Evaluating different passive optical clearing protocols for two-photon deep tissue imaging in adult intact visceral and neuronal organs

Abstract

Imaging cellular activities in an entire intact whole organ with light is a grand challenge in optical microscopy. To date, most passive clearing techniques were shown to transform brain, neuronal and embryo tissue into near transparent state for deep tissue imaging. Here, we expand these passive clearing protocols from neuronal tissue (brain and spinal cord) to other visceral organs such as liver and colon and further evaluate their \"depth-clearing performance\" based on image contrast of endogenous fluorescence structures. We found that SeeDB achieves the highest depth in brain, 3DISCO is adept at clearing liver and spinal cord and ScaleViewA2 in colon. Overall, 3DISCO clears more rapidly than other agents but at a higher cost, while ScaleViewA2 is the most economical however at a slower rate. This study, for the first time, provide a direct evaluation of imaging depth, cost and time amongst passive tissue clearing protocols for different intact organs. In addition, we discuss the possible roles of tissue composition in clearing.