Abstract
Tissue optical clearing techniques have provided important tools for large‐volume imaging. Aqueous‐based clearing methods are known for good fluorescence preservation and scalable size maintenance, but are limited by long incubation time, insufficient clearing performance, or requirements for specialized devices. Additionally, few clearing methods are compatible with widely used lipophilic dyes while maintaining high clearing performance. Here, to address these issues, m‐xylylenediamine (MXDA) is firstly introduced into tissue clearing and used to develop a rapid, highly efficient aqueous clearing method with robust lipophilic dyes compatibility, termed MXDA‐based Aqueous Clearing System (MACS). MACS can render whole adult brains highly transparent within 2.5 days and is also applicable for other intact organs. Meanwhile, MACS possesses ideal compatibility with multiple probes, especially for lipophilic dyes. MACS achieves 3D imaging of the intact neural structures labeled by various techniques. Combining MACS with DiI labeling, MACS allows reconstruction of the detailed vascular structures of various organs and generates 3D pathology of glomeruli tufts in healthy and diabetic kidneys. Therefore, MACS provides a useful method for 3D mapping of intact tissues and is expected to facilitate morphological, physiological, and pathological studies of various organs.
Highlights
Tissue optical clearing techniques have provided important tools for largevolume imaging
We developed a rapid aqueous clearing method based on m-xylylenediamine (MXDA), which was firstly introduced into tissue clearing, termed the MXDA-based Aqueous Clearing System (MACS)
Urea-based clearing methods show good clearing performance owing to urea's strong hyperhydration ability, which is derived from two NH2 groups.[12]
Summary
Urea-based clearing methods show good clearing performance owing to urea's strong hyperhydration ability, which is derived from two NH2 groups.[12]. MXDA solution (40 vol%) efficiently rendered 1-mm-thick brain sections transparent within 30 min with preservation of signals from enhanced green fluorescent protein (EGFP) (Figure S1b, Supporting Information). 30% w/v sorbitol mixed with 40 vol% MXDA provided increased performance in both clearing effect and fluorescence preservation (Figure S1c–h, Supporting Information). We refer to this reagent as MACS-R1. We introduced MACS-R0, which has low concentrations of both MXDA and sorbitol (half concentration of MACS-R1), as the first step of the clearing It could penetrate quickly into large tissue and lead to high-performance tissue hyperhydration, making the tissue become swollen and more permeable. Due to the good membrane integrity after MACS treatment, both the DiI and CM-DiI signals were well maintained without any obvious loss (Figure S6e, Supporting Information)
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