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The objective of this research is to evaluate the usefulness of a macroscopic, fluorescent, imaging technique to quantify spatiotemporal mass transport parameters in in vitro solid tumor tissues taken from rat models. Fluorescent images captured during the experiments are digitally analyzed to determine the concentration of a fluorescent marker dye as it diffuses into tissue specimens taken from rat tumors. The collected concentration data are used to estimate local diffusion coefficients. An analysis of the distribution of the local diffusion data indicates that the local diffusion coefficient is spatially dependent within the tumor tissue. When mass transfer is restricted to one dimension, the current technique can be used to determine the concentration distribution of fluorescent molecules on the tissue surface and to estimate the mass transfer parameters within the heterogeneous tumor tissue.