Fluorescence measurement of localized, deeply embedded physiological processes

Intrinsic and exogenous fluorescent molecules may be used as specific markers of disease processes, or metabolic status. A variety of fluorescent markers have been successfully used for transparent tissue, in-vitro studies, and in cases where the markers are located close to the tissue surface. For example, given fluorescence lifetime measurements of a fluorophore such as bis(carboxylic acid) dye, the known relationship of pH on its lifetime may be used to determine the pH of tissue at the fluorophore's location. For fluorophore depths greater than approximately one millimeter in normal tissue, such as might be encountered in in vivo studies, multiple scattering makes it impossible to make direct measurements of characteristics such as fluorophore lifetime. In a multiple scattering environment, the collected intensity depends heavily on the scattering and absorption coefficients of the tissue at both the excitation and emission frequencies. Thus, to obtain values for specific fluorophore characteristics such as the lifetime, a theoretical description of the complex photon paths is required. We have applied Random-walk theory to successfully model photon migration in turbid media such as tissue. We show how time-resolved intensity measurements may be used to determine fluorophore location and lifetime even when the fluorophore site is located many mean photon scattering lengths from the emitter and detector.