Rapid and in-depth labeling biological tissue samples is invaluable for studying tissue organization in three-dimensions. The current strategy in finding optimal labeling parameters for dye molecules through tissues often requires trial and error testing. Difficulty in establishing a standard approach is due to lack of information on the diffusion parameters of dye through tissues. In this study, we investigate the temporal progression of dye penetration in different tissue types. By the combination of multiphoton imaging of labeled tissues and application of the theoretical model of diffusion with well-defined boundary conditions, we aim to arrive at systematic parameters for optimizing dye penetration. Determination of the effective diffusion coefficient of the dyes used will then be useful for further application.