Volumetric, Nanoscale Optical Imaging of Mouse and Human Kidney via Expansion Microscopy

Abstract

Although light microscopy is a powerful tool for the assessment of kidney physiology and pathology, it has traditionally been unable to resolve structures separated by less than the ~250 nm diffraction limit of visible light. Here, we report on the optimization, validation, and application of a recently developed super-resolution fluorescence microscopy method, called expansion microscopy (ExM), for volumetric interrogation of mouse and human kidney tissue with 70–75 nm lateral and ~250 nm axial spatial resolution. Using ExM with a standard confocal microscope, we resolve fine details of structures that have traditionally required visualization by electron microscopy, including podocyte foot processes, the glomerular basement membrane, and the cytoskeleton. This inexpensive and accessible approach to volumetric, nanoscale imaging enables visualization of fine structural details of kidney tissues that were previously difficult or impossible to measure by conventional methodologies.

Global transcriptomic changes occur in aged mouse podocytes

“…11 Podocyte ultrastructure analysis using kidney expansion and microscopy Expansion microscopy was performed using a previously described protocol. 37,38…”

“…The inducible podocyte-specific reporter mouse (NPHS2-rtTAjtetO-crejtdTomato) was used for RNA-sequencing (seq) studies, in which a cohort of podocytes permanently labeled in juvenile animals resulted in an irreversible and persistent lifetime labeling of a population of podocytes (Supplementary Figure S1). Microalbuminuria (Supplementary Figure S2A) and glomerulosclerosis (Supplementary Figure S2B) were higher, accompanied by ultrastructural changes in podocytes using super-resolution fluorescence microscopy 37,38 (Supplementary Figure S2C).…”

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

“…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 .…”

“…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.…”

“…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.…”