Name: Video: Creation of a Thrombus or a Puncture Wound on a Murine Jugular Vein/Femoral Artery for Montaged Wide-Area Electron Microscopy Analysis
Uploaded: 2024-05-24T13:00:00
Description: 29 Views. Creation of a Thrombus or a Puncture Wound on a Murine Jugular Vein/Femoral Artery for Montaged Wide-Area Electron Microscopy Analysis

creation of a thrombus or a puncture wound on a murine jugular vein/femoral artery for montaged wide-area electron microscopy analysis

Puncture Wound Hemostasis and Montaged Electron Microscopy 

The formation of a puncture wound thrombus that leads to bleeding cessation is one of the most essential events in life1. Yet despite that essentiality, knowledge of what occurs structurally during thrombus formation, be it in a vein, an artery, an atherosclerotic event, or an occlusive clot, has been limited by resolution and imaging depth. Conventional light microscopy is limited in depth when compared to a fully formed puncture wound thrombus, 200 to 300 &#181;m in Z1,&#160;and in resolution level when compared to the size of platelet organelles and their spacing, often less than 30 nm2. Two-photon light microscopy can yield the needed depth of imaging but does not improve resolution significantly. The most recent advances in light microscopy, for example, super-resolution techniques, are still resolution limited, in practice ~20 nm in XY and twice that in Z, and depth limited, no more than conventional light microscopy. Furthermore, super-resolution light microscopy, like much of research light microscopy, is based on fluorescence microscopy, a technique that is inherently biased to a small set of candidate proteins for which good antibodies exist or good tagged constructs3. In conclusion, conventional scanning electron microscopy can, at most, visualize the surface of the forming platelet-rich thrombus.
To overcome these technical limitations to characterizing thrombus structure, we had three goals. First, reproducibly produce a defined puncture wound in a mouse vein or artery that could then be readily stabilized in situ by chemical fixation. Second, apply a preparative procedure that emphasizes membrane preservation, a goal consistent with the aim of defining the position of individual platelets within the forming thrombus. Third, use an unbiased visualization technique that, in a single image, could be scaled between nanometer to near millimeter scale.
Montaged, wide-area electron microscopy was chosen as a major end visualization technique for a single important reason: in electron microscope imaging, one sees a vast array of features within a cell that outlines its organelles and features within the organelles. Small objects such as ribosomes can be recognized. This range of features is seen because the electron-dense heavy metal stains, uranyl, lead, and osmium, that are used for electron microscopy to yield contrast bind to a wide range of molecules. In an electron microscope image, one sees much of what is there, while with immunofluorescence and protein tagging approaches, one only sees what lights up. This means, for example, the antigen sites present on a given individual protein species. In the case of a tagged molecule, often a protein, it is the site(s) where that protein is. All other molecules are dark and not lit up. However, is this choice of electron microscopy practical? A puncture wound thrombus has a size of 300 by 500 &#181;m and, at a pixel size of 3 nm, that is an image of 100,000 by 167,000 pixels. A high-quality electron microscope camera has 4000 by 4000 pixels. That means that approximately 1000 frames must be stitched/blended to give a single image. That is a possibility that has been present in most electron microscopes manufactured in the last 15 years. The microscope stage is computerized, and the images can be stitched together with a computer. That is the rationale that led to choices underlying the formulation of the presented protocol.
Conclusively, we present below a series of steps that give a reproducible wound in the vein or artery of mice that then, following in situ fixation steps and later embedding steps, can be visualized by montaged, wide-area transmission electron microscopy at nm scale and in the stitched image visualized at near mm scale, the scale of the actual in situ fixed thrombus. Scalability of this kind is required for understanding thrombus formation as both a problem in hematology/health and as a developmental biology system in which platelets are the major cell type. These advances deliver a major virtue of electron microscopy, namely, one sees what is there, not only what lights up. For a detailed protocol on the preparation of samples for serial block face scanning electron microscopy (SBF-SEM), the reader is directed to a recent article by Joshi et al.4.

Author Spotlight: Revealing Platelet Dynamics Through Advances in Structural Hematology

Puncture Wound Hemostasis and Preparation of Samples for Montaged Wide-Area Electron Microscopy Analysis

Creation of a Thrombus or a Puncture Wound on a Murine Jugular Vein/Femoral Artery for Montaged Wide-Area Electron Microscopy Analysis

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Research

JoVE Journal

Medicine

29 Views. Creation of a Thrombus or a Puncture Wound on a Murine Jugular Vein/Femoral Artery for Montaged Wide-Area Electron Microscopy Analysis

Video: Creation of a Thrombus or a Puncture Wound on a Murine Jugular Vein/Femoral Artery for Montaged Wide-Area Electron Microscopy Analysis

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside. Detailed characterization of this structure could lead to distinctions between hemostasis and thrombosis, a case of excessive platelet aggregation leading to occlusive clotting. An imaging-based approach to puncture wound thrombus structure is presented here that draws upon the ability of thin-section electron microscopy to visualize the interior of hemostatic thrombi. The most basic step in any imaging-based experimental protocol is good sample preparation. The protocol provides detailed procedures for preparing puncture wounds and platelet-rich thrombi in mice for subsequent electron microscopy. A detailed procedure is given for in situ fixation of the forming puncture wound thrombus and its subsequent processing for staining and embedding for electron microscopy. Electron microscopy is presented as the end imaging technique because of its ability, when combined with sequential sectioning, to visualize the details of the thrombus interior at high resolution. As an imaging method, electron microscopy gives unbiased sampling and an experimental output that scales from nanometer to millimeters in 2 or 3 dimensions. Appropriate freeware electron microscopy software is cited that will support wide-area electron microscopy in which hundreds of frames can be blended to give nanometer-scale imaging of entire puncture wound thrombi cross-sections. Hence, any subregion of the image file can be placed easily into the context of the full cross-section.

A puncture wound procedure for hemostatic thrombus formation is presented here. The formed thrombi are large and are hundreds of microns in diameter. Hence, volume imaging approaches are appropriate. We suggest montaged wide-area transmission electron microscopy as a high-resolution approach available to many and detail a preparative protocol.

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds ...