Broadband coherent anti-Stokes Raman scattering (BCARS), a powerful spectroscopy combining high signal intensity with spectral sensitivity for vibrational imaging of biological systems

Broadband coherent anti-Stokes Raman scattering (BCARS), a powerful spectroscopy combining high signal intensity with spectral sensitivity for vibrational imaging of biological systemsCsilla GERGELY, Thierry CLOITRELaboratoire Charles Coulomb (L2C), Université de Montpellier, CNRS, Montpellier, FranceThe high chemical specificity, minimal sample preparation and the ability to use advanced optical technologies in the visible or near-infrared spectral range have led in the last years to an increase in the biomedical applications of Raman spectroscopy. Raman spectroscopy measures the inelastic scattering of light by vibrating molecules, providing the chemical fingerprints of cells, tissues or biofluids. However, as Raman scattering efficiency is quite low, requiring relatively long acquisition times, new technologies based on Broadband Coherent Anti-Stokes Raman scattering (BCARS) was proposed [1]. This coherent Raman imaging technique is based on the detection of the fingerprint region of molecules (500-1800 cm-1), as well as the higher-energy CH-/OH-stretch region (~2,700 cm−1 to 3,300 cm−1) and could probe multiple Raman transitions simultaneously to allow imaging of biological tissues with improved molecular contrast. When compared to spontaneous Raman microscopy, BCARS microscopy provided 10-100x faster image acquisition for quantitative and qualitative assessment of pharmaceuticals at much higher spatiochemical resolution and with spectra of much higher signal-to noise ratio [2]. In this talk, I will present the principles of the BCARS microscopy we’ve developed in our laboratory and some examples of biological applications. BCARS imaging is used to evaluate changes underlying the lesion of a spinal cord injury in a murine model. After injury the axonal regeneration in spinal cord is inhibited by a glial and a fibrous scar, acting as a physical and chemical barrier. Recently we have reported that the signal exhibited by fibrillar collagen enabled to specifically monitor it as a biomarker of a spinal cord lesion [3]. However, the injury and the scarring process involves also other elements as the myelin sheets, microglia, astrocytes and extracellular matrix components. We demonstrate the versatility of BCARS imaging to assess the additional structural and metabolic modifications of the injured tissue in excised murine spinal cord samples at different time points (1, 2, 4, 6 weeks) after injury. 1) Camp Jr CH, Lee YJ, Heddleston JM et al. High-speed coherent Raman fingerprint imaging of biological tissues. Nat. Photonics 8(8), 627–634 (2014).2) Hartshorn CM, Lee YJ, Camp Jr CH et al. Multicomponent chemical imaging of pharmaceutical solid dosage forms with broadband CARS microscopy. Anal. Chem. 85(17), 8102–8111 (2013)3) C. Manesco, O. Saavedra-Villanueva, M. Martin, J.de Lizaraga, B.Varga, T. Cloitre, Y.N. Gerber, F.E. Perrin, C. Gergely. Organization of collagen fibers and tissue hardening: Markers of fibrotic scarring after spinal cord injury in mice revealed by multiphoton-atomic force microscopy imaging. Nanomedicine: Nanotechnology, Biology, and Medicine 53 (2023) 102699.