Sealing of Immersion Deuterium Dioxide and Its Application to Signal Maintenance for Ex-Vivo and In-Vivo Multiphoton Microscopy Excited at the 1700-nm Window

Excitation at the 1700-nm window is an effective means for extending imaging depth and imaging modalities in multiphoton microscopy (MPM). To enhance multiphoton signal levels and enable deep-tissue penetration, water immersion has to be replaced by deuterium dioxide (D₂O) immersion to boost transmittance at the 1700-nm window. The key problem facing this D₂O immersion technique is the hygroscopic nature of D₂O, which leads to decrease of MPM signals as time lapses. Here, we demonstrate a simple, yet very effective technique to isolate D₂ O from the ambient environment, by sealing it with the paraffin liquid. We demonstrate the application of this technique to MPM signal maintenance in both three-photon fluorescence generation in a fluorescent dye and third-harmonic generation (THG) imaging of biological tissue, excited at the 1700-nm window. Ex-vivo imaging results show that during an imaging session of 5 h, multiphoton signals of both modalities can be maintained with no deterioration due to absorption of water vapor from the environment. Furthermore, we demonstrate in-vivo deep-tissue mouse brain imaging using this technique, in which THG signals can be maintained for at least 5 h. This justifies the applicability and effectiveness of our D₂O sealing technique for long-span in-vivo imaging.