Light Sheet Fluorescence Microscopy of Human Kidney Using Clearing with CUBIC v1

Light sheet fluorescence microscopy (LSFM) is a method to cover micro-mesoscale (µm-cm) areas of tissue while achieving depth of several millimeters to enable 3D-volumetric imaging of tissue structures or organs. By using fluorescence tagged primary or secondary immunofluorescence it is capable of provides cellular resolution and anatomical maps. Compared to traditional laser confocal microscopy imaging LSFM, LSFM provides rapid acquisition of images over large optically cleared tissues while maintaining high resolution. As compared to serial 3D reconstruction, In toto imaging by LSFM provides a more accurate 3D dataset that captures the spatial relationships between structures and cells. Application of LSFM to adult human tissues, particularly solid tissues such kidney, is challenging due to its complex anatomical organization and composition, heterogeneity and interstitium. The relationships between extracellular and extrarenal structures have not been defined. This knowledge is critical to understand how different functional tissue units of the kidney are organized in 3D as key interactions and signaling necessary for its physiological roles cannot be correctly derived from 2D images. Here we describe a protocol we developed to aid in 3D visualization of kidney cells, functional tissue units and their relationship with nerves, vasculature and lymphatics as a goal in HuBMAP consortium to create 3D reference maps. After testing several protocols, here we describe a clearing method on tissue slices in conjunction with immunofluorescence staining o visualize deep kidney volumes (1-3mm depth) when imaged by light sheet microscope. We provide several tips during tissue preparation for clearing, clearing procedure and an example of imaging the tissue using LSFM. We also briefly discuss various outputs of imaging and metadata files that can be used for data submission or for publishing or hosting on external servers. The imaging section contains the following: 1)Refractive Index (RI) matching tissue with Refractive Index Matching Solution (RIMS), 2) Capturing a full-volume 5x image, 3) Capturing 20X images of regions of interest (ROIs), 4) Stitching and down sampling images prior to analysis, 5) Different file formats. All imaging was completed using a Zeiss Lightsheet 7 microscope.