Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease

Pullman, James; Nylk, Jonathan; Campbell, Elaine C.; Gunn-Moore, Frank J.; Prystowsky, Michael B.; Dholakia, Kishan

doi:10.1364/boe.7.000302

Cited by 30 publications

(29 citation statements)

References 15 publications

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“…Compared to previously published protocols using conventional microscopy, our fast and simple protocol outperformed them on all evaluated parameters. The protocol presented by Pullman et al 2 is slightly faster in total, but with the drawbacks that the protocol requires super-resolution SIM microscopy, more hands-on labor, more sample interactions and number of reagents. Further, the later protocol is performed on thin cryo-sections, thus lacking access to large in situ imaging depths.…”

Section: Optical Clearing and Mild Swelling Of Kidney Tissuementioning

confidence: 99%

“…In recent years, a range of novel optical imaging and sample preparation protocols have been presented both by our group and others, handing kidney researchers and pathologists the opportunity to use light microscopy for the analysis of renal ultrastructure [1][2][3][4][5][6][7][8] , reviewed in 9,10 .…”

Section: Introductionmentioning

confidence: 99%

“…Although powerful, all of these optical protocols require the use of advanced diffractionunlimited microscopy techniques, such as stimulated emission depletion (STED) microscopy 5 , single-molecule localization microscopy (SMLM) 4 or structured illumination microscopy (SIM) 2,3 , or involve the introduction of complex polymer chemistry into the sample to clear and/or physically expand it 1,6,7 . Many of the protocols are also very tedious in duration and require a substantial amount of hands-on labor.…”

Section: Introductionmentioning

confidence: 99%

“…This extra layer of complexity might be what is keeping these new methods from being routinely used by both researchers and clinical pathologists. Further, many of the protocols lack access to deep 3D imaging of foot processes (FPs) beyond 5-15 µm in depth [1][2][3]7,8 . Therefore, we set out to develop a drastically simplified and fast all-optical protocol, which clears and spatially swells kidney tissue samples sufficiently to allow for 3D high resolution analysis of renal anatomy and pathology using a conventional diffraction-limited microscope available in almost all lifescience and pathology institutes.…”

Section: Introductionmentioning

confidence: 99%

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Imaging Renal Ultrastructure using a Fast and Simple Optical Clearing and Swelling Protocol

Unnersjoe-Jess

Butt

Hoehne

et al. 2020

Preprint

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Many light-microscopy protocols have in recent years been published for visualization of ultrastructure 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 GBM. However, these protocols either involve the application of different complicated super-resolution microscopes or lengthy sample preparation protocols. We here present a fast and simple, 5-hour long procedure for the full three-dimensional visualization of podocyte foot processes using conventional confocal microscopy. The protocol combines and optimizes different optical clearing and tissue expansion concepts to produce a mild swelling, sufficient for resolving foot processes using a diffraction-limited confocal microscope. We further show that the protocol can be used for common visualization of large-scale renal histology, pathology, and kidney protein distributions. Thus, we believe that our fast and simple protocol can be beneficial for routine conventional microscopic evaluation of kidney tissue integrity both in research and in the clinic.

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Section: Optical Clearing and Mild Swelling Of Kidney Tissuementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Imaging Renal Ultrastructure using a Fast and Simple Optical Clearing and Swelling Protocol

Unnersjoe-Jess

Butt

Hoehne

et al. 2020

Preprint

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“…Interestingly, another recent paper described the use of super-resolution microscopy in the diagnosis of kidney disorders [19]. As with the diagnosis of platelet granule deficiencies, TEM is normally required to diagnose nephrotic disease, where the structure of the podocyte cells of the glomerulus may be disturbed.…”

Section: Future Prospectsmentioning

confidence: 99%

Super-resolution microscopy in the diagnosis of platelet granule disorders

Knight

Gomez

Cutler

2017

Expert Review of Hematology

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Introduction: Platelet granule deficiencies lead to bleeding disorders, but their specific diagnosis typically requires whole mount transmission electron microscopy, which is often not available and has a number of important limitations. We recently proposed the use of advanced forms of fluorescence microscopy – the so-called ‘super-resolution’ microscopies – as a possible solution.Areas covered: In this special report, we review the diagnosis of platelet granule deficiencies, and discuss how recent developments in fluorescence microscopy may be useful in improving diagnostic approaches to these and related disorders. In particular, we conclude that super-resolution fluorescence microscopy may have advantages over transmission electron microscopy in this application.Expert commentary: The value of the super-resolution microscopies has been amply demonstrated in research; however, their potential in diagnostic applications is ripe for further exploration. Hematology is a field particularly likely to benefit because of the relative simplicity of sample preparation. We anticipate that the costs of the necessary instrumentation will continue to fall rapidly, making these technologies widely accessible to clinicians.

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Application of direct stochastic optical reconstruction microscopy (dSTORM) to the histological analysis of human glomerular disease

García

Lightley

Kumar

et al. 2021

The Journal of Pathology CR

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Electron microscopy (EM) following immunofluorescence (IF) imaging is a vital tool for the diagnosis of human glomerular diseases, but the implementation of EM is limited to specialised institutions and it is not available in many countries. Recent progress in fluorescence microscopy now enables conventional widefield fluorescence microscopes to be adapted at modest cost to provide resolution below 50 nm in biological specimens. We show that stochastically switched single‐molecule localisation microscopy can be applied to clinical histological sections stained with standard IF techniques and that such super‐resolved IF may provide an alternative means to resolve ultrastructure to aid the diagnosis of kidney disease where EM is not available. We have implemented the direct stochastic optical reconstruction microscopy technique with human kidney biopsy frozen sections stained with clinically approved immunofluorescent probes for the basal laminae and immunoglobulin G deposits. Using cases of membranous glomerulonephritis, thin basement membrane lesion, and lupus nephritis, we compare this approach to clinical EM images and demonstrate enhanced imaging compared to conventional IF microscopy. With minor modifications in established IF protocols of clinical frozen renal biopsies, we believe the cost‐effective adaptation of conventional widefield microscopes can be widely implemented to provide super‐resolved image information to aid diagnosis of human glomerular disease.

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Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease

Cited by 30 publications

References 15 publications

Imaging Renal Ultrastructure using a Fast and Simple Optical Clearing and Swelling Protocol

Imaging Renal Ultrastructure using a Fast and Simple Optical Clearing and Swelling Protocol

Super-resolution microscopy in the diagnosis of platelet granule disorders

Application of direct stochastic optical reconstruction microscopy (dSTORM) to the histological analysis of human glomerular disease

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