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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), ScaleS, ClearT2, 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, ScaleS and SeeDB rendered moderate transparency, whereas ClearT2 performed the worst. uDISCO and 3DISCO induced substantial size reduction on brain sections, and PACT expanded the mouse brain most seriously. Among those methods, ScaleS performed best on fluorescence retention, 3DISCO induced the biggest decline of the fluorescence. PACT achieved the highest increase of imaging depth, and SeeDB and ClearT2 possessed the shallowest imaging depth. This study is expected to provide important reference for users in choosing the most suitable brain optical clearing method.
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