Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

作者：Felix A. Kyere, Ian Curtin, Oleh Krupa, Carolyn M. McCormick, Mustafa Dere, Sarah Khan, Minjeong Kim, Tzu-Wen Winnie Wang, Qiuhong He, Guorong Wu, Yen‐Yu Ian Shih, Jason L. Stein · 发表于：Journal of Visualized Experiments · 年份：2022 · DOI：10.3791/64096-v · 被引用次数：1 · 研究领域：Cell Image Analysis Techniques、Single-cell and spatial transcriptomics、Advanced Fluorescence Microscopy Techniques

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei. Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple cha...