Broadband CARS microscopy in the entire Raman-active region of biological samples via supercontinuum generation in bulk media

Abstract

Coherent anti-Stokes Raman Scattering (CARS) microscopy is a label-free vibrational imaging technique that delivers chemical maps of cells and tissues. CARS employs two narrowband picosecond pulses (pump and Stokes) that are spatiotemporally superimposed at the sample plane to probe a single vibrational mode. Broadband CARS (BCARS) combines narrowband pump pulses with broadband Stokes pulses to record broad vibrational spectra. Despite many technological advancements, BCARS microscopes still struggle to image biological samples spanning the entire Raman active region of biological samples (400-3100 cm -1 ).Here, we demonstrate a novel BCARS method to answer this need. Our experimental setup is based on a femtosecond fiber laser at 1035 nm and 2 MHz repetition rate, thus delivering high energy pulses used for generating sub-20 fs broadband Stokes pulses by white-light continuum in a bulk YAG crystal, a compact and alignment-insensitive technique. Combining them with narrowband picosecond pulses, we can generate a CARS signal with high (< 10 cm -1 ) spectral resolution in the entire Raman window exploiting both two-color and three-color excitation mechanisms. The system is equipped with a home-made transmission microscope to image cells and tissue at high-speed (< 3 ms) and large field of views. Using a post-processing pipeline, we deliver high-quality chemical maps, identifying the main chemical compounds in cancer cells and discriminating tumorous from healthy regions in liver slices of mouse models, unveiling the path for applications in histopathological settings.