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

Abstract

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.

Efficient Tissue Clearing and Multi-Organ Volumetric Imaging Enable Quantitative Visualization of Sparse Immune Cell Populations During Inflammation

“…By looking at a 7 µm thick optical section, we also found only little IgG deposits and no cellular structure of MHC-II expressing cells in glomeruli, hence making it difficult to determine cell numbers and to address distances of MHC-II expressing cells to glomeruli, again demonstrating the limitations of this 2D approach. To overcome these issues, 3D imaging of the cleared kidney using LSFM ( 19 ) or imaging of the cleared kidney by slicing and confocal or 2-photon microscopy ( 17 , 35 , 36 ) has been developed recently. Using EMOVI, we here demonstrated a homogenous staining of CD31 positive endothelial cells throughout half a kidney (about 1.5 mm depth) after fixation, which would also allow determining glomeruli counts and volume using algorithms and analysis tools described before ( 17 , 19 ).…”