Analysis of sampling volume and tissue heterogeneity on the in vivo detection of fluorescence

The effect of sampling region size and tissue heterogeneity is examined using fluorescence histogram assessment in a rat prostate tumor model with benzoporphyrin derivative fluorophore. Spatial heterogeneity in the fluorescence signal occurs on both macroscopic and microscopic scales. The periphery of the tumor is more fluorescent than the center. Fluorescence is also highest nearest the blood vessels immediately after injection, but over time this fluorescence becomes uniform through the tumor tissue. Using microscopy analysis, the fluorescence intensity histogram distributions follow a normal distribution, yet as the sampling area is increased from the micron scale to the millimeter scale, the variance of the distribution decreases. The mean fluorescence intensity is accurately measured with a millimeter size scale, but this cannot provide accurate measurements of the microscopic variance of drug in tissue. Fiber probe measurements taken in vivo are used to confirm that the variance observed is smaller than would be expected with microscopic sampling, but that the average fluorescence can be measured with fibers. Sampling tissue with fibers smaller than the intercapillary spacing could provide a way to estimate the spatial variance more accurately. In summary, sampling fiber size affects the fluorescence intensities detected and use of multiple region microscopic sampling could provide better information about the distribution of values that occur.