Two-photon fluorescence biosensing with conventional and photonic crystal fibers

Fluorescence is a powerful tool for biosensing, but conventional fluorescence measurements are limited because solid tumors are highly scattering media. To obtain quantitative in vivo fluorescence information from tumors, we have developed a two-photon optical fiber fluorescence (TPOFF) probe where excitation light is delivered and the two-photon fluorescence (TPF) excited at the tip of the fiber is collected back through the same fiber. In order to determine whether this system can provide quantitative information, we measured the fluorescence from a variety of systems including mouse tumors (both ex vivo and in vivo) which were transfected with the gene to express varying amounts of green fluorescence protein (GFP), and tumors which were labeled with targeted dendrimer-based drug delivery agents. The TPOFF technique showed results quantitatively in agreement with those from flow cytometry and confocal microscopy. In order to improve the sensitivity of our fiber probe, we developed a dual-clad photonic-crystal fiber which allowed single-mode excitation and multimode (high numerical aperture) collection of TPF. These experiments indicate that the TPOFF technique is highly promising for real-time, in vivo, quantitative fluorescence measurements.