Multiphoton fluorescence excitation: New spectral windows for biological imaging

Nonlinear excitation of fluorophores through molecular absorption of two or three near infra-red photons from the tightly focused femtosecond pulses of a mode-locked laser offers the cellular biologist an unprecedented panoply of biomolecular indicators for microscopic imaging and cellular analysis. Measurements of the two-photon excitation spectra of more than twenty ultra-violet and visible absorbing fluorophores from 690 to 1050 nm reveal useful cross sections for near infra-red excitation, providing an artist's palette of emission markers and chemical indicators for living biological preparations. Measurements of three-photon fluorophore excitation spectra now define alternative windows of relatively benign wavelength to excite deeper UV fluorophores. The three-photon excitation spectrum of the amino acid tryptophan, measured 700-900 nm, delivers native fluorescence for imaging and assay of proteins and neurotransmitters in living tissues. The inherent optical sectioning capabilities of focused nonlinear excitation provides three-dimensional resolution for imaging and avoids out of focus background. Here, we describe the characteristics of the measured nonlinear excitation spectra and define the resulting features of nonlinear microscopy for biological imaging.