We developed an advanced clearing method with superior fluorescence-preserving capability for 3D imaging of whole organs.

Recently, a variety of tissue optical clearing techniques have been developed to reduce light scattering for imaging deeper and three-dimensional reconstruction of tissue structures. Combined with optical imaging techniques and diverse labeling methods, these clearing methods have significantly promoted the development of neuroscience. Each of them has its own characteristics with certain advantages and disadvantages. Though there are some comparison results, the clearing methods covered are limited and the evaluation indices lack uniformity, which made it difficult to select a best-fit protocol from numerous methods for clearing in practical applications. Hence, it is necessary to systematically assess and compare these clearing methods. We evaluated the performance of seven typical clearing methods, including 3-D imaging of solvent-cleared organs (3DISCO), ultimate DISCO (uDISCO), see deep brain (SeeDB), ScaeS, , clear, unobstructed brain imaging cocktails and computational analysis, and passive CLARITY technique (PACT), on mouse brain samples. First, we compared the clearing effect and clearing time as well as size deformation on brain tissues. Further, we evaluated the fluorescence preservation and the increase of imaging depth induced by different methods. The results showed that 3DISCO, uDISCO, and PACT possessed excellent clearing capability on mouse brains, ScaeS and SeeDB rendered moderate transparency, whereas performed the worst. uDISCO and 3DISCO induced substantial size reduction on brain sections, and PACT expanded the mouse brain most seriously. Among those methods, ScaeS performed best on fluorescence retention, 3DISCO induced the biggest decline of the fluorescence. PACT achieved the highest increase of imaging depth, and SeeDB and possessed the shallowest imaging depth. This study is expected to provide important reference for users in choosing the most suitable brain optical clearing method.

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.