Optical clearing is a useful tool for investigating large biological tissues in 3D, but it has not been widely adapted in regular biomedical research community, partially due to the high complexity and low speed of the current optical clearing methods. Therefore, we developed an optical clearing technique, termed lipid-preserving index matching for prolonged imaging depth (LIMPID), that simplifies the clearing procedure while maintaining the advantages of the state of the art clearing methods. (1) LIMPID is designed as an aqueous solution that directly diffuses into the tissue and makes the refractive indices uniform. It is capable of clearing the tissue in a single step, simply by immersing fixed and pre-labeled samples in the clearing solution. In contrast, most current clearing techniques involve multiple steps and some of steps are complicated and time consuming. (2) LIMPID clears the tissue quickly. The solution has low viscosity and rapidly diffuses into the tissue at room temperature. For samples with submillimeter thickness, it clears the tissue within an hour. Clearing times for larger samples are also impressive. (3) LIMPID preserves fluorescence and tissue morphology while maintaining high transparency. No dehydration, organic solvent exchange or lipid extraction is required. We have used LIMPID to study several animal and disease models. For instance, it revealed abnormal peripheral nerve innervation in the embryonic quail heart in a fetal alcohol syndrome model. We believe this simple, quick method with no discernable disadvantages could become the optical clearing protocol of choice for many microscopy applications.
Yehe Liu, Michiko Watanabe, Andrew M. Rollins, and Michael W. Jenkins, "Simple lipid-preserving optical clearing for fluorescent imaging (Conference Presentation)," Proc. SPIE 10499, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXV, 104991J (Presented at SPIE BiOS: February 01, 2018; Published: 14 March 2018); https://doi.org/10.1117/12.2291174.5751635026001.
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