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.
Highlights
A range of established super-resolution fluorescence microscopy methods are capable of analyzing 3D molecular distributions at length scales below 250 nm and have recently been applied to the study of kidney
It should be noted that treatment with heat and detergent, which is used in the magnified analysis of the proteome (MAP) protocol under different fixation and hydrogel embedding conditions[9,10], did not successfully homogenize the specimen (Supplementary Fig. 1)
Interdigitated podocyte foot processes that were separated by
Summary
A range of established super-resolution fluorescence microscopy methods are capable of analyzing 3D molecular distributions at length scales below 250 nm and have recently been applied to the study of kidney. Single molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy, and structured illumination microscopy (SIM) have been used to study separate components of the GFB such as GBM composition[3], slit diaphragm structure[4], and podocyte effacement in diseased tissue[5], respectively. Each of these methods currently suffers from certain limitations which still hinder widespread implementation. SMLM, and to a lesser extent SIM (in its most common, commercial implementations) typically have poor resolution beyond a few micrometers from a coverglass substrate All of these methods require expensive, specialized instruments, and substantial technical and interpretive expertise. Because imaging is performed with a confocal microscope, multichannel data collection is straightforward
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