Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy

Abstract

The total number of glomeruli is a fundamental parameter of kidney function but very difficult to determine using standard methodology. Here, we counted all individual glomeruli in murine kidneys and sized the capillary tufts by combining in vivo fluorescence labeling of endothelial cells, a novel tissue-clearing technique, lightsheet microscopy, and automated registration by image analysis. Total hands-on time per organ was <1 hour, and automated counting/sizing was finished in <3 hours. We also investigated the novel use of ethyl-3-phenylprop-2-enoate (ethyl cinnamate) as a nontoxic solvent-based clearing reagent that can be handled without specific safety measures. Ethyl cinnamate rapidly cleared all tested organs, including calcified bone, but the fluorescence of proteins and immunohistochemical labels was maintained over weeks. Using ethyl cinnamate-cleared kidneys, we also quantified the average creatinine clearance rate per glomerulus. This parameter decreased in the first week of experimental nephrotoxic nephritis, whereas reduction in glomerular numbers occurred much later. Our approach delivers fundamental parameters of renal function, and because of its ease of use and speed, it is suitable for high-throughput analysis and could greatly facilitate studies of the effect of kidney diseases on whole-organ physiology.

Multi-Fluorescence High-Resolution Episcopic Microscopy (MF-HREM) for Three-Dimensional Imaging of Adult Murine Organs

“…The MF-HREM analysis revealed a glomeruli distribution that was consistent with their known spatial distribution and with other measures for wild-type (WT) adult mice performed with light sheet or in vivo MRI (39,43) MF-HREM pixel size was 2.17 μm lateral and 2.58 μm axial, enabling identify glomeruli which had a minimum volume of 24×10 5 μm 3 .…”

“…The final step is the outcome of the connected components analysis and hence quantification of glomeruli number, and volume distribution. These data show the expected distribution and size of glomeruli for heathy WT mouse as compared with other techniques(39), (43).…”

Panoptic vDISCO imaging reveals neuronal connectivity, remote trauma effects and meningeal vessels in intact transparent mice

“…However, mammalian tissues are naturally opaque, hindering high-resolution imaging in any tissue deeper than a few hundred micrometers-a major reason why sectioning is needed for histological examination of target organs 1 . Recent innovations in tissue clearing technology now allow 3D histological examination of intact organs [2][3][4][5][6][7][8][9][10][11][12][13][14][15] . Tissue clearing is a chemical process aiming to match refractive indices throughout intact tissues, thus rendering them transparent and allowing deep tissue fluorescent microscopy.…”

Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

“…3a and Supplementary Videos 3 and 4 ). We found that ECi-clearing ( n = 1.56) is well-suited for this application due to its clearing efficacy, as well as low toxicity [19]. A custom HIVEX holder ( n = 1.55) was machined with channels to accommodate multiple biopsies (13 in this case) and to keep them aligned and parallel.…”