In vivo deep-brain 2-photon fluorescent microscopy labeled with near-infrared dyes excited at the 1700 nm window

Abstract

2-Photon fluorescence microscopy (2PFM) is an indispensable imaging technology for neuroscience. However, the imaging depth is usually limited to the cortical layer in mouse brain in vivo. Here, we demonstrate deep brain 2PFM in vivo excited at the 1700 nm window, using IR780 and aza-IR780 as fluorescent labels. Our detailed characterization of the multiphoton excitation and emission properties of IR780 and aza-IR780 show that: (1) IR780 or aza-IR780 generate 2-photon fluorescence excited at the 1700 nm window and are promising for 2PFM; (2) aza-IR780 exhibits a larger ησ2 with better anti-photobleaching property compared to IR780; The 2-photon action cross-sections of IR780 and aza-IR780 in plasma are an order-of-magnitude larger than those in PBS; (3) In vivo 2-photon emission spectra for both dyes show a notable red shift compared to those in vitro. Based on these characterization results, we demonstrate deep brain 2PFM labeled by them. A maximum imaging depth of 1585 μm (labeled by IR780) and 1800 μm (labeled by aza-IR780) into the mouse brain in vivo readily penetrates the subcortical region of hippocampus. Besides, a maximum of 1528 μm hemodynamic imaging depth is realized via 2PFM with aza-IR780 labeling, enabling us to measure blood flow speed in the hippocampus.