Measurement of 3-photon excitation and emission spectra and verification of Kasha’s rule for selected fluorescent proteins excited at the 1700-nm window

Fluorescent proteins are widely used to visualize structures and dynamics in various biological samples. Multiphoton microscopy is especially suitable for imaging structures expressing fluorescent proteins with subcellular resolution. 3-photon microscopy (3PM) excited at the 1700-nm window has proven to be promising for deep-tissue (such as brain) imaging expressing red fluorescent proteins. However, the 3-photon excitation and emission spectra of fluorescent proteins suitable at this window remain largely unknown, hampering protein selection and detection optimization. Here we demonstrate detailed measurement of 3-photon excitation and emission spectra for selected fluorescent proteins, suitable for 3-photon excitation at the 1700-nm window. The measured 3-photon excitation spectra will provide guidelines for protein and excitation wavelength selection. The measured 3-photon emission spectra and comparison with the 1-photon emission spectra, on one hand proves that the fundamental Kasha's rule is still valid for 3-photon fluorescence in these fluorescent proteins, on the other hand will be helpful for efficient fluorescence signal detection.