Selection of proper objective lens for the higher-order multiphoton microscopy at the 1700-nm window

Abstract

The 1700-nm window has emerged as a promising excitation window for multiphoton microscopy (MPM). On one hand, the combined low tissue absorption and scattering make this window well suited for deep-tissue MPM; on the other hand, the long excitation wavelength makes higher-order MPM in biological tissues feasible, e.g., recently 4-photon fluorescence MPM in mouse brain has been demonstrated. Objective lens is a key optical component in the entire MPM setup. Multiphoton signal levels are largely dependent on the transmittance of objective lens. Here we demonstrate experimental results of transmittance measurement of two water immersion objective lenses commonly used for MPM at the 1700-nm window, covering both the excitation and the signal window. Our target application is MPM of even higher order excited at this window, i.e., 4th harmonic generation (FHG) imaging and 5-photon fluorescence generation. Our results show that, although the customized objective lens offers higher transmittance at the excitation window, it suffers from dramatically degraded transmittance at the signal window, compared with the non-customized objective lens. These results will offer guidelines for selection of proper objective lens for higher-order MPM at the 1700-nm window.