High-sensitive and Background-free Coherent Anti-Stokes Raman Scattering Microscopy Using Delay Modulation

Abstract Coherent anti-Stokes Raman scattering (CARS) microscopy has been demonstrated to be a powerful platform for label-free, non-invasive, and chemically specific imaging of biological samples. Yet, the non-resonant background hinders its sensitive detection of weak Raman bands. Here, we demonstrate an approach to remove the non-resonant background in CARS based on delay modulation (DM), enabled by an acousto-optic modulator and spectral focusing. We show that the DM-CARS reduces the background by 10 times and achieves a detection sensitivity of 3ⅹ105 molecules at a time constant of 7 μs, a 100 fold improvement over normal CARS. We demonstrated the potential clinical applications of DM-CARS in tracking heavy water metabolism in bacteria for antimicrobial susceptibility testing, which is challenging using normal CARS, and a fiber-delivered handheld DM-CARS device for liver tumor tissue imaging.