In Vivo Deep-Brain 3- and 4-Photon Fluorescence Imaging of Subcortical Structures Labeled by Quantum Dots Excited at the 2200 nm Window

作者：Shen Tong, Jincheng Zhong, Xinlin Chen, Xiangquan Deng, Jie Huang, Yingxian Zhang, Mengyuan Qin, Zhenhui Li, Hui Cheng, Wanjian Zhang, Lei Zheng, Weixin Xie, Ping Qiu, Ke Wang · 发表于：ACS Nano · 年份：2023 · DOI：10.1021/acsnano.2c10724 · 被引用次数：31 · 研究领域：Advanced Fluorescence Microscopy Techniques、Photoacoustic and Ultrasonic Imaging、Optical Imaging and Spectroscopy Techniques

Multiphoton microscopy (MPM) is an enabling technology for visualizing deep-brain structures at high spatial resolution in vivo . Within the low tissue absorption window, shifting to longer excitation wavelengths reduces tissue scattering and boosts penetration depth. Recently, the 2200 nm excitation window has emerged as the last and longest window suitable for deep-brain MPM. However, multiphoton fluorescence imaging at this window has not been demonstrated, due to the lack of characterization of multiphoton properties of fluorescent labels. Here we demonstrate technologies for measuring both the multiphoton excitation and emission properties of fluorescent labels at the 2200 nm window, using (1) 3-photon ( ησ 3 ) and 4-photon action cross sections ( ησ 4 ) and (2) 3-photon and 4-photon emission spectra both ex vivo and in vivo of quantum dots. Our results show that quantum dots have exceptionally large ησ 3 and ησ 4 for efficient generation of multiphoton fluorescence. Besides, the 3-photon and 4-photon emission spectra of quantum dots are essentially identical to those of one-photon emission, which change negligibly subject to the local environment of circulating blood. Based on these characterization results, we further demonstrate deep-brain vasculature imaging in vivo . Due to the superb multiphoton properties of quantum dots, 3-photon and 4-photon fluorescence imaging reaches a maximum brain imaging depth of 1060 and 940 μm below the surface of a mouse brain, respecti...