Superresolving the kidney\u2014a practical comparison of fluorescence nanoscopy of the glomerular filtration barrier

Lucia C S Wunderlich,Florian Ströhl,Luca Mascheroni,Stefan Ströhl,Oliver Vanderpoorten,Clemens F Kaminski

doi:10.1007/s00216-020-03084-8

Abstract

Immunofluorescence microscopy is routinely used in the diagnosis of and research on renal impairments. However, this highly specific technique is restricted in its maximum resolution to about 250 nm in the lateral and 700 nm in the axial directions and thus not sufficient to investigate the fine subcellular structure of the kidney’s glomerular filtration barrier. In contrast, electron microscopy offers high resolution, but this comes at the cost of poor preservation of immunogenic epitopes and antibody penetration alongside a low throughput. Many of these drawbacks were overcome with the advent of super-resolution microscopy methods. So far, four different super-resolution approaches have been used to study the kidney: single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and expansion microscopy (ExM), however, using different preservation methods and widely varying labelling strategies. In this work, all four methods were applied and critically compared on kidney slices obtained from samples treated with the most commonly used preservation technique: fixation by formalin and embedding in paraffin (FFPE). Strengths and weaknesses, as well as the practicalities of each method, are discussed to enable users of super-resolution microscopy in renal research make an informed decision on the best choice of technique. The methods discussed enable the efficient investigation of biopsies stored in kidney banks around the world.Graphical abstract

Highlights

The glomerular filtration barrier (GFB) resembles a molecular sieve and is one of the main components of the renal corpuscle [1]
They were immersed in pure paraffin for several days before embedding them in cassettes filled with paraffin at ambient
Optical super-resolution microscopy enables the nanoscale imaging of labelled samples and combines advantages of both light and electron microscopy

Summary

Introduction

The glomerular filtration barrier (GFB) resembles a molecular sieve and is one of the main components of the renal corpuscle [1]. Mutations of the NPHS1 gene encoding for nephrin lead to congenital nephrotic syndrome of the Finnish type (CNF) In this disease, massive proteinuria is already present at the foetal stage. Conventional immunofluorescence microscopy, on the other hand, allows the labelling and detection of proteins of interest with unparalleled specificity and sensitivity, albeit at a relatively modest resolution. The latter is about 250 nm in the lateral direction due to optical diffraction as already recognised by Ernst Abbe around 1870 [20, 21]. The principles of each method are briefly introduced, and respective benefits and disadvantages are outlined in the Methods section

Methods

Findings

Discussion

Conclusion