Deep-brain three-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, D₂ O has been exclusively used as the immersion medium. However, the hygroscopic property of D₂ O 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 D₂ O from time to time, wrapping both the objective lens and the immersion D₂ O, and sealing D₂ O 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 D₂ O for deep-brain imaging. Excited at 1600 nm, our comparative deep-brain imaging using both D₂ O 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 μm (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.