Abstract
Kidney glomerular diseases, such as the minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), and other nephrotic syndromes, are typically diagnosed or confirmed via electron microscopy. Although optical microscopy has been a vital tool to examine clinical specimens for diagnoses in pathology for decades, the optical resolution is constricted by the physical diffraction limit of the optical microscope, which prevents high-resolution investigation of subcellular anatomy, such as of the podocyte tertiary foot processes. Here, we describe a simple, fast, and inexpensive protocol for nanoscale optical imaging of kidney glomeruli. The protocol is based on Expansion Pathology (ExPath), a new principle of microscopy that overcomes optical diffraction limit by chemically embedding specimens into a swellable polymer and physically expanding it homogenously prior to imaging. Our method uses only commercially available reagents, a conventional fluorescence microscope and it can be applied to both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. It requires minimal operative experience in a wet lab, optical microscopy and imaging processing. Finally, we also discuss challenges, limitations and prospective applications for ExPath-based imaging of glomeruli.
Highlights
In pathology, examination of cellular structures and molecular composition using diffraction-limited microscopy has long been key to the diagnosis of a wide variety of pre-disease and disease states [1, 2].Kidney podocytes and their foot processes are a key component of the ultrafiltration system in the glomerulus, where they comprise the filtration barrier together with endothelial cells and the glomerular basement membrane (GBM) [3, 4]
Human kidney contains an abundance of structural proteins, such as collagen and elastin [15], which posts a challenge to mechanically homogenization and expansion of gelled kidney tissues using earlier versions of Expansion Microscopy (ExM) protocols, such as the original ExM [13], proExM [16], etc
For staining of the podocyte foot processes within the kidney’s glomeruli and investigating with precision the ultrafine structure of the tertiary food processes of the podocytes, kidney tissues fixed by non-cross-linking fixatives provide the optimal results (Figure 5)
Summary
Examination of cellular structures and molecular composition using diffraction-limited microscopy has long been key to the diagnosis of a wide variety of pre-disease and disease states [1, 2] Kidney podocytes and their foot processes are a key component of the ultrafiltration system in the glomerulus, where they comprise the filtration barrier together with endothelial cells and the glomerular basement membrane (GBM) [3, 4]. ExPath protocol allows biomolecules that are covalently linked to the polymer chain within diffraction limited space to be isotropically separated from each other throughout the tissue by hydrogel expansion It facilities optical imaging with sub-diffraction limit resolution using only a conventional optical microscope.
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