1600至1820nm波段三光子荧光成像已成为实现深层生物组织、特别是脑组织成像的重要光学手段。荧光信号强度优化是增加成像深度、提高成像速度的必要条件，它正比于作用截面，因此应当选择具有最大作用截面的荧光标记物。目前为止，还没有对1600至1820nm波段各种荧光标记物三光子作用截面的系统化实验测量结果，严重影响标记物选择以及定量分析。本项目拟在实验上系统测量多种荧光标记物，包括荧光染料、钙指示剂、荧光蛋白等在该波段不同波长处的三光子作用截面，建立作用截面的数据库，并在生物组织，例如小鼠大脑内进行对比成像研究。实验手段上，我们将采用可调谐高能飞秒光纤激光作为激发光源，以荧光信号强度探测为手段，获得三光子作用截面。通过本项目的研究，可以得到多种荧光标记物三光子作用截面这一基本物理量。研究结果将用于三光子深层生物组织成像，为实验中荧光标记物的选择、荧光信号的优化及定量分析提供必要的实验依据。

Three-photon (3P) fluorescence imaging in the 1600-1820 nm window has emerged as a valuable tool for deep-tissue imaging, especially for imaging brain tissues in vivo. To increase imaging depth and imaging speed, optimization of the detected fluorescence signal is the key. 3P fluorescence signal is proportional to 3P action cross section, consequently, fluorescent labels with maximum 3P action cross sections should be chosen in imaging applications. However, so far there has been no systematic measurement of 3P action cross sections of various fluorescent labels in this spectral window, hampering the choice of fluorescent labels and quantitative analysis in imaging. Our proposal aims to experimentally measure 3P cross sections of various fluorescent labels, including fluorescent dyes, calcium indicators, fluorescent proteins, etc., in the 1600-1820 nm spectral window. We will build a database of 3P cross sections of various fluorescent labels with the measured results, and apply these results in 3P comparative imaging in biological tissues, such as the mouse brain. Experimentally, we will use a high-energy, tunable femtosecond fiber laser source covering this entire spectral window as the excitation source, and use fluorescence signal intensity measurement to determine the 3P cross sections. Upon completion of this proposed project, the basic physical quantities, i.e., 3P action cross sections of various fluorescent labels can be quantitatively determined. Our measured results, in the form of a 3P cross section database, will find direct application in 3P deep-tissue imaging in the 1600-1820 nm window, offering guidelines for choosing fluorescent labels, optimizing fluorescence signals and quantitative analysis.