<meta charset="utf8"></head><body>穿刺伤口止血和蒙太奇电子显微镜

<ol>
	<li>Rhee, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venous+puncture+and+wound+hemostasis+results+in+a+vaulted+thrombus+structured+by+locally+nucleated+platelet+aggregates.">Venous puncture and wound hemostasis results in a vaulted thrombus structured by locally nucleated platelet aggregates.</a> Comm Biol. 4 (1), 1090 (2021).</li><li>Pokrovskaya, I. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tethered+platelet+capture+provides+a+mechanism+for+restricting+circulating+platelet+activation+in+the+wound+site.">Tethered platelet capture provides a mechanism for restricting circulating platelet activation in the wound site.</a> Res Pract Thromb Haemost. 7 (2), 100058 (2023).</li><li>Yadav, S., Storrie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+basis+of+platelet+secretion:+emerging+structure/function+relationships.">The cellular basis of platelet secretion: emerging structure/function relationships.</a> Platelets. 28 (2), 108-118 (2017).</li><li>Joshi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferric+chloride-induced+arterial+thrombosis+and+sample+collection+for+3D+electron+microscopy+analysis.">Ferric chloride-induced arterial thrombosis and sample collection for 3D electron microscopy analysis.</a> J Vis Exp. (193), e64985 (2023).</li><li>Guerrero, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+the+von+Willebrand+factor/glycoprotein+Ib-IX+axis+with+a+platelet-type+von+Willebrand+disease+mutation.">Visualizing the von Willebrand factor/glycoprotein Ib-IX axis with a platelet-type von Willebrand disease mutation.</a> Blood. 114 (27), 5541-5546 (2009).</li><li>Kato, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+glycoprotein+VI+to+stable+platelet+adhesion+and+thrombus+formation+illustrated+by+targeted+gene+deletion.">The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation illustrated by targeted gene deletion.</a> Blood. 102 (5), 1701-1707 (2003).</li><li>Tomaiuolo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrelationships+between+structure+and+function+during+the+hemostatic+response+to+injury.">Interrelationships between structure and function during the hemostatic response to injury.</a> Proc NatlAcad Sci U S A. 116 (6), 2243-2252 (2019).</li><li>Cocchiaro, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+lipid+droplets+are+translocated+into+the+lumen+of+the+Chlamydia+trachomatis+parasitophorous+vacuole.">Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole.</a> Proc Natl Acad Sci. 105 (27), 9379-9384 (2008).</li><li>Pokrovskaya, I. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydia+trachomatis+hijacks+intra+-+Golgi+COG+complex+-+dependent+vesicle+trafficking+pathway.">Chlamydia trachomatis hijacks intra - Golgi COG complex - dependent vesicle trafficking pathway.</a> Cell Microbiol. 14 (5), 656-668 (2012).</li><li>Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+electron+microscope+tomography+using+robust+prediction+of+specimen+movements.">Automated electron microscope tomography using robust prediction of specimen movements.</a> J Str Biol. 152 (1), 36-51 (2005).</li><li>Kremer, J. R., Mastronarde, D. N., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+visualization+of+three-dimensional+image+data+using+IMOD.">Computer visualization of three-dimensional image data using IMOD.</a> J Str Biol. 116 (1), 71-76 (1996).</li><li>Chan, N. C., Eikelboom, J. W., Weitz, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolving+treatments+for+arterial+and+venous+thrombosis:+role+of+the+direct+oral+anticoagulants.">Evolving treatments for arterial and venous thrombosis: role of the direct oral anticoagulants.</a> Circ Res. 118 (9), 1409-1424 (2016).</li><li>Kiernan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde,+formalin,+paraformaldehyde,+and+glutaraldehyde:+what+they+are+and+what+they+do.">Formaldehyde, formalin, paraformaldehyde, and glutaraldehyde: what they are and what they do.</a> Microscopy Today. 8 (1), 8-13 (2000).</li><li>Storrie, B., Attardi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+mitochondrial+genome+in+HeLa+cells:+XV.+Effect+of+inhibition+of+mitochondrial+protein+synthesis+on+mitochondrial+formation.">Expression of the mitochondrial genome in HeLa cells: XV. Effect of inhibition of mitochondrial protein synthesis on mitochondrial formation.</a> J CellBiol. 56, 819-831 (1973).</li><li>Liu, S., Pokrovskaya, I. D., Storrie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+-+pressure+freezing+followed+by+freeze+substitution:+an+optimal+electron+microscope+technique+to+study+Golgi+apparatus+organization+and+membrane+trafficking.">High - pressure freezing followed by freeze substitution: an optimal electron microscope technique to study Golgi apparatus organization and membrane trafficking.</a> Meth Mol Biol. 2557, 211-223 (2023).</li><li>Klarhofer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+blood+flow+velocity+measurements+in+the+human+finger.">High-resolution blood flow velocity measurements in the human finger.</a> Mag Res Med. 45 (4), 716-719 (2001).</li><li>Chaudry, R., Miao, J. H., Rehman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology,+cardiovascular.">Physiology, cardiovascular.</a> StatPearls. , (2022).</li><li>Pokrovskaya, I. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNARE+-+dependent+membrane+fusion+initiates+a-granule+matrix+decondensation+in+mouse+platelets.">SNARE - dependent membrane fusion initiates a-granule matrix decondensation in mouse platelets.</a> Blood Adv. 2 (21), 2947-2958 (2018).</li><li>Pokrovskaya, I. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+ultrastructural+analysis+of+a-granule,+dense+granule,+mitochondria,+and+canalicular+system+arrangement+in+resting+human+platelets.">3D ultrastructural analysis of a-granule, dense granule, mitochondria, and canalicular system arrangement in resting human platelets.</a> Res Pract Thrombosis Haemostasis. 4 (1), 72-85 (2019).</li></ol>

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</table>

puncture wound hemostasis and preparation of samples for montaged wide-area electron microscopy analysis

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.

<meta charset="utf8"></head><body>作者聚焦：通过结构血液学的进步揭示血小板动力学

穿刺伤口止血和蒙太奇广域电子显微镜分析样品的制备

We're a lab that does volume EM, structural work, to say how do platelets activate, how do they aggregate? How do they form a thrombus that causes bleeding cessation and how in the case of over stimulation, they bring about occlusive clotting. In doing this, the premise is that if we know the structure, we can then better target drugs and bring about better outcomes.We use mouse models in our research, as many researchers do, and we all have the challenge of establishing how our findings in mice and in our case in the arteries and veins in mice, carry over to the mechanisms in the much larger arteries and veins in humans. We all hope that the principles are the same in mice and humans. If you take our work and using volume EM approaches, the big things that we've contributed are one, to say that platelets are not just suicide bombers.They retain granules. They do things in response to signaling. They have stable states, and these stable states are part of a normal thrombus and they're also part of an inclusive clot.Two, we've brought the sense that there is really structure to the thrombus that forms, it's not just simply a pile of bricks, but rather it's an ordered structure in which platelets are ordered with respect to their activation, et cetera. And three, we've brought the sense that one should be able to apply and understand from structure what needs to be either stimulated to get a good outcome or what needs to be suppressed to give a outcome that will protect people from bad outcomes. We collect sequential images at near nanometer scale of a near millimeter distances.We assemble those into the single montaged image. Hence, we can observe any given spot within the image at a scale of near nanometers to near millimeter, which is sufficient to place individual features inside of the whole thrombus. Researchers now have a powerful tool, and they can ask the new question, how do coagulation cascades and cellular signals or drugs act spatially at local to global levels in thrombi?

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

puncture-wound-hemostasis-preparation-samples-for-montaged-wide-area

Research

JoVE Journal

Medicine

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

167 次观看.  University of Arkansas for Medical Sciences. 我们是一个从事体积 EM 的实验室，结构工作，要说血小板是如何激活的，它们是如何聚集的？它们如何形成导致止血的血栓，以及在过度刺激的情况下如何导致闭塞性凝血。这样做的前提是，如果我们知道结构，我们就可以更好地靶向药物并带来更好的结果。和许多研究人员一样，我们在研究中使用小鼠模型，我们都面临着确定我们在小鼠中?...