Open AccessMultiphoton excitation cross‐sections of molecular fluorophores
Author(s) -
Xu Chris,
Williams R M,
Zipfel Warren,
Webb Watt W
Publication year - 1996
Publication title -
bioimaging
Language(s) - English
Resource type - Journals
eISSN - 1361-6374
pISSN - 0966-9051
DOI - 10.1002/1361-6374(199609)4:3<198::aid-bio10>3.0.co;2-x
Subject(s) - femtosecond , excitation , fluorophore , microscopy , two photon excitation microscopy , laser , optics , chemistry , materials science , fluorescence , physics , quantum mechanics
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.