Pope Iestyn, Langbein Wolfgang, Borri Paola, Watson Peter
School of Biosciences, Cardiff University, Cardiff, United Kingdom.
Methods Enzymol. 2012;504:273-91. doi: 10.1016/B978-0-12-391857-4.00014-8.
Live cell microscopy using fluorescent proteins and small fluorescent probes is a well-established and essential tool for cell biology; however, there is a considerable need for noninvasive techniques able to study tissue and cell dynamics without the need to introduce chemical or genetically encoded probes. Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging tool for cell biologists to examine live cell dynamics with chemical specificity in a label-free, noninvasive way. CARS is a multiphoton process offering intrinsic three-dimensional submicron resolution, where the image contrast is obtained from light inelastically scattered by the vibrations of endogenous chemical bonds. CARS is particularly well suited to study lipid biology, since the CARS signal of localized lipids (exhibiting a large amount of identical bonds in the focal volume) is very strong. Conversely, photostable, lipid-specific markers for fluorescence microscopy are difficult to produce and the process of labeling often affects lipid localization and function, making imaging lipids in live cells challenging, and accurate quantification often impossible. Here, we describe in detail the principles behind our experimental setup for performing CARS microscopy of lipid droplets on live cells. Since typical vibrational resonances in liquid have coherence times in the picosecond range, CARS is preferably implemented with picosecond lasers which are however expensive and less efficient than femtosecond lasers, which could also be used for other multiphoton techniques such as two-photon fluorescence. In our setup, we show that femtosecond lasers can be spectrally focused in a simple, alignment insensitive, and cost-effective way to achieve a vibrational excitation similar to picosecond lasers. This opens the way to integrate CARS and two-photon fluorescence in a single multimodal instrument for its widespread application. We also describe our dual frequency CARS system which eliminates the nonresonant CARS background offering superior sensitivity and image contrast.
利用荧光蛋白和小型荧光探针进行的活细胞显微镜检查是细胞生物学中一项成熟且必不可少的工具；然而，迫切需要能够在无需引入化学或基因编码探针的情况下研究组织和细胞动态的非侵入性技术。相干反斯托克斯拉曼散射（CARS）显微镜是一种新兴工具，可供细胞生物学家以无标记、非侵入的方式检查具有化学特异性的活细胞动态。CARS是一个多光子过程，具有固有的三维亚微米分辨率，其图像对比度来自内源性化学键振动产生的非弹性散射光。CARS特别适合研究脂质生物学，因为局部脂质的CARS信号（在焦体积中表现出大量相同的键）非常强。相反，用于荧光显微镜的光稳定、脂质特异性标记物很难制备，而且标记过程常常会影响脂质的定位和功能，这使得对活细胞中的脂质进行成像具有挑战性，而且往往无法进行准确的定量分析。在此，我们详细描述了在活细胞上对脂滴进行CARS显微镜检查的实验装置背后的原理。由于液体中的典型振动共振具有皮秒级的相干时间，CARS最好用皮秒激光器来实现，然而皮秒激光器价格昂贵且效率低于飞秒激光器，而飞秒激光器也可用于其他多光子技术，如双光子荧光。在我们的装置中，我们展示了飞秒激光器可以通过一种简单、对校准不敏感且经济高效的方式进行光谱聚焦，以实现与皮秒激光器类似的振动激发。这为将CARS和双光子荧光集成到单一的多模态仪器中以实现广泛应用开辟了道路。我们还描述了我们的双频CARS系统，该系统消除了非共振CARS背景，提供了卓越的灵敏度和图像对比度。
