Breaking the detection limit for coherent anti-Stokes Raman (CARS) signals by using doubly resonant coherent anti-Stokes Raman (DR-CARS) signal generation with Acousto-Optical Tunable Filter (AOTF) signal modulation

Fluorescence microscopy has long been the driving force in the cellular biology field. However, its methodology has had a weakness since it requires exogenic fluorescent probes to select the molecules of interest. Those probes are not photostable and can potentially become toxic or interfere with the cellular environment. In the last decade, some label-free microscopy techniques have been shown to successfully overcome those problems. One of the most promising one is CARS microscopy. It is based on a resonant inelastic scattering between photons and molecules, and provides label-free, chemically specific information from the sample in video rate. However, one of the drawbacks of CARS microscopy is the strong signal dependence on the molecular concentration. We developed a technique that is based on a doubly resonant enhanced signal generation that allows pushing the concentration sensitivity by few orders of magnitude. In this work we show the implementation of a modified signal acquisition setup that theoretically allows increasing the concentration sensitivity to few μM. To overcome the signal detection limit, the double resonant signals are rapidly modulated using an Acousto-Optic Tunable Filter (AOTF),(Gooch&Housego, UK) and detected on a conventional photomultiplier tube (PMT),(Hamamatsu, JP) in conjunction with a Lock-In-detection scheme. The entire signal acquisition setup is controlled via a LabView code.