Clarifying CLARITY: Quantitative Optimization of the Diffusion Based Delipidation Protocol for Genetically Labeled Tissue.

Tissue clarification has been recently proposed to allow deep tissue imaging without light scattering. The clarification parameters are somewhat arbitrary and dependent on tissue type, source and dimension: every laboratory has its own protocol, but a quantitative approach to determine the optimum clearing time is still lacking. Since the use of transgenic mouse lines that express fluorescent proteins to visualize specific cell populations is widespread, a quantitative approach to determine the optimum clearing time for genetically labeled neurons from thick murine brain slices using CLARITY2 is described. In particular, as the main objective of the delipidation treatment is to clarify tissues, while limiting loss of fluorescent signal, the "goodness" of clarification was evaluated by considering the bulk tissue clarification index (BTCi) and the fraction of the fluorescent marker retained in the slice as easily quantifiable macroscale parameters. Here we describe the approach, illustrating an example of how it can be used to determine the optimum clearing time for 1 mm-thick cerebellar slice from transgenic L7GFP mice, in which Purkinje neurons express the GFP (green fluorescent protein) tag. To validate the method, we evaluated confocal stacks of our samples using standard image processing indices (i.e., the mean pixel intensity of neurons and the contrast-to-noise ratio) as figures of merit for image quality. The results show that detergent-based delipidation for more than 5 days does not increase tissue clarity but the fraction of GFP in the tissue continues to diminish. The optimum clearing time for 1 mm-thick slices was thus identified as 5 days, which is the best compromise between the increase in light penetration depth due to removal of lipids and a decrease in fluorescent signal as a consequence of protein loss: further clearing does not improve tissue transparency, but only leads to more protein removal or degradation. The rigorous quantitative approach described can be generalized to any clarification method to identify the moment when the clearing process should be terminated to avoid useless protein loss.