Department of Chemistry, Purdue University.
Department of Chemistry, Purdue University;
J Vis Exp. 2022 Apr 28(182). doi: 10.3791/63677.
Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.
受激拉曼散射（SRS）和相干反斯托克斯拉曼散射（CARS）显微镜是最广泛使用的相干拉曼散射成像技术。高光谱 SRS 和 CARS 成像在每个像素都提供拉曼光谱信息，这使得不同化学成分的更好分离成为可能。尽管这两种技术都需要两个激发激光，但它们的信号检测方案和光谱特性却有很大的不同。本方案的目的是在单个平台上同时进行高光谱 SRS 和 CARS 成像，并比较两种显微镜技术对不同生物样本的成像效果。本方案采用光谱聚焦方法使用飞秒激光获取光谱信息。通过使用标准化学样品，在相同的激发条件（即样品上的功率、像素停留时间、物镜、脉冲能量）下比较 SRS 和 CARS 的灵敏度、空间分辨率和光谱分辨率。并列比较生物样品的 CARS 和 SRS 成像对比度。CARS 和 SRS 性能的直接比较可以为化学成像模式的选择提供最佳方案。
