Optimization of the design of a multiple-photon excitation laser scanning fluorescence imaging system

Multi-photon (two or more photon) excitation imaging offers three significant advantages compared to laser-scanning confocal fluorescence microscopy for 3-D and 4-D fluorescence microscopy: considerable reduction in total sample excitation, increased depth penetration, and increased detection sensitivity. All-solid-state ultra-fast lasers offer tremendous potential for affordable, reliable, "turn-key" multi-photon excitation sources. We have been developing a multi-photon system that utilizes an all-solid- state Nd:YLF excitation source. We have been evaluating the potential of this source for biological microscopy and have been optimizing system parameters for this application area. We have found that the 1047 nm radiation from these lasers can excite by two-photon fluorescence many commonly used fluorophores that are normally excited from blue to yellow light. In addition, we have found that this wavelength readily excites several normally UV excited fluorophores by the mechanism of three-photon excitation. The Nd:YLF laser has proven reliable in operation with nearly 6000 hours logged without significant loss of power. However, the original system produced rather long pulses for multi-photon excitation (300 fs) and a beam shape that was not ideal. We have recently commissioned the development of an improved pulse compressor from the manufacturers that gives narrower pulses (120 fs), improved beam shape, and a smaller insertion loss. This optimized excitation system has 6 times more potential two-photon excited fluorescence and 22 times more potential three-photon excited fluorescence than the prototype system. In addition, by optimizing coatings in the excitation and signal paths, we have improved the descanned detection sensitivity by 20% for two-photon excited fluorescence and 315% for three-photon excited fluorescence. The excitation optical transfer efficiency (1047 nm) of our imaging system is currently 60% to the back aperture of the objective. The emission optical transfer efficiency (670 nm) is currently 47% for descanned detection and 83% for non- descanned detection; both from the objective back aperture. Surprisingly, we find there is a signal-to-background increase of a factor of 17 between descanned and non- descanned modes of detection using a Nile Red solution sample.