A fast and simple clearing and swelling protocol for 3D in-situ imaging of the kidney across scales
Authors: David Unnersjö-Jess, Linus Butt, Martin Höhne, Anna Witasp, Lucas Kühne, Peter F. Hoyer, Jaakko Patrakka, Paul T. Brinkkötter, Annika Wernerson, Bernhard Schermer, Thomas Benzing, Lena Scott, Hjalmar Brismar, Hans Blom
Journal: Kidney International (2020)

In recent years, many light-microscopy protocols have been published for visualization of nanoscale structures in the kidney. These protocols present researchers with new tools to evaluate both foot process anatomy and effacement, as well as protein distributions in foot processes, the slit diaphragm and in the glomerular basement membrane. However, these protocols either involve the application of different complicated super resolution microscopes or lengthy sample preparation protocols. Here, we present a fast and simple, five-hour long procedure for three-dimensional visualization of kidney morphology on all length scales. The protocol combines optical clearing and tissue expansion concepts to produce a mild swelling, sufficient for resolving nanoscale structures using a conventional confocal microscope. We show that the protocol can be applied to visualize a wide variety of pathologic features in both mouse and human kidneys. Thus, our fast and simple protocol can be beneficial for conventional microscopic evaluation of kidney tissue integrity both in research and possibly in future clinical routines. In recent years, many light-microscopy protocols have been published for visualization of nanoscale structures in the kidney. These protocols present researchers with new tools to evaluate both foot process anatomy and effacement, as well as protein distributions in foot processes, the slit diaphragm and in the glomerular basement membrane. However, these protocols either involve the application of different complicated super resolution microscopes or lengthy sample preparation protocols. Here, we present a fast and simple, five-hour long procedure for three-dimensional visualization of kidney morphology on all length scales. The protocol combines optical clearing and tissue expansion concepts to produce a mild swelling, sufficient for resolving nanoscale structures using a conventional confocal microscope. We show that the protocol can be applied to visualize a wide variety of pathologic features in both mouse and human kidneys. Thus, our fast and simple protocol can be beneficial for conventional microscopic evaluation of kidney tissue integrity both in research and possibly in future clinical routines. Numerous optical protocols for imaging nanoscale structures in the kidney have been presented in recent years.1Chozinski T.J. Mao C. Halpern A.R. et al.Volumetric, nanoscale optical imaging of mouse and human kidney via expansion microscopy.Sci Rep. 2018; 8: 10396Crossref PubMed Scopus (18) Google Scholar, 2Pullman J.M. Nylk J. Campbell E.C. et al.Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease.Biomed Opt Express. 2016; 7: 302-311Crossref PubMed Scopus (13) Google Scholar, 3Siegerist F. Ribback S. Dombrowski F. et al.Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement.Sci Rep. 2017; 7: 11473Crossref PubMed Scopus (26) Google Scholar, 4Suleiman H. Zhang L. Roth R. et al.Nanoscale protein architecture of the kidney glomerular basement membrane.Elife. 2013; 2e01149PubMed Google Scholar, 5Unnersjö-Jess D. Scott L. Blom H. Brismar H. Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue.Kidney Int. 2016; 89: 243-247Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 6Unnersjö-Jess D. Scott L. Sevilla S.Z. et al.Confocal super-resolution imaging of the glomerular filtration barrier enabled by tissue expansion.Kidney Int. 2018; 93: 1008-1013Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 7Zhao Y. Bucur O. Irshad H. et al.Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy.Nat Biotechnol. 2017; 35: 757-764Crossref PubMed Scopus (86) Google Scholar, 8Bucur O. Fu F. Calderon M. et al.Nanoscale imaging of clinical specimens using conventional and rapid-expansion pathology.Nat Protoc. 2020; 15: 1649-1672Crossref PubMed Scopus (5) Google Scholar The power of these new techniques as tools for quantifying filtration barrier anatomy and pathology is exemplified in recent publications from our group and others.3Siegerist F. Ribback S. Dombrowski F. et al.Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement.Sci Rep. 2017; 7: 11473Crossref PubMed Scopus (26) Google Scholar59Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar,10Motrapu M. Świderska M.K. Mesas I. et al.Drug testing for residual progression of diabetic kidney disease in mice beyond therapy with metformin, ramipril, and empagliflozin.J Am Soc Nephrol. 2020; 31: 1729-1745Crossref PubMed Scopus (7) Google Scholar Although powerful, all these optical protocols require the use of advanced diffraction-unlimited microscopy or involve the introduction of complex polymer chemistry into the sample to clear and/or physically expand it. Many of the protocols are also time-consuming and require a significant amount of hands-on labor. This extra layer of complexity might be what is keeping these new methods from being routinely used by both researchers and clinical pathologists. Furthermore, many of the protocols lack access to deep 3-dimensional (3D) imaging of foot processes (FPs) beyond 5–15 μm in depth.1Chozinski T.J. Mao C. Halpern A.R. et al.Volumetric, nanoscale optical imaging of mouse and human kidney via expansion microscopy.Sci Rep. 2018; 8: 10396Crossref PubMed Scopus (18) Google Scholar, 2Pullman J.M. Nylk J. Campbell E.C. et al.Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease.Biomed Opt Express. 2016; 7: 302-311Crossref PubMed Scopus (13) Google Scholar, 3Siegerist F. Ribback S. Dombrowski F. et al.Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement.Sci Rep. 2017; 7: 11473Crossref PubMed Scopus (26) Google Scholar, 4Suleiman H. Zhang L. Roth R. et al.Nanoscale protein architecture of the kidney glomerular basement membrane.Elife. 2013; 2e01149PubMed Google Scholar,7Zhao Y. Bucur O. Irshad H. et al.Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy.Nat Biotechnol. 2017; 35: 757-764Crossref PubMed Scopus (86) Google Scholar,8Bucur O. Fu F. Calderon M. et al.Nanoscale imaging of clinical specimens using conventional and rapid-expansion pathology.Nat Protoc. 2020; 15: 1649-1672Crossref PubMed Scopus (5) Google Scholar Therefore, we aimed to develop a drastically simplified and fast all-optical protocol that clears and spatially swells kidney tissue samples sufficiently to allow for in situ 3D high-resolution analysis of renal anatomy and pathology using a conventional diffraction-limited microscope available in almost all life science and pathology institutions. Previous protocol simplifications have shown that the introduction of acrylamide polymers into paraformaldehyde-fixed samples can be totally omitted while still preserving tissue integrity on sodium dodecyl sulfate (SDS) clearance.11Xu N. Tamadon A. Liu Y. et al.Fast free-of-acrylamide clearing tissue (FACT)—an optimized new protocol for rapid, high-resolution imaging of three-dimensional brain tissue.Sci Rep. 2017; 7: 9895Crossref PubMed Scopus (26) Google Scholar Another study has shown that tissue samples can be expanded by more than a factor of 2 using a simple polymer-free protocol based on immersion in different aqueous solutions.12Murakami T.C. Mano T. Saikawa S. et al.A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing.Nat Neurosci. 2018; 21: 625-637Crossref PubMed Scopus (115) Google Scholar Thus, we removed acrylamide infusion from our more tedious and complex published protocols5Unnersjö-Jess D. Scott L. Blom H. Brismar H. Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue.Kidney Int. 2016; 89: 243-247Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar,6Unnersjö-Jess D. Scott L. Sevilla S.Z. et al.Confocal super-resolution imaging of the glomerular filtration barrier enabled by tissue expansion.Kidney Int. 2018; 93: 1008-1013Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar and changed the sample embedding medium to allow for a mild (1.3-fold linear) swelling of kidney tissue samples. By also optimizing imaging parameters, we achieved a sufficiently high effective resolution for 3D visualization of podocyte FPs and the glomerular basement membrane (GBM) using conventional confocal microscopy. The simple clearing/swelling protocol is depicted schematically in Figure 1a. Note that the SDS delipidation step was added not primarily for optical transparency, but rather to ensure sufficient swelling of samples.12Murakami T.C. Mano T. Saikawa S. et al.A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing.Nat Neurosci. 2018; 21: 625-637Crossref PubMed Scopus (115) Google Scholar Furthermore, when omitting delipidation, the staining quality is poor (Supplementary Figure S1A). We show that rapid delipidation at 70 °C for 1 hour produced good results without the sample degradation occurring at higher temperatures (Supplementary Figure S1B). As reported by Murakami et al.,12Murakami T.C. Mano T. Saikawa S. et al.A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing.Nat Neurosci. 2018; 21: 625-637Crossref PubMed Scopus (115) Google Scholar urea and urea-like molecules can effectively swell tissue samples, resulting in increased effective spatial resolution. We thus added 4 M urea in a mixture with 80% (wt/wt) fructose (dubbed FRUIT),13Hou B. Zhang D. Zhao S. et al.Scalable and DiI-compatible optical clearance of the mammalian brain.Front Neuroanat. 2015; 9: 19Crossref PubMed Scopus (109) Google Scholar which resulted in a linear swelling factor of ∼1.3 (Figure 1b). To shorten the labeling time, we used dye-conjugated primary antibodies. Initially, when using dye-conjugated nephrin antibodies in a standard staining buffer (phosphate-buffered saline with Tween 20), the signal and labeling efficiency were not high enough to outline individual FPs. However, by using an alternative staining buffer, HEPES-TSC,14Susaki E.A. Shimizu C. Kuno A. et al.Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues.Nat Commun. 2020; 11: 1982Crossref PubMed Scopus (36) Google Scholar staining quality was increased to a satisfactory degree (Supplementary Figure S1C). Compared with previously published protocols using conventional microscopy, our fast and simple 5-hour protocol outperformed them on several essential parameters (Figure 1c–f). The protocol presented by Pullman et al.2Pullman J.M. Nylk J. Campbell E.C. et al.Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease.Biomed Opt Express. 2016; 7: 302-311Crossref PubMed Scopus (13) Google Scholar is slightly faster overall but has the drawbacks of requiring super-resolution structured illumination microscopy microscopy and using thin cryosections, precluding access to large imaging depths. Of note, our protocol is less complex than standard paraffin-embedding or cryosectioning when all evaluated parameters are considered. Because our protocol gives only a moderate swelling factor of 1.3, confocal imaging parameters had to be optimized to achieve sufficient resolving power for imaging FPs. Mouse kidney samples were treated according to the protocol and stained for nephrin using different fluorophores. The resolution of a confocal microscope depends on both the pinhole size and the wavelength of the excitation/emission light.15Wilson T. Resolution and optical sectioning in the confocal microscope.J Microsc. 2011; 244: 113-121Crossref PubMed Scopus (122) Google Scholar Thus, we used a smaller pinhole diameter of 0.3 Airy units (AU) together with a green-emitting dye, Alexa Fluor 488, and in this way achieved sufficient resolution to resolve FPs in a wild-type (WT) mouse kidney (Figure 2a). Thus, whenever mouse FPs are to be visualized, fluorophores with a maximum excitation wavelength of 488 nm are used. Under these optimized imaging conditions, we were able to visualize whole mouse glomeruli in 3D at an effective resolution of ∼115 nm, good enough to resolve individual FPs (Figure 2b–e). Moreover, we show that an antibody penetration depth of at least 70 μm is achieved with fast labeling (Supplementary Figure S2A–C). Using a glycerol objective with a correction collar, this provides access to larger Z-depths than any of the previously developed protocols, with the exception of clearing/stimulated emission depletion protocol.5Unnersjö-Jess D. Scott L. Blom H. Brismar H. Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue.Kidney Int. 2016; 89: 243-247Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Because the axial resolution of a confocal microscope is roughly 3 times worse than the lateral resolution,15Wilson T. Resolution and optical sectioning in the confocal microscope.J Microsc. 2011; 244: 113-121Crossref PubMed Scopus (122) Google Scholar capillaries should be oriented in the x-y plane for optimally resolving FPs. We show that with our protocol, we can find plenty (up to 25) areas for imaging in each glomerulus (Supplementary Figure S2D), allowing us to gather large amounts of data to also detect subtle variations in filtration barrier structures.9Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar This demonstrates the importance of having access to the depth dimension when performing high-throughput quantitative analyses of glomerular health. Importantly, we also validated the protocol for resolving FPs in human patient material (Figure 2f and g), which is less challenging compared with mouse, owing to the larger dimensions of human FPs.9Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar Moreover, using wheat germ agglutinin (WGA) lectin with affinity toward the glycocalyx of FPs, we were also able to resolve FPs in a cross-sectional manner in both mouse and human samples (Figure 2h and i). In addition, we costained for nephrin, with the expected localization in the slit membrane, showing that we can get data on protein localization in addition to morphologic information. As reported previously,1Chozinski T.J. Mao C. Halpern A.R. et al.Volumetric, nanoscale optical imaging of mouse and human kidney via expansion microscopy.Sci Rep. 2018; 8: 10396Crossref PubMed Scopus (18) Google Scholar, 2Pullman J.M. Nylk J. Campbell E.C. et al.Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease.Biomed Opt Express. 2016; 7: 302-311Crossref PubMed Scopus (13) Google Scholar, 3Siegerist F. Ribback S. Dombrowski F. et al.Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement.Sci Rep. 2017; 7: 11473Crossref PubMed Scopus (26) Google Scholar,5Unnersjö-Jess D. Scott L. Blom H. Brismar H. Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue.Kidney Int. 2016; 89: 243-247Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar,6Unnersjö-Jess D. Scott L. Sevilla S.Z. et al.Confocal super-resolution imaging of the glomerular filtration barrier enabled by tissue expansion.Kidney Int. 2018; 93: 1008-1013Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar,9Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar,10Motrapu M. Świderska M.K. Mesas I. et al.Drug testing for residual progression of diabetic kidney disease in mice beyond therapy with metformin, ramipril, and empagliflozin.J Am Soc Nephrol. 2020; 31: 1729-1745Crossref PubMed Scopus (7) Google Scholar en face FP imaging of the slit diaphragm can be readily used to visualize and quantify FP effacement. We validate that this approach also can be applied with our new protocol using a recently published mouse model for focal segmental glomerulosclerosis (FSGS)9Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar (Figure 3). By staining for nephrin, the slit diaphragm pattern could be visualized in 3 dimensions in whole glomeruli of WT and mutated mice (Figure 3a and b). As reported previously,3Siegerist F. Ribback S. Dombrowski F. et al.Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement.Sci Rep. 2017; 7: 11473Crossref PubMed Scopus (26) Google Scholar59Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar the slit diaphragm length per area can be quantified semiautomatically, and this analysis provides a result consistent with what has been published previously for this mouse line9Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar (Figure 3c). Furthermore, glomerular capillaries were imaged in cross-section showing both effacement of FPs as well as alterations to the GBM (Figure 3d–f); see Supplementary Methods and Figure S3 for details of GBM measurements. To further validate our protocol for possible clinical use, we applied it to human tissue from patients diagnosed with different types of kidney disease. Two patients with congenital nephrotic syndrome, either with a homozygous mutation in the NPHS1 gene (encoding for nephrin), or trans-associated mutations in the TRPC6 and NPHS2 genes (encoding for the TRPC6 channel and podocin). We also analyzed 2 patients diagnosed with FSGS and minimal change disease. We could visualize and quantify typical hallmarks of kidney disease, such as FP effacement, as well as GBM alterations (Figure 4). Images could be acquired both en face (Figure 4a–e) and from the side (Figure 4g–k). Moreover, we observed the expected absence of nephrin in slit diaphragms of the patient with an NPHS1 mutation16Tryggvasson K. Unraveling the mechanisms of glomerular ultrafiltration, a key component of the slit diaphragm.J Am Soc Nephrol. 1999; 10: 2440-2445Crossref PubMed Google Scholar (Figure 4b and h). As for mouse samples (Figure 3), imaging data could be evaluated quantitatively (Figure 4f and l and Supplementary Figure S4A and B). Of note, unexpectedly mild GBM thickening was observed in the diagnosed patients compared with controls. The nephrectomized control kidney in Figure 4 was taken from an older patient, whereas all the other samples were from infants or children. Because GBM thickness increases with age (Supplementary Figure S5A–E), a more significant thickening would probably be observed if compared with age-matched control patients. We also validated that no age-dependent swelling artifact was introduced by our protocol (Supplementary Figure S5F–H), and also that a different GBM measurement strategy yielded similar results (Supplementary Figure S5I and J). Because not only filtration barrier scale, but also histological changes to renal morphology, are of high interest to both researchers and clinicians, we investigated the possibility of using our protocol to assess large-scale morphology through fluorescence-based histologic analysis. We show that using lower magnification objectives, we could detect large-scale pathologies and differences in protein expression in both human samples (Supplementary Figure S6) and mouse samples (Supplementary Figure S7). To further exemplify the possible use of the protocol in patient samples, we investigated whether we could detect pathologic features of FSGS of the cellular type. We were able to visualize most pathologic features mentioned in the statement from the pathologist, including focal total/segmental sclerosis, tubular atrophy, and endothelial hypercellularity in glomerular capillaries compared with present gold standard methods (Figure 5a–j). A qualitative and quantitative side-by-side comparison with electron microscopy (EM) showed similar results using both methods (Figure 5k and l and Supplementary Figure S4; see Supplementary Methods for a discussion of FP width measurements). We also applied the protocol to a biopsy specimen from a patient diagnosed with IgA nephropathy (IgAN) (Figure 6) and were able to confirm the visualization of many diagnostic features. No severe pathologies were evident in the filtration barrier (Figure 6a and b), and a comparative analysis resulted in no obvious differences between EM and our protocol (Figure 6c and d). Next, the histology of the IgAN kidney was evaluated with both standard histology (Figure 6e) and "fluorescence histology" (Figure 6f). We could visualize the alterations (or lack of alterations) mentioned in the statement from the pathologist, such as interstitial infiltration of cells (Figure 6g and h), normal glomerular capillaries (Figure 6i), and mesangial expansion/hypercellularity (Figure 6j). Immunostaining using an IgA antibody clearly revealed deposits both on the large scale (Figure 6k and l) and in detail (Figure 6m) with a pattern equivalent to that seen on standard immunofluorescence (Figure 6n and o). Finally, FP imaging showed partial mild effacement (Figure 6p and q). The pathologic features that we could detect with our protocol are summarized in Supplementary Table S1. Here we present a protocol for high-throughput visualization of renal structures on all length scales that is both faster and simpler than previously published protocols for FP analysis and glomerular filtration barrier imaging in the kidney. We show that the resolution we achieve is high enough to draw conclusions about pathologies in the filtration barrier using simple confocal microscopy, a standard tool already available in most research facilities and pathology laboratories. The 3D capability of the protocol is important when performing imaging of FPs because a large imaging volume is accessible, allowing for identification and screening of suitable regions of the sample for quantitative analysis. An important finding of our study is that our protocol not only performs complementary and beneficially compared with the standard protocols currently used for standard analysis of kidney tissue. It could also merge these workflows into one protocol, with apparent savings in terms of working hours and material/reagents/equipment needed. However, as the analysis is based on antibodies to a large extent, the cost of labeling reagents is reasonably high (around 20€ per sample), which is true also for the other fluorescence-based protocols presented in this article (including regular immunofluorescence). In the fastest version of the protocol, we use dye-conjugated primary antibodies that yield sufficient staining quality for resolving FPs. However, the resulting signal is always lower compared with that from a 2-step primary/secondary approach. Moreover, the primary conjugated antibodies used in the study are not commercially available and must be conjugated in-house. Therefore, for some applications, a 2-step primary/secondary staining protocol still might be preferable, adding 2 hours to the protocol. Recent studies have applied Airyscan microscopy on renal tissue, which uses variable pinhole settings on a small camera array detector in combination with deconvolution and signal reassignment image processing.17Sachs W. Sachs M. Krüger E. et al.Distinct modes of balancing glomerular cell proteostasis in mucolipidosis type II and III prevent proteinuria.J Am Soc Nephrol. 2020; 31: 1796-1814Crossref PubMed Scopus (5) Google Scholar,18Suleiman H.Y. Roth R. Jain S. et al.Injury-induced actin cytoskeleton reorganization in podocytes revealed by super-resolution microscopy.JCI Insight. 2017; 2e94137Crossref PubMed Scopus (32) Google Scholar In this study, we show that with mild swelling and subsequent superb staining and imaging conditions, conventional confocal imaging with a closed-down pinhole provides sufficient resolution without the need for mathematical postprocessing of images. In this work, we present comparative data for our protocol and standard methods for the evaluation of pathology in patient samples. However, the analysis was carried out on a small number of patients, and further studies are crucial to investigate whether the protocol can be used to detect the plethora of pathological parameters present in other types of kidney diseases as well. To achieve this, a focused collaborative effort involving both pathologists, sample preparation experts, and imaging experts must be initiated. This thorough validation was not within the scope of this proof-of-principle study, in which the aim was to develop a fast, optical protocol for high-resolution imaging of the kidney. It also should be stressed that the resolution of our protocol is far from that of EM and cannot resolve fibrillary deposits, for example, and so for some cases, EM would still be required for accurate diagnosis. In this study, we used raw length measurements without correcting for sectioning artifacts arising from nonorthogonal optical or physical sectioning of the measured structure (GBM or FP). There are 2 main reasons for representing the data in this form. First, to correct for sectioning artifacts while retaining information regarding nanoscale alterations (such as an irregular "bumpy" GBM) is not straightforward. Second, in our transparent samples, we always chose an imaging plane in which the structure being studied is aligned as orthogonally as possible to the imaging plane. Thus, the sectioning is not random, further complicating sectioning artifact analysis. Consequentially, the mean values presented in this study probably are slightly overestimated, and sectioning artifacts contribute to the spread of data in scatterplots. For future sectioning-corrected measurements of structures without loss of real variation of data, modified versions of existing sectioning artifact correction methods, such as the orthogonal intercept method, potentially could be applied.19Fioretto P. Steffes M.W. Mauer M. Glomerular structure in nonproteinuric IDDM patients with various levels of albuminuria.Diabetes. 1994; 43: 1358-1364Crossref PubMed Google Scholar,20Jensen E.B. Gundersen H.J. Osterby R. Determination of membrane thickness distribution from orthogonal intercepts.J Microsc. 1979; 115: 19-33Crossref PubMed Scopus (319) Google Scholar The ability to extract pathological parameters from cleared kidney tissue using both nonlabeled (autofluorescence) and fluorescently labeled samples has been demonstrated recently.21Torres R. Velazquez H. Chang J.J. et al.Three-dimensional morphology by multiphoton microscopy with clearing in a model of cisplatin-induced CKD.J Am Soc Nephrol. 2016; 27: 1102-1112Crossref PubMed Scopus (48) Google Scholar22Puelles V.G. Fleck D. Ortz L. et al.Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis.Kidney Int. 2019; 96: 505-516Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Furthermore, a recent study has shown that "virtual histology" can be performed using deep learning algorithms on autofluorescence images,23Rivenson Y. Wang H. Wei Z. et al.Virtual histological staining of unlabelled tissue-autofluorescence images via deep learning.Nat Biomed Eng. 2019; 3: 466-477Crossref PubMed Scopus (130) Google Scholar and neural network-based segmentation and classification of kidney pathologies also has been shown.25Hermsen M. Bel T. Boer M Den et al.Deep learning-based histopathologic assessment of kidney tissue.J Am Soc Nephrol. 2019; 30: 1968-1979Crossref PubMed Scopus (70) Google Scholar26Ginley B. Lutnick B. Jen K.Y. et al.Computational segmentation and classification of diabetic glomerulosclerosis.J Am Soc Nephrol. 2019; 30: 1953-1967Crossref PubMed Scopus (51) Google Scholar Merging novel sample preparation methods (such as reported here) with new image analysis tools poses many interesting digital possibilities in the future, and we will likely see changes in how we image and analyze kidney samples both in research and in the clinic. All mouse experiments were approved by the German State Office of North Rhine-Westphalia, Department of Nature, Environment, and Consumer Protection and were performed in accordance with European, national, and institutional guidelines. Mice of 100% C57BL/6N background were used. After induction of anesthesia with ketamine and xylazine, the mice were euthanized by cardiac perfusion with Hank's balanced salt solution and fixated as described below. Patient material was obtained from 2 children suffering from steroid-resistant nephrotic syndromes due to either point mutations in NPHS1 or compound-heterozygous point mutations in TRPC6 and NPHS2, as well as 3 patients diagnosed with focal segmental glomerulosclerosis, minimal change disease, and IgA nephropathy, respectively. Control human tissue was collected from patients who were nephrectomized due to renal tumors. Tissue samples dissected from the nontumorous pole of the kidney showed a normal histologic picture on routine histologic examination. All procedures were approved by the Ethics Commission of Cologne and the regional Ethical Committee of Stockholm and conducted in accordance with the Declaration of Helsinki. When applicable, the patient or patient's guardian provided informed consent. Mice with 2 compound-heterozygous point mutations, PodR231Q/A286V, were generated as described previously.9Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar Mice were sacrificed at age 20 weeks as stated above. Human and mouse kidneys were fixed in 4% paraformaldehyde in 1× phosphate-buffered saline at room temperature for 1–3 hours. The nephrectomized human kidney was fixed in Duboscq–Brasil solution for 3 hours. Samples were embedded in 3% agarose and sectioned to 300 μm thickness using a vibratome. Samples were incubated in clearing solution (200 mM boric acid and 4% SDS, pH 8.5) at 70 °C for 1 hour with shaking at 500 rpm. The labeling protocol and a list of antibodies/probes are provided in the Supplementary Material. Samples were incubated in 80% wt/wt fructose (1 ml of dH20 added to 4 g of fructose) containing 4 M urea at 37 °C with shaking at 500 rpm for 15 minutes, and then placed in a MatTek dish (MatTek, Ashland, MA) with a cover slip on top (to prevent evaporation) before imaging. Images were acquired using a Leica SP8 confocal system (objectives stated in the figure legends) (Leica Microsystems, Buffalo Grove, IL). Imaging with the 100× objective always implies a pinhole setting of 0.3 AU (as calculated for 594-nm light) if not stated otherwise. Standard histology imaging was performed using a Leica DM3000 system (Figure 5) or an Olympus BX45 system (Olympus, Tokyo, Japan) (Figure 6). Standard immunofluorescence was performed using a Nikon Microphot-FXA (Nikon, Tokyo, Japan), and EM was performed with a Zeiss 912 (Figure 5) or Hitachi 7700 (Hitachi, Tokyo, Japan) (Figure 6) transmission electron microscope. Unless stated otherwise, images were smoothed by replacing each pixel value by the mean of its 3×3 neighborhood using Fiji/ImageJ (National Institutes of Health, Bethesda, MD). The morphometric analyses are described in Supplementary Methods and shown in Supplementary Figure S4. The scheme in Figure 1a was produced by DU-J, apart from the vector graphics for the Eppendorf tube and the razor blade, which were downloaded from https://pixabay.com/vectors/vial-tube-fluid-laboratory-41375/ and https://pixabay.com/illustrations/razor-blade-blade-sharp-cutting-220323/, respectively (free to use). All the authors declared no competing interests. This study was supported by grants from the Swedish Research Council ( VR 2013-6041 ), the Swedish Foundation for Strategic Research ( RIF14-0091 ), and Clinical Research Unit 329 of the Deutsche Forschungsgemeinschaft . We thank the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) Imaging Facility for their support in the acquisition of microscopy data. Download .pdf (2.14 MB) Help with pdf files Supplementary File (PDF)