29] Multiphoton excitation microscopy, confocal microscopy, and spectroscopy of living cells and tissues; functional metabolic imaging of human skin in vivo

Abstract

The chapter discusses multiphoton excitation microscopy based on NAD(P)H functional imaging. Progress is also being made in synthesizing molecules with very large two-photon absorption cross sections. For multiphoton excitation processes, the number of photon pairs absorbed for each laser pulse is related inversely to the pulse width. The passage of ultrashort pulses from the mode-locked laser through a dielectric medium results in pulse broadening because of group velocity dispersion. This pulse broadening, called “pulse dispersion,” is a serious problem in multiphoton excitation microscopy because it results in a reduction in the probability of multiphoton excitation. Multiphoton excitation microscopy has the following important advantages: (1) reduced phototoxicity, (2) reduced photobleaching, (3) increased penetration depth, (4) ability to perform uncaging or photobleaching in a diffraction limited volume, (5) ability to excite fluorophores in the ultraviolet without a ultraviolet laser, (6) the excitation and the fuorescence wavelengths are well separated, and (7) no spatial filter is required. The chapter discusses functional metabolic imaging of cellular metabolism based on NAD(P)H fluorescence, ultraviolet confocal fluorescence microscopy compared with multiphoton excitation microscopy, laser sources for multiphoton excitation microscopy, and pulse compensation techniques.