Deep-brain 3-photon microscopy excited at 1600 nm with silicone oil immersion.

Three-photon microscopy excited at the 1700-nm window (roughly covering 1600-1840 nm) is especially suitable for deep-brain imaging in living animals. To match the brain refractive index, D2O has been exclusively used as the immersion medium. However, the hygroscopic property of D2O leads to a decrease of transmittance of the excitation light and as a result a decrease in three-photon signals over time. Solutions such as replacing D2O from time to time, wrapping both the objective lens and the immersion D2O, and sealing D2O with paraffin liquid have all been demonstrated, which add to the system complexity. Based on our recent characterization of immersion oils, we propose using silicone oil as a potential alternative to D2O for deep-brain imaging. Excited at 1600 nm, our comparative deep-brain imaging using both D2O and silicone oil immersion show that silicone oil immersion yields 17% higher three-photon signal in third-harmonic generation imaging within the white matter. Besides, silicone oil immersion also enables three-photon fluorescence imaging of vasculature up to 1460 mu m (mechanical depth) into the mouse brain in vivo acquired at 2 seconds/frame. Together with the nonhygroscopic physical property, silicone oil is promising for long-span three-photon brain imaging excited at the 1700-nm window.