Coherent Stokes Raman scattering microscopy (CSRS)

Coherent Stokes Raman scattering microscopy (CSRS) Jitendra Acharyyaa, Gina Bennia, Hervé Rigneaulta and Sandro Heukea a Aix Marseille University, CNRS, Centrale Marseille, Institut Fresnel, Marseille, France We report on the first implementation of laser-scanning coherent Stokes Raman scattering (CSRS) microscopy [1]. Blue-excited CSRS has the potential to become the coherent Raman technique with the highest spatial resolution, since the emitted wavelength is red-shifted and lies in the visible range. Consequently, CSRS avoids the low transmission issues of ultraviolet-emitted coherent anti-Stokes Raman scattering (CARS) and is not affected by absorption artefacts typical of stimulated Raman scattering (SRS). Historically, CSRS has not been used for biomedical imaging because of a strong fluorescence background obscuring the coherent signal. We show that this fluorescence background can be effectively suppressed even under visible excitation by combining narrow bandpass filtering with lock-in-based demodulation. Furthermore, we demonstrate both numerically and experimentally that CSRS overcomes a major limitation of all coherent Raman microscopy techniques: under tight focusing conditions, a significant portion (up to 100%) of the CSRS photons is emitted directly in the backward direction. The experimental data confirming this effect have been obtained only recently. This finding paves the way for efficient investigations of thick, non-transparent samples and enables future technological advances such as epi-detected coherent Raman multifocus imaging, real-time laser-scanning-based spectroscopy, and compact endoscopic implementations. References [1] Heuke, S., Rigneault, H. Coherent Stokes Raman scattering microscopy (CSRS). Nat Commun 14, 3337 (2023).