Navas-Moreno Maria, Chan James W
Center for Biophotonics, University of California, Davis, Sacramento, CA, USA.
Department of Pathology and Laboratory Medicine, Center for Biophotonics, University of California, Davis, Sacramento, CA, USA.
Methods Mol Biol. 2018;1745:219-257. doi: 10.1007/978-1-4939-7680-5_13.
Laser tweezers Raman spectroscopy (LTRS) is a variation of micro-Raman spectroscopy that is used to analyze single cells and biological particles suspended in an aqueous environment. The Raman spectrum of the cell/particle reflects its intrinsic biochemical composition and molecular structures. The technique utilizes a laser trap generated by a tightly focused Gaussian laser beam to physically manipulate individual cells and immobilize them in the laser focal volume. The same laser that is used for optical trapping also simultaneously excites Raman signals from the trapped cell, which are detected using a spectrometer and a confocal detection setup. LTRS offers unique capabilities not commonly found in other optical cytometry methods, such as label-free chemical analysis, multi-parametric chemical detection with a single excitation laser, and a non-photobleaching signal that can be used to quantitate and monitor dynamic chemical changes. This chapter provides guidelines on the design of a single beam LTRS microscope and methods for building and aligning the system. Operating procedures for trapping particles and acquiring spectra and a summary of data analysis techniques are provided.
激光镊子拉曼光谱技术（LTRS）是显微拉曼光谱技术的一种变体，用于分析悬浮在水环境中的单细胞和生物颗粒。细胞/颗粒的拉曼光谱反映了其内在的生化组成和分子结构。该技术利用由紧密聚焦的高斯激光束产生的激光阱来物理操纵单个细胞并将其固定在激光焦斑体积内。用于光学捕获的同一束激光同时也激发被捕获细胞的拉曼信号，这些信号使用光谱仪和共聚焦检测装置进行检测。LTRS具有其他光学细胞计数方法中不常见的独特功能，例如无标记化学分析、用单一激发激光进行多参数化学检测以及可用于定量和监测动态化学变化的非光漂白信号。本章提供了单光束LTRS显微镜设计指南以及构建和校准该系统的方法。还提供了捕获颗粒和获取光谱的操作程序以及数据分析技术总结。
