Optical clearing is an effective tool for investigating spatial distribution of molecules in embryonic tissue. Unfortunately, it has not been broadly adapted in the field of development biology. One reason is that most current optical clearing methods involve complicated procedures that are more difficult compared to common lab procedures. To address this problem, we developed an easy and convenient optical clearing method, termed lipid-preserving index matching for prolonged imaging depth (LIMPID), that involves only one major step in the entire procedure. Since all LIMPID ingredient are water-soluble, it can directly diffuse into the fixed tissue and match the refractive index. Because no dehydration, organic solvent exchange or lipid extraction is required, LIMPID also well preserves fluorescent signal and tissue morphology while maintaining high clearing capability. In addition, LIMPID clearing solution has low viscosity that allows fast diffusion of the chemicals into the tissue. We have tested LIMPID using embryonic quail tissue at various developmental stages. By simply immersing the fixed and stained tissue in excess LIMPID solution, it is capable of clearing whole-mount stage 20 quail embryos in 10 minutes, stage 36 quail hearts overnight, and stage 36 quail brains in 24 hours. Verified by confocal microscopy, fluorescent signals from eYFP, DAPI, LysoTracker and several Alexa Fluor tagged primary antibodies were all well preserved. Imaging depth of LIMPID is only limited by the working distance of the optical system, up to multiple millimeters. Many small embryo tissues can be imaged all the way through using common confocal setups.
Yehe Liu, Michael W. Jenkins, Michiko Watanabe, and Andrew M. Rollins, "A simple optical clearing method for investigating molecular distribution in intact embryonic tissues (Conference Presentation)," Proc. SPIE 10472, Diagnosis and Treatment of Diseases in the Breast and Reproductive System IV, 104720P (Presented at SPIE BiOS: January 28, 2018; Published: 14 March 2018); https://doi.org/10.1117/12.2291193.5751469799001.
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