scholarly journals Platelet Imaging

Platelets ◽  
2020 ◽  
Author(s):  
Zachary A. Matthay ◽  
Lucy Zumwinkle Kornblith
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Electron Tomography ◽  
Thrombus Formation ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Experimental Systems ◽  
Health And Disease ◽  
Fluorescence Light

The knowledge gained through imaging platelets has formed the backbone of our understanding of their biology in health and disease. Early investigators relied on conventional light microscopy with limited resolution and were primarily able to identify the presence and basic morphology of platelets. The advent of high resolution technologies, in particular, electron microscopy, accelerated our understanding of the dynamics of platelet ultrastructure dramatically. Further refinements and improvements in our ability to localize and reliably identify platelet structures have included the use of immune-labeling techniques, correlative-fluorescence light and electron microscopy, and super-resolution microscopies. More recently, the expanded development and application of intravital microscopy in animal models has enhanced our knowledge of platelet functions and thrombus formation in vivo, as these experimental systems most closely replicate native biological environments. Emerging improvements in our ability to characterize platelets at the ultrastructural and organelle levels include the use of platelet cryogenic electron tomography with quantitative, unbiased imaging analysis, and the ability to genetically label platelet features with electron dense markers for analysis by electron microscopy.

scholarly journals AutoCLEM: An Automated Workflow for Correlative Live-Cell Fluorescence Microscopy and Cryo-Electron Tomography

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Xiaofeng Fu ◽  
Jiying Ning ◽  
Zhou Zhong ◽  
Zandrea Ambrose ◽  
Simon Charles Watkins ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Speed ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Live Cell ◽  
Host Cells ◽  
Spatiotemporal Information ◽  
Fluorescence Light ◽  
Correlation Process ◽  
Electron Imaging

AbstractCorrelative light and electron microscopy (CLEM) combines the strengths of both light and electron imaging modalities and enables linking of biological spatiotemporal information from live-cell fluorescence light microscopy (fLM) to high-resolution cellular ultra-structures from cryo-electron microscopy and tomography (cryoEM/ET). This has been previously achieved by using fLM signals to localize the regions of interest under cryogenic conditions. The correlation process, however, is often tedious and time-consuming with low throughput and limited accuracy, because multiple correlation steps at different length scales are largely carried out manually. Here, we present an experimental workflow, AutoCLEM, which overcomes the existing limitations and improves the performance and throughput of CLEM methods, and associated software. The AutoCLEM system encompasses a high-speed confocal live-cell imaging module to acquire an automated fLM grid atlas that is linked to the cryoEM grid atlas, followed by cryofLM imaging after freezing. The fLM coordinates of the targeted areas are automatically converted to cryoEM/ET and refined using fluorescent fiducial beads. This AutoCLEM workflow significantly accelerates the correlation efficiency between live-cell fluorescence imaging and cryoEM/ET structural analysis, as demonstrated by visualizing human immunodeficiency virus type 1 (HIV-1) interacting with host cells.

scholarly journals Cryogenic Super-Resolution Fluorescence and Electron Microscopy Correlated at the Nanoscale

2021 ◽  
Vol 72 (1) ◽  
Author(s):  
Peter D. Dahlberg ◽  
W.E. Moerner
Keyword(s):  
Electron Microscopy ◽  
Single Molecule ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Nanometer Scale ◽  
Annual Review ◽  
Publication Date ◽  
Fluorescence And Electron Microscopy ◽  
Cellular Context

We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications. Expected final online publication date for the Annual Review of Physical Chemistry, Volume 72 is April 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marie Fuest ◽  
Miroslava Schaffer ◽  
Giovanni Marco Nocera ◽  
Rodrigo I. Galilea-Kleinsteuber ◽  
Jan-Erik Messling ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Light Microscope ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Live Imaging ◽  
Cryo Electron Tomography

AbstractWe present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

scholarly journals Dense HRP filling in pre-fixed brain tissue for light and electron microscopy.

1987 ◽  
Vol 35 (10) ◽  
pp. 1127-1136 ◽  
Author(s):  
G H Kageyama ◽  
R L Meyer
Keyword(s):  
Electron Microscopy ◽  
Optic Nerve ◽  
High Resolution ◽  
Light Microscopy ◽  
Glial Cells ◽  
Light And Electron Microscopy ◽  
Polyethylene Tube ◽  
Fixed Tissue ◽  
Cat Brain

The use of neuroanatomical markers in tissues that have been pre-fixed has been virtually ignored, even though this approach could offer certain advantages over in vivo methods, in terms of convenience of application and choice of markers. We have found that HRP can be used on well-fixed brains of cats and goldfish to fill neurons, dendrites, axons, terminals, glial cells, and glial processes for high-resolution light microscopy and electron microscopy. Best results were obtained using brains that were perfusion-fixed with 2.5% depolymerized paraformaldehyde and 1.5% glutaraldehyde. Two methods of HRP application were used: optically guided injections of microliter quantities into various regions of cat brain, and optic nerve fills in goldfish by attaching an HRP-filled polyethylene tube for periods of 1 day to 2 weeks. HRP applied in these ways to pre-fixed tissue was found to fill neurons or glial cells with solid label in the anterograde and retrograde directions.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

Solving the mechanisms responsible for organelle behavior in vivo by same-cell correlative light and electron microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 14-15
Author(s):  
Conly L. Rieder
Keyword(s):  
Electron Microscopy ◽  
Quantitative Analysis ◽  
Light Microscopy ◽  
Living Cell ◽  
Light And Electron Microscopy ◽  
Time Behavior ◽  
Dynamic Interactions ◽  
Positive Identification ◽  
Cellular Components

The behavior of many cellular components, and their dynamic interactions, can be characterized in the living cell with considerable spatial and temporal resolution by video-enhanced light microscopy (video-LM). Indeed, under the appropriate conditions video-LM can be used to determine the real-time behavior of organelles ≤ 25-nm in diameter (e.g., individual microtubules—see). However, when pushed to its limit the structures and components observed within the cell by video-LM cannot be resolved nor necessarily even identified, only detected. Positive identification and a quantitative analysis often requires the corresponding electron microcopy (EM).

Thrombus formation on artificial surfaces: Correlative microscopy

1990 ◽  
Vol 48 (3) ◽  
pp. 840-841
Author(s):  
Quintin J. Lai ◽  
Stuart L. Cooper ◽  
Ralph M. Albrecht
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Low Voltage ◽  
Thrombus Formation ◽  
Blood Platelets ◽  
Polymer Surfaces ◽  
Flow Conditions ◽  
Transmission Electron ◽  
Adhesion Of Platelets ◽  
Microscopic Techniques

Thrombus formation and embolization are significant problems for blood-contacting biomedical devices. Two major components of thrombi are blood platelets and the plasma protein, fibrinogen. Previous studies have examined interactions of platelets with polymer surfaces, fibrinogen with platelets, and platelets in suspension with spreading platelets attached to surfaces. Correlative microscopic techniques permit light microscopic observations of labeled living platelets, under static or flow conditions, followed by the observation of identical platelets by electron microscopy. Videoenhanced, differential interference contrast (DIC) light microscopy permits high-resolution, real-time imaging of live platelets and their interactions with surfaces. Interference reflection microscopy (IRM) provides information on the focal adhesion of platelets on surfaces. High voltage, transmission electron microscopy (HVEM) allows observation of platelet cytoskeletal structure of whole mount preparations. Low-voltage, high resolution, scanning electron microscopy allows observation of fine surface detail of platelets. Colloidal gold-labeled fibrinogen, used to identify the Gp Ilb/IIIa membrane receptor for fibrinogen, can be detected in all the above microscopies.

scholarly journals Analytical High-resolution Electron Microscopy Reveals Organ-specific Nanoceria Bioprocessing

2017 ◽  
Vol 46 (1) ◽  
pp. 47-61 ◽  
Author(s):  
Uschi M. Graham ◽  
Robert A. Yokel ◽  
Alan K. Dozier ◽  
Lawrence Drummy ◽  
Krishnamurthy Mahalingam ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
High Resolution ◽  
Analytical Electron Microscopy ◽  
High Resolution Electron Microscopy ◽  
Dark Field ◽  
Transmission Electron ◽  
Analytical Electron ◽  
Organ Specific

This is the first utilization of advanced analytical electron microscopy methods, including high-resolution transmission electron microscopy, high-angle annular dark field scanning transmission electron microscopy, electron energy loss spectroscopy, and energy-dispersive X-ray spectroscopy mapping to characterize the organ-specific bioprocessing of a relatively inert nanomaterial (nanoceria). Liver and spleen samples from rats given a single intravenous infusion of nanoceria were obtained after prolonged (90 days) in vivo exposure. These advanced analytical electron microscopy methods were applied to elucidate the organ-specific cellular and subcellular fate of nanoceria after its uptake. Nanoceria is bioprocessed differently in the spleen than in the liver.

scholarly journals Dynamics of in vivo ASC speck formation

2017 ◽  
Vol 216 (9) ◽  
pp. 2891-2909 ◽  
Author(s):  
Paola Kuri ◽  
Nicole L. Schieber ◽  
Thomas Thumberger ◽  
Joachim Wittbrodt ◽  
Yannick Schwab ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Death ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Inflammasome Activation ◽  
Caspase Activation ◽  
Endogenous Gene ◽  
Pyrin Domain ◽  
Effector Caspase

Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signaling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested that speck-containing, pyroptotic debris. A three-dimensional, ultrastructural reconstruction, based on correlative light and electron microscopy of the in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas pyrin domain (PYD) or caspase activation and recruitment domain alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis but only after substantial delay. Therefore, formation of a single, compact speck and rapid cell-death induction in vivo requires a full-length ASC.

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