基于飞秒激光脉冲整形的高光谱相干拉曼显微成像研究

Coherent Raman scattering microscopy, based on either coherent anti-StokesRaman scattering (CARS) or stimulated Raman scattering(SRS),employsmolecular vibrations as image contrast and thus could be applied to the study of small biomolecules that can not be labeled by the bulky fluorescent tags. Traditional single-color imaging requires isolated Raman bands to ensure the chemical specificity and therefore might not satisfy the imaging applications of some complicated biological samples. Hyperspectral coherent Raman scattering microscopy offers spectral information at each pixel, and as such could separate molecules with overlapped Raman bands, providing better chemical specificity and much more abundant chemical information.In this project, we aim to develop a broadband(> 1000 cm-1), high spectral resolution (~10 cm-1) coherent Raman spectroscopic imaging system with a single femtosecond oscillator. Silouette MIIPS will be deployed to perform precise pulse shaping in thetime-domain, and the dispersion of different frequency components within the ultra-broadband fs pulses (~ 100 nm) will be carefully manipulated so as to match the chirp of the pump and Stokes pulsesexactly. Based on this, rapid Raman shift tuning will be achieved via the spectral focusing method. Meanwhile, a frequency modulation method will be proposed to suppress the nonresonant backgrounds in coherent Raman scatting microscopy (e.g. four wave mixing backgrounds in CARS and pump-probe backgrounds in SRS) and to improve the image quality and detection sensitivity.

传统的单频相干拉曼散射显微成像为了保证化学特异性往往要求被探测成分的拉曼振动相对分立，因而难以满足复杂生物样品的成像需求。高光谱相干拉曼显微成像在每一个像素点都拥有一段光谱，不仅可以区分拥有重叠拉曼谱带的不同分子，还能提供更加丰富的化学信息，通过分析光谱即可检测样品化学成分，因而具有更好的化学选择性和化学特异性。本项目提出利用单个超宽带飞秒激光振荡器光源（~100 nm），结合Silouette MIIPS 时域脉冲精准整形，通过严格控制宽带飞秒激光脉冲内部不同频率成分的色散来精确匹配泵浦光和斯托克斯光的线性啁啾，运用光谱聚焦方法扫描拉曼频移实现超宽带(1000 cm-1)、高光谱分辨率（~10 cm-1）相干拉曼光谱显微成像，同时提出一种频率调制方法用来降低相干拉曼散射显微成像中非共振背景（如CARS成像中四波混频背景以及SRS成像中泵浦探测背景等），进而提高成像质量及探测灵敏度。

本项目搭建了多个非线性光学光谱探测系统以及显微成像系统，并对目标化学样品开展了光谱探测实验，对多种化学样品以及生物样品开展了非线性显微成像实验，其中光谱探测系统包括高波数区及指纹区飞秒时间分辨相干反斯特克斯(Coherent anti-stokes raman scattering, CARS)光谱探测系统、高光谱分辨率光谱聚焦CARS光谱探测系统，显微成像系统包括双光子荧光(two-photon fluorescence, TPF)显微成像系统、指纹区宽带CARS成像系统、Time-lens 多元CARS显微成像系统，在实验方面，对Rhodamine B染料样品、Caski宫颈癌细胞样品以及乳鼠成骨细胞样品开展了二维双光子荧光显微成像实验以及三维重构成像实验，以二氧化硅、聚苯乙烯为样品，采集了其在高波数区以及非指纹区的飞秒时间分辨CARS光谱，并通过玻璃棒对飞秒脉冲引入啁啾，测量了聚苯乙烯在指纹区的光谱聚焦CARS光谱，观察到了明显提升的光谱分辨率以及拉曼模式之间的切换。并以三聚氰胺、聚甲基丙烯酸甲酯(PMMA)为样品开展了宽带CARS显微成像实验，利用二分之一波片改变Pump光脉冲、Stokes光脉冲的偏振特性，实现了一定程度上抑制宽带CARS显微成像结果中的非共振背景，以PMMA、聚苯乙烯颗粒以及小鼠耳朵为样品，开展了Time-lens 多元CARS显微成像，很大程度上抑制了CARS成像结果中的非共振背景，并观察到了小鼠耳朵样品中富脂区与富蛋白区的相对分布，此外还以双光子荧光显微成像为例，通过改变探测窗口增加成像深度，利用前向双光子信号与后向双光子信号同时进行成像从而提升成像精度，并分析了双轴振镜扫描显微成像系统中信号强度畸变产生原理以及如何通过数学方式消除这种强度畸变。