Fluorescence measurement in thick tissue layers by linear or nonlinear long-wavelength excitation

Samples of different animal tissues containing, at variable depth, a thin fluorescent sheet are irradiated with continuous violet or red light or with nonlinearly absorbed pulsed infrared light. The fluorescence intensity measured at the tissue surface as a function of the location of the fluorescent sheet exhibits, after a transition zone close to the tissue surface, an exponential decrease, the slope of which depends on the optical penetration depths of the exciting and the fluorescent light. From these results the total fluorescence output is determined for specific fluorophor distributions. It is seen that considerably deeper tissue layers are explored by use of excitation with red instead of violet light. Nonlinear excitation by infrared light can provide a further improvement, especially in liver tissue.