Artigo Acesso aberto Revisado por pares

Modern field emission scanning electron microscopy provides new perspectives for imaging kidney ultrastructure

2018; Elsevier BV; Volume: 94; Issue: 3 Linguagem: Inglês

10.1016/j.kint.2018.05.017

ISSN

1523-1755

Autores

Carsten Dittmayer, Eckhard Völcker, Irene Wacker, Rasmus R. Schröder, Sebastian Bachmann,

Tópico(s)

Renal cell carcinoma treatment

Resumo

Recent progress in electron microscopy (EM) techniques has opened new pathways to study renal tissue in research and pathology. Modern field emission scanning EM may be utilized to scan thin sections of resin-embedded tissue mounted on a conductive support. Here we sought to achieve automated imaging without the typical limitations of transmission EM with equivalent or superior quality. Extended areas of tissue were either imaged in two (nanotomy) or in three dimensions (volume EM) by serial-section-based array tomography. Single-beam and fast-recording multi-beam field emission scanning EM instruments were compared using perfusion-fixed rodent kidneys. High-resolution scans produced excellent images of tissue, cells, and organelles down to macromolecular complexes. Digital stitching of image tiles in both modes allowed seamless Google Earth–like zooming from overview to regions of interest at the nanoscale. Large datasets were created that can be rapidly shared between scientists of different disciplines or pathologists using open source software. Three-dimensional array tomography of thin sections was followed by segmentation to visualize selected features in a large volume. Furthermore, correlative light-EM enabled the identification of functional information in a structural context. Thus, limitations in biomedical transmission EM can be overcome by introducing field emission scanning EM-based technology that permits high-quality, large field-of-view nanotomy, volume EM, and correlative light-EM modes. Advantages of virtual microscopy in clinical and experimental nephrology are illustrated. Recent progress in electron microscopy (EM) techniques has opened new pathways to study renal tissue in research and pathology. Modern field emission scanning EM may be utilized to scan thin sections of resin-embedded tissue mounted on a conductive support. Here we sought to achieve automated imaging without the typical limitations of transmission EM with equivalent or superior quality. Extended areas of tissue were either imaged in two (nanotomy) or in three dimensions (volume EM) by serial-section-based array tomography. Single-beam and fast-recording multi-beam field emission scanning EM instruments were compared using perfusion-fixed rodent kidneys. High-resolution scans produced excellent images of tissue, cells, and organelles down to macromolecular complexes. Digital stitching of image tiles in both modes allowed seamless Google Earth–like zooming from overview to regions of interest at the nanoscale. Large datasets were created that can be rapidly shared between scientists of different disciplines or pathologists using open source software. Three-dimensional array tomography of thin sections was followed by segmentation to visualize selected features in a large volume. Furthermore, correlative light-EM enabled the identification of functional information in a structural context. Thus, limitations in biomedical transmission EM can be overcome by introducing field emission scanning EM-based technology that permits high-quality, large field-of-view nanotomy, volume EM, and correlative light-EM modes. Advantages of virtual microscopy in clinical and experimental nephrology are illustrated. Structural features of the vertebrate kidney including its large number of distinct cell types have been illustrated with the help of electron microscopy (EM).1Kriz W. Kaissling B. Structural organization of the mammalian kidney.in: Seldin and Giebisch's: Physiology and Pathophysiology. 5. Elsevier, Boston, MA2013: 595-691Google Scholar, 2Grahammer F. Wigge C. Schell C. et al.A flexible, multilayered protein scaffold maintains the slit in between glomerular podocytes.JCI Insight. 2016; 1Crossref PubMed Scopus (56) Google Scholar EM was equally successful in exploring and classifying renal disease.3Zuppan C. Role of electron microscopy in the diagnosis of nonneoplastic renal disease in children.Ultrastruct Pathol. 2011; 35: 240-244Crossref PubMed Scopus (8) Google Scholar, 4Liapis H. Gaut J.P. The renal biopsy in the genomic era.Pediatr Nephrol. 2013; 28: 1207-1219Crossref PubMed Scopus (17) Google Scholar A recent report on the diagnosis of glomerulonephritis has continued to classify EM as a crucial tool in diagnosis; EM processing for all native renal biopsies was recommended for evaluating glomerular deposits, podocyte effacement, early changes in diabetes or transplantation, or matrix abnormalities.5Sethi S. Haas M. Markowitz G.S. et al.Mayo Clinic/Renal Pathology Society consensus report on pathologic classification, diagnosis, and reporting of GN.J Am Soc Nephrol. 2016; 27: 1278-1287Crossref PubMed Scopus (143) Google Scholar With its open-view character, not filtered by, for example, selective (immuno)staining, EM may as well serve to answer unasked questions, such as detecting multiple abnormalities in a single biopsy.6Pavlisko E.N. Howell D.N. The continued vital role of electron microscopy in the diagnosis of renal disease/dysfunction.Ultrastruct Pathol. 2013; 37: 1-8Crossref PubMed Scopus (12) Google Scholar However, shortcomings of transmission EM (TEM) such as the long preparatory turnaround time, laborious examination, and limited interpretation due to pre-selection of images have led to some dwindling in the use of EM in spite of its diagnostic value. A variety of recent advancements in EM technology has now revolutionized not only the sector of structural biology but also of digitizing biological and diagnostic samples in 2-dimensional (2D) and 3-dimenstional (3D) form with a wide range of automated procedures. Field emission scanning EM (FESEM) using single- or multi-beam techniques now facilitates the examination of large areas of ultrathin sections by automated stitching of high-resolution image tiles to a coherent 2D data set, also termed nanotomy.7Kuipers J. Kalicharan R.D. Wolters A.H. et al.Large-scale scanning transmission electron microscopy (nanotomy) of healthy and injured zebrafish brain.J Vis Exp. 2016; 111: 53635Google Scholar, 8Wacker I. Chockley P. Bartels C. et al.Array tomography: characterizing FAC-sorted populations of zebrafish immune cells by their 3D ultrastructure.J Microsc. 2015; 259: 105-113Crossref PubMed Scopus (18) Google Scholar, 9Lee K.C. Mak L.S. Virtual electron microscopy: a simple implementation creating a new paradigm in ultrastructural examination.Int J Surg Pathol. 2011; 19: 570-575Crossref PubMed Scopus (8) Google Scholar, 10Sokol E. Kramer D. Diercks G.F.H. et al.Large-scale electron microscopy maps of patient skin and mucosa provide insight into pathogenesis of blistering diseases.J Invest Dermatol. 2015; 135: 1763-1770Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Array tomography (AT), on the other hand, allows analysis of large volumes by combining serial sections into a 3D data set.7Kuipers J. Kalicharan R.D. Wolters A.H. et al.Large-scale scanning transmission electron microscopy (nanotomy) of healthy and injured zebrafish brain.J Vis Exp. 2016; 111: 53635Google Scholar, 11Swanson L.W. Lichtman J.W. From Cajal to Connectome and beyond.Annu Rev Neurosci. 2016; 39: 197-216Crossref PubMed Scopus (104) Google Scholar, 12Eberle A.L. Mikula S. Schalek R. et al.High-resolution, high-throughput imaging with a multibeam scanning electron microscope.J Microsc. 2015; 259: 114-120Crossref PubMed Scopus (147) Google Scholar, 13Peddie C.J. Collinson L.M. Exploring the third dimension: volume electron microscopy comes of age.Micron. 2014; 61: 9-19Crossref PubMed Scopus (204) Google Scholar Obtained data sets may be analyzed site-independently with a graphic workstation, a process termed virtual microscopy.7Kuipers J. Kalicharan R.D. Wolters A.H. et al.Large-scale scanning transmission electron microscopy (nanotomy) of healthy and injured zebrafish brain.J Vis Exp. 2016; 111: 53635Google Scholar 2D and 3D structural data may further be enriched by combining light microscopy (LM) and EM (correlative light-electron microscopy, CLEM) to study cellular function at the nanoscale.11Swanson L.W. Lichtman J.W. From Cajal to Connectome and beyond.Annu Rev Neurosci. 2016; 39: 197-216Crossref PubMed Scopus (104) Google Scholar, 14Peddie C.J. Domart M.C. Snetkov X. et al.Correlative super-resolution fluorescence and electron microscopy using conventional fluorescent proteins in vacuo.J Struct Biol. 2017; 199: 120-131Crossref PubMed Scopus (37) Google Scholar Here, we demonstrate how limitations in biomedical TEM can be overcome by introducing nondestructive FESEM-based technology enabling large field-of-view nanotomy, volume EM, and CLEM in biomedical practice. Currently available EM techniques were compared in perfusion-fixed rodent kidneys. Standard FESEM illustrated 3D podocyte ultrastructure of a critical point-dried glomerular bulk sample (Figure 1a and b ). Standard TEM of ultrathin sections produced well-resolved detail of the filtration barrier (Figure 1c and d). Alternatively, FESEM analysis of ultrathin sections mounted on conductive support produced a very sharp image by scanning the surface of only the section's topmost layer (range: 1–3 nm); the result is comparable or even superior to what is obtainable by TEM imaging (Figure 1e and f). When comparing tubular epithelial morphology in TEM (Figure 1g and i) versus FESEM (Figure 1h and j) imaging, FESEM provided TEM-equivalent visual quality with clear-cut appearance of membrane boundaries and cytoplasmic structures. High-quality imaging of large regions on ultrathin kidney sections, or nanotomy, already became feasible with FESEM at intermediate spatial resolution (pixel size: 60 nm), excelling with drastic reduction of the typical artifacts of TEM wide-field analysis and absence of restrictions by mesh grid bars (Figure 2).13Peddie C.J. Collinson L.M. Exploring the third dimension: volume electron microscopy comes of age.Micron. 2014; 61: 9-19Crossref PubMed Scopus (204) Google Scholar Scans at high resolution produced excellent images of glomerular structures down to the nanoscale range (Figure 3). To economize time, sections were evaluated in a hierarchical mode using an initial, large-scale survey scan at low resolution (1000 nm image pixels), followed by higher resolution (e.g., 100 nm image pixels) to define regions of interest. Then glomerular or tubular profiles of interest were imaged at the nanoscale range using the highest resolution (5–3 nm image pixels; Supplementary Figure S1). With the need for larger, highly resolved regions of interest, single images, or tiles, were digitally stitched together, which allowed Google Earth–like zooming from overview to detail (Supplementary Movie S1). Such data sets permit offline examination of large digitized tissue areas secured in a local intranet server with rapid zooming in and out and comprehensive analysis without sitting at the EM in a time-limited instrument session. The required digital tools (e.g., open source software such as Fiji/TrakEM2 and IIPImage server) are available for any standard operating system and computer performance level (Figure 3 and Supplementary Figure S2).Figure 2Field emission scanning electron microscopy can generate large fields of view. Glomerulus with adjacent tubular segments (thick ascending limb of the loop of Henle [TAL], macula densa [MD], proximal tubule [PT], distal convoluted tubule [DCT]); a field of view of approximately 180 x 140 μm (60-nm pixel size, 3072 x 2303 pixels) produces a clear, well-resolving structural overview. Artifacts such as wrinkles, precipitates, and holes are absent owing to the stable adherence of the thin section to the conductive substrate. Bar = 20 μm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Large-scale imaging of a glomerulus, single-beam field emission scanning electron microscopy. Stitching of 2 x 2 tiles into a mosaic with a total field of view of approximately 75 x 75 μm allows imaging of an entire glomerulus at highest possible resolution (3-nm pixel size, 25,000 x 25,000 pixels). (a) Overview and (b) detail are from the same data set: (b) is a digital zoom to the square marked in (a). Mesangial cell (arrow) and podocytes (arrowheads) in (a); cell components such as Golgi apparatus, multivesicular bodies (MVB), centrioles (circle), and foot processes (arrows) in (b) are well resolved. Bar = 15 μm (a) and 500 nm (b). To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In an attempt to reduce scanning time we have made use of a MultiSEM 505 (Zeiss Microscopy GmbH, Jena, Germany), which uses 61 electron beams, whose secondary electron signals are imaged onto a multidetector with individual detection units for each electron beam. Multiple pixels instead of 1 single pixel at a time may thus be acquired in a high-throughput approach.12Eberle A.L. Mikula S. Schalek R. et al.High-resolution, high-throughput imaging with a multibeam scanning electron microscope.J Microsc. 2015; 259: 114-120Crossref PubMed Scopus (147) Google Scholar Scanning a sectioned glomerulus at high resolution (65,000 x 65,000 pixels, 4 nm image pixels) was thus completed at a rapid pace (approximately 1 minute); the resulting ultrastructural detail was comparable to the single-beam procedure (Figure 4). In order to obtain 3D information about tissue volume, first a series of semi-thin sections (300 nm), cut through Epon-embedded rat renal cortex, was used for LM-based AT. 3D survey of glomerular capillaries and adjacent tissue is shown in a movie (Supplementary Figure S2 and Supplementary Movie S2). The model file of the glomerular capillaries was further processed for 3D printing, creating an informative view over the entire glomerular capillary tuft (Supplementary Figure S3). Then for FESEM-based AT a series of ultrathin sections (50–70 nm) was recorded with 5 nm image pixels to reconstruct a distal epithelial portion in 3D with ultrastructural resolution (Supplementary Figure S4 and Supplementary Movie S3). CLEM was applied for the correlative localization of protein epitopes by immunofluorescence and ultrastructural analysis by FESEM using formaldehyde-perfused renal tissue embedded in hydrophilic resin. Overlay of the 4′,6-diamidino-2-phenylindole–labeled nuclei with their morphological counterpart in the FESEM image showed that the immunofluorescence signal for the distal Na,K,Cl-cotransporter (NKCC2) was indeed localized at the luminal plasma membrane of the thick ascending limb. Observing both imaging modes on the same section thus identifies the signal of a target molecule within its fine structural environment (Figure 5). Properties of the different techniques demonstrated here have been summarized in Table 1.Table 1Properties of the distinct EM approaches discussedTEMaWide-field TEM up to 200 kV electron energy, not a spot-scanning instrument.Single-beam FESEMMulti-beam FESEMInstrumentationMax. resolution∼ 2 nm; limited by sample preparation∼ 2 nm; limited by instrument8 nm, fixed; limited by instrumentImaging 100 x 100 μm∼ 10 min∼ 20 min∼ 15 sec; suitable for high throughputMounting samplesSample holder typically for 1 to 2 grids of 3 mm diameterLarge chamber; 9 individual samples of 10 mm x 10 mm each or a single wafer of 100 mm diameterDetector choiceTransmission, amplitude & phase contrastSecondary & back-scattered electronsSecondary electrons onlyLimitationsSmall field of viewModerate imaging speedFixed, reduced resolutionPreparationSamplesStandard EM processing; fixation by immersion or perfusion, optional osmication, resin embeddingSection substrateMesh or slot EM grids, with or without filmConductive material; silicon wafer, carbon/metal-coated glassStainingStandard EM contrasting protocols; uranyl acetate and lead citrateAdvantagesImmunostaining of sections from both sides feasibleScanning option of bulk samples; stable adherence of sections to substrate with few artifacts; fast and easy handling of single or serial sections; multiple immunostaining and elutions for CLEM; full-scale examination of sections free of obstructions by EM gridLimitationsInstability of sections Grid bars obstruct view Serial section handling tediousNATechniques discussed in paper2D nanotomy = large area imagingSingle ultrathin sections, 50–100 nm thickSingle ultrathin sections, 50–200 nm thickSecondary and back-scattered electron detectionSingle ultrathin sections, 50–100 nm thickSecondary electron detection only3D electron tomographyConventional tilt series of sections up to 300 nm thickNA3D array tomographyNAArrays of serial sections, 50–200 nm thickArrays of serial sections, 50–100 nm thickCLEM, correlative light-electron microscopy; FESEM, field emission scanning electron microscope; NA, not applicable; TEM, transmission electron microscope.a Wide-field TEM up to 200 kV electron energy, not a spot-scanning instrument. Open table in a new tab CLEM, correlative light-electron microscopy; FESEM, field emission scanning electron microscope; NA, not applicable; TEM, transmission electron microscope. Here we have employed FESEM technology to demonstrate its advantages in imaging renal tissue by 2D nanotomy at large scale. An area size of up to 5 x 5 mm may be achieved using advanced preparation techniques. Data sets of thin sections obtained using single-beam or, to economize scan time, multi-beam FESEM instruments enabled examination of samples with a quality equal or superior to classical TEM. Supreme quality of the FESEM images down to a resolution of a few nanometers resulted from scanning only the section's surface, which avoids blurring from underlying structures. The use of silicon wafers as carriers for the sections provided an unobscured, open view of the structure of renal tissue, cells, organelles, and macromolecules. Large data sets were produced by stitching mosaics of single image tiles using open source software.8Wacker I. Chockley P. Bartels C. et al.Array tomography: characterizing FAC-sorted populations of zebrafish immune cells by their 3D ultrastructure.J Microsc. 2015; 259: 105-113Crossref PubMed Scopus (18) Google Scholar, 11Swanson L.W. Lichtman J.W. From Cajal to Connectome and beyond.Annu Rev Neurosci. 2016; 39: 197-216Crossref PubMed Scopus (104) Google Scholar, 12Eberle A.L. Mikula S. Schalek R. et al.High-resolution, high-throughput imaging with a multibeam scanning electron microscope.J Microsc. 2015; 259: 114-120Crossref PubMed Scopus (147) Google Scholar, 14Peddie C.J. Domart M.C. Snetkov X. et al.Correlative super-resolution fluorescence and electron microscopy using conventional fluorescent proteins in vacuo.J Struct Biol. 2017; 199: 120-131Crossref PubMed Scopus (37) Google Scholar Previous examining of only a few selected TEM images may now be replaced by large data sets to be rapidly shared between interdisciplinary scientists or pathologists as HTML files in a pan-and-zoom manner, which allows selection of distinct regions of interest and statistical analysis in an off-instrument manner, using cloud-based systems.7Kuipers J. Kalicharan R.D. Wolters A.H. et al.Large-scale scanning transmission electron microscopy (nanotomy) of healthy and injured zebrafish brain.J Vis Exp. 2016; 111: 53635Google Scholar, 9Lee K.C. Mak L.S. Virtual electron microscopy: a simple implementation creating a new paradigm in ultrastructural examination.Int J Surg Pathol. 2011; 19: 570-575Crossref PubMed Scopus (8) Google Scholar, 10Sokol E. Kramer D. Diercks G.F.H. et al.Large-scale electron microscopy maps of patient skin and mucosa provide insight into pathogenesis of blistering diseases.J Invest Dermatol. 2015; 135: 1763-1770Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar LM AT imaging of entire glomeruli permitted selective segmentation of, for example, vascular or mesangial volumes with the perspective to perform 2D FESEM with histologically stained 300-nm sections for a multiscale approach.15Farahani N. Braun A. Jutt D. et al.Three-dimensional imaging and scanning: current and future applications for pathology.J Pathol Inform. 2017; 8: 36Crossref PubMed Scopus (39) Google Scholar, 16Koga D. Kusumi S. Shodo R. et al.High-resolution imaging by scanning electron microscopy of semithin sections in correlation with light microscopy.Microscopy (Oxf). 2015; 64: 387-394Crossref PubMed Scopus (32) Google Scholar Segmentation of small volumes from ultrathin section series imaged by FESEM (3D AT) illustrated that volume EM data may also be examined repeatedly for different purposes, used for statistics, and shared by others.7Kuipers J. Kalicharan R.D. Wolters A.H. et al.Large-scale scanning transmission electron microscopy (nanotomy) of healthy and injured zebrafish brain.J Vis Exp. 2016; 111: 53635Google Scholar, 8Wacker I. Chockley P. Bartels C. et al.Array tomography: characterizing FAC-sorted populations of zebrafish immune cells by their 3D ultrastructure.J Microsc. 2015; 259: 105-113Crossref PubMed Scopus (18) Google Scholar, 14Peddie C.J. Domart M.C. Snetkov X. et al.Correlative super-resolution fluorescence and electron microscopy using conventional fluorescent proteins in vacuo.J Struct Biol. 2017; 199: 120-131Crossref PubMed Scopus (37) Google Scholar, 17Wacker I. Schröder R.R. Schroeder J.A. Pathology goes 3D: exploring the potential of array tomography versus FIB nanotomography for a CADASIL sample.Ultrastruct Pathol. 2017; 41: 114-115Crossref Google Scholar The disadvantage of anisometric voxels caused by exclusively acquiring the topmost surface information of each thin section may be overcome by using focused ion beam milling technology, albeit at the cost of losing precious tissue.13Peddie C.J. Collinson L.M. Exploring the third dimension: volume electron microscopy comes of age.Micron. 2014; 61: 9-19Crossref PubMed Scopus (204) Google Scholar Preservation of the latter in nanotomy or 3D AT allowed us to perform CLEM, a rapidly expanding technology for conjugate structural and functional examination of cellular and subcellular structures.13Peddie C.J. Collinson L.M. Exploring the third dimension: volume electron microscopy comes of age.Micron. 2014; 61: 9-19Crossref PubMed Scopus (204) Google Scholar, 14Peddie C.J. Domart M.C. Snetkov X. et al.Correlative super-resolution fluorescence and electron microscopy using conventional fluorescent proteins in vacuo.J Struct Biol. 2017; 199: 120-131Crossref PubMed Scopus (37) Google Scholar In sum, FESEM opens up new perspectives in ultrastructural analysis, and facilitates correlative approaches and high-throughput workflows in kidney research. The advantages of the FESEM approach may equally well be used in human renal pathology. Perfusion-fixed rodent kidneys were embedded in Epon or in hydrophilic LR white for CLEM experiments. Semi-thin sections were stained with Richardson's solution for LM, ultrathin sections with uranyl acetate and lead citrate for EM analysis. For a description of methodological details see Supplementary Information. Image data were generated on different microscopes (see Supplementary Information) and processed using commercial and open source software. All the authors declared no competing interests. The authors wish to thank Kerstin Riskowsky, John Horn, Petra Schrade, Marcus Mildner, and Robert Labes (Charité); Ulrich Gernert (ZELMI, TU Berlin); Anna Lena Eberle, Tomasz Garbowski, and Stephan Nickell (Zeiss MultiSEM team); Geertje Bammert (Zeiss application laboratory); and Stephan Saalfeld (Janelia, Ashburn, VA) for expert help in sample preparation, imaging, and data processing, and Martin Thomson (Charité) for reading the manuscript. Sources of support: Charité foundation (Stiftung Charité, Max Rubner award 2016), Deutsche Forschungsgemeinschaft (MU 2924/2-1,2 und BA 700/22-1,2; INST 335/596-1 FUGG), MorphiQuant-3D-FKZ13GW0044, BMBF. Download .docx (.02 MB) Help with docx files Supplementary InformationDetailed methods regarding fixation, embedding, section preparation, imaging and data processing; legends to supplementary figures and movies, docx format.eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJjYWUzYWU4OTQ1OTkwMDlhNjFjN2UzOWMwZjY3ZmU4MSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc3ODQ5NDYyfQ.Ks2EqpTRC-4pgRiadmI6rQjBjiJZirkgApl22qfzA4fD39ly7gPInkUEXeoDXMdytA0mjGVnr7TjDJemkxXqYE0lR6cIxSz9v1ORMUBpxv6VQYxwleF0BFED3a0_cQIwFo9JiAGQtYoljD4vH2wsOJxgp6B5NSuOj1M6uUJW1VuOYQV964nuNNzNEZEwIMKgTxx8ORCbfgle0p08ThJubtDQ43YBUxLE1afIfSKjBm0yWEhwnNIJXFAK52IWuVjajAySWpa_8NBJJDRCwsMcHHTmFc3rIG3Fj5_uwRXK8ot5mQ1hAoiUSP7ssm0XsMRE8jTJPxY0eHHOEN6GYJh0Rw Download .mp4 (0.65 MB) Help with .mp4 files Movie S1Hierarchical imaging concept. A fly-in movie shows virtual zooming and moving around on pre-recorded data. Starting from 3 sections recorded with 1000 nm image pixels (original magnification ×288) we first zoom in to a trapezoid region of interest containing 2 glomeruli, recorded with 100 nm image pixels (original magnification ×2880), and in a second step to a square region of interest around the left glomerulus. This region of interest consists of 4 tiles, recorded with 3 nm image pixels (original magnification ×95,800). Further zooming, first to a podocyte and finally to a pair of centrioles within that cell, illustrates the image quality obtainable at that scan resolution. Further zooming out and in again shows a synapse with numerous vesicles at the edge of the high-resolution region of interest and then at the same zoom level a neighboring smooth muscle cell with numerous caveolae.Figure S2LM-based array tomography of semithin sections for light microscopy. Three-hundred and fifty serial sections (300 nm thick), cut through Epon-embedded, perfusion-fixed rat kidney cortex and stained with Richardson's histological staining (A), were imaged in an LM. After alignment of the recorded image stack, lumina were segmented to show glomerular capillaries (brown; B), interstitial capillaries (blue), and distal tubule (green; C). Modeled structures are shown in relation to the original xy image plane plus the reconstructed orthogonal planes (D); bars = 50 µm (A) and 100 µm (B–D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJlY2FmZjVhODM0MmM4ZjZmNmE4ZDZkYTllMmM1NWRkMSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc3ODQ5NDYyfQ.eItoiKySKKqJFsr_b1lXHgm-3dupi16FhFXYwaOq3dcXNIPaVI6cn043kE3TksymGD1SVf_qHWS2S4_P69kZY8jx-g1mRJo0WjQ0YH0JbFTC470IglhO4E_FU0QkysmZ-aQJpvJ77LOeg6Cbw-7jfWqHMw9YQtdhhVndHLiY3DqJXlTO40KI_Xc4TSw2eRbNSAwG-27v6DDuf3pRjGaC40Ocy1ZY09b7QJyvlFesfgfFKisWXrwnYu5WDZlifGWxOC8Sip-QIvseobbSvnUqhkDmlT9GOFGkpvUN2sVHElt18-lr7Z5kEa__Rb_FjV-BGXY_D02cNX0OwIxrmyqXtg Download .mp4 (4.83 MB) Help with .mp4 files Movie S2Light microscopy (LM)-based array tomography (cf. Figure S2). Moving through the original image planes from top to bottom of the recorded image stack, then in reverse direction through the glomerular capillaries, followed by a rotation of the whole structure.Figure S3Printed glomerulus (cf. Figure S2). Model file of glomerular capillaries was exported to STL file format and 3D-printed by fused deposition modeling.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure S4Three-dimensional (3D) reconstruction of tubular portion by field emission scanning electron microscopy (FESEM)-based array tomography. (A) A thick ascending limb portion (volume of interest) is highlighted, with its inner perimeter labeled yellow and the outer one green. (B) Segmentation of mitochondria (red) near nucleus (blue), along with (C) a selected portion of the endoplasmic reticulum (green) and an adjacent phagosome (yellow), enables a 3D view of a selected region of interest. (D) Detail of a phagosome is exemplified in a series of 5 consecutive sections. In 3D view, the spatial relation between endoplasmic reticulum and the phagosome is illustrated from 3 different angles (E). Bar = 5 μm (A), 2 μm (B,C), and 500 nm (D,E).View Large Image Figure ViewerDownload Hi-res image Download (PPT)eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI4YmU5NzZlZGYxYzBjZjA5ZmU5NDA3NzY4NmU5NzhjOCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc3ODQ5NDYyfQ.H1ygRB2SgrQvjdkkO5orU1bdl1xWwzEBZcxUbxN2_3ZxS2ByDVUcGRrVpKoqLACVPqFZqbcV1mtR-fg6EOT3Wf7GqkFosp53ocTDlxSVtRFonDSfXcNWlZZUWIQxmRPdvnQu8G0kIW-74CLwurZd3HpHrsj55A8YqvoWgzs46PZvOr4UTLq0jmZCaEkVBsoh4gfF5b2KHC0b-ue5HisS1b72yb-qC_kSPTUbGtwInlpEDyErFExTQykpib0EWOmrwnPX_1uDLv__x9axymA1knkRYAoYOB02sXRp89qRJRwV4dzK-nUZz2Zm3TUAcGe0bOyMhujqDsKoEMNe1ZgFwA Download .mp4 (0.8 MB) Help with .mp4 files Movie S3FESEM-based array tomography (cf. Figure S4). The virtual reconstruction of a volume of interest containing a thick ascending limb cell's inventory of perinuclear mitochondria. The original image plane, 2 reconstructed image planes, and 3-dimensional models of segmented organelles are shown.

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