Multi-photon Molecular Excitation to Illuminate Non-linear Laser Microscopy

Abstract

Molecular excitation by two or more infra red photons simultaneously absorbed from strongly focused 100 femtosecond mode locked laser pulses provides intrinsic 3-d resolution for fluorescence microscopy and photochemical micro pharmacology in living biological cells. Confinement of non-linear excitation to the focal volume illuminated by ~1016 photons/nm2s eliminates out of focus photobleaching and photodamage and provides intrinsic 3-d resolution. To facilitate this technology new methods have developed for accurate measurements of multi-photon excitation cross sections and spectra in the absence of adequate data to guide non-linear laser microscopy applications. Absolute cross section measurements have been facilitated by development of a method that takes advantage of the interference of excitation pulse trains that are time shifted by a conventional Michaelson interferometer to allow variable relative delay of two spatially superimposed beams. Two photon excitation with single mode CW lasers provides an independent absolute calibration of cross sections. Measurements of more than twenty useful two-photon excitation spectra (690-1050nm) have revealed some with large blue shifts of the excitation peak. Two photon excitation spectra of some asymmetric molecules superimpose on the one photon absorption spectra (with wavelength doubled) presumably due to relaxation of parity selection rules in these cases. Most of the tested fluorescent molecules show blue shifted excitation peaks with strongly enhanced cross sections exceeding those observed at twice the one photon absorption. No red shifts of the excitation spectra have been detected. Three photon excitation spectra tend to mimic the one photon absorption spectra. Multi- photon excited emission spectra appear to duplicate the one photon fluorescence emission spectra.