Ex and in vivo characterization of the wavelength‐dependent 3‐photon action cross‐sections of red fluorescent proteins covering the 1700‐nm window

Abstract

Multiphoton action cross-sections are the prerequisite for excitation light selection. At the 1700-nm window suitable for deep-tissue imaging, wavelength-dependent 3-photon action cross-sections ησ₃ for RFPs are unknown, preventing wavelength selection. Here we demonstrate: (1) ex vivo measurement of wavelength-dependent ησ₃ for purified RFPs; (2) a multiphoton imaging guided measurement system for in vivo measurement; and (3) in vivo measurement of wavelength-dependent ησ₃ in RFP labeled cells. These fundamental results will provide guidelines for excitation wavelength selection for 3-photon fluorescence imaging of RFPs at the 1700-nm window, and augment the existing database of multiphoton action cross-sections for fluorophores.