Three-Dimensional Cell and Tissue Reconstruction by Serial Block Face SEM

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

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Abstract

Our laboratory is equipped with (1) a serial block-face scanning electron microscope comprising a Zeiss SIGMA-VP SEM coupled with a Gatan 3View in situ ultramicrotomy system for determining the 3D ultrastructure of cells and tissues at a lateral (x,y) resolution of 5 to 10 nanometers and a z-resolution of 25 nm to 50 nm; and (2) a Zeiss Crossbeam 550 focused ion beam scanning electron microscope for determining the 3D ultrastructure of cells and tissues at a lateral (x,y) resolution of 3 to 5 nanometers and a z-resolution of 5 nm to 10 nm. We have applied SBEM extensively to determine the 3-D ultrastructure of human and mouse blood platelets that were rapidly fixed prior to purification to minimize activation (1-3). One objective was to determine the 3D organization of granules, dense granules, mitochondria, and canalicular system in resting human platelets and map their spatial relationships. We found that granule number correlated linearly with platelet size, whereas dense granule and mitochondria number had little correlation with platelet size. 3D data from 30 platelets indicated only limited spatial intermixing of the different organelle classes. Interestingly, almost 70% of granules came within 35 nm of each other, a distance associated in other cell systems with protein mediated contact sites. Size and shape analysis of the 1,500 granules analyzed revealed no more variation than that expected for a Gaussian distribution. We have also used SBF-SEM to investigate the organization and activation of platelets in the formation of blood clots in a mouse model. Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like diabetes and obesity, and chronic inflammatory diseases like atherosclerosis, cancer, and autoimmune diseases. Upon vascular injury, usually the coagulation system, platelets, and endothelium act in an orchestrated manner to prevent bleeding by forming a clot at the site of the injury. Abnormalities in this process lead to either excessive bleeding or uncontrolled thrombosis/insufficient anti-thrombotic activity, which translates into vessel occlusion and its sequelae. Such models involve endothelial damage and subsequent clot formation at the injured site, and provide a sensitive, quantitative assay to monitor vascular damage and clot formation in response to different degrees of vascular damage. Once optimized, this standard technique can be used to study the molecular mechanisms underlying thrombosis, as well as the ultrastructural changes in platelets in a growing thrombus (Joshi et al., J. Vis. Exp; 2023). It has been established that primary hemostasis after vascular injury results in a platelet-rich thrombus, which has been assumed to form a solid plug. Unexpectedly, our 3D electron microscopy of mouse jugular vein puncture wounds revealed that the resulting thrombi were structured about localized, nucleated platelet aggregates, containing pedestals and columns, that produced a vaulted thrombus capped by extravascular platelet adherence. Pedestal and column surfaces were lined by pro-coagulant platelets. Furthermore, early steps in thrombus assembly were sensitive to the ADP chemoreceptor P2Y12 inhibition as well as to thrombin inhibition. Based on these results, a cap and build paradigm has been proposed, which might have translational implications for bleeding control and hemostasis. Further work revealed that initial platelet anchoring to the exposed adventitia resulted in localized patches of degranulated, highly activated, procoagulant-like platelets bound directly to collagen (initiation step). Activation to a procoagulant state was sensitive to dabigatran, a direct protease-activated receptor (PAR) inhibitor, but not to cangrelor, a P2Y12 receptor inhibitor. Sustained thrombus growth (propagation step) was accompanied by the capture of strings of loosely associated discoid platelets tethered one to another. Spatial examination indicated that staged platelet activation resulted in a discoid platelet tethering zone that was pushed progressively outward. Overall thrombus growth was sensitive to cangrelor. As thrombus growth slowed, discoid platelet recruitment became rare and loosely adherent intravascular platelets failed to convert to tightly adherent platelets. Our 3D image data support a model in which initial high platelet activation is limited to 5 microns distance from the adventitia; subsequent tethered discoid platelet recruitment is followed by platelet activation to produce tightly adherent platelets; and decreased signaling intensity over time results in self-limiting, intravascular platelet activation (Pokrovskaya et al., Res. Pract. Thromb. Haemost. 7: 100058; 2023). Based on our collaboration with the Storrie laboratory, University of Arkansas for Medical Sciences, and the Whiteheart laboratory, University of Kentucky, we have performed an ultrastructural study of human blood platelets from hospitalized COVID-19 patients at University of Kentucky HealthCare, Lexington, KY. Platelets contribute to a wide variety of COVID-19 clinical manifestations of which microclotting in the pulmonary vasculature, in particular, has been a prominent contributor to respiratory deficits. To investigate the potential diagnostic contribution of overall platelet morphology and their alpha-granules and mitochondria to understanding platelet hyperactivation and microclotting seen in COVID-19 patients, we took a 3D ultrastructural approach using both serial SBEM and FIB-SEM. Because differences might be small, we employed deep learning computational methods for the evaluation of nearly 600 individual platelets and nearly 30,000 included organelles within three controls and three severely ill COVID-19 patients. Statistical analysis reveals that the alpha-granule/mitochondrial-to-platelet volume ratio is significantly greater in COVID patient platelets indicating a more densely organelle packed, more compact platelet. The COVID patient platelets were significantly smaller, 35%, and most of difference in organelle packing density was due to decreased platelet size, rather than differences in organelle count or volume. There was little to no 3D ultrastructural evidence for differential activation of the platelets from COVID patients versus control donors. These studies, although limited in blood donor number suggest that factors outside of the platelets themselves are likely responsible for such COVID complications as microclotting. In addition, we suggest that deep learning 3D methodology developed in these studies moves the gold standard for 3D ultrastructural studies of platelets to a new level (Matharu et al., Platelets, in press). We are now combining FIB-SEM with super-resolution optical imaging and machine learning techniques to understand the biogenesis of fenestrated endothelial cells line the blood vessels of liver vasculature. We anticipate that such an approach can address important cell biological questions related to liver tumors and liver vascular diseases by linking cell morphology with specific expressed proteins.