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.