光梳相干反斯托克斯拉曼散射显微成像光谱技术

Cells, the functional units of life, are best studied in vivo. To further reveal the mystery of life and conquer critical illness, it is often required to localize proteins in a live cell to a certain accuracy to study their localization-related function, and the development of a non-invasive imaging method with molecular recognition and quantitative analysis capabilities are needed. Based on the measurement of molecular bond vibration, coherent anti-Stokes Raman scattering (CARS) is a kind of non-invasive optical microscopic imaging technique, and has been widely used in spectroscopy imaging and medical examination. In recent years, the significant breakthrough in femtosecond frequency comb can provide a new thought for improving microscopic imaging techniques. Initially conceived for frequency metrology, femtosecond frequency combs are now becoming enabling tools for the rapid and sensitive acquisition of molecular linear absorption spectra over a vast spectral span. Since the intensity of ultra-short pulses can easily be large enough to produce some nonlinear transient response in the observed medium, frequency combs may be harnessed for broad spectral bandwidth nonlinear molecular spectroscopy and imaging. Therefore, this project intends to develop a frequency-comb based CARS microscopy imaging methods. The method will take the advantage of optical frequency comb and optical coherent detection to realize fast CARS microscopic imaging with broad spectral bandwidth and high spectral resolution.

在分子水平上研究细胞的基本生命活动规律是当代医学和生命科学的重要研究内容。在不影响样本的生命过程和疾病机理的前提下，发展无外源性标记的、非侵入式的、具有分子识别和定量分析能力的显微成像技术是进一步揭示生命奥秘、改造生命体和征服疾病的关键。相干反斯托克斯拉曼散射(CARS)是一种基于测量分子键振动非标记的光学显微成像技术，一直以来都作为具有高光谱分辨率、高探测灵敏度的光谱成像方法，成为推动生命科学及众多交叉学科发展的重要方法与手段。近年，飞秒光梳研究的重大进展可为完善多种显微成像方法提供独特的技术途径，飞秒光梳能实现高精度、高灵敏度的频率传递，并用于光谱的标定和测量。本项目拟开发基于光梳光谱的CARS显微成像方法，该方法将实现飞秒光梳、CARS显微成像、光梳相干灵敏探测的有机融合，完成全波段、快捷、实时、高精度的CARS显微成像过程，获得光谱分辨率高、信息丰富的显微图谱。

如何实现基于分子键振动的非标记的光学显微成像，如何在较宽光谱范围、一次性快速获取分子完整特征的CARS信号，是当前CARS显微成像技术发展的重要方面。双光梳光谱具有高精度的时间分辨和频率分辨能力，在物质指纹光谱分析、痕量气体检测、绝对距离测量等研究领域有重要应用。光梳可为完善多种显微成像方法提供独特的技术途径，用于光谱的标定和测量。本项目开发了基于双光梳光谱技术的相干反斯托克斯拉曼散射(CARS)成像研究。项目期间，发展了低阈值自启动的锁模技术、超短脉冲非线性放大技术、重复频率和载波位相高精度锁定技术、双光梳光谱CARS显微成像技术，实现了飞秒光梳、CARS显微成像、光梳相干灵敏探测的有机融合。在超短脉冲激光研究方面，克服了保偏光纤激光器在弱泵浦条件下启动时间不确定的难题，获得了低阈值的锁模脉冲激光输出，锁模脉冲启动时间低于1ms；在超短脉冲功率放大方面，采用非线性分离脉冲放大技术，在1.55um波段获得了脉冲宽度100fs、单脉冲能量20nJ、重复频率80MHz的脉冲输出；基于泵浦调制非线性原理，在非线性放大环镜锁模激光器上获得了高的重复频率锁定精度，1s积分时间下的精度为70uHz；在双光梳光谱成像方面，获得了光谱分辨率为8cm-1增强的反斯托克斯拉曼光谱信号，单次采样时间仅为32.8μs。