Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.

TLDR

Nonlinear laser‑scanning fluorescence microscopy enables intrinsic, three‑dimensional, high‑resolution imaging of living specimens while suppressing out‑of‑focus background and photodamage. The study reports measured nonlinear excitation spectra and their photophysical properties to enable biological imaging with nonlinear laser microscopy. The authors identified over 25 two‑photon and several three‑photon excitation spectra for UV/visible fluorophores—including NADH, flavins, and GFP—providing new, near‑infrared and benign wavelength windows for deep‑tissue biological imaging.

Abstract

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.