Live cell imaging with chemical specificity using dual frequency CARS microscopy.

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.