Illumination and fluorescence collection volumes for fiber optic probes in tissue

Optical fibers can deliver light to, and collect it from, regions deep in tissue. However, reported illumination and fluorescence collection volumes adjacent to the fiber tip have been inconsistent, and systematic data on this topic are not available. Illumination and fluorescence collection profiles were characterized with high spatial resolution for different optical fibers in tissue and various fluids using two-photon flash photolysis and excitation. We confirm that illumination and fluorescence collection volumes for optical fibers are near identical. Collection volume is determined by the core dimensions and numerical aperture (NA) of the fiber and the scattering properties of the medium. For a multimode optical fiber with 100 microm core diam and NA=0.22, 80% of the total fluorescence is collected from a depth of 170 microm in tissue and 465 microm in nonscattering fluid. A semiempirical mathematical description of photon flux adjacent to the fiber tip was also developed and validated. This was used to quantify the extent of temporal blurring associated with propagation of a wavefront of altered fluorescence emission across the region addressed by fiber optic probes. We provide information that will facilitate the design of optical probes for tissue imaging or therapeutic applications.