Despite the fact that laser scanning confocal microscopy (LSCM) has become an important tool in modern biological laboratories, it is bulky, inflexible and has limited field of view, thus limiting its applications. To overcome these drawbacks, we report the development of a compact dual-clad photonic-crystal-fiber (DCPCF) based multiphoton scanning microscope. In this novel microscope, beam-scanning is achieved by directly scanning an optical fiber, in contrast to conventional beam scanning achieved by varying the incident angle of a laser beam at an objective entrance pupil. The fiber delivers femtosecond laser pulses for two-photon excitation and collects fluorescence back through the same fiber. Conventional fibers, either single-mode fiber (SMF) or multimode fiber (MMF), are not suitable for this detection configuration because of the low collection efficiency for a SMF and low excitation rate for a MMF. Our newly invented DCPCF allows one to optimize collection and excitation efficiency at the same time. In addition, when a gradient-index (GRIN) lens is used to focus the fiber output to a tight spot, the fluorescence signal collected back through the GRIN lens forms a large spot at the fiber tip because of the chromatic aberrations of the GRIN lens. This problem prevents a standard fiber from being applicable, but is completely overcome by the DCPCF. We demonstrate that this next generation scanning confocal microscope has an extremely simple structure and a number of unique features owing to its fundamentally different scanning mechanism: high flexibility, arbitrarily large scan range, aberration-free imaging, and low cost.
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.