MULTIPHOTON FLUORESCENCE EXCITATION - NEW SPECTRAL WINDOWS FOR BIOLOGICAL NONLINEAR MICROSCOPY

Intrinsic, three-dimensionally resolved, microscopic imaging of dynami cal structures and biochemical processes In living preparations has be en realized by nonlinear laser scanning fluorescence microscopy. The s earch for useful two-photon and three-photon excitation spectra, motiv ated by the emergence of nonlinear microscopy as a powerful biophysica l instrument, has now discovered a virtual artist's palette of chemica l indicators, fluorescent markers, and native biological fluorophores, including NADH, flavins, and green fluorescent proteins, that are app licable to living biological preparations, More than 25 two-photon exc itation spectra of ultraviolet and visible absorbing molecules reveal useful cross sections, some conveniently blue-shifted, for near-infrar ed absorption, Measurements of three-photon fluorophore excitation spe ctra now define alternative windows at relatively benign wavelengths t o excite deeper ultraviolet fluorophores. The inherent optical section ing capability of nonlinear excitation provides three-dimensional reso lution for imaging and avoids out-of-focus background and photodamage. Here, the measured nonlinear excitation spectra and their photophysic al characteristics that empower nonlinear laser microscopy for biologi cal imaging are described.