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

Abstract

Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging nonlinear vibrational imaging technique that delivers label-free chemical maps of cells and tissues. In narrowband CARS, two spatiotemporally superimposed picosecond pulses, pump and Stokes, illuminate the sample to interrogate a single vibrational mode. Broadband CARS (BCARS) combines narrowband pump pulses with broadband Stokes pulses to record broad vibrational spectra. Despite recent technological advancements, BCARS microscopes still struggle to image biological samples over the entire Raman-active region (400–3100 cm–1). Here, we demonstrate a robust BCARS platform that answers this need. Our system is based on a femtosecond ytterbium laser at a 1035 nm wavelength and a 2 MHz repetition rate, which delivers high-energy pulses used to produce broadband Stokes pulses by white-light continuum generation in a bulk YAG crystal. Combining such pulses, pre-compressed to sub-20 fs duration, with narrowband pump pulses, we generate a CARS signal with a high (<9 cm–1) spectral resolution in the whole Raman-active window, exploiting both the two-color and three-color excitation mechanisms. Aided by an innovative post-processing pipeline, our microscope allows us to perform high-speed (≈1 ms pixel dwell time) imaging over a large field of view, identifying the main chemical compounds in cancer cells and discriminating tumorous from healthy regions in liver slices of mouse models, paving the way for applications in histopathological settings.

Pinpointing Fat Molecules: Advances in Coherent Raman Scattering Microscopy for Lipid Metabolism

“…In another example, BCARS imaging has been applied to image liver cancer tissue. By leveraging the broad spectrum provided by BCARS in both C–H stretching and fingerprint regions, discrimination of tumor regions from healthy tissue in liver tissue slices of mouse models became possible …”

Vibrational spectroscopy identifies myocardial chemical modifications in heart failure with preserved ejection fraction

“…However, there are still challenges that need to be addressed, such as low signal and tissue autofluorescence for Raman scattering, and the interference from the strong water signals in FTIR spectra. Techniques derived from the Raman effect or IR absorption (like Optical Photothermal IR [ 28 ], Surface Enhanced Raman Scattering [ 29 ] or Coherent anti-Stokes Raman scattering [ 30 ]), have the potential to enhance the sensitivity and selectivity of tissue analysis and implement their clinical use.…”