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Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue

2015; Elsevier BV; Volume: 89; Issue: 1 Linguagem: Inglês

10.1038/ki.2015.308

1523-1755

David Unnersjö‐Jess, Lena Scott, Hans Blom, Hjalmar Brismar,

Photosynthetic Processes and Mechanisms

The glomerular filtration barrier, consisting of podocyte foot processes with bridging slit diaphragm, glomerular basement membrane, and endothelium, is a key component for renal function. Previously, the subtlest elements of the filtration barrier have only been visualized using electron microscopy. However, electron microscopy is mostly restricted to ultrathin two-dimensional samples, and the possibility to simultaneously visualize multiple different proteins is limited. Therefore, we sought to implement a super-resolution immunofluorescence microscopy protocol for the study of the filtration barrier in the kidney. Recently, several optical clearing methods have been developed making it possible to image through large volumes of tissue and even whole organs using light microscopy. Here we found that hydrogel-based optical clearing is a beneficial tool to study intact renal tissue at the nanometer scale. When imaging samples using super-resolution STED microscopy, the staining quality was critical in order to assess correct nanoscale information. The signal-to-noise ratio and immunosignal homogeneity were both improved in optically cleared tissue. Thus, STED of slit diaphragms in fluorescently labeled, optically cleared, intact kidney samples is a new tool for studying the glomerular filtration barrier in health and disease. The glomerular filtration barrier, consisting of podocyte foot processes with bridging slit diaphragm, glomerular basement membrane, and endothelium, is a key component for renal function. Previously, the subtlest elements of the filtration barrier have only been visualized using electron microscopy. However, electron microscopy is mostly restricted to ultrathin two-dimensional samples, and the possibility to simultaneously visualize multiple different proteins is limited. Therefore, we sought to implement a super-resolution immunofluorescence microscopy protocol for the study of the filtration barrier in the kidney. Recently, several optical clearing methods have been developed making it possible to image through large volumes of tissue and even whole organs using light microscopy. Here we found that hydrogel-based optical clearing is a beneficial tool to study intact renal tissue at the nanometer scale. When imaging samples using super-resolution STED microscopy, the staining quality was critical in order to assess correct nanoscale information. The signal-to-noise ratio and immunosignal homogeneity were both improved in optically cleared tissue. Thus, STED of slit diaphragms in fluorescently labeled, optically cleared, intact kidney samples is a new tool for studying the glomerular filtration barrier in health and disease. Traditionally, electron microscopy has been the only method available to visualize the glomerular filtration barrier, as the dimensions of the podocyte foot processes and the slit diaphragm are on the nanometer scale.1Grahammer F. Schnell C. Huber T.B. The podocyte slit diaphragm – from a thin grey line to a complex signaling hub.Nat Rev Nephrol. 2013; 9: 587-598Crossref PubMed Scopus (161) Google Scholar In terms of spatial resolution, electron microscopy is superior to light microscopy, due to the short effective wavelengths of electrons used to reconstruct images.2Rose H.H. Optics of high performance electron microscopes.Sci Technol Adv Mater. 2008; 9: 014107Crossref Scopus (66) Google Scholar Even if the morphology of renal foot processes has been dissected with electron microscopy, many questions remain regarding the functional distribution of proteins.1Grahammer F. Schnell C. Huber T.B. The podocyte slit diaphragm – from a thin grey line to a complex signaling hub.Nat Rev Nephrol. 2013; 9: 587-598Crossref PubMed Scopus (161) Google Scholar The possibility to stain for proteins using immunostaining or by expression of fluorescent proteins would here be beneficial, if high enough spatial resolution using light microscopy could be generated. With the use of super-resolution stimulated emission depletion (STED) microscopy,3Hell S. Wichmann J. Breaking the diffraction resolution limit by stimulated emission: stimulated emission depletion microscopy.Opt Lett. 1994; 19: 780-782Crossref PubMed Scopus (4315) Google Scholar we demonstrate that the nanoscale localization of proteins at the slit diaphragm is possible to study. Our results show that super-resolution imaging in intact tissue can be difficult due to unspecific/inhomogeneous staining and autofluorescence background seen in paraformaldehyde (PFA) fixed samples. These issues are probably reflected in the limited number of high-resolution studies performed in renal tissue to date. To the best of our knowledge, only two superresolution studies have been carried out,4Suleiman H. Zhang L. Roth R. et al.Nanoscale protein architecture of the kidney glomerular basement membrane.Elife. 2013; 2: e01149Google Scholar, 5Yu H. Suleiman H. Kim A.H. et al.Rac1 Activation in podocytes induces Rapid Foot Process Effacement and Proteinuria.Mol Cell Biol. 2013; 33: 4755-4764Crossref PubMed Scopus (91) Google Scholar both performed on ultrathin sections using a combination of transmission electron microscopy and super-resolution single molecule localization microscopy. High-resolution confocal microscopy has been used in one study of podocyte foot processes, where genetic labeling was used to label a sparse subset of podocytes.6Grgic I. Brooks C.R. Hofmeister A.F. et al.Imaging of podocyte foot processes by fluorescence microscopy.J Am Soc Nephrol. 2012; 23: 785-791Crossref PubMed Scopus (28) Google Scholar On this background, we have identified the need for improved sample preparation protocols for superresolution microscopy in order to study functional structures in kidneys using immunofluorescence at the nanoscale. By applying an optical clearing protocol based on the CLARITY7Chung K. Wallace J. Kim S.Y. et al.Structural and molecular interrogation of intact biological systems.Nature. 2013; 497: 332-337Crossref PubMed Scopus (1386) Google Scholar, 8Chung K. Deisseroth K. CLARITY for mapping the nervous system.Nat Methods. 2013; 10: 508-513Crossref PubMed Scopus (514) Google Scholar, 9Tomer R. Ye L. Hsueh B. et al.Advanced CLARITY for rapid and high resolution imaging of intact tissues.Nat Protoc. 2014; 9: 1682-1697Crossref PubMed Scopus (576) Google Scholar and SeeDB10Ke M.T. Fujimoto S. Imai T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.Nat Neurosci. 2013; 16: 1154-1161Crossref PubMed Scopus (629) Google Scholar methods, we show that background as well as staining problems can be significantly decreased, making it possible to study the spatial distribution of proteins at the slit diaphragm in intact tissue. Rat kidneys were optically cleared using a hydrogel-based protocol,9Tomer R. Ye L. Hsueh B. et al.Advanced CLARITY for rapid and high resolution imaging of intact tissues.Nat Protoc. 2014; 9: 1682-1697Crossref PubMed Scopus (576) Google Scholar followed by immunostaining and mounting in fructose solution.10Ke M.T. Fujimoto S. Imai T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.Nat Neurosci. 2013; 16: 1154-1161Crossref PubMed Scopus (629) Google Scholar The optical transparency (Figure 1a) and antibody penetration depth (Figure 1b and c) were sufficient for imaging samples on the mm-scale using confocal microscopy, allowing for a global view of kidney morphology and protein expression (Figure 1e and f). Further, we show a substantial increase in fluorescence contrast or signal-to-noise ratio by a factor of ∼100 (2.6 ± 0.44 to 230 ± 43) in cleared samples by applying standard immunostaining protocols to both PFA fixed and cleared samples (Figure 1d). For super-resolution STED microscopy, samples were prepared with the same protocol as for millimeter-scale imaging. Immunostaining in cleared samples stained for podocin was specific and highly localized (Figure 2d and e), which allows elucidating the localization on both sides of the slit diaphragm (Figure 2f). Comparison with non-cleared control samples show that optical clearing was crucial to reveal the spatial distribution of podocin on the nanometer scale. Control samples were stained following the same immunoprotocol as cleared samples, but here the immunosignal was incomplete (Figure 2g and h) and the nanoscale distribution of podocin could not be revealed (Figure 2i). Furthermore, the resolution as a function of depth is kept constant up to at least 30 μm using oil immersion objectives (Figure 2j). This allows for three-dimensional STED imaging, producing volumetric representations of foot processes on the nanometer scale (Figure 2b and c). Kidney samples were additionally counterstained for nephrin, an abundant anchoring protein at the filtration slit. Localizations of podocin (at podocyte membranes proximal to slit diaphragms) and nephrin (spanning the slit diaphragm; Figure 2a) could clearly be resolved (Figure 2k and l). The protocol was also applied to quantitative analysis of glomerular pathology. Podocin expression was analyzed in kidney samples from rats with passive Heymann nephritis (PHN), a rat model of human membranous nephropathy.11Saran A.M. Yuang H. Takeuchi E. et al.Compliment mediates nephrin redistribution and actin dissociation in experimental membranous nephropathy.Kidney Int. 2003; 64: 2072-2078Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar We observed a distinct alteration of foot process morphology in PHN compared with control rats (Figure 3a–d, Supplementary Movies S1–S2 online). These alterations were quantified in terms of the effacement fraction and the foot process coverage (Figure 3e and f). We found a significant increase in the effacement fraction and a significant decrease in the foot process coverage in PHN rats, in line with what has previously been observed using electron microscopy.11Saran A.M. Yuang H. Takeuchi E. et al.Compliment mediates nephrin redistribution and actin dissociation in experimental membranous nephropathy.Kidney Int. 2003; 64: 2072-2078Abstract Full Text Full Text PDF PubMed Scopus (61) Google ScholarFigure 2Super-resolution stimulated emission depletion (STED) imaging in optically cleared kidney samples. All samples were stained for podocin with Abberior STAR 635P. For STED/Confocal imaging, a Leica SP8 3X microscope with a 100× NA 1.4 oil objective was used unless other stated. (a) Schematic representation of the glomerular filtration barrier and the localization of podocin and nephrin at the slit diaphragm. (b) Eighty micrometer deep confocal stack of a whole glomerulus in a cleared slice of kidney tissue acquired using a Zeiss LSM780 system and a 63× NA 1.4 oil objective. Scale bar = 20 μm. (c) Three-dimensional (3D) renderings (Amira software) of a 3D STED z-stack display foot processes at the surface of a glomerular capillary. Scale bar = 2 μm. (d–i) Deconvolved (SVI Huygen's) STED/Confocal images of glomerular foot processes in cleared (d–e) and uncleared (g–h) kidney samples with intensity profiles (f, i) along marked arrows in STED/Confocal images, showing that podocin expression at both sides of the slit diaphragm is revealed only in cleared samples. Scale bars = 500 nm. (j) Plot of measured slit diaphragm width as a function of imaging depth. STED images were acquired at 6 different depths, and for each image the minimum resolvable slit diaphragm width was measured at 8 different positions. Error bars show SD of measured values. (k) Two-color STED image of a cleared kidney sample stained for podocin (green) with Abberior STAR635P and nephrin (magenta) with Alexa-594. (l) Intensity profiles along the marked arrow, showing the 2-sided expression of podocin and the central expression of nephrin at the slit diaphragm.View Large Image Figure ViewerDownload (PPT)Figure 3Superresolved glomerular pathology in passive Heymann nephritis (PHN) rats. All samples were stained for podocin with Atto 594 and imaged using a Leica SP8 3X microscope with a 100 × NA 1.4 oil objective. All images were deconvolved using SVI Huygens software. (a–b) Three-dimensional (3D) renderings (Amira software) of 3D stimulated emission depletion (STED) z-stacks showing inside views of glomerular capillaries from control (a) and PHN (b) rats. (c–d) STED images of glomerular foot processes in control (c) and PHN (d) rats. (e) Quantification of slit diaphragm effacement in control and PHN rats. The total length where slit diaphragms could not be resolved was measured and divided by the total length of the filtration slit. This was carried out along 5 randomly selected segments, showing a significant (*P < 0.0001) increase in effacement for PHN rats. Error bars show SD. (f) Quantification of filtration slit coverage in control and PHN rats. The total length of the filtration slit was divided by the total area of interest. This was carried out for 5 randomly selected areas of at least 14 μm2, showing a significant (*P < 0.00001) decrease for PHN rats. Error bars show SD.View Large Image Figure ViewerDownload (PPT) We demonstrate that hydrogel-based optical clearing enables volumetric high-resolution fluorescence imaging of thick kidney samples.12Yang B. Treweek J.B. Kulkarni R.P. et al.Single-cell phenotyping within transparent intact tissue through whole-body clearing.Cell. 2014; 158: 945-958Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar Further, we show that optical clearing is an important procedure in order to reveal the finest structures of the filtration barrier. Conventionally prepared PFA fixated samples give staining quality that is insufficient at the detailed level now available in super-resolution microscopy. The improved staining quality is due to the removal of lipids, which results in an increased antibody penetration and exposure of binding epitopes. Even if samples can be mechanically sectioned for studies of fine structures, sample transparency is beneficial in order to give access to large imaging volumes. A large imaging volume facilitates identification of regions of interest, and large quantities of imaging data can be extracted from the same sample. Also, the risk of mechanically damaging the sample by physical sectioning is eliminated. Concern has been raised that clearing tissue by removing lipids may introduce artifacts where the fine localization of proteins is lost. By the use of STED microscopy, we found that this is not an issue in the presented protocol. Tissue morphology is well preserved even at the nanoscale, as demonstrated by the organization of podocin and nephrin in the slit diaphragm. To conclude, the presented sample preparation protocol adds a novel tool for high- and super-resolution microscopy studies of protein localization with an impact for understanding pathologies occurring at the glomerular filtration barrier. Rats from Charles River, Germany, were used in all experiments. PHN was induced in 40-day-old rats as described by Saran et al.11Saran A.M. Yuang H. Takeuchi E. et al.Compliment mediates nephrin redistribution and actin dissociation in experimental membranous nephropathy.Kidney Int. 2003; 64: 2072-2078Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar 16 weeks before euthanization. Animals were anesthetized (intraperitoneal injection of pentobarbital), the aorta cut, and kidneys dissected. All experiments were performed in accordance with animal welfare guidelines set forth by Karolinska Institutet and were approved by Stockholm North Ethical Evaluation Board for Animal Research. Kidneys were dissected and immediately incubated at 4°C in hydrogel solution (1–2% v/v acrylamide, 0.0125–0.025% v/v bisacrylamide, 0.25% w/v VA-044 initiator, 4% PFA, 1 × phosphatebuffered saline (PBS)) for 5 days. The gel was polymerized at 37 °C for 3 h, and the presence of oxygen was minimized by filling tubes to the top with hydrogel solution. Samples were removed from the hydrogel solution and immersed in clearing solution (200 mmol/l boric acid, 4% SDS, pH 8.5) and incubated for 1 day. Kidneys were cut in 0.8–1.5 mm thick slices using a Vibratome (myNeurolab, St Louis, MO) and incubated at 50 °C for 2 weeks with clearing solution changed every 3 days. Before immunolabeling, samples were incubated in PBST (0.1% Triton-X in 1 × PBS) for 1 day. For PHN and PHN-negative control, kidneys were fixed in Duboscq-Brasil solution for 6 h, then hydrated and immersed in hydrogel solution (4% acrylamide, 0% bis-acrylamide) without PFA, and then the above protocol was followed. For all steps PBST was used as dilutant. Samples were incubated in primary antibody for 24 h at 37 °C and then washed in PBST for 8 h at 37 °C followed by secondary antibody incubation for 24 h at 37 °C and washed for 8 h at 37 °C prior to mounting. To stain for E-cadherin, a mouse anti-E-cadherin primary antibody (BD Biosciences, San Jose, CA, 610182, 1:50) and a goat anti-mouse Alexa-647-conjugated secondary antibody (1:100) were used. To stain for podocin, a rabbit anti-podocin primary antibody (Sigma-Aldrich, St Louis, MO, P0372, 1:50) and a goat anti-rabbit Alexa-546 (1:200), Abberior STAR635P (1:100), or Atto-594 (1:100) secondary antibody were used. To stain for nephrin, a goat anti-nephrin primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, sc19000 1:100) and a donkey anti-goat Alexa-594 secondary antibody (1:200) were used. Samples were immersed in 40% (w/v) fructose with 0.5% (v/v) 1-thioglycerol for 2 h and then transferred to 80% (w/w) fructose with 0.5% (v/v) 1-thioglycerol for 24 h. Samples were mounted in a glass bottom dish (MatTek, Ashland, MA, P35G-1.5-14-C) before imaging. All the authors declared no competing interests. This study was supported by grants from the Swedish Research Council (VR 2013-6041). We thank Evgeniya Burlaka for support with PHN induction. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI0ZDE1NmZkNmM4ZDdhZTZiYTE4OGRmN2FkM2I1YTYxNyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NDY5ODc0fQ.c9Src8AV2O4aWZAkYaMxMnvekR6tvyP2GUyPFpICKbAzef0a38996AL2bUiBlKzteo7I0wq2-uKzRSfzTggxJP7VNy4V7KHK5t88k7twz8g6s1EARcy6vSGinCVx6Ra15rnVqVYgWj4Q0ShWNddZF6oOS3CuUrRkTidGo9tWpT1iAiqz7dDdnIQPGSaQirPpPrEA1hFZvQwQfyCbx2yFIRlLcRKWgipji6kbyjtq5FVsKxcWgEuRbRXgXe0bggo5zRzqJ5c3WMkZgLk6AQ4MpWZ9TVkBE0u6jLe6dmQSBSHYVBcLak4QJdSLxLqL7w8cMthv9GcABZFSZH0akvKd-g Download .mp4 (21.57 MB) Help with .mp4 files Movie S1Foot processes in healthy rat.eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI4MGIwZjFjOWU1ZmIyNDEwNjA5ZWE4NmUxYmZiMTFhMCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NDY5ODc0fQ.e9n-jkmZbM-EVVL7r0PeNF9dUcfew-UC00Dsoo8OuwPJIpjDyi_D0KEDqmDVku_47IEGvxh2CIZVs8K-kSGtYNarpLIYJMz_cNQUwZ7vVM4Jwzxgvo40vIE6_FSlPxwk2QhKluHlQOtkYaRPlauHE-y5NgzHnSzbkNJgWND-EVTciwCSzCZ4igeJ5u9Pbt-wIJX4fX2zExtE4GpeveQSlB-BVFtVwX-aDuMvK_BlfeaCWAhsc2EJFKadIW43Z76vWPQBiJx_jAvsS4NjxCsWJ69yBINrbhoctEJf7Ps0oMczoAvv8EliSnS_lKX9X5Z-YGBfl6Scmjj1anEuIwVHRw Download .mp4 (18.13 MB) Help with .mp4 files Movie S2Foot processes in rat with PHN renal disease.