Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.
Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's palette of chemical indicators, fluorescent markers, and native biological fluorophores, including NADH, flavins, and green fluorescent proteins, that are applicable to living biological preparations. More than 25 two-photon excitation spectra of ultraviolet and visible absorbing molecules reveal useful cross sections, some conveniently blue-shifted, for near-infrared absorption. Measurements of three-photon fluorophore excitation spectra now define alternative windows at relatively benign wavelengths to excite deeper ultraviolet fluorophores. The inherent optical sectioning capability of nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background and photodamage. Here, the measured nonlinear excitation spectra and their photophysical characteristics that empower nonlinear laser microscopy for biological imaging are described.
Xu
C
C
Cornell University, Ithaca, NY 14853, USA.
Zipfel
W
W
Shear
J B
JB
Williams
R M
RM
Webb
W W
WW
D000818:Animals; D005486:Flavin Mononucleotide / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins; D007834:Lasers; D008164:Luminescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D009243:NAD / Q000737:chemistry; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D009243:NAD; D049452:Green Fluorescent Proteins; D005486:Flavin Mononucleotide