Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen, 518060, China.
Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, Hubei International Scientific and Technological Cooperation Base of Pesticide and Green Synthesis, International Joint Research Center for Intelligent Biosensing Technology and Health, College of Chemistry, Central China Normal University, Wuhan, 430079, China.
Anal Chim Acta. 2023 May 15;1255:341118. doi: 10.1016/j.aca.2023.341118. Epub 2023 Mar 24.
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 ησ 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.
双光子荧光显微镜（2PFM）是神经科学中不可或缺的成像技术。然而，其成像深度通常局限于活体小鼠大脑的皮层层。在这里，我们演示了使用 IR780 和 aza-IR780 作为荧光标记物，在 1700nm 窗口激发的深部脑 2PFM 活体成像。我们对 IR780 和 aza-IR780 的多光子激发和发射特性进行了详细表征，结果表明：（1）IR780 或 aza-IR780 在 1700nm 窗口激发产生双光子荧光，是 2PFM 的理想选择；（2）与 IR780 相比，aza-IR780 具有更大的 ησ 和更好的抗光漂白性；IR780 和 aza-IR780 在等离子体中的双光子作用截面比在 PBS 中大一个数量级；（3）两种染料的活体 2 光子发射光谱与体外相比有明显的红移。基于这些特性，我们展示了它们标记的深部脑 2PFM。通过它们标记的活体小鼠大脑的最大成像深度为 1585μm（IR780 标记）和 1800μm（aza-IR780 标记），很容易穿透海马体的皮质下区域。此外，通过 aza-IR780 标记的 2PFM 实现了最大 1528μm 的血液动力学成像深度，使我们能够测量海马体中的血流速度。
