NIR-II-Excitable Dye-Loaded Nanoemulsions for Two-Photon Microscopy Imaging of Capillary Blood Vessels in the Entire Hippocampal CA1 Region of Living Mice

For in vivo two-photon fluorescence microscopy (2PM) imaging, the development of techniques that can improve the observable depth and temporal resolution is an important challenge to address biological and biomedical concerns such as vascular dynamics in the deep brain (typically the hippocampal region) of living animals. Improvements have been achieved through two approaches: an optical approach using a highly tissue-penetrating excitation laser oscillating in the second near-infrared wavelength region (NIR-II, 1100–1350 nm) and a chemical approach employing fluorescent probes with high two-photon brightness (characterized by the product of the two-photon absorption cross section, σ², and the fluorescence quantum yield, Φ). To integrate these two approaches, we developed a fluorescent dye exhibiting a sufficiently high σ²Φ value of 68 Goeppert-Mayer units at 1100 nm. When a nanoemulsion encapsulating >1000 dye molecules per particle and a 1100 nm laser were employed for 2PM imaging, capillary blood vessels in almost the entire hippocampal CA1 region of the mouse brain (approximately 1.1–1.5 mm below the surface) were clearly visualized at a frame rate of 30 frames s^–1 (averaged over eight frames, practically 3.75 frames s^–1). This observable depth and frame rate are much higher than those in previous reports on 2PM imaging. Furthermore, this nanoemulsion allowed for the visualization of blood vessels at a depth of 1.8 mm, corresponding to the hippocampal dentate gyrus. These results highlight the advantage of combining bright probes with NIR-II lasers. Our probe is a promising tool for studying the vascular dynamics of living animals and related diseases.