Chemical Landscape for Tissue Clearing Based on Hydrophilic Reagents

Abstract

We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues.

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

“…Aqueous-based protocols typically involve removing lipids using detergents, followed by immersion in diluted water-soluble reagents, resulting in a refractive index range of n = 1.38 to 1.49 [21]. For most aqueous-based protocols, there is an optically compatible glass such that Δ n ≤ 0.01.…”

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

“…This included the development of a MF-HREM tissue-processing, acquisition and post-processing pipeline, with the use of an opacifying agent and image deconvolution to recover axial image resolution. As MF-HREM does not need tissues to be optically cleared, it is advantageous for imaging tissue morphology (13) and can be used in conjunction with lipophilic dyes that cannot be used in solvent based clearing (26,27). For MF-HREM, lateral resolution is determined directly by the microscope objective, while axial resolution is related to both section thickness and light penetration into the block.…”

A hybrid open-top light-sheet microscope for multi-scale imaging of cleared tissues

“…Recent advances in tissue-clearing protocols, which homogenize the refractive-index composition of tissues to reduce light scattering (i.e. promoting tissue transparency), have enabled deep optical access within intact tissues to yield new insights in many fields, including neuroscience, developmental biology, and anatomic pathology [1][2][3][4][5][6][7][8][9]. Although cleared tissues can be imaged using any optical approach, light-sheet microscopy has emerged as the preferred means for high-resolution volumetric imaging due to its unrivaled speed and optical efficiency (low photobleaching).…”