides a method to investigate the cellular metabolism of fully differentiated plant cell types in vivo. PMID:20150518

Building quantitative, three-dimensional atlases of gene expression and morphology at cellular resolution.

PubMed

Knowles, David W; Biggin, Mark D

2013-01-01

Animals comprise dynamic three-dimensional arrays of cells that express gene products in intricate spatial and temporal patterns that determine cellular differentiation and morphogenesis. A rigorous understanding of these developmental processes requires automated methods that quantitatively record and analyze complex morphologies and their associated patterns of gene expression at cellular resolution. Here we summarize light microscopy-based approaches to establish permanent, quantitative datasets-atlases-that record this information. We focus on experiments that capture data for whole embryos or large areas of tissue in three dimensions, often at multiple time points. We compare and contrast the advantages and limitations of different methods and highlight some of the discoveries made. We emphasize the need for interdisciplinary collaborations and integrated experimental pipelines that link sample preparation, image acquisition, image analysis, database design, visualization, and quantitative analysis. Copyright © 2013 Wiley Periodicals, Inc.

High sensitivity contrast enhanced optical coherence tomography for functional in vivo imaging

NASA Astrophysics Data System (ADS)

Liba, Orly; SoRelle, Elliott D.; Sen, Debasish; de la Zerda, Adam

2017-02-01

In this study, we developed and applied highly-scattering large gold nanorods (LGNRs) and custom spectral detection algorithms for high sensitivity contrast-enhanced optical coherence tomography (OCT). We were able to detect LGNRs at a concentration as low as 50 pM in blood. We used this approach for noninvasive 3D imaging of blood vessels deep in solid tumors in living mice. Additionally, we demonstrated multiplexed imaging of spectrally-distinct LGNRs that enabled observations of functional drainage in lymphatic networks. This method, which we call MOZART, provides a platform for molecular imaging and characterization of tissue noninvasively at cellular resolution.

47 CFR 22.972 - Interference resolution procedures.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 47 Telecommunication 2 2012-10-01 2012-10-01 false Interference resolution procedures. 22.972... than February 22, 2005. (3) Cellular Radiotelephone licensees must respond to the initial notification... knowledge or belief after due diligence; (iv) Is not proffered by a contractor or other third party; and (v...

47 CFR 22.972 - Interference resolution procedures.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 47 Telecommunication 2 2011-10-01 2011-10-01 false Interference resolution procedures. 22.972... than February 22, 2005. (3) Cellular Radiotelephone licensees must respond to the initial notification... knowledge or belief after due diligence; (iv) Is not proffered by a contractor or other third party; and (v...

47 CFR 22.972 - Interference resolution procedures.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 47 Telecommunication 2 2013-10-01 2013-10-01 false Interference resolution procedures. 22.972... than February 22, 2005. (3) Cellular Radiotelephone licensees must respond to the initial notification... knowledge or belief after due diligence; (iv) Is not proffered by a contractor or other third party; and (v...

47 CFR 22.972 - Interference resolution procedures.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 47 Telecommunication 2 2014-10-01 2014-10-01 false Interference resolution procedures. 22.972... than February 22, 2005. (3) Cellular Radiotelephone licensees must respond to the initial notification... knowledge or belief after due diligence; (iv) Is not proffered by a contractor or other third party; and (v...

Quantitative measurement of zinc secretion from pancreatic islets with high temporal resolution using droplet-based microfluidics.

PubMed

Easley, Christopher J; Rocheleau, Jonathan V; Head, W Steven; Piston, David W

2009-11-01

We assayed glucose-stimulated insulin secretion (GSIS) from live, murine islets of Langerhans in microfluidic devices by the downstream formation of aqueous droplets. Zinc ions, which are cosecreted with insulin from beta-cells, were quantitatively measured from single islets with high temporal resolution using a fluorescent indicator, FluoZin-3. Real-time storage of secretions into droplets (volume of 0.470 +/- 0.009 nL) effectively preserves the temporal chemical information, allowing reconstruction of the secretory time record. The use of passive flow control within the device removes the need for syringe pumps, requiring only a single hand-held syringe. Under stimulatory glucose levels (11 mM), bursts of zinc as high as approximately 800 fg islet(-1) min(-1) were measured. Treatment with diazoxide effectively blocked zinc secretion, as expected. High temporal resolution reveals two major classes of oscillations in secreted zinc, with predominate periods at approximately 20-40 s and approximately 5-10 min. The more rapid oscillation periods match closely with those of intraislet calcium oscillations, while the slower oscillations are consistent with insulin pulses typically measured in bulk islet experiments or in the bloodstream. This droplet sampling technique should be widely applicable to time-resolved cellular secretion measurements, either in real-time or for postprocessing.

Quantitative measurement of zinc secretion from pancreatic islets with high temporal resolution using droplet-based microfluidics

PubMed Central

Easley, Christopher J.; Rocheleau, Jonathan V.; Head, W. Steven; Piston, David W.

2009-01-01

We assayed glucose-stimulated insulin secretion (GSIS) from live, murine islets of Langerhans in microfluidic devices by the downstream formation of aqueous droplets. Zinc ions, which are co-secreted with insulin from β-cells, were quantitatively measured from single islets with high temporal resolution using a fluorescent indicator, FluoZin-3. Real-time storage of secretions into droplets (volume of 0.470 ± 0.009 nL) effectively preserves the temporal chemical information, allowing reconstruction of the secretory time record. The use of passive flow control within the device removes the need for syringe pumps, requiring only a single handheld syringe. Under stimulatory glucose levels (11 mM), bursts of zinc as high as ~800 fg islet−1 min−1 were measured. Treatment with diazoxide effectively blocked zinc secretion, as expected. High temporal resolution reveals two major classes of oscillations in secreted zinc, with predominate periods at ~20-40 s and ~ 5-10 min. The more rapid oscillation periods match closely with those of intraislet calcium oscillations, while the slower oscillations are consistent with insulin pulses typically measured in bulk islet experiments or in the bloodstream. This droplet sampling technique should be widely applicable to time-resolved cellular secretion measurements, either in real-time or for post-processing. PMID:19874061

Cryogenic x-ray diffraction microscopy utilizing high-pressure cryopreservation

NASA Astrophysics Data System (ADS)

Lima, Enju; Chushkin, Yuriy; van der Linden, Peter; Kim, Chae Un; Zontone, Federico; Carpentier, Philippe; Gruner, Sol M.; Pernot, Petra

2014-10-01

We present cryo x-ray diffraction microscopy of high-pressure-cryofixed bacteria and report high-convergence imaging with multiple image reconstructions. Hydrated D. radiodurans cells were cryofixed at 200 MPa pressure into ˜10-μm-thick water layers and their unstained, hydrated cellular environments were imaged by phasing diffraction patterns, reaching sub-30-nm resolutions with hard x-rays. Comparisons were made with conventional ambient-pressure-cryofixed samples, with respect to both coherent small-angle x-ray scattering and the image reconstruction. The results show a correlation between the level of background ice signal and phasing convergence, suggesting that phasing difficulties with frozen-hydrated specimens may be caused by high-background ice scattering.

Bessel beam fluorescence lifetime tomography of live embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Peng, Leilei

2016-03-01

Optical tomography allows isotropic 3D imaging of embryos. Scanning-laser optical tomography (SLOT) has superior light collecting efficiency than wide-field optical tomography, making it ideal for fluorescence imaging of live embryos. We previously reported an imaging system that combines SLOT with a novel Fourier-multiplexed fluorescence lifetime imaging (FmFLIM) technique named FmFLIM-SLOT. FmFLIM-SLOT performs multiplexed FLIM-FRET readout of multiple FRET sensors in live embryos. Here we report a recent effort on improving the spatial resolution of the FmFLIM-SLOT system in order to image complex biochemical processes in live embryos at the cellular level. Optical tomography has to compromise between resolution and the depth of view. In SLOT, the commonly-used focused Gaussian beam diverges quickly from the focal plane, making it impossible to achieve high resolution imaging in a large volume specimen. We thus introduce Bessel beam laser-scanning tomography, which illuminates the sample with a spatial-light-modulator-generated Bessel beam that has an extended focal depth. The Bessel beam is scanned across the whole specimen. Fluorescence projection images are acquired at equal angular intervals as the sample rotates. Reconstruction artifacts due to annular-rings of the Bessel beam are removed by a modified 3D filtered back projection algorithm. Furthermore, in combination of Fourier-multiplexing fluorescence lifetime imaging (FmFLIM) method, the Bessel FmFLIM-SLOT system is capable of perform 3D lifetime imaging of live embryos at cellular resolution. The system is applied to in-vivo imaging of transgenic Zebrafish embryos. Results prove that Bessel FmFLIM-SLOT is a promising imaging method in development biology research.

In situ imaging of the mouse cochlea using two-photon microscopy

NASA Astrophysics Data System (ADS)

Yang, Xin; Pu, Ye; Psaltis, Demetri; Stankovic, Konstantina M.

2013-04-01

Intracochlear imaging is of great interest clinically because cochlea is the central organ of hearing. However, intracochlear imaging is technologically challenging due to the cochlea's small size and encasement in bone. The state-of- the-art imaging techniques are not adequate for high resolution cellular imaging to establish diagnosis without destroying the cochlea. We report in situ imaging of intact mouse cochlea using endogenous two-photon excitation fluorescence (TPEF) as the contrast mechanism. TPEF eliminates the need for exogenous labeling and eradicating the staining-induced artifacts. We used a natural, membranous opening into the cochlea, the round window, as the optical access to reach the organ of Corti, requiring no additional slicing or opening. Our approach provides the maximum non-invasiveness in the imaging process. TPEF exhibits strong contrast allowing deep imaging of mouse cochlea with cellular and even subcellular resolution. Inner hair cell, outer hair cell and supporting cell are clearly identifiable in TPEF images. Distinct morphological differences are observed between healthy and noise-exposed cochleae, allowing detection of specific, noise-induced pathologic changes. The TPEF images taken through the round window are correlated with the whole mount sections, verifying their reliability. Compared with one-photon excitation fluorescence (OPEF) confocal microscope and wide-field transmission microscope images taken under the same magnification and resolution, TPEF images demonstrate clear advantages in terms of sharpness, signal to noise ratio and contrast. These capabilities provide a working foundation for microendoscopy-based clinical diagnostics of sensorineural hearing loss.

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

PubMed Central

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

2013-01-01

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

Prospects and challenges of quantitative phase imaging in tumor cell biology

NASA Astrophysics Data System (ADS)

Kemper, Björn; Götte, Martin; Greve, Burkhard; Ketelhut, Steffi

2016-03-01

Quantitative phase imaging (QPI) techniques provide high resolution label-free quantitative live cell imaging. Here, prospects and challenges of QPI in tumor cell biology are presented, using the example of digital holographic microscopy (DHM). It is shown that the evaluation of quantitative DHM phase images allows the retrieval of different parameter sets for quantification of cellular motion changes in migration and motility assays that are caused by genetic modifications. Furthermore, we demonstrate simultaneously label-free imaging of cell growth and morphology properties.

The Effect of Rainfall Measurement Technique and Its Spatiotemporal Resolution on Discharge Predictions in the Netherlands

NASA Astrophysics Data System (ADS)

Uijlenhoet, R.; Brauer, C.; Overeem, A.; Sassi, M.; Rios Gaona, M. F.

2014-12-01

Several rainfall measurement techniques are available for hydrological applications, each with its own spatial and temporal resolution. We investigated the effect of these spatiotemporal resolutions on discharge simulations in lowland catchments by forcing a novel rainfall-runoff model (WALRUS) with rainfall data from gauges, radars and microwave links. The hydrological model used for this analysis is the recently developed Wageningen Lowland Runoff Simulator (WALRUS). WALRUS is a rainfall-runoff model accounting for hydrological processes relevant to areas with shallow groundwater (e.g. groundwater-surface water feedback). Here, we used WALRUS for case studies in a freely draining lowland catchment and a polder with controlled water levels. We used rain gauge networks with automatic (hourly resolution but low spatial density) and manual gauges (high spatial density but daily resolution). Operational (real-time) and climatological (gauge-adjusted) C-band radar products and country-wide rainfall maps derived from microwave link data from a cellular telecommunication network were also used. Discharges simulated with these different inputs were compared to observations. We also investigated the effect of spatiotemporal resolution with a high-resolution X-band radar data set for catchments with different sizes. Uncertainty in rainfall forcing is a major source of uncertainty in discharge predictions, both with lumped and with distributed models. For lumped rainfall-runoff models, the main source of input uncertainty is associated with the way in which (effective) catchment-average rainfall is estimated. When catchments are divided into sub-catchments, rainfall spatial variability can become more important, especially during convective rainfall events, leading to spatially varying catchment wetness and spatially varying contribution of quick flow routes. Improving rainfall measurements and their spatiotemporal resolution can improve the performance of rainfall-runoff models, indicating their potential for reducing flood damage through real-time control.

CLIP-related methodologies and their application to retrovirology.

PubMed

Bieniasz, Paul D; Kutluay, Sebla B

2018-05-02

Virtually every step of HIV-1 replication and numerous cellular antiviral defense mechanisms are regulated by the binding of a viral or cellular RNA-binding protein (RBP) to distinct sequence or structural elements on HIV-1 RNAs. Until recently, these protein-RNA interactions were studied largely by in vitro binding assays complemented with genetics approaches. However, these methods are highly limited in the identification of the relevant targets of RBPs in physiologically relevant settings. Development of crosslinking-immunoprecipitation sequencing (CLIP) methodology has revolutionized the analysis of protein-nucleic acid complexes. CLIP combines immunoprecipitation of covalently crosslinked protein-RNA complexes with high-throughput sequencing, providing a global account of RNA sequences bound by a RBP of interest in cells (or virions) at near-nucleotide resolution. Numerous variants of the CLIP protocol have recently been developed, some with major improvements over the original. Herein, we briefly review these methodologies and give examples of how CLIP has been successfully applied to retrovirology research.

Compartmentalized microchannel array for high-throughput analysis of single cell polarized growth and dynamics

DOE PAGES

Geng, Tao; Bredeweg, Erin L.; Szymanski, Craig J.; ...

2015-11-04

Here, interrogating polarized growth is technologically challenging due to extensive cellular branching and uncontrollable environmental conditions in conventional assays. Here we present a robust and high-performance microfluidic system that enables observations of polarized growth with enhanced temporal and spatial control over prolonged periods. The system has built-in tunability and versatility to accommodate a variety of science applications requiring precisely controlled environments. Using the model filamentous fungus, Neurospora crassa, this microfluidic system enabled direct visualization and analysis of cellular heterogeneity in a clonal fungal cell population, nuclear distribution and dynamics at the subhyphal level, and quantitative dynamics of gene expression withmore » single hyphal compartment resolution in response to carbon source starvation and exchange experiments. Although the microfluidic device is demonstrated on filamentous fungi, our technology is immediately extensible to a wide array of other biosystems that exhibit similar polarized cell growth with applications ranging from bioenergy production to human health.« less

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

PubMed Central

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

2013-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2011-09-01

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

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

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.

Optical imaging with a high-resolution microendoscope to identify cholesteatoma of the middle ear.

PubMed

Levy, Lauren L; Jiang, Nancy; Smouha, Eric; Richards-Kortum, Rebecca; Sikora, Andrew G

2013-04-01

High-resolution optical imaging is an imaging modality that allows visualization of structural changes in epithelial tissue in real time. Our prior studies using contrast-enhanced microendoscopy to image squamous cell carcinoma in the head and neck demonstrated that the contrast agent, proflavine, has high affinity for keratinized tissue. Thus, high-resolution microendoscopy with proflavine provides a potential mechanism to identify ectopic keratin production, such as that associated with cholesteatoma formation, and distinguish between uninvolved mucosa and residual keratin at the time of surgery. Ex vivo imaging of histopathologically confirmed samples of cholesteatoma and uninvolved middle ear epithelium. Seven separate specimens collected from patients who underwent surgical treatment for cholesteatoma were imaged ex vivo with the fiberoptic endoscope after surface staining with proflavine. Following imaging, the specimens were submitted for hematoxylin and eosin staining to allow histopathological correlation. Cholesteatoma and surrounding middle ear epithelium have distinct imaging characteristics. Keratin-bearing areas of cholesteatoma lack nuclei and appear as confluent hyperfluorescence, whereas nuclei are easily visualized in specimens containing normal middle ear epithelium. Hyperfluorescence and loss of cellular detail is the imaging hallmark of keratin, allowing for discrimination of cholesteatoma from normal middle ear epithelium. This study demonstrates the feasibility of high-resolution optical imaging to discriminate cholesteatoma from uninvolved middle ear mucosa based on the unique staining properties of keratin. Use of real-time imaging may facilitate more complete extirpation of cholesteatoma by identifying areas of residual disease. Laryngoscope, 2012. Copyright © 2013 The American Laryngological, Rhinological, and Otological Society, Inc.

Anomalously Fast Diffusion of Targeted Carbon Nanotubes in Cellular Spheroids.

PubMed

Wang, Yichun; Bahng, Joong Hwan; Che, Quantong; Han, Jishu; Kotov, Nicholas A

2015-08-25

Understanding transport of carbon nanotubes (CNTs) and other nanocarriers within tissues is essential for biomedical imaging and drug delivery using these carriers. Compared to traditional cell cultures in animal studies, three-dimensional tissue replicas approach the complexity of the actual organs and enable high temporal and spatial resolution of the carrier permeation. We investigated diffusional transport of CNTs in highly uniform spheroids of hepatocellular carcinoma and found that apparent diffusion coefficients of CNTs in these tissue replicas are anomalously high and comparable to diffusion rates of similarly charged molecules with molecular weights 10000× lower. Moreover, diffusivity of CNTs in tissues is enhanced after functionalization with transforming growth factor β1. This unexpected trend contradicts predictions of the Stokes-Einstein equation and previously obtained empirical dependences of diffusivity on molecular mass for permeants in gas, liquid, solid or gel. It is attributed to the planar diffusion (gliding) of CNTs along cellular membranes reducing effective dimensionality of diffusional space. These findings indicate that nanotubes and potentially similar nanostructures are capable of fast and deep permeation into the tissue, which is often difficult to realize with anticancer agents.

3D printed phantoms of retinal photoreceptor cells for evaluating adaptive optics imaging modalities

NASA Astrophysics Data System (ADS)

Kedia, Nikita; Liu, Zhuolin; Sochol, Ryan; Hammer, Daniel X.; Agrawal, Anant

2018-02-01

Adaptive optics-enabled optical coherence tomography (AO-OCT) and scanning laser ophthalmoscopy (AO-SLO) devices can resolve retinal cones and rods in three dimensions. To evaluate the improved resolution of AO-OCT and AO-SLO, a phantom that mimics retinal anatomy at the cellular level is required. We used a two-photon polymerization approach to fabricate three-dimensional (3D) photoreceptor phantoms modeled on the central foveal cones. By using a femtosecond laser to selectively photocure precise locations within a liquid-based photoresist via two-photon absorption, we produced high-resolution phantoms with μm-level dimensions similar to true anatomy. In this work, we present two phantoms to evaluate the resolution limits of an AO imaging system: one that models only the outer segments of the photoreceptor cells at varying retinal eccentricities and another that contains anatomically relevant features of the full-length photoreceptor. With these phantoms we are able to quantitatively estimate transverse resolution of an AO system and produce images that are comparable to those found in the human retina.

High-Definition Medicine.

PubMed

Torkamani, Ali; Andersen, Kristian G; Steinhubl, Steven R; Topol, Eric J

2017-08-24

The foundation for a new era of data-driven medicine has been set by recent technological advances that enable the assessment and management of human health at an unprecedented level of resolution-what we refer to as high-definition medicine. Our ability to assess human health in high definition is enabled, in part, by advances in DNA sequencing, physiological and environmental monitoring, advanced imaging, and behavioral tracking. Our ability to understand and act upon these observations at equally high precision is driven by advances in genome editing, cellular reprogramming, tissue engineering, and information technologies, especially artificial intelligence. In this review, we will examine the core disciplines that enable high-definition medicine and project how these technologies will alter the future of medicine. Copyright © 2017 Elsevier Inc. All rights reserved.

Update of the FANTOM web resource: high resolution transcriptome of diverse cell types in mammals.

PubMed

Lizio, Marina; Harshbarger, Jayson; Abugessaisa, Imad; Noguchi, Shuei; Kondo, Atsushi; Severin, Jessica; Mungall, Chris; Arenillas, David; Mathelier, Anthony; Medvedeva, Yulia A; Lennartsson, Andreas; Drabløs, Finn; Ramilowski, Jordan A; Rackham, Owen; Gough, Julian; Andersson, Robin; Sandelin, Albin; Ienasescu, Hans; Ono, Hiromasa; Bono, Hidemasa; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R R; Kasukawa, Takeya; Kawaji, Hideya

2017-01-04

Upon the first publication of the fifth iteration of the Functional Annotation of Mammalian Genomes collaborative project, FANTOM5, we gathered a series of primary data and database systems into the FANTOM web resource (http://fantom.gsc.riken.jp) to facilitate researchers to explore transcriptional regulation and cellular states. In the course of the collaboration, primary data and analysis results have been expanded, and functionalities of the database systems enhanced. We believe that our data and web systems are invaluable resources, and we think the scientific community will benefit for this recent update to deepen their understanding of mammalian cellular organization. We introduce the contents of FANTOM5 here, report recent updates in the web resource and provide future perspectives. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

High resolution FTIR imaging provides automated discrimination and detection of single malaria parasite infected erythrocytes on glass.

PubMed

Perez-Guaita, David; Andrew, Dean; Heraud, Philip; Beeson, James; Anderson, David; Richards, Jack; Wood, Bayden R

2016-06-23

New highly sensitive tools for malaria diagnostics are urgently needed to enable the detection of infection in asymptomatic carriers and patients with low parasitemia. In pursuit of a highly sensitive diagnostic tool that can identify parasite infections at the single cell level, we have been exploring Fourier transform infrared (FTIR) microscopy using a Focal Plane Array (FPA) imaging detector. Here we report for the first time the application of a new optic configuration developed by Agilent that incorporates 25× condenser and objective Cassegrain optics with a high numerical aperture (NA = 0.81) along with additional high magnification optics within the microscope to provide 0.66 micron pixel resolution (total IR system magnification of 61×) to diagnose malaria parasites at the single cell level on a conventional glass microscope slide. The high quality images clearly resolve the parasite's digestive vacuole demonstrating sub-cellular resolution using this approach. Moreover, we have developed an algorithm that first detects the cells in the infrared image, and secondly extracts the average spectrum. The average spectrum is then run through a model based on Partial Least Squares-Discriminant Analysis (PLS-DA), which diagnoses unequivocally the infected from normal cells. The high quality images, and the fact this measurement can be achieved without a synchrotron source on a conventional glass slide, shows promise as a potential gold standard for malaria detection at the single cell level.

Rigid two-axis MEMS force plate for measuring cellular traction force

NASA Astrophysics Data System (ADS)

Takahashi, Hidetoshi; Jung, Uijin G.; Kan, Tetsuo; Tsukagoshi, Takuya; Matsumoto, Kiyoshi; Shimoyama, Isao

2016-10-01

Cellular traction force is one of the important factors for understanding cell behaviors, such as spreading, migration and differentiation. Cells are known to change their behavior according to the mechanical stiffness of the environment. However, the measurement of cell traction forces on a rigid environment has remained difficult. This paper reports a micro-electromechanical systems (MEMS) force plate that provides a cellular traction force measurement on a rigid substrate. Both the high force sensitivity and high stiffness of the substrate were obtained using piezoresistive sensing elements. The proposed force plate consists of a 70 µm  ×  15 µm  ×  5 µm base as the substrate for cultivating a bovine aortic smooth muscle cell, and the supporting beams with piezoresistors on the sidewall and the surface were used to measure the forces in both the horizontal and vertical directions. The spring constant and force resolution of the fabricated force plate in the horizontal direction were 0.2 N m-1 and less than 0.05 µN, respectively. The cell traction force was measured, and the traction force increased by approximately 1 µN over 30 min. These results demonstrate that the proposed force plate is applicable as an effective traction force measurement.

Multiphoton Intravital Calcium Imaging.

PubMed

Cheetham, Claire E J

2018-06-26

Multiphoton intravital calcium imaging is a powerful technique that enables high-resolution longitudinal monitoring of cellular and subcellular activity hundreds of microns deep in the living organism. This unit addresses the application of 2-photon microscopy to imaging of genetically encoded calcium indicators (GECIs) in the mouse brain. The protocols in this unit enable real-time intravital imaging of intracellular calcium concentration simultaneously in hundreds of neurons, or at the resolution of single synapses, as mice respond to sensory stimuli or perform behavioral tasks. Protocols are presented for implantation of a cranial imaging window to provide optical access to the brain and for 2-photon image acquisition. Protocols for implantation of both open skull and thinned skull windows for single or multi-session imaging are described. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.

Multiphoton tomography to detect chemo- and biohazards

NASA Astrophysics Data System (ADS)

König, Karsten

2015-03-01

In vivo high-resolution multiphoton/CARS tomography provides optical biopsies with 300 nm lateral resolution with chemical fingerprints. Thousands of volunteers and patients have been investigated for early cancer diagnosis, evaluation of anti-ageing cosmetic products, and changes of cellular metabolism by UV exposure and decreased oxygen supply. The skin as the outermost and largest organ is also the major target of CB agents. Current UV-based sensors are useful for bio-aerosol sensing but not for evaluating exposed in vivo skin. Here we evaluate the use of 4D multiphoton/CARS tomographs based on near infrared femtosecond laser radiation, time-correlated single photon counting (FLIM) and white light generation by photonic crystal fibers to detect bio- and chemohazards in human in vivo skin using twophoton fluorescence, SHG, and Raman signals.

Ligand Binding Induces Conformational Changes in Human Cellular Retinol-binding Protein 1 (CRBP1) Revealed by Atomic Resolution Crystal Structures.

PubMed

Silvaroli, Josie A; Arne, Jason M; Chelstowska, Sylwia; Kiser, Philip D; Banerjee, Surajit; Golczak, Marcin

2016-04-15

Important in regulating the uptake, storage, and metabolism of retinoids, cellular retinol-binding protein 1 (CRBP1) is essential for trafficking vitamin A through the cytoplasm. However, the molecular details of ligand uptake and targeted release by CRBP1 remain unclear. Here we report the first structure of CRBP1 in a ligand-free form as well as ultra-high resolution structures of this protein bound to either all-trans-retinol or retinylamine, the latter a therapeutic retinoid that prevents light-induced retinal degeneration. Superpositioning of human apo- and holo-CRBP1 revealed major differences within segments surrounding the entrance to the retinoid-binding site. These included α-helix II and hairpin turns between β-strands βC-βD and βE-βF as well as several side chains, such as Phe-57, Tyr-60, and Ile-77, that change their orientations to accommodate the ligand. Additionally, we mapped hydrogen bond networks inside the retinoid-binding cavity and demonstrated their significance for the ligand affinity. Analyses of the crystallographic B-factors indicated several regions with higher backbone mobility in the apoprotein that became more rigid upon retinoid binding. This conformational flexibility of human apo-CRBP1 facilitates interaction with the ligands, whereas the more rigid holoprotein structure protects the labile retinoid moiety during vitamin A transport. These findings suggest a mechanism of induced fit upon ligand binding by mammalian cellular retinol-binding proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A 3D Image Filter for Parameter-Free Segmentation of Macromolecular Structures from Electron Tomograms

PubMed Central

Ali, Rubbiya A.; Landsberg, Michael J.; Knauth, Emily; Morgan, Garry P.; Marsh, Brad J.; Hankamer, Ben

2012-01-01

3D image reconstruction of large cellular volumes by electron tomography (ET) at high (≤5 nm) resolution can now routinely resolve organellar and compartmental membrane structures, protein coats, cytoskeletal filaments, and macromolecules. However, current image analysis methods for identifying in situ macromolecular structures within the crowded 3D ultrastructural landscape of a cell remain labor-intensive, time-consuming, and prone to user-bias and/or error. This paper demonstrates the development and application of a parameter-free, 3D implementation of the bilateral edge-detection (BLE) algorithm for the rapid and accurate segmentation of cellular tomograms. The performance of the 3D BLE filter has been tested on a range of synthetic and real biological data sets and validated against current leading filters—the pseudo 3D recursive and Canny filters. The performance of the 3D BLE filter was found to be comparable to or better than that of both the 3D recursive and Canny filters while offering the significant advantage that it requires no parameter input or optimisation. Edge widths as little as 2 pixels are reproducibly detected with signal intensity and grey scale values as low as 0.72% above the mean of the background noise. The 3D BLE thus provides an efficient method for the automated segmentation of complex cellular structures across multiple scales for further downstream processing, such as cellular annotation and sub-tomogram averaging, and provides a valuable tool for the accurate and high-throughput identification and annotation of 3D structural complexity at the subcellular level, as well as for mapping the spatial and temporal rearrangement of macromolecular assemblies in situ within cellular tomograms. PMID:22479430

Axon Regeneration in C. elegans

PubMed Central

Hammarlund, Marc; Jin, Yishi

2014-01-01

Single axon transection by laser surgery has made C. elegans a new model for axon regeneration. Multiple conserved molecular signaling modules have been discovered through powerful genetic screening. in vivo imaging with single cell and axon resolution has revealed unprecedented cellular dynamics in regenerating axons. Information from C. elegans has greatly expanded our knowledge of the molecular and cellular mechanisms of axon regeneration. PMID:24794753

Plasmonic Nanobubbles as Tunable Cellular Probes for Cancer Theranostics

PubMed Central

Lapotko, Dmitri

2011-01-01

This review is focused on a novel cellular probe, the plasmonic nanobubble (PNB), which has the dynamically tunable and multiple functions of imaging, diagnosis, delivery, therapy and, ultimately, theranostics. The concept of theranostics was recently introduced in order to unite the clinically important stages of treatment, namely diagnosis, therapy and therapy guidance, into one single, rapid and highly accurate procedure. Cell level theranostics will have far-reaching implications for the treatment of cancer and other diseases at their earliest stages. PNBs were developed to support cell level theranostics as a new generation of on-demand tunable cellular probes. A PNB is a transient vapor nanobubble that is generated within nanoseconds around an overheated plasmonic nanoparticle with a short laser pulse. In the short term, we expect that PNB technology will be rapidly adaptable to clinical medicine, where the single cell resolution it provides will be critical for diagnosing incipient or residual disease and eliminating cancer cells, while leaving healthy cells intact. This review discusses mechanisms of plasmonic nanobubbles and their biomedical applications with the focus on cancer cell theranostics. PMID:21442036

Gabor domain optical coherence microscopy

NASA Astrophysics Data System (ADS)

Murali, Supraja

Time domain Optical Coherence Tomography (TD-OCT), first reported in 1991, makes use of the low temporal coherence properties of a NIR broadband laser to create depth sectioning of up to 2mm under the surface using optical interferometry and point to point scanning. Prior and ongoing work in OCT in the research community has concentrated on improving axial resolution through the development of broadband sources and speed of image acquisition through new techniques such as Spectral domain OCT (SD-OCT). In SD-OCT, an entire depth scan is acquired at once with a low numerical aperture (NA) objective lens focused at a fixed point within the sample. In this imaging geometry, a longer depth of focus is achieved at the expense of lateral resolution, which is typically limited to 10 to 20 mum. Optical Coherence Microscopy (OCM), introduced in 1994, combined the advantages of high axial resolution obtained in OCT with high lateral resolution obtained by increasing the NA of the microscope placed in the sample arm. However, OCM presented trade-offs caused by the inverse quadratic relationship between the NA and the DOF of the optics used. For applications requiring high lateral resolution, such as cancer diagnostics, several solutions have been proposed including the periodic manual re-focusing of the objective lens in the time domain as well as the spectral domain C-mode configuration in order to overcome the loss in lateral resolution outside the DOF. In this research, we report for the first time, high speed, sub-cellular imaging (lateral resolution of 2 mum) in OCM using a Gabor domain image processing algorithm with a custom designed and fabricated dynamic focus microscope interfaced to a Ti:Sa femtosecond laser centered at 800 nm within an SD-OCM configuration. It is envisioned that this technology will provide a non-invasive replacement for the current practice of multiple biopsies for skin cancer diagnosis. The research reported here presents three important advances to this technology all of which have been demonstrated in full functional hardware conceived and built during the course of this research. First, it has been demonstrated that the coherence gate created by the femtosecond laser can be coupled into a scanning optical microscope using optical design methods to include liquid lens technology that enables scanning below the surface of skin with no moving parts and at high resolution throughout a 2x2x2 mm imaging cube. Second, the integration the variable-focus liquid lens technology within a fixed-optics microscope custom optical design helped increase the working NA by an order of magnitude over the limitation imposed by the liquid lens alone. Thus, this design has enabled homogenous axial and lateral resolution at the micron-level (i.e., 2 mum) while imaging in the spectral domain, and still maintaining in vivo speeds. The latest images in biological specimens clearly demonstrate sub-cellular resolution in all dimensions throughout the imaging volume. Third, this new modality for data collection has been integrated with an automated Gabor domain image registration and fusion algorithm to provide full resolution images across the data cube in real-time. We refer to this overall OCM method as Gabor domain OCM (GD-OCM). These advantages place GD-OCM in a unique position with respect to the diagnosis of cancer, because when fully developed, it promises to enable fast and accurate screening for early symptoms that could lead to prevention. The next step for this technology is to apply it directly, in a clinical environment. This step is underway and is expected to be reported by the next generation of researchers within this group.

Design and validation of a diffuse reflectance and spectroscopic microendoscope with poly(dimethylsioxane)-based phantoms

PubMed Central

Greening, Gage J.; Powless, Amy J.; Hutcheson, Joshua A.; Prieto, Sandra P.; Majid, Aneeka A.; Muldoon, Timothy J.

2015-01-01

Many cases of epithelial cancer originate in basal layers of tissue and are initially undetected by conventional microendoscopy techniques. We present a bench-top, fiber-bundle microendoscope capable of providing high resolution images of surface cell morphology. Additionally, the microendoscope has the capability to interrogate deeper into material by using diffuse reflectance and broadband diffuse reflectance spectroscopy. The purpose of this multimodal technique was to overcome the limitation of microendoscopy techniques that are limited to only visualizing morphology at the tissue or cellular level. Using a custom fiber optic probe, high resolution surface images were acquired using topical proflavine to fluorescently stain non-keratinized epithelia. A 635 nm laser coupled to a 200 μm multimode fiber delivers light to the sample and the diffuse reflectance signal was captured by a 1 mm image guide fiber. Finally, a tungsten-halogen lamp coupled to a 200 μm multimode fiber delivers broadband light to the sample to acquire spectra at source-detector separations of 374, 729, and 1051 μm. To test the instrumentation, a high resolution proflavine-induced fluorescent image of resected healthy mouse colon was acquired. Additionally, five monolayer poly(dimethylsiloxane)-based optical phantoms with varying absorption and scattering properties were created to acquire diffuse reflectance profiles and broadband spectra. PMID:25983372

Design and validation of a diffuse reflectance and spectroscopic microendoscope with poly(dimethylsiloxane)-based phantoms

NASA Astrophysics Data System (ADS)

Greening, Gage J.; Powless, Amy J.; Hutcheson, Joshua A.; Prieto, Sandra P.; Majid, Aneeka A.; Muldoon, Timothy J.

2015-03-01

Many cases of epithelial cancer originate in basal layers of tissue and are initially undetected by conventional microendoscopy techniques. We present a bench-top, fiber-bundle microendoscope capable of providing high resolution images of surface cell morphology. Additionally, the microendoscope has the capability to interrogate deeper into material by using diffuse reflectance and broadband diffuse reflectance spectroscopy. The purpose of this multimodal technique was to overcome the limitation of microendoscopy techniques that are limited to only visualizing morphology at the tissue or cellular level. Using a custom fiber optic probe, high resolution surface images were acquired using topical proflavine to fluorescently stain non-keratinized epithelia. A 635 nm laser coupled to a 200 μm multimode fiber delivers light to the sample and the diffuse reflectance signal was captured by a 1 mm image guide fiber. Finally, a tungsten-halogen lamp coupled to a 200 μm multimode fiber delivers broadband light to the sample to acquire spectra at source-detector separations of 374, 729, and 1051 μm. To test the instrumentation, a high resolution proflavine-induced fluorescent image of resected healthy mouse colon was acquired. Additionally, five monolayer poly(dimethylsiloxane)-based optical phantoms with varying absorption and scattering properties were created to acquire diffuse reflectance profiles and broadband spectra.

Identification of high-confidence RNA regulatory elements by combinatorial classification of RNA-protein binding sites.

PubMed

Li, Yang Eric; Xiao, Mu; Shi, Binbin; Yang, Yu-Cheng T; Wang, Dong; Wang, Fei; Marcia, Marco; Lu, Zhi John

2017-09-08

Crosslinking immunoprecipitation sequencing (CLIP-seq) technologies have enabled researchers to characterize transcriptome-wide binding sites of RNA-binding protein (RBP) with high resolution. We apply a soft-clustering method, RBPgroup, to various CLIP-seq datasets to group together RBPs that specifically bind the same RNA sites. Such combinatorial clustering of RBPs helps interpret CLIP-seq data and suggests functional RNA regulatory elements. Furthermore, we validate two RBP-RBP interactions in cell lines. Our approach links proteins and RNA motifs known to possess similar biochemical and cellular properties and can, when used in conjunction with additional experimental data, identify high-confidence RBP groups and their associated RNA regulatory elements.

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.

Electron tomography of whole cultured cells using novel transmission electron imaging technique.

PubMed

Okumura, Taiga; Shoji, Minami; Hisada, Akiko; Ominami, Yusuke; Ito, Sukehiro; Ushiki, Tatsuo; Nakajima, Masato; Ohshima, Takashi

2018-01-01

Since a three-dimensional (3D) cellular ultrastructure is significant for biological functions, it has been investigated using various electron microscopic techniques. Although transmission electron microscopy (TEM)-based techniques are traditionally used, cells must be embedded in resin and sliced into ultrathin sections in sample preparation processes. Block-face observation using a scanning electron microscope (SEM) has also been recently applied to 3D observation of cellular components, but this is a destructive inspection and does not allow re-examination. Therefore, we developed electron tomography using a transmission electron imaging technique called Plate-TEM. With Plate-TEM, the cells cultured directly on a scintillator plate are inserted into a conventional SEM equipped with a Plate-TEM observation system, and their internal structures are observed by detecting scintillation light produced by electrons passing through the cells. This technology has the following four advantages. First, the cells cultured on the plate can be observed at electron-microscopic resolution since they remain on the plate. Second, both surface and internal information can be obtained simultaneously by using electron- and photo-detectors, respectively, because a Plate-TEM detector is installed in an SEM. Third, the cells on the scintillator plate can also be inspected using light microscopy because the plate has transparent features. Finally, correlative observation with other techniques, such as conventional TEM, is possible after Plate-TEM observation because Plate-TEM is a non-destructive analysis technique. We also designed a sample stage to tilt the samples for tomography with Plate-TEM, by which 3D organization of cellular structures can be visualized as a whole cell. In the present study, Mm2T cells were investigated using our tomography system, resulting in 3D visualization of cell organelles such as mitochondria, lipid droplets, and microvilli. Correlative observations with various imaging techniques were also conducted by successive observations with light microscopy, SEM, Plate-TEM, and conventional TEM. Consequently, the Plate-TEM tomography technique encourages understanding of cellular structures at high resolution, which can contribute to cellular biological research. Copyright © 2017 Elsevier Ltd. All rights reserved.

Investigation of FANCA gene in Fanconi anaemia patients in Iran

PubMed Central

Saffar Moghadam, Ali Akbar; Mahjoubi, Frouzandeh; Reisi, Nahid; Vosough, Parvaneh

2016-01-01

Background & objectives: Fanconi anaemia (FA) is a syndrome with a predisposition to bone marrow failure, congenital anomalies and malignancies. It is characterized by cellular hypersensitivity to cross-linking agents such as mitomycin C (MMC). In the present study, a new approach was selected to investigate FANCA (Fanconi anaemia complementation group A) gene in patients clinically diagnosed with cellular hypersensitivity to DNA cross-linking agent MMC. Methods: Chromosomal breakage analysis was performed to prove the diagnosis of Fanconi anaemia in 318 families. Of these, 70 families had a positive result. Forty families agreed to molecular genetic testing. In total, there were 27 patients with unknown complementary types. Genomic DNA was extracted and total RNA was isolated from fresh whole blood of the patients. The first-strand cDNA was synthesized and the cDNA of each patient was then tested with 21 pairs of overlapping primers. High resolution melting curve analysis was used to screen FANCA, and LinReg software version 1.7 was utilized for analysis of expression. Results: In total, six sequence alterations were identified, which included two stop codons, two frames-shift mutations, one large deletion and one amino acid exchange. FANCA expression was downregulated in patients who had sequence alterations. Interpretation & conclusions: The results of the present study show that high resolution melting (HRM) curve analysis may be useful in the detection of sequence alteration. It is simpler and more costeffective than the multiplex ligation-dependent probe amplification (MLPA) procedure. PMID:27121516

Investigation of FANCA gene in Fanconi anaemia patients in Iran.

PubMed

Moghadam, Ali Akbar Saffar; Mahjoubi, Frouzandeh; Reisi, Nahid; Vosough, Parvaneh

2016-02-01

Fanconi anaemia (FA) is a syndrome with a predisposition to bone marrow failure, congenital anomalies and malignancies. It is characterized by cellular hypersensitivity to cross-linking agents such as mitomycin C (MMC). In the present study, a new approach was selected to investigate FANCA (Fanconi anaemia complementation group A) gene in patients clinically diagnosed with cellular hypersensitivity to DNA cross-linking agent MMC. Chromosomal breakage analysis was performed to prove the diagnosis of Fanconi anaemia in 318 families. Of these, 70 families had a positive result. Forty families agreed to molecular genetic testing. In total, there were 27 patients with unknown complementary types. Genomic DNA was extracted and total RNA was isolated from fresh whole blood of the patients. The first-strand cDNA was synthesized and the cDNA of each patient was then tested with 21 pairs of overlapping primers. High resolution melting curve analysis was used to screen FANCA, and LinReg software version 1.7 was utilized for analysis of expression. In total, six sequence alterations were identified, which included two stop codons, two frames-shift mutations, one large deletion and one amino acid exchange. FANCA expression was downregulated in patients who had sequence alterations. The results of the present study show that high resolution melting (HRM) curve analysis may be useful in the detection of sequence alteration. It is simpler and more cost-effective than the multiplex ligation-dependent probe amplification (MLPA) procedure.

Comparison of high-resolution microendoscope images and histopathological sections in ex vivo middle ear cholesteatomas and surrounding tissue

NASA Astrophysics Data System (ADS)

Bradley, James; Levy, Lauren; Richards-Kortum, Rebecca; Sikora, Andrew G.; Smouha, Eric

2013-03-01

Objective: To investigate the concordance between optical images obtained with high-resolution microendoscopy (HRME) and conventional histopathology for ex vivo cholesteatoma specimens and surrounding middle ear epithelium. Methods: After resection of cholesteatoma and surrounding middle ear epithelium from surgical patients, tissues were stained with a contrast agent, proflavine, and the HRME fiberoptic scope was placed directly on each tissue specimen. 4- 10 short movie clips were recorded for both the cholesteatoma and surrounding middle ear epithelium specimens. The imaged areas were sent for standard histopathology, and the stained specimens were correlated with the HRME images. IRB approval was obtained, and each patient was consented for the study. Results: Ten cholesteatoma specimens and 9 middle ear specimens were collected from 10 patients. In each case, cholesteatoma was easily discriminated from normal middle ear epithelium by its hyperfluorescence and loss of cellular detail. Qualitative analysis for concordance between HRME images and histological images from the same surgical specimen yielded a strong correlation between imaging modalities. Conclusions: Keratinizing cholesteatoma and surrounding middle ear epithelium have distinct imaging characteristics. Loss of cellular detail and hyperfluorescence with proflavine are the hallmark characteristics of cholesteatoma which allow for differentiation from normal middle ear epithelium. Real-time optical imaging can potentially improve the results of otologic surgery by allowing for extirpation of cholesteatomas while eliminating residual disease. We anticipate performing an in vivo study to test this hypothesis.

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy.

PubMed

Lemcke, Heiko; Voronina, Natalia; Steinhoff, Gustav; David, Robert

2017-06-19

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Use of MALDI Mass Spectrometry for Identification of Microbes

NASA Astrophysics Data System (ADS)

Wilkins, C. L.; Stump, M.; Jones, J.; Lay, J. O.; Fleming, R.

2003-12-01

Recently, it has been demonstrated that bacteria can be characterized using whole cells and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). However, identification of specific bacterial proteins usually requires analysis of cellular fractions or purified extracts. This presentation will discuss the first application of Fourier transform mass spectrometry (FTMS) to analysis of bacterial proteins directly from whole cells. In this research it is seen that accurate mass MALDI-FTMS can be used to characterize specific ribosomal proteins directly from Escherichia coli cells. Using the high-accuracy mass measurements and high resolution isotope profile data thus available it is possible to confirm posttranslational modifications proposed previously on the basis of low resolution mass measurements. In our initial work, ribosomal proteins from E. coli whole cells were observed with errors of less than 27 ppm. This was accomplished directly from whole cells without fractionation, concentration, or overt overexpression of characteristic cellular proteins. More recently, by use of carbon and nitrogen isotopically-depleted growth media additional E. coli proteins have been identified with even smaller mass measurement errors. MALDI FTMS also provided information regarding E. coli lipids in the low-mass region. Although ions with m/z values below 1000 were previously observed by FTMS of whole cells, the work to be presented was the first report of detection of ions in the 5000 to 10 000 m/z range by MALDI-FTMS using whole cells. The implications of these results for genus, species, and strain assignments of such organisms will be discussed.

Cellular structure of the healthy and keratoconic human cornea imaged in-vivo with sub-micrometer axial resolution OCT(Conference Presentation)

NASA Astrophysics Data System (ADS)

Bizheva, Kostadinka; Tan, Bingyao; Mason, Erik; Carter, Kirsten; Haines, Lacey; Sorbara, Luigina

2017-02-01

Keratoconus causes progressive morphological changes in the corneal epithelium (EPI), Bowman's membrane (BM) and anterior stroma. However, it is still not well understood if KC originates in the corneal epithelium and propagates to the anterior stroma through disruptions of the BM, or vice versa. In this study we used a sub-micrometer axial resolution OCT system to image in-vivo the cellular structure of the EPI layer and the fibrous structure of the BM and the anterior stroma in mild to advanced keratoconics, as well as healthy subjects. The imaging study was approved by the University of Waterloo Human Research Ethics Committee. The OCT system operates in the 800 nm spectral region at 34 kHz image acquisition rate and provides 0.95 um axial and < 2 um lateral resolution in corneal tissue, which is sufficient to visualize the cellular structure of the corneal epithelium and the fibrous structure of the BM. In some subjects, localized thinning and thickening of the EPI layer was observed, while there was no visible damage to the BM or anterior stroma. In other subjects, localized breakage of the stromal collagen fibrils was observed with no significant morphological changes of the corneal EPI.

The Rich Club of the C. elegans Neuronal Connectome

PubMed Central

Vértes, Petra E.; Ahnert, Sebastian E.; Schafer, William R.; Bullmore, Edward T.

2013-01-01

There is increasing interest in topological analysis of brain networks as complex systems, with researchers often using neuroimaging to represent the large-scale organization of nervous systems without precise cellular resolution. Here we used graph theory to investigate the neuronal connectome of the nematode worm Caenorhabditis elegans, which is defined anatomically at a cellular scale as 2287 synaptic connections between 279 neurons. We identified a small number of highly connected neurons as a rich club (N = 11) interconnected with high efficiency and high connection distance. Rich club neurons comprise almost exclusively the interneurons of the locomotor circuits, with known functional importance for coordinated movement. The rich club neurons are connector hubs, with high betweenness centrality, and many intermodular connections to nodes in different modules. On identifying the shortest topological paths (motifs) between pairs of peripheral neurons, the motifs that are found most frequently traverse the rich club. The rich club neurons are born early in development, before visible movement of the animal and before the main phase of developmental elongation of its body. We conclude that the high wiring cost of the globally integrative rich club of neurons in the C. elegans connectome is justified by the adaptive value of coordinated movement of the animal. The economical trade-off between physical cost and behavioral value of rich club organization in a cellular connectome confirms theoretical expectations and recapitulates comparable results from human neuroimaging on much larger scale networks, suggesting that this may be a general and scale-invariant principle of brain network organization. PMID:23575836

Genetic interaction analysis of point mutations enables interrogation of gene function at a residue-level resolution

PubMed Central

Braberg, Hannes; Moehle, Erica A.; Shales, Michael; Guthrie, Christine; Krogan, Nevan J.

2014-01-01

We have achieved a residue-level resolution of genetic interaction mapping – a technique that measures how the function of one gene is affected by the alteration of a second gene – by analyzing point mutations. Here, we describe how to interpret point mutant genetic interactions, and outline key applications for the approach, including interrogation of protein interaction interfaces and active sites, and examination of post-translational modifications. Genetic interaction analysis has proven effective for characterizing cellular processes; however, to date, systematic high-throughput genetic interaction screens have relied on gene deletions or knockdowns, which limits the resolution of gene function analysis and poses problems for multifunctional genes. Our point mutant approach addresses these issues, and further provides a tool for in vivo structure-function analysis that complements traditional biophysical methods. We also discuss the potential for genetic interaction mapping of point mutations in human cells and its application to personalized medicine. PMID:24842270

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

NASA Astrophysics Data System (ADS)

Higgins, Laura M.; Pierce, Mark C.

2014-08-01

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

Submicron-resolution photoacoustic microscopy of endogenous light-absorbing biomolecules

NASA Astrophysics Data System (ADS)

Zhang, Chi

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

Cellular Telephones Measure Activity and Lifespace in Community-Dwelling Adults: Proof of Principle

PubMed Central

Schenk, Ana Katrin; Witbrodt, Bradley C.; Hoarty, Carrie A.; Carlson, Richard H.; Goulding, Evan H.; Potter, Jane F.; Bonasera, Stephen J.

2011-01-01

OBJECTIVES To describe a system that uses off-the-shelf sensor and telecommunication technologies to continuously measure individual lifespace and activity levels in a novel way. DESIGN Proof of concept involving three field trials of 30, 30, and 21 days. SETTING Omaha, Nebraska, metropolitan and surrounding rural region. PARTICIPANTS Three participants (48-year-old man, 33-year-old woman, and 27-year-old male), none with any functional limitations. MEASUREMENTS Cellular telephones were used to detect in-home position and in-community location and to measure physical activity. Within the home, cellular telephones and Bluetooth transmitters (beacons) were used to locate participants at room-level resolution. Outside the home, the same cellular telephones and global positioning system (GPS) technology were used to locate participants at a community-level resolution. Physical activity was simultaneously measured using the cellular telephone accelerometer. RESULTS This approach had face validity to measure activity and lifespace. More importantly, this system could measure the spatial and temporal organization of these metrics. For example, an individual’s lifespace was automatically calculated across multiple time intervals. Behavioral time budgets showing how people allocate time to specific regions within the home were also automatically generated. CONCLUSION Mobile monitoring shows much promise as an easily deployed system to quantify activity and lifespace, important indicators of function, in community-dwelling adults. PMID:21288235

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

NASA Astrophysics Data System (ADS)

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

2016-09-01

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

Structural, biological and biophysical properties of glycated and glycoxidized phosphatidylethanolamines

PubMed Central

Annibal, Andrea; Riemer, Thomas; Jovanovic, Olga; Westphal, Dennis; Griesser, Eva; Pohl, Elena E.; Schiller, Jürgen; Hoffmann, Ralf; Fedorova, Maria

2018-01-01

Glycation and glycoxidation of proteins and peptides have been intensively studied and are considered as reliable diagnostic biomarkers of hyperglycemia and early stages of type II diabetes. However, glucose can also react with primary amino groups present in other cellular components, such as aminophospholipids (aminoPLs). Although it is proposed that glycated aminoPLs can induce many cellular responses and contribute to the development and progression of diabetes, the routes of their formation and their biological roles are only partially revealed. The same is true for the influence of glucose-derived modifications on the biophysical properties of PLs. Here we studied structural, signaling, and biophysical properties of glycated and glycoxidized phosphatidylethanolamines (PEs). By combining high resolution mass spectrometry and nuclear magnetic resonance spectroscopy it was possible to deduce the structures of several intermediates indicating an oxidative cleavage of the Amadori product yielding glycoxidized PEs including advanced glycation end products, such as carboxyethyl- and carboxymethyl-ethanolamines. The pro-oxidative role of glycated PEs was demonstrated and further associated with several cellular responses including activation of NFκB signaling pathways. Label free proteomics indicated significant alterations in proteins regulating cellular metabolisms. Finally, the biophysical properties of PL membranes changed significantly upon PE glycation, such as melting temperature (Tm), membrane surface charge, and ion transport across the phospholipid bilayer. PMID:27012418

High-frame-rate imaging of biological samples with optoacoustic micro-tomography

NASA Astrophysics Data System (ADS)

Deán-Ben, X. Luís.; López-Schier, Hernán.; Razansky, Daniel

2018-02-01

Optical microscopy remains a major workhorse in biological discovery despite the fact that light scattering limits its applicability to depths of ˜ 1 mm in scattering tissues. Optoacoustic imaging has been shown to overcome this barrier by resolving optical absorption with microscopic resolution in significantly deeper regions. Yet, the time domain is paramount for the observation of biological dynamics in living systems that exhibit fast motion. Commonly, acquisition of microscopy data involves raster scanning across the imaged volume, which significantly limits temporal resolution in 3D. To overcome these limitations, we have devised a fast optoacoustic micro-tomography (OMT) approach based on simultaneous acquisition of 3D image data with a high-density hemispherical ultrasound array having effective detection bandwidth around 25 MHz. We performed experiments by imaging tissue-mimicking phantoms and zebrafish larvae, demonstrating that OMT can provide nearly cellular resolution and imaging speed of 100 volumetric frames per second. As opposed to other optical microscopy techniques, OMT is a hybrid method that resolves optical absorption contrast acoustically using unfocused light excitation. Thus, no penetration barriers are imposed by light scattering in deep tissues, suggesting it as a powerful approach for multi-scale functional and molecular imaging applications.

Development and application of Fourier-transform infrared chemical imaging of tumour in human tissue.

PubMed

Petter, C H; Heigl, N; Rainer, M; Bakry, R; Pallua, J; Bonn, G K; Huck, C W

2009-01-01

Fourier-transform infrared (FT-IR) based mapping and imaging is a fast emerging technology which is being increasingly applied to investigate tissues in the high-throughput mode. The high resolution close to the cellular level, the possibility to determine the bio-distribution of molecules of interest (proteins, peptides, lipids, carbohydrates) without any pre-treatment and the offer to yield molecular structure information have brought evidence that this technique allows to gain new insights in cancer pathology. Thus, several individual mainly protein and peptide cancer markers ("biomarkers") can be identified from FT-IR tissue images, enabling accurate discrimination between healthy and tumour areas. Optimal data acquisition (spatial resolution, spectral resolution, signal to noise ratio), classification, and validation are necessary to establish practical protocols that can be translated to the qualitative and quantitative clinical routine analysis. Thereby, the development of modern fast infrared imaging systems has strongly supported its acceptance in clinical histopathology. In this review, the necessity of analysis based on global cancer statistics, instrumental setups and developments, experimental state of the art are summarised and applications to investigate different kinds of cancer (e.g., prostate, breast, cervical, colon, oral cavity) are shown and discussed in detail.

Efficient Exploration of Membrane-Associated Phenomena at Atomic Resolution.

PubMed

Vermaas, Josh V; Baylon, Javier L; Arcario, Mark J; Muller, Melanie P; Wu, Zhe; Pogorelov, Taras V; Tajkhorshid, Emad

2015-06-01

Biological membranes constitute a critical component in all living cells. In addition to providing a conducive environment to a wide range of cellular processes, including transport and signaling, mounting evidence has established active participation of specific lipids in modulating membrane protein function through various mechanisms. Understanding lipid-protein interactions underlying these mechanisms at a sufficiently high resolution has proven extremely challenging, partly due to the semi-fluid nature of the membrane. In order to address this challenge computationally, multiple methods have been developed, including an alternative membrane representation termed highly mobile membrane mimetic (HMMM) in which lateral lipid diffusion has been significantly enhanced without compromising atomic details. The model allows for efficient sampling of lipid-protein interactions at atomic resolution, thereby significantly enhancing the effectiveness of molecular dynamics simulations in capturing membrane-associated phenomena. In this review, after providing an overview of HMMM model development, we will describe briefly successful application of the model to study a variety of membrane processes, including lipid-dependent binding and insertion of peripheral proteins, the mechanism of phospholipid insertion into lipid bilayers, and characterization of optimal tilt angle of transmembrane helices. We conclude with practical recommendations for proper usage of the model in simulation studies of membrane processes.

Technical Note: Novel method for water vapour monitoring using wireless communication networks measurements

NASA Astrophysics Data System (ADS)

David, N.; Alpert, P.; Messer, H.

2009-04-01

We propose a new technique that overcomes the obstacles of the existing methods for monitoring near-surface water vapour, by estimating humidity from data collected through existing wireless communication networks. Weather conditions and atmospheric phenomena affect the electromagnetic channel, causing attenuations to the radio signals. Thus, wireless communication networks are in effect built-in environmental monitoring facilities. The wireless microwave links, used in these networks, are widely deployed by cellular providers for backhaul communication between base stations, a few tens of meters above ground level. As a result, if all available measurements are used, the proposed method can provide moisture observations with high spatial resolution and potentially high temporal resolution. Further, the implementation cost is minimal, since the data used are already collected and saved by the cellular operators. In addition - many of these links are installed in areas where access is difficult such as orographic terrain and complex topography. As such, our method enables measurements in places that have been hard to measure in the past, or have never been measured before. The technique is restricted to weather conditions which exclude rain, fog or clouds along the propagation path. Strong winds that may cause movement of the link transmitter or receiver (or both) may also interfere with the ability to conduct accurate measurements. We present results from real-data measurements taken from two microwave links used in a backhaul cellular network that show convincing correlation to surface station humidity measurements. The measurements were taken daily in two sites, one in northern Israel (28 measurements), the other in central Israel (29 measurements). The correlation between the microwave link measurements and the humidity gauges were 0.9 and 0.82 for the north and central sites, respectively. The Root Mean Square Differences (RMSD) were 1.8 g/m3 and 3.4 g/m3 for the northern and central site measurements, respectively.

Interferometric scattering (iSCAT) microscopy: studies of biological membrane dynamics

NASA Astrophysics Data System (ADS)

Reina, Francesco; Galiani, Silvia; Shrestha, Dilip; Sezgin, Erdinc; Lagerholm, B. Christoffer; Cole, Daniel; Kukura, Philipp; Eggeling, Christian

2018-02-01

The study of the organization and dynamics of molecules in model and cellular membranes is an important topic in contemporary biophysics. Imaging and single particle tracking in this particular field, however, proves particularly demanding, as it requires simultaneously high spatio-temporal resolution and high signal-to-noise ratios. A remedy to this challenge might be Interferometric Scattering (iSCAT) microscopy, due to its fast sampling rates, label-free imaging capabilities and, most importantly, tuneable signal level output. Here we report our recent advances in the imaging and molecular tracking on phase-separated model membrane systems and live-cell membranes using this technique.

An essential role for UTX in resolution and activation of bivalent promoters

PubMed Central

Dhar, Shilpa S.; Lee, Sung-Hun; Chen, Kaifu; Zhu, Guangjing; Oh, WonKyung; Allton, Kendra; Gafni, Ohad; Kim, Young Zoon; Tomoiga, Alin S.; Barton, Michelle Craig; Hanna, Jacob H.; Wang, Zhibin; Li, Wei; Lee, Min Gyu

2016-01-01

Trimethylated histone H3 lysine 27 (H3K27me3) is linked to gene silencing, whereas H3K4me3 is associated with gene activation. These two marks frequently co-occupy gene promoters, forming bivalent domains. Bivalency signifies repressed but activatable states of gene expression and can be resolved to active, H3K4me3-prevalent states during multiple cellular processes, including differentiation, development and epithelial mesenchymal transition. However, the molecular mechanism underlying bivalency resolution remains largely unknown. Here, we show that the H3K27 demethylase UTX (also called KDM6A) is required for the resolution and activation of numerous retinoic acid (RA)-inducible bivalent genes during the RA-driven differentiation of mouse embryonic stem cells (ESCs). Notably, UTX loss in mouse ESCs inhibited the RA-driven bivalency resolution and activation of most developmentally critical homeobox (Hox) a–d genes. The UTX-mediated resolution and activation of many bivalent Hox genes during mouse ESC differentiation were recapitulated during RA-driven differentiation of human NT2/D1 embryonal carcinoma cells. In support of the importance of UTX in bivalency resolution, Utx-null mouse ESCs and UTX-depleted NT2/D1 cells displayed defects in RA-driven cellular differentiation. Our results define UTX as a bivalency-resolving histone modifier necessary for stem cell differentiation. PMID:26762983

Vibrational imaging of glucose uptake activity in live cells and tissues by stimulated Raman scattering microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hu, Fanghao; Chen, Zhixing; Zhang, Luyuan; Shen, Yihui; Wei, Lu; Min, Wei

2016-03-01

Glucose is consumed as an energy source by virtually all living organisms, from bacteria to humans. Its uptake activity closely reflects the cellular metabolic status in various pathophysiological transformations, such as diabetes and cancer. Extensive efforts such as positron emission tomography, magnetic resonance imaging and fluorescence microscopy have been made to specifically image glucose uptake activity but all with technical limitations. Here, we report a new platform to visualize glucose uptake activity in live cells and tissues with subcellular resolution and minimal perturbation. A novel glucose analogue with a small alkyne tag (carbon-carbon triple bond) is developed to mimic natural glucose for cellular uptake, which can be imaged with high sensitivity and specificity by targeting the strong and characteristic alkyne vibration on stimulated Raman scattering (SRS) microscope to generate a quantitative three dimensional concentration map. Cancer cells with differing metabolic characteristics can be distinguished. Heterogeneous uptake patterns are observed in tumor xenograft tissues, neuronal culture and mouse brain tissues with clear cell-cell variations. Therefore, by offering the distinct advantage of optical resolution but without the undesirable influence of bulky fluorophores, our method of coupling SRS with alkyne labeled glucose will be an attractive tool to study energy demands of living systems at the single cell level.

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

PubMed

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

2015-11-26

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

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

PubMed Central

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

2015-01-01

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

Single photon counting fluorescence lifetime detection of pericellular oxygen concentrations

NASA Astrophysics Data System (ADS)

Hosny, Neveen A.; Lee, David A.; Knight, Martin M.

2012-01-01

Fluorescence lifetime imaging microscopy offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods are either invasive, require custom-made systems, or show limited spatial resolution. Therefore, these methods are unsuitable for investigation of pericellular oxygen concentrations. This study describes an adaptation of commercially available equipment which has been optimized for quantitative extracellular oxygen detection with high lifetime accuracy and spatial resolution while avoiding systematic photon pile-up. The oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)3]2+, was excited using a two-photon excitation laser. Lifetime was measured using a Becker & Hickl time-correlated single photon counting, which will be referred to as a TCSPC card. [Ru(bipy)3]2+ characterization studies quantified the influences of temperature, pH, cellular culture media and oxygen on the fluorescence lifetime measurements. This provided a precisely calibrated and accurate system for quantification of pericellular oxygen concentration based on measured lifetimes. Using this technique, quantification of oxygen concentrations around isolated viable chondrocytes, seeded in three-dimensional agarose gel, revealed a subpopulation of cells that exhibited significant spatial oxygen gradients such that oxygen concentration reduced with increasing proximity to the cell. This technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.

Single photon counting fluorescence lifetime detection of pericellular oxygen concentrations.

PubMed

Hosny, Neveen A; Lee, David A; Knight, Martin M

2012-01-01

Fluorescence lifetime imaging microscopy offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods are either invasive, require custom-made systems, or show limited spatial resolution. Therefore, these methods are unsuitable for investigation of pericellular oxygen concentrations. This study describes an adaptation of commercially available equipment which has been optimized for quantitative extracellular oxygen detection with high lifetime accuracy and spatial resolution while avoiding systematic photon pile-up. The oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)(3)](2+), was excited using a two-photon excitation laser. Lifetime was measured using a Becker & Hickl time-correlated single photon counting, which will be referred to as a TCSPC card. [Ru(bipy)(3)](2+) characterization studies quantified the influences of temperature, pH, cellular culture media and oxygen on the fluorescence lifetime measurements. This provided a precisely calibrated and accurate system for quantification of pericellular oxygen concentration based on measured lifetimes. Using this technique, quantification of oxygen concentrations around isolated viable chondrocytes, seeded in three-dimensional agarose gel, revealed a subpopulation of cells that exhibited significant spatial oxygen gradients such that oxygen concentration reduced with increasing proximity to the cell. This technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

A Highly Accurate Face Recognition System Using Filtering Correlation

NASA Astrophysics Data System (ADS)

Watanabe, Eriko; Ishikawa, Sayuri; Kodate, Kashiko

2007-09-01

The authors previously constructed a highly accurate fast face recognition optical correlator (FARCO) [E. Watanabe and K. Kodate: Opt. Rev. 12 (2005) 460], and subsequently developed an improved, super high-speed FARCO (S-FARCO), which is able to process several hundred thousand frames per second. The principal advantage of our new system is its wide applicability to any correlation scheme. Three different configurations were proposed, each depending on correlation speed. This paper describes and evaluates a software correlation filter. The face recognition function proved highly accurate, seeing that a low-resolution facial image size (64 × 64 pixels) has been successfully implemented. An operation speed of less than 10 ms was achieved using a personal computer with a central processing unit (CPU) of 3 GHz and 2 GB memory. When we applied the software correlation filter to a high-security cellular phone face recognition system, experiments on 30 female students over a period of three months yielded low error rates: 0% false acceptance rate and 2% false rejection rate. Therefore, the filtering correlation works effectively when applied to low resolution images such as web-based images or faces captured by a monitoring camera.

A quick and simple FISH protocol with hybridization-sensitive fluorescent linear oligodeoxynucleotide probes

PubMed Central

Wang, Dan Ohtan; Matsuno, Hitomi; Ikeda, Shuji; Nakamura, Akiko; Yanagisawa, Hiroyuki; Hayashi, Yasunori; Okamoto, Akimitsu

2012-01-01

Fluorescence in situ hybridization (FISH) is a powerful tool used in karyotyping, cytogenotyping, cancer diagnosis, species specification, and gene-expression analysis. Although widely used, conventional FISH protocols are cumbersome and time consuming. We have now developed a FISH method using exciton-controlled hybridization-sensitive fluorescent oligodeoxynucleotide (ECHO) probes. ECHO–FISH uses a 25-min protocol from fixation to mounting that includes no stringency washing steps. We use ECHO–FISH to detect both specific DNA and RNA sequences with multicolor probes. ECHO–FISH is highly reproducible, stringent, and compatible with other fluorescent cellular labeling techniques. The resolution allows detection of intranuclear speckles of poly(A) RNA in HeLa cells and dissociated hippocampal primary cultures, and mRNAs in the distal dendrites of hippocampal neurons. We also demonstrate detection of telomeric and centromeric DNA on metaphase mouse chromosomes. The simplicity of the ECHO–FISH method will likely accelerate cytogenetic and gene-expression analysis with high resolution. PMID:22101241

Fluorescent in vivo imaging of reactive oxygen species and redox potential in plants.

PubMed

Ortega-Villasante, Cristina; Burén, Stefan; Blázquez-Castro, Alfonso; Barón-Sola, Ángel; Hernández, Luis E

2018-04-05

Reactive oxygen species (ROS) are by-products of aerobic metabolism, and excessive production can result in oxidative stress and cell damage. In addition, ROS function as cellular messengers, working as redox regulators in a multitude of biological processes. Understanding ROS signalling and stress responses requires methods for precise imaging and quantification to monitor local, subcellular and global ROS dynamics with high selectivity, sensitivity and spatiotemporal resolution. In this review, we summarize the present knowledge for in vivo plant ROS imaging and detection, using both chemical probes and fluorescent protein-based biosensors. Certain characteristics of plant tissues, for example high background autofluorescence in photosynthetic organs and the multitude of endogenous antioxidants, can interfere with ROS and redox potential detection, making imaging extra challenging. Novel methods and techniques to measure in vivo plant ROS and redox changes with better selectivity, accuracy, and spatiotemporal resolution are therefore desirable to fully acknowledge the remarkably complex plant ROS signalling networks. Copyright © 2018 Elsevier Inc. All rights reserved.

Imaging inflammation in mouse colon using a rapid stage-scanning confocal fluorescence microscope

NASA Astrophysics Data System (ADS)

Saldua, Meagan A.; Olsovsky, Cory A.; Callaway, Evelyn S.; Chapkin, Robert S.; Maitland, Kristen C.

2012-01-01

Large area confocal microscopy may provide fast, high-resolution image acquisition for evaluation of tissue in pre-clinical studies with reduced tissue processing in comparison to histology. We present a rapid beam and stage-scanning confocal fluorescence microscope to image cellular and tissue features along the length of the entire excised mouse colon. The beam is scanned at 8,333 lines/sec by a polygon scanning mirror while the specimen is scanned in the orthogonal axis by a motorized translation stage with a maximum speed of 7 mm/sec. A single 1×60 mm2 field of view image spanning the length of the mouse colon is acquired in 10 s. Z-projection images generated from axial image stacks allow high resolution imaging of the surface of non-flat specimens. In contrast to the uniform size, shape, and distribution of colon crypts in confocal images of normal colon, confocal images of chronic bowel inflammation exhibit heterogeneous tissue structure with localized severe crypt distortion.

The Structure of an Infectious Human Polyomavirus and Its Interactions with Cellular Receptors.

PubMed

Hurdiss, Daniel L; Frank, Martin; Snowden, Joseph S; Macdonald, Andrew; Ranson, Neil A

2018-06-05

BK polyomavirus (BKV) causes polyomavirus-associated nephropathy and hemorrhagic cystitis in immunosuppressed patients. These are diseases for which we currently have limited treatment options, but potential therapies could include pre-transplant vaccination with a multivalent BKV vaccine or therapeutics which inhibit capsid assembly or block attachment and entry into target cells. A useful tool in such efforts would be a high-resolution structure of the infectious BKV virion and how this interacts with its full repertoire of cellular receptors. We present the 3.4-Å cryoelectron microscopy structure of native, infectious BKV in complex with the receptor fragment of GT1b ganglioside. We also present structural evidence that BKV can utilize glycosaminoglycans as attachment receptors. This work highlights features that underpin capsid stability and provides a platform for rational design and development of urgently needed pharmacological interventions for BKV-associated diseases. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Imaging of oxygenation in 3D tissue models with multi-modal phosphorescent probes

NASA Astrophysics Data System (ADS)

Papkovsky, Dmitri B.; Dmitriev, Ruslan I.; Borisov, Sergei

2015-03-01

Cell-penetrating phosphorescence based probes allow real-time, high-resolution imaging of O2 concentration in respiring cells and 3D tissue models. We have developed a panel of such probes, small molecule and nanoparticle structures, which have different spectral characteristics, cell penetrating and tissue staining behavior. The probes are compatible with conventional live cell imaging platforms and can be used in different detection modalities, including ratiometric intensity and PLIM (Phosphorescence Lifetime IMaging) under one- or two-photon excitation. Analytical performance of these probes and utility of the O2 imaging method have been demonstrated with different types of samples: 2D cell cultures, multi-cellular spheroids from cancer cell lines and primary neurons, excised slices from mouse brain, colon and bladder tissue, and live animals. They are particularly useful for hypoxia research, ex-vivo studies of tissue physiology, cell metabolism, cancer, inflammation, and multiplexing with many conventional fluorophors and markers of cellular function.

Application of differential interference contrast with inverted microscopes to the in vitro perfused nephron.

PubMed

Horster, M; Gundlach, H

1979-12-01

The study of in vitro perfused individual nephron segments requires a microscope which provides: (1) easy access to the specimen for measurement of cellular solute flux and voltage; (2) an image with high resolution and contrast; (3) optical sectioning of the object at different levels; and (4) rapid recording of the morphological phenomena. This paper describes an example of commercially available apparatus meeting the above requirements, and illustrates its efficiency. The microscope is of the inverted type (Zeiss IM 35) equipped with differential-interference-contrast (DIC) with a long working distance, and an automatically controlled camera system. The microscopic image exhibits cellular and intercellular details in the unstained transporting mammalian nephron segments despite their tubular structure and great thickness and makes obvious function-structure correlations (e.g. cell volume changes); luminal and contraluminal cell borders are well resolved for controlled microelectrode impalement.

Visualizing Cochlear Mechanics Using Confocal Microscopy

NASA Astrophysics Data System (ADS)

Ulfendahl, M.; Boutet de Monvel, J.; Fridberger, A.

2003-02-01

The sound-evoked vibration pattern of the hearing organ is based on complex mechanical interactions between different cellular structures. To explore the structural changes occurring within the organ of Corti during basilar-membrane motion, stepwise alterations of the scala tympani pressure were applied in an in vitro preparation of the guinea-pig temporal bone. Confocal images were acquired at each pressure level. In this way, the motion of several structures could be simultaneously observed with high resolution in a nearly intact system. Images were analyzed using a novel wavelet-based optical-flow estimation algorithm. Under the present experimental conditions, the reticular lamina moved as a stiff plate with a center of rotation in the region of the inner hair cells. The outer hair cells appeared non-rigid and the basal, synaptic regions of these cells displayed significant radial motion indicative of cellular bending and internal shearing.

Intraoperative intravital microscopy permits the study of human tumour vessels

PubMed Central

Fisher, Daniel T.; Muhitch, Jason B.; Kim, Minhyung; Doyen, Kurt C.; Bogner, Paul N.; Evans, Sharon S.; Skitzki, Joseph J.

2016-01-01

Tumour vessels have been studied extensively as they are critical sites for drug delivery, anti-angiogenic therapies and immunotherapy. As a preclinical tool, intravital microscopy (IVM) allows for in vivo real-time direct observation of vessels at the cellular level. However, to date there are no reports of intravital high-resolution imaging of human tumours in the clinical setting. Here we report the feasibility of IVM examinations of human malignant disease with an emphasis on tumour vasculature as the major site of tumour-host interactions. Consistent with preclinical observations, we show that patient tumour vessels are disorganized, tortuous and ∼50% do not support blood flow. Human tumour vessel diameters are larger than predicted from immunohistochemistry or preclinical IVM, and thereby have lower wall shear stress, which influences delivery of drugs and cellular immunotherapies. Thus, real-time clinical imaging of living human tumours is feasible and allows for detection of characteristics within the tumour microenvironment. PMID:26883450

Emerging Imaging and Genomic Tools for Developmental Systems Biology.

PubMed

Liu, Zhe; Keller, Philipp J

2016-03-21

Animal development is a complex and dynamic process orchestrated by exquisitely timed cell lineage commitment, divisions, migration, and morphological changes at the single-cell level. In the past decade, extensive genetic, stem cell, and genomic studies provided crucial insights into molecular underpinnings and the functional importance of genetic pathways governing various cellular differentiation processes. However, it is still largely unknown how the precise coordination of these pathways is achieved at the whole-organism level and how the highly regulated spatiotemporal choreography of development is established in turn. Here, we discuss the latest technological advances in imaging and single-cell genomics that hold great promise for advancing our understanding of this intricate process. We propose an integrated approach that combines such methods to quantitatively decipher in vivo cellular dynamic behaviors and their underlying molecular mechanisms at the systems level with single-cell, single-molecule resolution. Copyright © 2016 Elsevier Inc. All rights reserved.

Three-Dimensional Unstained Live-Cell Imaging Using Stimulated Parametric Emission Microscopy

NASA Astrophysics Data System (ADS)

Dang, Hieu M.; Kawasumi, Takehito; Omura, Gen; Umano, Toshiyuki; Kajiyama, Shin'ichiro; Ozeki, Yasuyuki; Itoh, Kazuyoshi; Fukui, Kiichi

2009-09-01

The ability to perform high-resolution unstained live imaging is very important to in vivo study of cell structures and functions. Stimulated parametric emission (SPE) microscopy is a nonlinear-optical microscopy based on ultra-fast electronic nonlinear-optical responses. For the first time, we have successfully applied this technique to archive three-dimensional (3D) images of unstained sub-cellular structures, such as, microtubules, nuclei, nucleoli, etc. in live cells. Observation of a complete cell division confirms the ability of SPE microscopy for long time-scale imaging.

ASSESSMENT OF VENOUS THROMBOSIS IN ANIMAL MODELS

PubMed Central

SP, Grover; CE, Evans; AS, Patel; B, Modarai; P, Saha; A, Smith

2016-01-01

Deep vein thrombosis and common complications, including pulmonary embolism and post thrombotic syndrome, represent a major source of morbidity and mortality worldwide. Experimental models of venous thrombosis have provided considerable insight into the cellular and molecular mechanisms that regulate thrombus formation and subsequent resolution. Here we critically appraise the ex vivo and in vivo techniques used to assess venous thrombosis in these models. Particular attention is paid to imaging modalities, including magnetic resonance imaging, micro computed tomography and high frequency ultrasound that facilitate longitudinal assessment of thrombus size and composition. PMID:26681755

Assessment of Venous Thrombosis in Animal Models.

PubMed

Grover, Steven P; Evans, Colin E; Patel, Ashish S; Modarai, Bijan; Saha, Prakash; Smith, Alberto

2016-02-01

Deep vein thrombosis and common complications, including pulmonary embolism and post-thrombotic syndrome, represent a major source of morbidity and mortality worldwide. Experimental models of venous thrombosis have provided considerable insight into the cellular and molecular mechanisms that regulate thrombus formation and subsequent resolution. Here, we critically appraise the ex vivo and in vivo techniques used to assess venous thrombosis in these models. Particular attention is paid to imaging modalities, including magnetic resonance imaging, micro-computed tomography, and high-frequency ultrasound that facilitate longitudinal assessment of thrombus size and composition. © 2015 American Heart Association, Inc.

Agent-based modeling of autophagy reveals emergent regulatory behavior of spatio-temporal autophagy dynamics.

PubMed

Börlin, Christoph S; Lang, Verena; Hamacher-Brady, Anne; Brady, Nathan R

2014-09-10

Autophagy is a vesicle-mediated pathway for lysosomal degradation, essential under basal and stressed conditions. Various cellular components, including specific proteins, protein aggregates, organelles and intracellular pathogens, are targets for autophagic degradation. Thereby, autophagy controls numerous vital physiological and pathophysiological functions, including cell signaling, differentiation, turnover of cellular components and pathogen defense. Moreover, autophagy enables the cell to recycle cellular components to metabolic substrates, thereby permitting prolonged survival under low nutrient conditions. Due to the multi-faceted roles for autophagy in maintaining cellular and organismal homeostasis and responding to diverse stresses, malfunction of autophagy contributes to both chronic and acute pathologies. We applied a systems biology approach to improve the understanding of this complex cellular process of autophagy. All autophagy pathway vesicle activities, i.e. creation, movement, fusion and degradation, are highly dynamic, temporally and spatially, and under various forms of regulation. We therefore developed an agent-based model (ABM) to represent individual components of the autophagy pathway, subcellular vesicle dynamics and metabolic feedback with the cellular environment, thereby providing a framework to investigate spatio-temporal aspects of autophagy regulation and dynamic behavior. The rules defining our ABM were derived from literature and from high-resolution images of autophagy markers under basal and activated conditions. Key model parameters were fit with an iterative method using a genetic algorithm and a predefined fitness function. From this approach, we found that accurate prediction of spatio-temporal behavior required increasing model complexity by implementing functional integration of autophagy with the cellular nutrient state. The resulting model is able to reproduce short-term autophagic flux measurements (up to 3 hours) under basal and activated autophagy conditions, and to measure the degree of cell-to-cell variability. Moreover, we experimentally confirmed two model predictions, namely (i) peri-nuclear concentration of autophagosomes and (ii) inhibitory lysosomal feedback on mTOR signaling. Agent-based modeling represents a novel approach to investigate autophagy dynamics, function and dysfunction with high biological realism. Our model accurately recapitulates short-term behavior and cell-to-cell variability under basal and activated conditions of autophagy. Further, this approach also allows investigation of long-term behaviors emerging from biologically-relevant alterations to vesicle trafficking and metabolic state.

Spatially resolved RNA-sequencing of the embryonic heart identifies a role for Wnt/β-catenin signaling in autonomic control of heart rate

PubMed Central

Burkhard, Silja Barbara

2018-01-01

Development of specialized cells and structures in the heart is regulated by spatially -restricted molecular pathways. Disruptions in these pathways can cause severe congenital cardiac malformations or functional defects. To better understand these pathways and how they regulate cardiac development we used tomo-seq, combining high-throughput RNA-sequencing with tissue-sectioning, to establish a genome-wide expression dataset with high spatial resolution for the developing zebrafish heart. Analysis of the dataset revealed over 1100 genes differentially expressed in sub-compartments. Pacemaker cells in the sinoatrial region induce heart contractions, but little is known about the mechanisms underlying their development. Using our transcriptome map, we identified spatially restricted Wnt/β-catenin signaling activity in pacemaker cells, which was controlled by Islet-1 activity. Moreover, Wnt/β-catenin signaling controls heart rate by regulating pacemaker cellular response to parasympathetic stimuli. Thus, this high-resolution transcriptome map incorporating all cell types in the embryonic heart can expose spatially restricted molecular pathways critical for specific cardiac functions. PMID:29400650

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

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.

Imaging and characterizing cells using tomography

PubMed Central

Do, Myan; Isaacson, Samuel A.; McDermott, Gerry; Le Gros, Mark A.; Larabell, Carolyn A.

2015-01-01

We can learn much about cell function by imaging and quantifying sub-cellular structures, especially if this is done non-destructively without altering said structures. Soft x-ray tomography (SXT) is a high-resolution imaging technique for visualizing cells and their interior structure in 3D. A tomogram of the cell, reconstructed from a series of 2D projection images, can be easily segmented and analyzed. SXT has a very high specimen throughput compared to other high-resolution structure imaging modalities; for example, tomographic data for reconstructing an entire eukaryotic cell is acquired in a matter of minutes. SXT visualizes cells without the need for chemical fixation, dehydration, or staining of the specimen. As a result, the SXT reconstructions are close representations of cells in their native state. SXT is applicable to most cell types. The deep penetration of soft x-rays allows cells, even mammalian cells, to be imaged without being sectioned. Image contrast in SXT is generated by the differential attenuation soft x-ray illumination as it passes through the specimen. Accordingly, each voxel in the tomographic reconstruction has a measured linear absorption coefficient (LAC) value. LAC values are quantitative and give rise to each sub-cellular component having a characteristic LAC profile, allowing organelles to be identified and segmented from the milieu of other cell contents. In this chapter, we describe the fundamentals of SXT imaging and how this technique can answer real world questions in the study of the nucleus. We also describe the development of correlative methods for the localization of specific molecules in a SXT reconstruction. The combination of fluorescence and SXT data acquired from the same specimen produces composite 3D images, rich with detailed information on the inner workings of cells. PMID:25602704

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.

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

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

PubMed

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

2006-06-30

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

Integrative, Dynamic Structural Biology at Atomic Resolution—It’s About Time

PubMed Central

van den Bedem, Henry; Fraser, James S.

2015-01-01

Biomolecules adopt a dynamic ensemble of conformations, each with the potential to interact with binding partners or perform the chemical reactions required for a multitude of cellular functions. Recent advances in X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, and other techniques are helping us realize the dream of seeing—in atomic detail—how different parts of biomolecules exchange between functional sub-states using concerted motions. Integrative structural biology has advanced our understanding of the formation of large macromolecular complexes and how their components interact in assemblies by leveraging data from many low-resolution methods. Here, we review the growing opportunities for integrative, dynamic structural biology at the atomic scale, contending there is increasing synergistic potential between X-ray crystallography, NMR, and computer simulations to reveal a structural basis for protein conformational dynamics at high resolution. PMID:25825836

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

PubMed Central

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

2016-01-01

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

Bringing the light to high throughput screening: use of optogenetic tools for the development of recombinant cellular assays

NASA Astrophysics Data System (ADS)

Agus, Viviana; Di Silvio, Alberto; Rolland, Jean Francois; Mondini, Anna; Tremolada, Sara; Montag, Katharina; Scarabottolo, Lia; Redaelli, Loredana; Lohmer, Stefan

2015-03-01

The use of light-activated proteins represents a powerful tool to control biological processes with high spatial and temporal precision. These so called "optogenetic" technologies have been successfully validated in many recombinant systems, and have been widely applied to the study of cellular mechanisms in intact tissues or behaving animals; to do that, complex, high-intensity, often home-made instrumentations were developed to achieve the optimal power and precision of light stimulation. In our study we sought to determine if this optical modulation can be obtained also in a miniaturized format, such as a 384-well plate, using the instrumentations normally dedicated to fluorescence analysis in High Throughput Screening (HTS) activities, such as for example the FLIPR (Fluorometric Imaging Plate Reader) instrument. We successfully generated optogenetic assays for the study of different ion channel targets: the CaV1.3 calcium channel was modulated by the light-activated Channelrhodopsin-2, the HCN2 cyclic nucleotide gated (CNG) channel was modulated by the light activated bPAC adenylyl cyclase, and finally the genetically encoded voltage indicator ArcLight was efficiently used to measure potassium, sodium or chloride channel activity. Our results showed that stable, robust and miniaturized cellular assays can be developed using different optogenetic tools, and efficiently modulated by the FLIPR instrument LEDs in a 384-well format. The spatial and temporal resolution delivered by this technology might enormously advantage the early stages of drug discovery, leading to the identification of more physiological and effective drug molecules.

High-resolution spatiotemporal strain mapping reveals non-uniform deformation in micropatterned elastomers

NASA Astrophysics Data System (ADS)

Aksoy, B.; Rehman, A.; Bayraktar, H.; Alaca, B. E.

2017-04-01

Micropatterns are generated on a vast selection of polymeric substrates for various applications ranging from stretchable electronics to cellular mechanobiological systems. When these patterned substrates are exposed to external loading, strain field is primarily affected by the presence of microfabricated structures and similarly by fabrication-related defects. The capturing of such nonhomogeneous strain fields is of utmost importance in cases where study of the mechanical behavior with a high spatial resolution is necessary. Image-based non-contact strain measurement techniques are favorable and have recently been extended to scanning tunneling microscope and scanning electron microscope images for the characterization of mechanical properties of metallic materials, e.g. steel and aluminum, at the microscale. A similar real-time analysis of strain heterogeneity in elastomers is yet to be achieved during the entire loading sequence. The available measurement methods for polymeric materials mostly depend on cross-head displacement or precalibrated strain values. Thus, they suffer either from the lack of any real-time analysis, spatiotemporal distribution or high resolution in addition to a combination of these factors. In this work, these challenges are addressed by integrating a tensile stretcher with an inverted optical microscope and developing a subpixel particle tracking algorithm. As a proof of concept, the patterns with a critical dimension of 200 µm are generated on polydimethylsiloxane substrates and strain distribution in the vicinity of the patterns is captured with a high spatiotemporal resolution. In the field of strain measurement, there is always a tradeoff between minimum measurable strain value and spatial resolution. Current noncontact techniques on elastomers can deliver a strain resolution of 0.001% over a minimum length of 5 cm. More importantly, inhomogeneities within this quite large region cannot be captured. The proposed technique can overcome this challenge and provides a displacement measurement resolution of 116 nm and a strain resolution of 0.04% over a gage length of 300 µm. Similarly, the ability to capture inhomogeneities is demonstrated by mapping strain around a thru-hole. The robustness of the technique is also evaluated, where no appreciable change in strain measurement is observed despite the significant variations imposed on the measurement mesh. The proposed approach introduces critical improvements for the determination of displacement and strain gradients in elastomers regarding the real-time nature of strain mapping with a microscale spatial resolution.

A 17-kDa Fragment of Lactoferrin Associates With the Termination of Inflammation and Peptides Within Promote Resolution

PubMed Central

Lutaty, Aviv; Soboh, Soaad; Schif-Zuck, Sagie; Zeituni-Timor, Orly; Rostoker, Ran; Podolska, Malgorzata J.; Schauer, Christine; Herrmann, Martin; Muñoz, Luis E.; Ariel, Amiram

2018-01-01

During the resolution of inflammation, macrophages engulf apoptotic polymorphonuclear cells (PMN) and can accumulate large numbers of their corpses. Here, we report that resolution phase macrophages acquire the neutrophil-derived glycoprotein lactoferrin (Lf) and fragments thereof in vivo and ex vivo. During the onset and resolving phases of inflammation in murine peritonitis and bovine mastitis, Lf fragments of 15 and 17 kDa occurred in various body fluids, and the murine fragmentation, accumulation, and release were mediated initially by neutrophils and later by efferocytic macrophages. The 17-kDa fragment contained two bioactive tripeptides, FKD and FKE that promoted resolution phase macrophage conversion to a pro-resolving phenotype. This resulted in a reduction in peritoneal macrophage numbers and an increase in the CD11blow subset of these cells. Moreover, FKE, but not FKD, peptides enhanced efferocytosis of apoptotic PMN, reduced TNFα and interleukin (IL)-6, and increased IL-10 secretion by lipopolysaccharide-stimulated macrophages ex vivo. In addition, FKE promoted neutrophil-mediated resolution at high concentrations (100 µM) by enhancing the formation of cytokine-scavenging aggregated NETs (tophi) at a low cellular density. Thus, PMN Lf is processed, acquired, and “recycled” by neutrophils and macrophages during inflammation resolution to generate fragments and peptides with paramount pro-resolving activities. PMID:29643857

Adaptive optics and the eye (super resolution OCT).

PubMed

Miller, D T; Kocaoglu, O P; Wang, Q; Lee, S

2011-03-01

The combination of adaptive optics (AO) and optical coherence tomography (OCT) was first reported 8 years ago and has undergone tremendous technological advances since then. The technical benefits of adding AO to OCT (increased lateral resolution, smaller speckle, and enhanced sensitivity) increase the imaging capability of OCT in ways that make it well suited for three-dimensional (3D) cellular imaging in the retina. Today, AO-OCT systems provide ultrahigh 3D resolution (3 × 3 × 3 μm³) and ultrahigh speed (up to an order of magnitude faster than commercial OCT). AO-OCT systems have been used to capture volume images of retinal structures, previously only visible with histology, and are being used for studying clinical conditions. Here, we present representative examples of cellular structures that can be visualized with AO-OCT. We overview three studies from our laboratory that used ultrahigh-resolution AO-OCT to measure the cross-sectional profiles of individual bundles in the retinal nerve fiber layer; the diameters of foveal capillaries that define the terminal rim of the foveal avascular zone; and the spacing and length of individual cone photoreceptor outer segments as close as 0.5° from the fovea center.

Endoscopic probe optics for spectrally encoded confocal microscopy.

PubMed

Kang, Dongkyun; Carruth, Robert W; Kim, Minkyu; Schlachter, Simon C; Shishkov, Milen; Woods, Kevin; Tabatabaei, Nima; Wu, Tao; Tearney, Guillermo J

2013-01-01

Spectrally encoded confocal microscopy (SECM) is a form of reflectance confocal microscopy that can achieve high imaging speeds using relatively simple probe optics. Previously, the feasibility of conducting large-area SECM imaging of the esophagus in bench top setups has been demonstrated. Challenges remain, however, in translating SECM into a clinically-useable device; the tissue imaging performance should be improved, and the probe size needs to be significantly reduced so that it can fit into luminal organs of interest. In this paper, we report the development of new SECM endoscopic probe optics that addresses these challenges. A custom water-immersion aspheric singlet (NA = 0.5) was developed and used as the objective lens. The water-immersion condition was used to reduce the spherical aberrations and specular reflection from the tissue surface, which enables cellular imaging of the tissue deep below the surface. A custom collimation lens and a small-size grating were used along with the custom aspheric singlet to reduce the probe size. A dual-clad fiber was used to provide both the single- and multi- mode detection modes. The SECM probe optics was made to be 5.85 mm in diameter and 30 mm in length, which is small enough for safe and comfortable endoscopic imaging of the gastrointestinal tract. The lateral resolution was 1.8 and 2.3 µm for the single- and multi- mode detection modes, respectively, and the axial resolution 11 and 17 µm. SECM images of the swine esophageal tissue demonstrated the capability of this device to enable the visualization of characteristic cellular structural features, including basal cell nuclei and papillae, down to the imaging depth of 260 µm. These results suggest that the new SECM endoscopic probe optics will be useful for imaging large areas of the esophagus at the cellular scale in vivo.

Tissue vascularization through 3D printing: Will technology bring us flow?

PubMed

Paulsen, S J; Miller, J S

2015-05-01

Though in vivo models provide the most physiologically relevant environment for studying tissue function, in vitro studies provide researchers with explicit control over experimental conditions and the potential to develop high throughput testing methods. In recent years, advancements in developmental biology research and imaging techniques have significantly improved our understanding of the processes involved in vascular development. However, the task of recreating the complex, multi-scale vasculature seen in in vivo systems remains elusive. 3D bioprinting offers a potential method to generate controlled vascular networks with hierarchical structure approaching that of in vivo networks. Bioprinting is an interdisciplinary field that relies on advances in 3D printing technology along with advances in imaging and computational modeling, which allow researchers to monitor cellular function and to better understand cellular environment within the printed tissue. As bioprinting technologies improve with regards to resolution, printing speed, available materials, and automation, 3D printing could be used to generate highly controlled vascularized tissues in a high throughput manner for use in regenerative medicine and the development of in vitro tissue models for research in developmental biology and vascular diseases. © 2015 Wiley Periodicals, Inc.

Living Cell Microarrays: An Overview of Concepts

PubMed Central

Jonczyk, Rebecca; Kurth, Tracy; Lavrentieva, Antonina; Walter, Johanna-Gabriela; Scheper, Thomas; Stahl, Frank

2016-01-01

Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays. PMID:27600077

Cellular Force Microscopy for in Vivo Measurements of Plant Tissue Mechanics1[W][OA

PubMed Central

Routier-Kierzkowska, Anne-Lise; Weber, Alain; Kochova, Petra; Felekis, Dimitris; Nelson, Bradley J.; Kuhlemeier, Cris; Smith, Richard S.

2012-01-01

Although growth and morphogenesis are controlled by genetics, physical shape change in plant tissue results from a balance between cell wall loosening and intracellular pressure. Despite recent work demonstrating a role for mechanical signals in morphogenesis, precise measurement of mechanical properties at the individual cell level remains a technical challenge. To address this challenge, we have developed cellular force microscopy (CFM), which combines the versatility of classical microindentation techniques with the high automation and resolution approaching that of atomic force microscopy. CFM’s large range of forces provides the possibility to map the apparent stiffness of both plasmolyzed and turgid tissue as well as to perform micropuncture of cells using very high stresses. CFM experiments reveal that, within a tissue, local stiffness measurements can vary with the level of turgor pressure in an unexpected way. Altogether, our results highlight the importance of detailed physically based simulations for the interpretation of microindentation results. CFM’s ability to be used both to assess and manipulate tissue mechanics makes it a method of choice to unravel the feedbacks between mechanics, genetics, and morphogenesis. PMID:22353572

Dynamic maps of UV damage formation and repair for the human genome

PubMed Central

Hu, Jinchuan; Adebali, Ogun; Adar, Sheera; Sancar, Aziz

2017-01-01

Formation and repair of UV-induced DNA damage in human cells are affected by cellular context. To study factors influencing damage formation and repair genome-wide, we developed a highly sensitive single-nucleotide resolution damage mapping method [high-sensitivity damage sequencing (HS–Damage-seq)]. Damage maps of both cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] from UV-irradiated cellular and naked DNA revealed that the effect of transcription factor binding on bulky adducts formation varies, depending on the specific transcription factor, damage type, and strand. We also generated time-resolved UV damage maps of both CPDs and (6-4)PPs by HS–Damage-seq and compared them to the complementary repair maps of the human genome obtained by excision repair sequencing to gain insight into factors that affect UV-induced DNA damage and repair and ultimately UV carcinogenesis. The combination of the two methods revealed that, whereas UV-induced damage is virtually uniform throughout the genome, repair is affected by chromatin states, transcription, and transcription factor binding, in a manner that depends on the type of DNA damage. PMID:28607063

Dynamic maps of UV damage formation and repair for the human genome.

PubMed

Hu, Jinchuan; Adebali, Ogun; Adar, Sheera; Sancar, Aziz

2017-06-27

Formation and repair of UV-induced DNA damage in human cells are affected by cellular context. To study factors influencing damage formation and repair genome-wide, we developed a highly sensitive single-nucleotide resolution damage mapping method [high-sensitivity damage sequencing (HS-Damage-seq)]. Damage maps of both cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] from UV-irradiated cellular and naked DNA revealed that the effect of transcription factor binding on bulky adducts formation varies, depending on the specific transcription factor, damage type, and strand. We also generated time-resolved UV damage maps of both CPDs and (6-4)PPs by HS-Damage-seq and compared them to the complementary repair maps of the human genome obtained by excision repair sequencing to gain insight into factors that affect UV-induced DNA damage and repair and ultimately UV carcinogenesis. The combination of the two methods revealed that, whereas UV-induced damage is virtually uniform throughout the genome, repair is affected by chromatin states, transcription, and transcription factor binding, in a manner that depends on the type of DNA damage.

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

PubMed

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

2014-08-01

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

Mass spectrometry-based proteomics: from cancer biology to protein biomarkers, drug targets, and clinical applications.

PubMed

Jimenez, Connie R; Verheul, Henk M W

2014-01-01

Proteomics is optimally suited to bridge the gap between genomic information on the one hand and biologic functions and disease phenotypes at the other, since it studies the expression and/or post-translational modification (especially phosphorylation) of proteins--the major cellular players bringing about cellular functions--at a global level in biologic specimens. Mass spectrometry technology and (bio)informatic tools have matured to the extent that they can provide high-throughput, comprehensive, and quantitative protein inventories of cells, tissues, and biofluids in clinical samples at low level. In this article, we focus on next-generation proteomics employing nanoliquid chromatography coupled to high-resolution tandem mass spectrometry for in-depth (phospho)protein profiling of tumor tissues and (proximal) biofluids, with a focus on studies employing clinical material. In addition, we highlight emerging proteogenomic approaches for the identification of tumor-specific protein variants, and targeted multiplex mass spectrometry strategies for large-scale biomarker validation. Below we provide a discussion of recent progress, some research highlights, and challenges that remain for clinical translation of proteomic discoveries.

Multimodal optoacoustic and multiphoton fluorescence microscopy

NASA Astrophysics Data System (ADS)

Sela, Gali; Razansky, Daniel; Shoham, Shy

2013-03-01

Multiphoton microscopy is a powerful imaging modality that enables structural and functional imaging with cellular and sub-cellular resolution, deep within biological tissues. Yet, its main contrast mechanism relies on extrinsically administered fluorescent indicators. Here we developed a system for simultaneous multimodal optoacoustic and multiphoton fluorescence 3D imaging, which attains both absorption and fluorescence-based contrast by integrating an ultrasonic transducer into a two-photon laser scanning microscope. The system is readily shown to enable acquisition of multimodal microscopic images of fluorescently labeled targets and cell cultures as well as intrinsic absorption-based images of pigmented biological tissue. During initial experiments, it was further observed that that detected optoacoustically-induced response contains low frequency signal variations, presumably due to cavitation-mediated signal generation by the high repetition rate (80MHz) near IR femtosecond laser. The multimodal system may provide complementary structural and functional information to the fluorescently labeled tissue, by superimposing optoacoustic images of intrinsic tissue chromophores, such as melanin deposits, pigmentation, and hemoglobin or other extrinsic particle or dye-based markers highly absorptive in the NIR spectrum.

The resolution of ambiguity as the basis for life: A cellular bridge between Western reductionism and Eastern holism.

PubMed

Torday, John S; Miller, William B

2017-12-01

Boundary conditions enable cellular life through negentropy, chemiosmosis, and homeostasis as identifiable First Principles of Physiology. Self-referential awareness of status arises from this organized state to sustain homeostatic imperatives. Preferred homeostatic status is dependent upon the appraisal of information and its communication. However, among living entities, sources of information and their dissemination are always imprecise. Consequently, living systems exist within an innate state of ambiguity. It is presented that cellular life and evolutionary development are a self-organizing cellular response to uncertainty in iterative conformity with its basal initiating parameters. Viewing the life circumstance in this manner permits a reasoned unification between Western rational reductionism and Eastern holism. Copyright © 2017 Elsevier Ltd. All rights reserved.

Line-Scanning Particle Image Velocimetry: An Optical Approach for Quantifying a Wide Range of Blood Flow Speeds in Live Animals

PubMed Central

Kim, Tyson N.; Goodwill, Patrick W.; Chen, Yeni; Conolly, Steven M.; Schaffer, Chris B.; Liepmann, Dorian; Wang, Rong A.

2012-01-01

Background The ability to measure blood velocities is critical for studying vascular development, physiology, and pathology. A key challenge is to quantify a wide range of blood velocities in vessels deep within living specimens with concurrent diffraction-limited resolution imaging of vascular cells. Two-photon laser scanning microscopy (TPLSM) has shown tremendous promise in analyzing blood velocities hundreds of micrometers deep in animals with cellular resolution. However, current analysis of TPLSM-based data is limited to the lower range of blood velocities and is not adequate to study faster velocities in many normal or disease conditions. Methodology/Principal Findings We developed line-scanning particle image velocimetry (LS-PIV), which used TPLSM data to quantify peak blood velocities up to 84 mm/s in live mice harboring brain arteriovenous malformation, a disease characterized by high flow. With this method, we were able to accurately detect the elevated blood velocities and exaggerated pulsatility along the abnormal vascular network in these animals. LS-PIV robustly analyzed noisy data from vessels as deep as 850 µm below the brain surface. In addition to analyzing in vivo data, we validated the accuracy of LS-PIV up to 800 mm/s using simulations with known velocity and noise parameters. Conclusions/Significance To our knowledge, these blood velocity measurements are the fastest recorded with TPLSM. Partnered with transgenic mice carrying cell-specific fluorescent reporters, LS-PIV will also enable the direct in vivo correlation of cellular, biochemical, and hemodynamic parameters in high flow vascular development and diseases such as atherogenesis, arteriogenesis, and vascular anomalies. PMID:22761686

Ultrasonic three-dimensional on-chip cell culture for dynamic studies of tumor immune surveillance by natural killer cells.

PubMed

Christakou, Athanasia E; Ohlin, Mathias; Önfelt, Björn; Wiklund, Martin

2015-08-07

We demonstrate a simple method for three-dimensional (3D) cell culture controlled by ultrasonic standing waves in a multi-well microplate. The method gently arranges cells in a suspension into a single aggregate in each well of the microplate and, by this, nucleates 3D tissue-like cell growth for culture times between two and seven days. The microplate device is compatible with both high-resolution optical microscopy and maintenance in a standard cell incubator. The result is a scaffold- and coating-free method for 3D cell culture that can be used for controlling the cellular architecture, as well as the cellular and molecular composition of the microenvironment in and around the formed cell structures. We demonstrate the parallel production of one hundred synthetic 3D solid tumors comprising up to thousands of human hepatocellular carcinoma (HCC) HepG2 cells, we characterize the tumor structure by high-resolution optical microscopy, and we monitor the functional behavior of natural killer (NK) cells migrating, docking and interacting with the tumor model during culture. Our results show that the method can be used for determining the collective ability of a given number of NK cells to defeat a solid tumor having a certain size, shape and composition. The ultrasound-based method itself is generic and can meet any demand from applications where it is advantageous to monitor cell culture from production to analysis of 3D tissue or tumor models using microscopy in one single microplate device.

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

PubMed

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

2017-01-01

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

Helium ion microscopy and ultra-high-resolution scanning electron microscopy analysis of membrane-extracted cells reveals novel characteristics of the cytoskeleton of Giardia intestinalis.

PubMed

Gadelha, Ana Paula Rocha; Benchimol, Marlene; de Souza, Wanderley

2015-06-01

Giardia intestinalis presents a complex microtubular cytoskeleton formed by specialized structures, such as the adhesive disk, four pairs of flagella, the funis and the median body. The ultrastructural organization of the Giardia cytoskeleton has been analyzed using different microscopic techniques, including high-resolution scanning electron microscopy. Recent advances in scanning microscopy technology have opened a new venue for the characterization of cellular structures and include scanning probe microscopy techniques such as ultra-high-resolution scanning electron microscopy (UHRSEM) and helium ion microscopy (HIM). Here, we studied the organization of the cytoskeleton of G. intestinalis trophozoites using UHRSEM and HIM in membrane-extracted cells. The results revealed a number of new cytoskeletal elements associated with the lateral crest and the dorsal surface of the parasite. The fine structure of the banded collar was also observed. The marginal plates were seen linked to a network of filaments, which were continuous with filaments parallel to the main cell axis. Cytoplasmic filaments that supported the internal structures were seen by the first time. Using anti-actin antibody, we observed a labeling in these filamentous structures. Taken together, these data revealed new surface characteristics of the cytoskeleton of G. intestinalis and may contribute to an improved understanding of the structural organization of trophozoites. Copyright © 2015 Elsevier Inc. All rights reserved.

Imaging and reconstruction of cell cortex structures near the cell surface

NASA Astrophysics Data System (ADS)

Jin, Luhong; Zhou, Xiaoxu; Xiu, Peng; Luo, Wei; Huang, Yujia; Yu, Feng; Kuang, Cuifang; Sun, Yonghong; Liu, Xu; Xu, Yingke

2017-11-01

Total internal reflection fluorescence microscopy (TIRFM) provides high optical sectioning capability and superb signal-to-noise ratio for imaging of cell cortex structures. The development of multi-angle (MA)-TIRFM permits high axial resolution imaging and reconstruction of cellular structures near the cell surface. Cytoskeleton is composed of a network of filaments, which are important for maintenance of cell function. The high-resolution imaging and quantitative analysis of filament organization would contribute to our understanding of cytoskeleton regulation in cell. Here, we used a custom-developed MA-TIRFM setup, together with stochastic photobleaching and single molecule localization method, to enhance the lateral resolution of TIRFM imaging to about 100 nm. In addition, we proposed novel methods to perform filament segmentation and 3D reconstruction from MA-TIRFM images. Furthermore, we applied these methods to study the 3D localization of cortical actin and microtubule structures in U373 cancer cells. Our results showed that cortical actins localize ∼ 27 nm closer to the plasma membrane when compared with microtubules. We found that treatment of cells with chemotherapy drugs nocodazole and cytochalasin B disassembles cytoskeletal network and induces the reorganization of filaments towards the cell periphery. In summary, this study provides feasible approaches for 3D imaging and analyzing cell surface distribution of cytoskeletal network. Our established microscopy platform and image analysis toolkits would facilitate the study of cytoskeletal network in cells.

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.

Removing Contamination-Induced Reconstruction Artifacts from Cryo-electron Tomograms

PubMed Central

Fernandez, Jose-Jesus; Laugks, Ulrike; Schaffer, Miroslava; Bäuerlein, Felix J.B.; Khoshouei, Maryam; Baumeister, Wolfgang; Lucic, Vladan

2016-01-01

Imaging of fully hydrated, vitrified biological samples by electron tomography yields structural information about cellular protein complexes in situ. Here we present a computational procedure that removes artifacts of three-dimensional reconstruction caused by contamination present in samples during imaging by electron microscopy. Applying the procedure to phantom data and electron tomograms of cellular samples significantly improved the resolution and the interpretability of tomograms. Artifacts caused by surface contamination associated with thinning by focused ion beam, as well as those arising from gold fiducial markers and from common, lower contrast contamination, could be removed. Our procedure is widely applicable and is especially suited for applications that strive to reach a higher resolution and involve the use of recently developed, state-of-the-art instrumentation. PMID:26743046

High resolution x-ray microtomography of biological samples: Requirements and strategies for satisfying them

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

Loo, B.W. Jr.

High resolution x-ray microscopy has been made possible in recent years primarily by two new technologies: microfabricated diffractive lenses for soft x-rays with about 30-50 nm resolution, and high brightness synchrotron x-ray sources. X-ray microscopy occupies a special niche in the array of biological microscopic imaging methods. It extends the capabilities of existing techniques mainly in two areas: a previously unachievable combination of sub-visible resolution and multi-micrometer sample size, and new contrast mechanisms. Because of the soft x-ray wavelengths used in biological imaging (about 1-4 nm), XM is intermediate in resolution between visible light and electron microscopies. Similarly, the penetrationmore » depth of soft x-rays in biological materials is such that the ideal sample thickness for XM falls in the range of 0.25 - 10 {mu}m, between that of VLM and EM. XM is therefore valuable for imaging of intermediate level ultrastructure, requiring sub-visible resolutions, in intact cells and subcellular organelles, without artifacts produced by thin sectioning. Many of the contrast producing and sample preparation techniques developed for VLM and EM also work well with XM. These include, for example, molecule specific staining by antibodies with heavy metal or fluorescent labels attached, and sectioning of both frozen and plastic embedded tissue. However, there is also a contrast mechanism unique to XM that exists naturally because a number of elemental absorption edges lie in the wavelength range used. In particular, between the oxygen and carbon absorption edges (2.3 and 4.4 nm wavelength), organic molecules absorb photons much more strongly than does water, permitting element-specific imaging of cellular structure in aqueous media, with no artifically introduced contrast agents. For three-dimensional imaging applications requiring the capabilities of XM, an obvious extension of the technique would therefore be computerized x-ray microtomography (XMT).« less

Fluorine-containing nanoemulsions for MRI cell tracking

PubMed Central

Janjic, Jelena M.; Ahrens, Eric T.

2009-01-01

In this article we review the chemistry and nanoemulsion formulation of perfluorocarbons used for in vivo 19F MRI cell tracking. In this application, cells of interest are labeled in culture using a perfluorocarbon nanoemulsion. Labeled cells are introduced into a subject and tracked using 19F MRI or NMR spectroscopy. In the same imaging session, a high-resolution, conventional (1H) image can be used to place the 19F-labeled cells into anatomical context. Perfluorocarbon-based 19F cell tracking is a useful technology because of the high specificity for labeled cells, ability to quantify cell accumulations, and biocompatibility. This technology can be widely applied to studies of inflammation, cellular regenerative medicine, and immunotherapy. PMID:19920872

Modification of measurement methods for evaluation of tissue-engineered cartilage function and biochemical properties using nanosecond pulsed laser

NASA Astrophysics Data System (ADS)

Ishihara, Miya; Sato, Masato; Kutsuna, Toshiharu; Ishihara, Masayuki; Mochida, Joji; Kikuchi, Makoto

2008-02-01

There is a demand in the field of regenerative medicine for measurement technology that enables determination of functions and components of engineered tissue. To meet this demand, we developed a method for extracellular matrix characterization using time-resolved autofluorescence spectroscopy, which enabled simultaneous measurements with mechanical properties using relaxation of laser-induced stress wave. In this study, in addition to time-resolved fluorescent spectroscopy, hyperspectral sensor, which enables to capture both spectral and spatial information, was used for evaluation of biochemical characterization of tissue-engineered cartilage. Hyperspectral imaging system provides spectral resolution of 1.2 nm and image rate of 100 images/sec. The imaging system consisted of the hyperspectral sensor, a scanner for x-y plane imaging, magnifying optics and Xenon lamp for transmmissive lighting. Cellular imaging using the hyperspectral image system has been achieved by improvement in spatial resolution up to 9 micrometer. The spectroscopic cellular imaging could be observed using cultured chondrocytes as sample. At early stage of culture, the hyperspectral imaging offered information about cellular function associated with endogeneous fluorescent biomolecules.

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

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

PubMed Central

Ortega, Richard; Devès, Guillaume; Carmona, Asunción

2009-01-01

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

Spatiotemporal endothelial cell - pericyte association in tumors as shown by high resolution 4D intravital imaging.

PubMed

Seynhaeve, Ann L B; Oostinga, Douwe; van Haperen, Rien; Eilken, Hanna M; Adams, Susanne; Adams, Ralf H; Ten Hagen, Timo L M

2018-06-25

Endothelial cells and pericytes are integral cellular components of the vasculature with distinct interactive functionalities. To study dynamic interactions between these two cells we created two transgenic animal lines. A truncated eNOS (endothelial nitric oxide synthase) construct was used as a GFP tag for endothelial cell evaluation and an inducible Cre-lox recombination, under control of the Pdgfrb (platelet derived growth factor receptor beta) promoter, was created for pericyte assessment. Also, eNOStag-GFP animals were crossed with the already established Cspg4-DsRed mice expressing DsRed fluorescent protein in pericytes. For intravital imaging we used tumors implanted in the dorsal skinfold of these transgenic animals. This setup allowed us to study time and space dependent complexities, such as distribution, morphology, motility, and association between both vascular cell types in all angiogenetic stages, without the need for additional labeling. Moreover, as fluorescence was still clearly detectable after fixation, it is possible to perform comparative histology following intravital evaluation. These transgenic mouse lines form an excellent model to capture collective and individual cellular and subcellular endothelial cell - pericyte dynamics and will help answer key questions on the cellular and molecular relationship between these two cells.

Cations Form Sequence Selective Motifs within DNA Grooves via a Combination of Cation-Pi and Ion-Dipole/Hydrogen Bond Interactions

PubMed Central

Stewart, Mikaela; Dunlap, Tori; Dourlain, Elizabeth; Grant, Bryce; McFail-Isom, Lori

2013-01-01

The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl+) and the polarized first hydration shell waters of divalent cations (Mg2+, Ca2+) form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves. PMID:23940752

Cations form sequence selective motifs within DNA grooves via a combination of cation-pi and ion-dipole/hydrogen bond interactions.

PubMed

Stewart, Mikaela; Dunlap, Tori; Dourlain, Elizabeth; Grant, Bryce; McFail-Isom, Lori

2013-01-01

The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl⁺) and the polarized first hydration shell waters of divalent cations (Mg²⁺, Ca²⁺) form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves.

Chronic cranial window with access port for repeated cellular manipulations, drug application, and electrophysiology

PubMed Central

Roome, Christopher J.; Kuhn, Bernd

2014-01-01

Chronic cranial windows have been instrumental in advancing optical studies in vivo, permitting long-term, high-resolution imaging in various brain regions. However, once a window is attached it is difficult to regain access to the brain under the window for cellular manipulations. Here we describe a simple device that combines long term in vivo optical imaging with direct brain access via glass or quartz pipettes and metal, glass, or quartz electrodes for cellular manipulations like dye or drug injections and electrophysiological stimulations or recordings while keeping the craniotomy sterile. Our device comprises a regular cranial window glass coverslip with a drilled access hole later sealed with biocompatible silicone. This chronic cranial window with access port is cheap, easy to manufacture, can be mounted just as the regular chronic cranial window, and is self-sealing after retraction of the pipette or electrode. We demonstrate that multiple injections can be performed through the silicone port by repetitively bolus loading calcium sensitive dye into mouse barrel cortex and recording spontaneous cellular activity over a period of weeks. As an example to the extent of its utility for electrophysiological recording, we describe how simple removal of the silicone seal can permit patch pipette access for whole-cell patch clamp recordings in vivo. During these chronic experiments we do not observe any infections under the window or impairment of animal health. PMID:25426027

Integrated hybrid polystyrene-polydimethylsiloxane device for monitoring cellular release with microchip electrophoresis and electrochemical detection

PubMed Central

Johnson, Alicia S.; Mehl, Benjamin T.; Martin, R. Scott

2015-01-01

In this work, a polystyrene (PS)-polydimethylsiloxane (PDMS) hybrid device was developed to enable the integration of cell culture with analysis by microchip electrophoresis and electrochemical detection. It is shown that this approach combines the fundamental advantages of PDMS devices (the ability to integrate pumps and valves) and PS devices (the ability to permanently embed fluidic tubing and electrodes). The embedded fused-silica capillary enables high temporal resolution measurements from off-chip cell culture dishes and the embedded electrodes provide close to real-time analysis of small molecule neurotransmitters. A novel surface treatment for improved (reversible) adhesion between PS and PDMS is described using a chlorotrimethylsilane stamping method. It is demonstrated that a Pd decoupler is efficient at handling the high current (and cathodic hydrogen production) resulting from use of high ionic strength buffers needed for cellular analysis; thus allowing an electrophoretic separation and in-channel detection. The separation of norepinephrine (NE) and dopamine (DA) in highly conductive biological buffers was optimized using a mixed surfactant system. This PS-PDMS hybrid device integrates multiple processes including continuous sampling from a cell culture dish, on-chip pump and valving technologies, microchip electrophoresis, and electrochemical detection to monitor neurotransmitter release from PC 12 cells. PMID:25663849

Cellular automata modeling depicts degradation of cellulosic material by a cellulase system with single-molecule resolution.

PubMed

Eibinger, Manuel; Zahel, Thomas; Ganner, Thomas; Plank, Harald; Nidetzky, Bernd

2016-01-01

Enzymatic hydrolysis of cellulose involves the spatiotemporally correlated action of distinct polysaccharide chain cleaving activities confined to the surface of an insoluble substrate. Because cellulases differ in preference for attacking crystalline compared to amorphous cellulose, the spatial distribution of structural order across the cellulose surface imposes additional constraints on the dynamic interplay between the enzymes. Reconstruction of total system behavior from single-molecule activity parameters is a longstanding key goal in the field. We have developed a stochastic, cellular automata-based modeling approach to describe degradation of cellulosic material by a cellulase system at single-molecule resolution. Substrate morphology was modeled to represent the amorphous and crystalline phases as well as the different spatial orientations of the polysaccharide chains. The enzyme system model consisted of an internally chain-cleaving endoglucanase (EG) as well as two processively acting, reducing and non-reducing chain end-cleaving cellobiohydrolases (CBHs). Substrate preference (amorphous: EG, CBH II; crystalline: CBH I) and characteristic frequencies for chain cleavage, processive movement, and dissociation were assigned from biochemical data. Once adsorbed, enzymes were allowed to reach surface-exposed substrate sites through "random-walk" lateral diffusion or processive motion. Simulations revealed that slow dissociation of processive enzymes at obstacles obstructing further movement resulted in local jamming of the cellulases, with consequent delay in the degradation of the surface area affected. Exploiting validation against evidence from atomic force microscopy imaging as a unique opportunity opened up by the modeling approach, we show that spatiotemporal characteristics of cellulose surface degradation by the system of synergizing cellulases were reproduced quantitatively at the nanometer resolution of the experimental data. This in turn gave useful prediction of the soluble sugar release rate. Salient dynamic features of cellulose surface degradation by different cellulases acting in synergy were reproduced in simulations in good agreement with evidence from high-resolution visualization experiments. Due to the single-molecule resolution of the modeling approach, the utility of the presented model lies not only in predicting system behavior but also in elucidating inherently complex (e.g., stochastic) phenomena involved in enzymatic cellulose degradation. Thus, it creates synergy with experiment to advance the mechanistic understanding for improved application.

Is the Cortical Deficit in Amblyopia Due to Reduced Cortical Magnification, Loss of Neural Resolution, or Neural Disorganization?

PubMed

Clavagnier, Simon; Dumoulin, Serge O; Hess, Robert F

2015-11-04

The neural basis of amblyopia is a matter of debate. The following possibilities have been suggested: loss of foveal cells, reduced cortical magnification, loss of spatial resolution of foveal cells, and topographical disarray in the cellular map. To resolve this we undertook a population receptive field (pRF) functional magnetic resonance imaging analysis in the central field in humans with moderate-to-severe amblyopia. We measured the relationship between averaged pRF size and retinal eccentricity in retinotopic visual areas. Results showed that cortical magnification is normal in the foveal field of strabismic amblyopes. However, the pRF sizes are enlarged for the amblyopic eye. We speculate that the pRF enlargement reflects loss of cellular resolution or an increased cellular positional disarray within the representation of the amblyopic eye. The neural basis of amblyopia, a visual deficit affecting 3% of the human population, remains a matter of debate. We undertook the first population receptive field functional magnetic resonance imaging analysis in participants with amblyopia and compared the projections from the amblyopic and fellow normal eye in the visual cortex. The projection from the amblyopic eye was found to have a normal cortical magnification factor, enlarged population receptive field sizes, and topographic disorganization in all early visual areas. This is consistent with an explanation of amblyopia as an immature system with a normal complement of cells whose spatial resolution is reduced and whose topographical map is disordered. This bears upon a number of competing theories for the psychophysical defect and affects future treatment therapies. Copyright © 2015 the authors 0270-6474/15/3514740-16$15.00/0.

Fluorescent scanning laser ophthalmoscopy for cellular resolution in vivo mouse retinal imaging: benefits and drawbacks of implementing adaptive optics (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zhang, Pengfei; Goswami, Mayank; Pugh, Edward N.; Zawadzki, Robert J.

2016-03-01

Scanning Laser Ophthalmoscopy (SLO) is a very important imaging tool in ophthalmology research. By combing with Adaptive Optics (AO) technique, AO-SLO can correct for ocular aberrations resulting in cellular level resolution, allowing longitudinal studies of single cells morphology in the living eyes. The numerical aperture (NA) sets the optical resolution that can be achieve in the "classical" imaging systems. Mouse eye has more than twice NA of the human eye, thus offering theoretically higher resolution. However, in most SLO based imaging systems the imaging beam size at mouse pupil sets the NA of that instrument, while most of the AO-SLO systems use almost the full NA of the mouse eye. In this report, we first simulated the theoretical resolution that can be achieved in vivo for different imaging beam sizes (different NA), assumingtwo cases: no aberrations and aberrations based on published mouse ocular wavefront data. Then we imaged mouse retinas with our custom build SLO system using different beam sizes to compare these results with theory. Further experiments include comparison of the SLO and AO-SLO systems for imaging different type of fluorescently labeled cells (microglia, ganglion, photoreceptors, etc.). By comparing those results and taking into account systems complexity and ease of use, the benefits and drawbacks of two imaging systems will be discussed.

Molecular monitoring of epithelial-to-mesenchymal transition in breast cancer cells by means of Raman spectroscopy.

PubMed

Marro, M; Nieva, C; Sanz-Pamplona, R; Sierra, A

2014-09-01

In breast cancer the presence of cells undergoing the epithelial-to-mesenchymal transition is indicative of metastasis progression. Since metabolic features of breast tumour cells are critical in cancer progression and drug resistance, we hypothesized that the lipid content of malignant cells might be a useful indirect measure of cancer progression. In this study Multivariate Curve Resolution was applied to cellular Raman spectra to assess the metabolic composition of breast cancer cells undergoing the epithelial to mesenchymal transition. Multivariate Curve Resolution analysis led to the conclusion that this transition affects the lipid profile of cells, increasing tryptophan but maintaining a low fatty acid content in comparison with highly metastatic cells. Supporting those results, a Partial Least Square-Discriminant analysis was performed to test the ability of Raman spectroscopy to discriminate the initial steps of epithelial to mesenchymal transition in breast cancer cells. We achieved a high level of sensitivity and specificity, 94% and 100%, respectively. In conclusion, Raman microspectroscopy coupled with Multivariate Curve Resolution enables deconvolution and tracking of the molecular content of cancer cells during a biochemical process, being a powerful, rapid, reagent-free and non-invasive tool for identifying metabolic features of breast cancer cell aggressiveness at first stages of malignancy. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparison of Cornea Module and DermaInspect for noninvasive imaging of ocular surface pathologies

NASA Astrophysics Data System (ADS)

Steven, Philipp; Müller, Maya; Koop, Norbert; Rose, Christian; Hüttmann, Gereon

2009-11-01

Minimally invasive imaging of ocular surface pathologies aims at securing clinical diagnosis without actual tissue probing. For this matter, confocal microscopy (Cornea Module) is in daily use in ophthalmic practice. Multiphoton microscopy is a new optical technique that enables high-resolution imaging and functional analysis of living tissues based on tissue autofluorescence. This study was set up to compare the potential of a multiphoton microscope (DermaInspect) to the Cornea Module. Ocular surface pathologies such as pterygia, papillomae, and nevi were investigated in vivo using the Cornea Module and imaged immediately after excision by DermaInspect. Two excitation wavelengths, fluorescence lifetime imaging and second-harmonic generation (SHG), were used to discriminate different tissue structures. Images were compared with the histopathological assessment of the samples. At wavelengths of 730 nm, multiphoton microscopy exclusively revealed cellular structures. Collagen fibrils were specifically demonstrated by second-harmonic generation. Measurements of fluorescent lifetimes enabled the highly specific detection of goblet cells, erythrocytes, and nevus-cell clusters. At the settings used, DermaInspect reaches higher resolutions than the Cornea Module and obtains additional structural information. The parallel detection of multiphoton excited autofluorescence and confocal imaging could expand the possibilities of minimally invasive investigation of the ocular surface toward functional analysis at higher resolutions.

Regulation of dopamine D1 receptor dynamics within the postsynaptic density of hippocampal glutamate synapses.

PubMed

Ladepeche, Laurent; Yang, Luting; Bouchet, Delphine; Groc, Laurent

2013-01-01

Dopamine receptor potently modulates glutamate signalling, synaptic plasticity and neuronal network adaptations in various pathophysiological processes. Although key intracellular signalling cascades have been identified, the cellular mechanism by which dopamine and glutamate receptor-mediated signalling interplay at glutamate synapse remain poorly understood. Among the cellular mechanisms proposed to aggregate D1R in glutamate synapses, the direct interaction between D1R and the scaffold protein PSD95 or the direct interaction with the glutamate NMDA receptor (NMDAR) have been proposed. To tackle this question we here used high-resolution single nanoparticle imaging since it provides a powerful way to investigate at the sub-micron resolution the dynamic interaction between these partners in live synapses. We demonstrate in hippocampal neuronal networks that dopamine D1 receptors (D1R) laterally diffuse within glutamate synapses, in which their diffusion is reduced. Disrupting the interaction between D1R and PSD95, through genetical manipulation and competing peptide, did not affect D1R dynamics in glutamatergic synapses. However, preventing the physical interaction between D1R and the GluN1 subunit of NMDAR abolished the synaptic stabilization of diffusing D1R. Together, these data provide direct evidence that the interaction between D1R and NMDAR in synapses participate in the building of the dopamine-receptor-mediated signalling, and most likely to the glutamate-dopamine cross-talk.

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

PubMed Central

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

2015-01-01

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

Cell lineage and cell cycling analyses of the 4d micromere using live imaging in the marine annelid Platynereis dumerilii

PubMed Central

Handberg-Thorsager, Mette; Vervoort, Michel

2017-01-01

Cell lineage, cell cycle, and cell fate are tightly associated in developmental processes, but in vivo studies at single-cell resolution showing the intricacies of these associations are rare due to technical limitations. In this study on the marine annelid Platynereis dumerilii, we investigated the lineage of the 4d micromere, using high-resolution long-term live imaging complemented with a live-cell cycle reporter. 4d is the origin of mesodermal lineages and the germline in many spiralians. We traced lineages at single-cell resolution within 4d and demonstrate that embryonic segmental mesoderm forms via teloblastic divisions, as in clitellate annelids. We also identified the precise cellular origins of the larval mesodermal posterior growth zone. We found that differentially-fated progeny of 4d (germline, segmental mesoderm, growth zone) display significantly different cell cycling. This work has evolutionary implications, sets up the foundation for functional studies in annelid stem cells, and presents newly established techniques for live imaging marine embryos. PMID:29231816

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

DOE PAGES

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

2015-09-01

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

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

PubMed

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

2015-09-01

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

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.

High-resolution resonant and nonresonant fiber-scanning confocal microscope.

PubMed

Hendriks, Benno H W; Bierhoff, Walter C J; Horikx, Jeroen J L; Desjardins, Adrien E; Hezemans, Cees A; 't Hooft, Gert W; Lucassen, Gerald W; Mihajlovic, Nenad

2011-02-01

We present a novel, hand-held microscope probe for acquiring confocal images of biological tissue. This probe generates images by scanning a fiber-lens combination with a miniature electromagnetic actuator, which allows it to be operated in resonant and nonresonant scanning modes. In the resonant scanning mode, a circular field of view with a diameter of 190 μm and an angular frequency of 127 Hz can be achieved. In the nonresonant scanning mode, a maximum field of view with a width of 69 μm can be achieved. The measured transverse and axial resolutions are 0.60 and 7.4 μm, respectively. Images of biological tissue acquired in the resonant mode are presented, which demonstrate its potential for real-time tissue differentiation. With an outer diameter of 3 mm, the microscope probe could be utilized to visualize cellular microstructures in vivo across a broad range of minimally-invasive procedures.

Vectorization with SIMD extensions speeds up reconstruction in electron tomography.

PubMed

Agulleiro, J I; Garzón, E M; García, I; Fernández, J J

2010-06-01

Electron tomography allows structural studies of cellular structures at molecular detail. Large 3D reconstructions are needed to meet the resolution requirements. The processing time to compute these large volumes may be considerable and so, high performance computing techniques have been used traditionally. This work presents a vector approach to tomographic reconstruction that relies on the exploitation of the SIMD extensions available in modern processors in combination to other single processor optimization techniques. This approach succeeds in producing full resolution tomograms with an important reduction in processing time, as evaluated with the most common reconstruction algorithms, namely WBP and SIRT. The main advantage stems from the fact that this approach is to be run on standard computers without the need of specialized hardware, which facilitates the development, use and management of programs. Future trends in processor design open excellent opportunities for vector processing with processor's SIMD extensions in the field of 3D electron microscopy.

Using quantum filters to process images of diffuse axonal injury

NASA Astrophysics Data System (ADS)

Pineda Osorio, Mateo

2014-06-01

Some images corresponding to a diffuse axonal injury (DAI) are processed using several quantum filters such as Hermite Weibull and Morse. Diffuse axonal injury is a particular, common and severe case of traumatic brain injury (TBI). DAI involves global damage on microscopic scale of brain tissue and causes serious neurologic abnormalities. New imaging techniques provide excellent images showing cellular damages related to DAI. Said images can be processed with quantum filters, which accomplish high resolutions of dendritic and axonal structures both in normal and pathological state. Using the Laplacian operators from the new quantum filters, excellent edge detectors for neurofiber resolution are obtained. Image quantum processing of DAI images is made using computer algebra, specifically Maple. Quantum filter plugins construction is proposed as a future research line, which can incorporated to the ImageJ software package, making its use simpler for medical personnel.

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

Pivotal Impacts of Retrotransposon Based Invasive RNAs on Evolution.

PubMed

Habibi, Laleh; Salmani, Hamzeh

2017-01-01

RNAs have long been described as the mediators of gene expression; they play a vital role in the structure and function of cellular complexes. Although the role of RNAs in the prokaryotes is mainly confined to these basic functions, the effects of these molecules in regulating the gene expression and enzymatic activities have been discovered in eukaryotes. Recently, a high-resolution analysis of the DNA obtained from different organisms has revealed a fundamental impact of the RNAs in shaping the genomes, heterochromatin formation, and gene creation. Deep sequencing of the human genome revealed that about half of our DNA is comprised of repetitive sequences (remnants of transposable element movements) expanded mostly through RNA-mediated processes. ORF2 encoded by L1 retrotransposons is a cellular reverse transcriptase which is mainly responsible for RNA invasion of various transposable elements (L1s, Alus, and SVAs) and cellular mRNAs in to the genomic DNA. In addition to increasing retroelements copy number; genomic expansion in association with centromere, telomere, and heterochromatin formation as well as pseudogene creation are the evolutionary consequences of this RNA-based activity. Threatening DNA integrity by disrupting the genes and forming excessive double strand breaks is another effect of this invasion. Therefore, repressive mechanisms have been evolved to control the activities of these invasive intracellular RNAs. All these mechanisms now have essential roles in the complex cellular functions. Therefore, it can be concluded that without direct action of RNA networks in shaping the genome and in the development of different cellular mechanisms, the evolution of higher eukaryotes would not be possible.

Pivotal Impacts of Retrotransposon Based Invasive RNAs on Evolution

PubMed Central

Habibi, Laleh; Salmani, Hamzeh

2017-01-01

RNAs have long been described as the mediators of gene expression; they play a vital role in the structure and function of cellular complexes. Although the role of RNAs in the prokaryotes is mainly confined to these basic functions, the effects of these molecules in regulating the gene expression and enzymatic activities have been discovered in eukaryotes. Recently, a high-resolution analysis of the DNA obtained from different organisms has revealed a fundamental impact of the RNAs in shaping the genomes, heterochromatin formation, and gene creation. Deep sequencing of the human genome revealed that about half of our DNA is comprised of repetitive sequences (remnants of transposable element movements) expanded mostly through RNA-mediated processes. ORF2 encoded by L1 retrotransposons is a cellular reverse transcriptase which is mainly responsible for RNA invasion of various transposable elements (L1s, Alus, and SVAs) and cellular mRNAs in to the genomic DNA. In addition to increasing retroelements copy number; genomic expansion in association with centromere, telomere, and heterochromatin formation as well as pseudogene creation are the evolutionary consequences of this RNA-based activity. Threatening DNA integrity by disrupting the genes and forming excessive double strand breaks is another effect of this invasion. Therefore, repressive mechanisms have been evolved to control the activities of these invasive intracellular RNAs. All these mechanisms now have essential roles in the complex cellular functions. Therefore, it can be concluded that without direct action of RNA networks in shaping the genome and in the development of different cellular mechanisms, the evolution of higher eukaryotes would not be possible. PMID:29067016

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

PubMed

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

2016-05-01

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

High-resolution handheld rigid endomicroscope based on full-field optical coherence tomography

NASA Astrophysics Data System (ADS)

Benoit a la Guillaume, Emilie; Martins, Franck; Boccara, Claude; Harms, Fabrice

2016-02-01

Full-field optical coherence tomography (FF-OCT) is a powerful tool for nondestructive assessment of biological tissue, i.e., for the structural examination of tissue in depth at a cellular resolution. Mostly known as a microscopy device for ex vivo analysis, FF-OCT has also been adapted to endoscopy setups since it shows good potential for in situ cancer diagnosis and biopsy guidance. Nevertheless, all the attempts to perform endoscopic FF-OCT imaging did not go beyond lab setups. We describe here, to the best of our knowledge, the first handheld FF-OCT endoscope based on a tandem interferometry assembly using incoherent illumination. A common-path passive imaging interferometer at the tip of an optical probe makes it robust and insensitive to environmental perturbations, and a low finesse Fabry-Perot processing interferometer guarantees a compact system. A good resolution (2.7 μm transverse and 6 μm axial) is maintained through the long distance, small diameter relay optics of the probe, and a good signal-to-noise ratio is achieved in a limited 100 ms acquisition time. High-resolution images and a movie of a rat brain slice have been recorded by moving the contact endoscope over the surface of the sample, allowing for tissue microscopic exploration at 20 μm under the surface. These promising ex vivo results open new perspectives for in vivo imaging of biological tissue, in particular, in the field of cancer and surgical margin assessment.

Multiplexed Nanoplasmonic Temporal Profiling of T-Cell Response under Immunomodulatory Agent Exposure

PubMed Central

2016-01-01

Immunomodulatory drugs—agents regulating the immune response—are commonly used for treating immune system disorders and minimizing graft versus host disease in persons receiving organ transplants. At the cellular level, immunosuppressant drugs are used to inhibit pro-inflammatory or tissue-damaging responses of cells. However, few studies have so far precisely characterized the cellular-level effect of immunomodulatory treatment. The primary challenge arises due to the rapid and transient nature of T-cell immune responses to such treatment. T-cell responses involve a highly interactive network of different types of cytokines, which makes precise monitoring of drug-modulated T-cell response difficult. Here, we present a nanoplasmonic biosensing approach to quantitatively characterize cytokine secretion behaviors of T cells with a fine time-resolution (every 10 min) that are altered by an immunosuppressive drug used in the treatment of T-cell-mediated diseases. With a microfluidic platform integrating antibody-conjugated gold nanorod (AuNR) arrays, the technique enables simultaneous multi-time-point measurements of pro-inflammatory (IL-2, IFN-γ, and TNF-α) and anti-inflammatory (IL-10) cytokines secreted by T cells. The integrated nanoplasmonic biosensors achieve precise measurements with low operating sample volume (1 μL), short assay time (∼30 min), heightened sensitivity (∼20–30 pg/mL), and negligible sensor crosstalk. Data obtained from the multicytokine secretion profiles with high practicality resulting from all of these sensing capabilities provide a comprehensive picture of the time-varying cellular functional state during pharmacologic immunosuppression. The capability to monitor cellular functional response demonstrated in this study has great potential to ultimately permit personalized immunomodulatory treatment. PMID:27478873

[18F](2S,4R)4-Fluoroglutamine PET Detects Glutamine Pool Size Changes in Triple Negative Breast Cancer in Response to Glutaminase Inhibition

PubMed Central

Pantel, Austin R.; Li, Shihong; Lieberman, Brian P.; Ploessl, Karl; Choi, Hoon; Blankemeyer, Eric; Lee, Hsiaoju; Kung, Hank F.; Mach, Robert H.

2017-01-01

Glutaminolysis is a metabolic pathway adapted by many aggressive cancers, including triple-negative breast cancers (TNBC), to utilize glutamine for survival and growth. In this study, we examined the utility of [18F](2S,4R)4-fluoroglutamine ([18F]4F-Gln) PET to measure tumor cellular glutamine pool size, whose change might reveal the pharmacodynamic (PD) effect of drugs targeting this cancer-specific metabolic pathway. High glutaminase (GLS) activity in TNBC tumors resulted in low cellular glutamine pool size assayed via high-resolution 1H magnetic resonance spectroscopy (MRS). GLS inhibition significantly increased glutamine pool size in TNBC tumors. MCF-7 tumors, with inherently low GLS activity compared to TNBC, displayed a larger baseline glutamine pool size that did not change as much in response to GLS inhibition. The tumor-to-blood-activity-ratios (T/B) obtained from [18F]4F-Gln PET images matched the distinct glutamine pool sizes of both tumor models at baseline. After a short course of GLS inhibitor treatment, the T/B values increased significantly in TNBC, but did not change in MCF-7 tumors. Across both tumor types and after GLS inhibitor or vehicle treatment, we observed a strong positive correlation between T/B values and tumor glutamine pool size measured using MRS (R2=0.71). In conclusion, [18F]4F-Gln PET tracked cellular glutamine pool size in breast cancers with differential GLS activity and detected increases in cellular glutamine pool size induced by GLS inhibitors. This study accomplished the first necessary step towards validating [18F]4F-Gln PET as a PD marker for glutaminase-targeting drugs. PMID:28202527

Applications of microscopy in Salmonella research.

PubMed

Malt, Layla M; Perrett, Charlotte A; Humphrey, Suzanne; Jepson, Mark A

2015-01-01

Salmonella enterica is a Gram-negative enteropathogen that can cause localized infections, typically resulting in gastroenteritis, or systemic infection, e.g., typhoid fever, in humans and many other animals. Understanding the mechanisms by which Salmonella induces disease has been the focus of intensive research. This has revealed that Salmonella invasion requires dynamic cross-talk between the microbe and host cells, in which bacterial adherence rapidly leads to a complex sequence of cellular responses initiated by proteins translocated into the host cell by a type 3 secretion system. Once these Salmonella-induced responses have resulted in bacterial invasion, proteins translocated by a second type 3 secretion system initiate further modulation of cellular activities to enable survival and replication of the invading pathogen. Elucidation of the complex and highly dynamic pathogen-host interactions ultimately requires analysis at the level of single cells and single infection events. To achieve this goal, researchers have applied a diverse range of microscopy techniques to analyze Salmonella infection in models ranging from whole animal to isolated cells and simple eukaryotic organisms. For example, electron microscopy and high-resolution light microscopy techniques such as confocal microscopy can reveal the precise location of Salmonella and its relationship to cellular components. Widefield light microscopy is a simpler approach with which to study the interaction of bacteria with host cells and often has advantages for live cell imaging, enabling detailed analysis of the dynamics of infection and cellular responses. Here we review the use of imaging techniques in Salmonella research and compare the capabilities of different classes of microscope to address specific types of research question. We also provide protocols and notes on some microscopy techniques used routinely in our own research.

Extracting microtubule networks from superresolution single-molecule localization microscopy data

PubMed Central

Zhang, Zhen; Nishimura, Yukako; Kanchanawong, Pakorn

2017-01-01

Microtubule filaments form ubiquitous networks that specify spatial organization in cells. However, quantitative analysis of microtubule networks is hampered by their complex architecture, limiting insights into the interplay between their organization and cellular functions. Although superresolution microscopy has greatly facilitated high-resolution imaging of microtubule filaments, extraction of complete filament networks from such data sets is challenging. Here we describe a computational tool for automated retrieval of microtubule filaments from single-molecule-localization–based superresolution microscopy images. We present a user-friendly, graphically interfaced implementation and a quantitative analysis of microtubule network architecture phenotypes in fibroblasts. PMID:27852898

In vivo multimodal nonlinear optical imaging of mucosal tissue

NASA Astrophysics Data System (ADS)

Sun, Ju; Shilagard, Tuya; Bell, Brent; Motamedi, Massoud; Vargas, Gracie

2004-05-01

We present a multimodal nonlinear imaging approach to elucidate microstructures and spectroscopic features of oral mucosa and submucosa in vivo. The hamster buccal pouch was imaged using 3-D high resolution multiphoton and second harmonic generation microscopy. The multimodal imaging approach enables colocalization and differentiation of prominent known spectroscopic and structural features such as keratin, epithelial cells, and submucosal collagen at various depths in tissue. Visualization of cellular morphology and epithelial thickness are in excellent agreement with histological observations. These results suggest that multimodal nonlinear optical microscopy can be an effective tool for studying the physiology and pathology of mucosal tissue.

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

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

Jun, Ji Hyun

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

Minimally invasive multimode optical fiber microendoscope for deep brain fluorescence imaging

PubMed Central

Ohayon, Shay; Caravaca-Aguirre, Antonio; Piestun, Rafael; DiCarlo, James J.

2018-01-01

A major open challenge in neuroscience is the ability to measure and perturb neural activity in vivo from well defined neural sub-populations at cellular resolution anywhere in the brain. However, limitations posed by scattering and absorption prohibit non-invasive multi-photon approaches for deep (>2mm) structures, while gradient refractive index (GRIN) endoscopes are relatively thick and can cause significant damage upon insertion. Here, we present a novel micro-endoscope design to image neural activity at arbitrary depths via an ultra-thin multi-mode optical fiber (MMF) probe that has 5–10X thinner diameter than commercially available micro-endoscopes. We demonstrate micron-scale resolution, multi-spectral and volumetric imaging. In contrast to previous approaches, we show that this method has an improved acquisition speed that is sufficient to capture rapid neuronal dynamics in-vivo in rodents expressing a genetically encoded calcium indicator (GCaMP). Our results emphasize the potential of this technology in neuroscience applications and open up possibilities for cellular resolution imaging in previously unreachable brain regions. PMID:29675297

Special Issue on Optochemical and Optogenetic Control of Cellular Processes.

PubMed

Deiters, Alexander

2018-06-06

Diverse optochemical and optobiological approaches are being developed and applied to the light-regulation of cellular processes with exquisite spatial and temporal resolution in cells and multicellular model organisms. In this special issue, experts report some of the latest progress in the expanding field of the optical control of biological systems and present an overview of the state of the art of select approaches. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Cellular Electron Cryotomography: Toward Structural Biology In Situ.

PubMed

Oikonomou, Catherine M; Jensen, Grant J

2017-06-20

Electron cryotomography (ECT) provides three-dimensional views of macromolecular complexes inside cells in a native frozen-hydrated state. Over the last two decades, ECT has revealed the ultrastructure of cells in unprecedented detail. It has also allowed us to visualize the structures of macromolecular machines in their native context inside intact cells. In many cases, such machines cannot be purified intact for in vitro study. In other cases, the function of a structure is lost outside the cell, so that the mechanism can be understood only by observation in situ. In this review, we describe the technique and its history and provide examples of its power when applied to cell biology. We also discuss the integration of ECT with other techniques, including lower-resolution fluorescence imaging and higher-resolution atomic structure determination, to cover the full scale of cellular processes.

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.

Geomorphological Mapping of Sputnik Planum on Pluto

NASA Astrophysics Data System (ADS)

White, Oliver; Moore, Jeffrey M.; Stern, S. Alan; Weaver, Harold A.; Olkin, Catherine B.; Ennico, Kimberly; Young, Leslie; Cheng, Andrew F.; New Horizons Geology, Geophysics and Imaging Theme Team, New Horizons Composition Theme Team

2016-10-01

The New Horizons flyby of Pluto in July 2015 provided extensive high-resolution coverage of its encounter hemisphere. The most prominent surface feature in this hemisphere is the high albedo region informally named Tombaugh Regio, the western portion of which is represented by the expansive nitrogen ice plains informally named Sputnik Planum. A large fraction of Sputnik Planum displays a distinct cellular pattern, with individual cells typically displaying ovoid planforms and shallow pitting on a scale of a few hundred meters. Troughs with medial ridges define the boundaries between cells. Prior studies have argued that this pattern is indicative of solid-state convection occurring within the nitrogen ice. The southern non-cellular plains are either featureless or display dense fields of often elongate and aligned pits typically reaching a few km across, which are interpreted to have formed via sublimation.The mapping that will be presented at DPS focuses on identifying the different plains units that compose Sputnik Planum and defining the boundaries between them, which aids in assessing their time sequencing and correlation to one another. The cellular plains are divided into bright and dark units; the nature of the contact between the two indicates that ice of the bright plains, interpreted to have been recently emplaced via glacial flow from the highlands to the east of Sputnik Planum, is overlying ice of the dark plains, interpreted to be an older ice mass with a higher abundance of entrained dark material. Reconciling the seemingly contradictory models of a layered and also convecting Sputnik Planum requires consideration of the timescale of lateral flow of the bright plains ice relative to the timescale of convective overturn. The non-cellular plains are universally bright and display evidence for southwards flow of the ice, based on the orientations of elongate sublimation pits as well as the presence of 'extinct cells' that appear to have migrated away from the zone of active convection. The larger pits that occur within the non-cellular plains imply that these plains are older than the cellular plains, where resurfacing via convection limits the maximum size attainable by sublimation pits.

Precise Protein Photolithography (P3): High Performance Biopatterning Using Silk Fibroin Light Chain as the Resist

PubMed Central

Liu, Wanpeng; Zhou, Zhitao; Zhang, Shaoqing; Shi, Zhifeng; Tabarini, Justin; Lee, Woonsoo; Zhang, Yeshun; Gilbert Corder, S. N.; Li, Xinxin; Dong, Fei; Cheng, Liang; Liu, Mengkun; Kaplan, David L.; Omenetto, Fiorenzo G.

2017-01-01

Precise patterning of biomaterials has widespread applications, including drug release, degradable implants, tissue engineering, and regenerative medicine. Patterning of protein‐based microstructures using UV‐photolithography has been demonstrated using protein as the resist material. The Achilles heel of existing protein‐based biophotoresists is the inevitable wide molecular weight distribution during the protein extraction/regeneration process, hindering their practical uses in the semiconductor industry where reliability and repeatability are paramount. A wafer‐scale high resolution patterning of bio‐microstructures using well‐defined silk fibroin light chain as the resist material is presented showing unprecedent performances. The lithographic and etching performance of silk fibroin light chain resists are evaluated systematically and the underlying mechanisms are thoroughly discussed. The micropatterned silk structures are tested as cellular substrates for the successful spatial guidance of fetal neural stems cells seeded on the patterned substrates. The enhanced patterning resolution, the improved etch resistance, and the inherent biocompatibility of such protein‐based photoresist provide new opportunities in fabricating large scale biocompatible functional microstructures. PMID:28932678

Chaperone-client complexes: A dynamic liaison

NASA Astrophysics Data System (ADS)

Hiller, Sebastian; Burmann, Björn M.

2018-04-01

Living cells contain molecular chaperones that are organized in intricate networks to surveil protein homeostasis by avoiding polypeptide misfolding, aggregation, and the generation of toxic species. In addition, cellular chaperones also fulfill a multitude of alternative functionalities: transport of clients towards a target location, help them fold, unfold misfolded species, resolve aggregates, or deliver clients towards proteolysis machineries. Until recently, the only available source of atomic resolution information for virtually all chaperones were crystal structures of their client-free, apo-forms. These structures were unable to explain details of the functional mechanisms underlying chaperone-client interactions. The difficulties to crystallize chaperones in complexes with clients arise from their highly dynamic nature, making solution NMR spectroscopy the method of choice for their study. With the advent of advanced solution NMR techniques, in the past few years a substantial number of structural and functional studies on chaperone-client complexes have been resolved, allowing unique insight into the chaperone-client interaction. This review summarizes the recent insights provided by advanced high-resolution NMR-spectroscopy to understand chaperone-client interaction mechanisms at the atomic scale.

Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window

PubMed Central

Holtmaat, Anthony; Bonhoeffer, Tobias; Chow, David K; Chuckowree, Jyoti; De Paola, Vincenzo; Hofer, Sonja B; Hübener, Mark; Keck, Tara; Knott, Graham; Lee, Wei-Chung A; Mostany, Ricardo; Mrsic-Flogel, Tom D; Nedivi, Elly; Portera-Cailliau, Carlos; Svoboda, Karel; Trachtenberg, Joshua T; Wilbrecht, Linda

2011-01-01

To understand the cellular and circuit mechanisms of experience-dependent plasticity, neurons and their synapses need to be studied in the intact brain over extended periods of time. Two-photon excitation laser scanning microscopy (2PLSM), together with expression of fluorescent proteins, enables high-resolution imaging of neuronal structure in vivo. In this protocol we describe a chronic cranial window to obtain optical access to the mouse cerebral cortex for long-term imaging. A small bone flap is replaced with a coverglass, which is permanently sealed in place with dental acrylic, providing a clear imaging window with a large field of view (∼0.8–12 mm2). The surgical procedure can be completed within ∼1 h. The preparation allows imaging over time periods of months with arbitrary imaging intervals. The large size of the imaging window facilitates imaging of ongoing structural plasticity of small neuronal structures in mice, with low densities of labeled neurons. The entire dendritic and axonal arbor of individual neurons can be reconstructed. PMID:19617885

Structural anomalies in undoped Gallium Arsenide observed in high resolution diffraction imaging with monochromatic synchrotron radiation

NASA Technical Reports Server (NTRS)

Steiner, B.; Kuriyama, M.; Dobbyn, R. C.; Laor, U.; Larson, D.; Brown, M.

1988-01-01

Novel, streak-like disruption features restricted to the plane of diffraction have recently been observed in images obtained by synchrotron radiation diffraction from undoped, semi-insulating gallium arsenide crystals. These features were identified as ensembles of very thin platelets or interfaces lying in (110) planes, and a structural model consisting of antiphase domain boundaries was proposed. We report here the other principal features observed in high resolution monochromatic synchrotron radiation diffraction images: (quasi) cellular structure; linear, very low-angle subgrain boundaries in (110) directions, and surface stripes in a (110) direction. In addition, we report systematic differences in the acceptance angle for images involving various diffraction vectors. When these observations are considered together, a unifying picture emerges. The presence of ensembles of thin (110) antiphase platelet regions or boundaries is generally consistent not only with the streak-like diffraction features but with the other features reported here as well. For the formation of such regions we propose two mechanisms, operating in parallel, that appear to be consistent with the various defect features observed by a variety of techniques.

Structural anomalies in undoped gallium arsenide observed in high-resolution diffraction imaging with monochromatic synchrotron radiation

NASA Technical Reports Server (NTRS)

Steiner, B.; Kuriyama, M.; Dobbyn, R. C.; Laor, U.; Larson, D.

1989-01-01

Novel, streak-like disruption features restricted to the plane of diffraction have recently been observed in images obtained by synchrotron radiation diffraction from undoped, semi-insulating gallium arsenide crystals. These features were identified as ensembles of very thin platelets or interfaces lying in (110) planes, and a structural model consisting of antiphase domain boundaries was proposed. We report here the other principal features observed in high resolution monochromatic synchrotron radiation diffraction images: (quasi) cellular structure; linear, very low-angle subgrain boundaries in (110) directions, and surface stripes in a (110) direction. In addition, we report systematic differences in the acceptance angle for images involving various diffraction vectors. When these observations are considered together, a unifying picture emerges. The presence of ensembles of thin (110) antiphase platelet regions or boundaries is generally consistent not only with the streak-like diffraction features but with the other features reported here as well. For the formation of such regions we propose two mechanisms, operating in parallel, that appear to be consistent with the various defect features observed by a variety of techniques.

Shedding light on cell compartmentation in the candidate phylum Poribacteria by high resolution visualisation and transcriptional profiling

NASA Astrophysics Data System (ADS)

Jahn, Martin T.; Markert, Sebastian M.; Ryu, Taewoo; Ravasi, Timothy; Stigloher, Christian; Hentschel, Ute; Moitinho-Silva, Lucas

2016-10-01

Assigning functions to uncultivated environmental microorganisms continues to be a challenging endeavour. Here, we present a new microscopy protocol for fluorescence in situ hybridisation-correlative light and electron microscopy (FISH-CLEM) that enabled, to our knowledge for the first time, the identification of single cells within their complex microenvironment at electron microscopy resolution. Members of the candidate phylum Poribacteria, common and uncultivated symbionts of marine sponges, were used towards this goal. Cellular 3D reconstructions revealed bipolar, spherical granules of low electron density, which likely represent carbon reserves. Poribacterial activity profiles were retrieved from prokaryotic enriched sponge metatranscriptomes using simulation-based optimised mapping. We observed high transcriptional activity for proteins related to bacterial microcompartments (BMC) and we resolved their subcellular localisation by combining FISH-CLEM with immunohistochemistry (IHC) on ultra-thin sponge tissue sections. In terms of functional relevance, we propose that the BMC-A region may be involved in 1,2-propanediol degradation. The FISH-IHC-CLEM approach was proven an effective toolkit to combine -omics approaches with functional studies and it should be widely applicable in environmental microbiology.

Wide-field and high-resolution optical imaging for early detection of oral neoplasia

NASA Astrophysics Data System (ADS)

Pierce, Mark C.; Schwarz, Richard A.; Rosbach, Kelsey; Roblyer, Darren; Muldoon, Tim; Williams, Michelle D.; El-Naggar, Adel K.; Gillenwater, Ann M.; Richards-Kortum, Rebecca

2010-02-01

Current procedures for oral cancer screening typically involve visual inspection of the entire tissue surface at risk under white light illumination. However, pre-cancerous lesions can be difficult to distinguish from many benign conditions when viewed under these conditions. We have developed wide-field (macroscopic) imaging system which additionally images in cross-polarized white light, narrowband reflectance, and fluorescence imaging modes to reduce specular glare, enhance vascular contrast, and detect disease-related alterations in tissue autofluorescence. We have also developed a portable system to enable high-resolution (microscopic) evaluation of cellular features within the oral mucosa in situ. This system is a wide-field epi-fluorescence microscope coupled to a 1 mm diameter, flexible fiber-optic imaging bundle. Proflavine solution was used to specifically label cell nuclei, enabling the characteristic differences in N/C ratio and nuclear distribution between normal, dysplastic, and cancerous oral mucosa to be quantified. This paper discusses the technical design and performance characteristics of these complementary imaging systems. We will also present data from ongoing clinical studies aimed at evaluating diagnostic performance of these systems for detection of oral neoplasia.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

PubMed Central

Malide, Daniela; Métais, Jean-Yves; Dunbar, Cynthia E.

2014-01-01

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner. PMID:25145579

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

PubMed

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

2015-04-01

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

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.

Microfluidic Approaches for Isolation, Detection, and Characterization of Extracellular Vesicles: Current Status and Future Directions

PubMed Central

Gholizadeh, Shima; Draz, Mohamed; Zarghooni, Maryam; Nezhad, Amir Sanati; Ghavami, Saeid; Shafiee, Hadi; Akbari, Mohsen

2017-01-01

Extracellular vesicles (EVs) are cell-derived vesicles present in body fluids that play an essential role in various cellular processes, such as intercellular communication, inflammation, cellular homeostasis, survival, transport, and regeneration. Their isolation and analysis from body fluids have a great clinical potential to provide information on a variety of disease states such as cancer, cardiovascular complication and inflammatory disorders. Despite increasing scientific and clinical interest in this field, at the time of writing there are still no standardized procedures available for the purification, detection, and characterization of EVs. Advances in microfluidics allow for chemical sampling with increasingly high spatial resolution and under precise manipulation down to single molecule level. In this review, our objective is to give a brief overview on the working principle and examples of the isolation and detection methods with the potential to be used for extracellular vesicles. This review will also highlight the integrated on-chip systems for isolation and characterization of EVs. PMID:28088752

Spectroscopic studies of anthracyclines: Structural characterization and in vitro tracking

NASA Astrophysics Data System (ADS)

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

2016-12-01

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

3D Printing of Tissue Engineered Constructs for In Vitro Modeling of Disease Progression and Drug Screening.

PubMed

Vanderburgh, Joseph; Sterling, Julie A; Guelcher, Scott A

2017-01-01

2D cell culture and preclinical animal models have traditionally been implemented for investigating the underlying cellular mechanisms of human disease progression. However, the increasing significance of 3D vs. 2D cell culture has initiated a new era in cell culture research in which 3D in vitro models are emerging as a bridge between traditional 2D cell culture and in vivo animal models. Additive manufacturing (AM, also known as 3D printing), defined as the layer-by-layer fabrication of parts directed by digital information from a 3D computer-aided design file, offers the advantages of simultaneous rapid prototyping and biofunctionalization as well as the precise placement of cells and extracellular matrix with high resolution. In this review, we highlight recent advances in 3D printing of tissue engineered constructs that recapitulate the physical and cellular properties of the tissue microenvironment for investigating mechanisms of disease progression and for screening drugs.

The conformational dynamics of the mitochondrial Hsp70 chaperone.

PubMed

Mapa, Koyeli; Sikor, Martin; Kudryavtsev, Volodymyr; Waegemann, Karin; Kalinin, Stanislav; Seidel, Claus A M; Neupert, Walter; Lamb, Don C; Mokranjac, Dejana

2010-04-09

Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments. 2010 Elsevier Inc. All rights reserved.

High-resolution mapping reveals topologically distinct cellular pools of phosphatidylserine

PubMed Central

Fairn, Gregory D.; Schieber, Nicole L.; Ariotti, Nicholas; Murphy, Samantha; Kuerschner, Lars; Webb, Richard I.; Grinstein, Sergio

2011-01-01

Phosphatidylserine (PS) plays a central role in cell signaling and in the biosynthesis of other lipids. To date, however, the subcellular distribution and transmembrane topology of this crucial phospholipid remain ill-defined. We transfected cells with a GFP-tagged C2 domain of lactadherin to detect by light and electron microscopy PS exposed on the cytosolic leaflet of the plasmalemma and organellar membranes. Cytoplasmically exposed PS was found to be clustered on the plasma membrane, and to be associated with caveolae, the trans-Golgi network, and endocytic organelles including intraluminal vesicles of multivesicular endosomes. This labeling pattern was compared with the total cellular distribution of PS as visualized using a novel on-section technique. These complementary methods revealed PS in the interior of the ER, Golgi complex, and mitochondria. These results indicate that PS in the lumenal monolayer of the ER and Golgi complex becomes exposed cytosolically at the trans-Golgi network. Transmembrane flipping of PS may contribute to the exit of cargo from the Golgi complex. PMID:21788369

Twenty-seven Years of Cerebral Pyruvate Recycling.

PubMed

Cerdán, Sebastián

2017-06-01

Cerebral pyruvate recycling is a metabolic pathway deriving carbon skeletons and reducing equivalents from mitochondrial oxaloacetate and malate, to the synthesis of mitochondrial and cytosolic pyruvate, lactate and alanine. The pathway allows both, to provide the tricarboxylic acid cycle with pyruvate molecules produced from alternative substrates to glucose and, to generate reducing equivalents necessary for the operation of NADPH requiring processes. At the cellular level, pyruvate recycling involves the activity of malic enzyme, or the combined activities of phosphoenolpyruvate carboxykinase and pyruvate kinase, as well as of those transporters of the inner mitochondrial membrane exchanging the corresponding intermediates. Its cellular localization between the neuronal or astrocytic compartments of the in vivo brain has been controversial, with evidences favoring either a primarily neuronal or glial localizations, more recently accepted to occur in both environments. This review provides a brief history on the detection and characterization of the pathway, its relations with the early developments of cerebral high resolution 13 C NMR, and its potential neuroprotective functions under hypoglycemic conditions or ischemic redox stress.

3D Printing of Tissue Engineered Constructs for in vitro Modeling of Disease Progression and Drug Screening

PubMed Central

Vanderburgh, Joseph; Sterling, Julie A.

2016-01-01

2D cell culture and preclinical animal models have traditionally been implemented for investigating the underlying cellular mechanisms of human disease progression. However, the increasing significance of 3D versus 2D cell culture has initiated a new era in cell culture research in which 3D in vitro models are emerging as a bridge between traditional 2D cell culture and in vivo animal models. Additive manufacturing (AM, also known as 3D printing), defined as the layer-by-layer fabrication of parts directed by digital information from a 3D computer-aided design (CAD) file, offers the advantages of simultaneous rapid prototyping and biofunctionalization as well as the precise placement of cells and extracellular matrix with high resolution. In this review, we highlight recent advances in 3D printing of tissue engineered constructs (TECs) that recapitulate the physical and cellular properties of the tissue microenvironment for investigating mechanisms of disease progression and for screening drugs. PMID:27169894

Ezrin enhances line tension along transcellular tunnel edges via NMIIa driven actomyosin cable formation

NASA Astrophysics Data System (ADS)

Stefani, Caroline; Gonzalez-Rodriguez, David; Senju, Yosuke; Doye, Anne; Efimova, Nadia; Janel, Sébastien; Lipuma, Justine; Tsai, Meng Chen; Hamaoui, Daniel; Maddugoda, Madhavi P.; Cochet-Escartin, Olivier; Prévost, Coline; Lafont, Frank; Svitkina, Tatyana; Lappalainen, Pekka; Bassereau, Patricia; Lemichez, Emmanuel

2017-06-01

Transendothelial cell macroaperture (TEM) tunnels control endothelium barrier function and are triggered by several toxins from pathogenic bacteria that provoke vascular leakage. Cellular dewetting theory predicted that a line tension of uncharacterized origin works at TEM boundaries to limit their widening. Here, by conducting high-resolution microscopy approaches we unveil the presence of an actomyosin cable encircling TEMs. We develop a theoretical cellular dewetting framework to interpret TEM physical parameters that are quantitatively determined by laser ablation experiments. This establishes the critical role of ezrin and non-muscle myosin II (NMII) in the progressive implementation of line tension. Mechanistically, fluorescence-recovery-after-photobleaching experiments point for the upstream role of ezrin in stabilizing actin filaments at the edges of TEMs, thereby favouring their crosslinking by NMIIa. Collectively, our findings ascribe to ezrin and NMIIa a critical function of enhancing line tension at the cell boundary surrounding the TEMs by promoting the formation of an actomyosin ring.

Ezrin enhances line tension along transcellular tunnel edges via NMIIa driven actomyosin cable formation

PubMed Central

Stefani, Caroline; Gonzalez-Rodriguez, David; Senju, Yosuke; Doye, Anne; Efimova, Nadia; Janel, Sébastien; Lipuma, Justine; Tsai, Meng Chen; Hamaoui, Daniel; Maddugoda, Madhavi P.; Cochet-Escartin, Olivier; Prévost, Coline; Lafont, Frank; Svitkina, Tatyana; Lappalainen, Pekka; Bassereau, Patricia; Lemichez, Emmanuel

2017-01-01

Transendothelial cell macroaperture (TEM) tunnels control endothelium barrier function and are triggered by several toxins from pathogenic bacteria that provoke vascular leakage. Cellular dewetting theory predicted that a line tension of uncharacterized origin works at TEM boundaries to limit their widening. Here, by conducting high-resolution microscopy approaches we unveil the presence of an actomyosin cable encircling TEMs. We develop a theoretical cellular dewetting framework to interpret TEM physical parameters that are quantitatively determined by laser ablation experiments. This establishes the critical role of ezrin and non-muscle myosin II (NMII) in the progressive implementation of line tension. Mechanistically, fluorescence-recovery-after-photobleaching experiments point for the upstream role of ezrin in stabilizing actin filaments at the edges of TEMs, thereby favouring their crosslinking by NMIIa. Collectively, our findings ascribe to ezrin and NMIIa a critical function of enhancing line tension at the cell boundary surrounding the TEMs by promoting the formation of an actomyosin ring. PMID:28643776

Ezrin enhances line tension along transcellular tunnel edges via NMIIa driven actomyosin cable formation.

PubMed

Stefani, Caroline; Gonzalez-Rodriguez, David; Senju, Yosuke; Doye, Anne; Efimova, Nadia; Janel, Sébastien; Lipuma, Justine; Tsai, Meng Chen; Hamaoui, Daniel; Maddugoda, Madhavi P; Cochet-Escartin, Olivier; Prévost, Coline; Lafont, Frank; Svitkina, Tatyana; Lappalainen, Pekka; Bassereau, Patricia; Lemichez, Emmanuel

2017-06-23

Transendothelial cell macroaperture (TEM) tunnels control endothelium barrier function and are triggered by several toxins from pathogenic bacteria that provoke vascular leakage. Cellular dewetting theory predicted that a line tension of uncharacterized origin works at TEM boundaries to limit their widening. Here, by conducting high-resolution microscopy approaches we unveil the presence of an actomyosin cable encircling TEMs. We develop a theoretical cellular dewetting framework to interpret TEM physical parameters that are quantitatively determined by laser ablation experiments. This establishes the critical role of ezrin and non-muscle myosin II (NMII) in the progressive implementation of line tension. Mechanistically, fluorescence-recovery-after-photobleaching experiments point for the upstream role of ezrin in stabilizing actin filaments at the edges of TEMs, thereby favouring their crosslinking by NMIIa. Collectively, our findings ascribe to ezrin and NMIIa a critical function of enhancing line tension at the cell boundary surrounding the TEMs by promoting the formation of an actomyosin ring.

Multiparametric, Longitudinal Optical Coherence Tomography Imaging Reveals Acute Injury and Chronic Recovery in Experimental Ischemic Stroke

PubMed Central

Srinivasan, Vivek J.; Mandeville, Emiri T.; Can, Anil; Blasi, Francesco; Climov, Mihail; Daneshmand, Ali; Lee, Jeong Hyun; Yu, Esther; Radhakrishnan, Harsha; Lo, Eng H.; Sakadžić, Sava; Eikermann-Haerter, Katharina; Ayata, Cenk

2013-01-01

Progress in experimental stroke and translational medicine could be accelerated by high-resolution in vivo imaging of disease progression in the mouse cortex. Here, we introduce optical microscopic methods that monitor brain injury progression using intrinsic optical scattering properties of cortical tissue. A multi-parametric Optical Coherence Tomography (OCT) platform for longitudinal imaging of ischemic stroke in mice, through thinned-skull, reinforced cranial window surgical preparations, is described. In the acute stages, the spatiotemporal interplay between hemodynamics and cell viability, a key determinant of pathogenesis, was imaged. In acute stroke, microscopic biomarkers for eventual infarction, including capillary non-perfusion, cerebral blood flow deficiency, altered cellular scattering, and impaired autoregulation of cerebral blood flow, were quantified and correlated with histology. Additionally, longitudinal microscopy revealed remodeling and flow recovery after one week of chronic stroke. Intrinsic scattering properties serve as reporters of acute cellular and vascular injury and recovery in experimental stroke. Multi-parametric OCT represents a robust in vivo imaging platform to comprehensively investigate these properties. PMID:23940761

Single-cell and subcellular pharmacokinetic imaging allows insight into drug action in vivo.

PubMed

Thurber, Greg M; Yang, Katy S; Reiner, Thomas; Kohler, Rainer H; Sorger, Peter; Mitchison, Tim; Weissleder, Ralph

2013-01-01

Pharmacokinetic analysis at the organ level provides insight into how drugs distribute throughout the body, but cannot explain how drugs work at the cellular level. Here we demonstrate in vivo single-cell pharmacokinetic imaging of PARP-1 inhibitors and model drug behaviour under varying conditions. We visualize intracellular kinetics of the PARP-1 inhibitor distribution in real time, showing that PARP-1 inhibitors reach their cellular target compartment, the nucleus, within minutes in vivo both in cancer and normal cells in various cancer models. We also use these data to validate predictive finite element modelling. Our theoretical and experimental data indicate that tumour cells are exposed to sufficiently high PARP-1 inhibitor concentrations in vivo and suggest that drug inefficiency is likely related to proteomic heterogeneity or insensitivity of cancer cells to DNA-repair inhibition. This suggests that single-cell pharmacokinetic imaging and derived modelling improve our understanding of drug action at single-cell resolution in vivo.