Multiphoton excitation cross-sections of molecular fluorophores

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 25 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, chemical indicators, and native cellular absorbers for living biological preparations. Measurements of three-photon fluorophore excitation spectra now suggest relatively benign wavelengths to excite deeper UV fluorophores. The inherent optical sectioning capabilities of focused nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background. Measured nonlinear excitation spectra are described and implications to nonlinear microscopy for biological imaging are defined.