Full-Spectrum CARS Microscopy of Cells and Tissues with Ultrashort White-Light Continuum Pulses

Coherent anti-StokesRaman scattering (CARS) microscopyis an emergingnonlinear vibrational imaging technique that delivers label-free chemicalmaps of cells and tissues. In narrowband CARS, two spatiotemporallysuperimposed picosecond pulses, pump and Stokes, illuminate the sampleto interrogate a single vibrational mode. Broadband CARS (BCARS) combinesnarrowband pump pulses with broadband Stokes pulses to record broadvibrational spectra. Despite recent technological advancements, BCARSmicroscopes still struggle to image biological samples over the entireRaman-active region (400-3100 cm(-1)). Here,we demonstrate a robust BCARS platform that answers this need. Oursystem is based on a femtosecond ytterbium laser at a 1035 nm wavelengthand a 2 MHz repetition rate, which delivers high-energy pulses usedto produce broadband Stokes pulses by white-light continuum generationin a bulk YAG crystal. Combining such pulses, pre-compressed to sub-20fs duration, with narrowband pump pulses, we generate a CARS signalwith a high (<9 cm(-1)) spectral resolution inthe whole Raman-active window, exploiting both the two-color and three-colorexcitation mechanisms. Aided by an innovative post-processing pipeline,our microscope allows us to perform high-speed (approximate to 1 ms pixeldwell time) imaging over a large field of view, identifying the mainchemical compounds in cancer cells and discriminating tumorous fromhealthy regions in liver slices of mouse models, paving the way forapplications in histopathological settings.