Abstract

Multiphoton Excitation (MPE) of fluorescence provides the optimum photophysics for microscopic imaging deep in living tissue with minimal photodamage, to depths so far ~ 400 µm. Tissue autofluorescence excited by two-photon or three-photon absorption to ultra-violet energies can provide specific indications of disease. Useful autofluorescence of serotonin (5HT), melatonin, indolamine breakdown products, NADH, collagen, elastin, and a number of yet-to-be-identified molecular species, some of which identify disease states are already being imaged routinely. For research in model animals, genetic constructs that label specific molecules with mutants of Green Fluorescent Protein (GFP) can be imaged deep in tissue with MPM. MPM excitation of GFP mutants at nanomolar concentrations for Fluorescence Correlation Spectroscopy (FCS) provides a robust, internally calibrated, new measure of pH in cells and tissues. Fluorescent labels that penetrate tissue can be usefully imaged in living animals and thick tissue cultures; for example, thioflavins in the beta amyloid plaques of Alzheimer’s Disease are being imaged deep in living transgenic mouse brains. Multiphoton imaging spectroscopy and fluorescence lifetime imaging (FLIM) provides useful molecular identification diagnostics. Some applications are shown in order to illustrate capability. However, the potential of MPM for in vivoimaging has barely been explored, and this technology should be regarded as providing a fertile opportunity that is yet to be fully exploited for biomedical research and for clinical applications.

© 2000 Optical Society of America