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

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.