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New type of Raman microscopy reported

Fluorescence suppression enables coherent Stokes Raman scattering microscopy

by Celia Henry Arnaud, special to C&EN
June 23, 2023

Image shows four panels of magnified human skin,
Credit: Nature Comms.
CSRS microscopy of human skin. At one demodulation frequency, the CSRS signal is mixed with significant fluorescence background. At another demodulation frequency, the fluorescence background is suppressed.

Nonlinear Raman microscopy techniques, such as coherent anti-Stokes Raman scattering and stimulated Raman scattering, are widely used to provide chemical and spatial information. A third nonlinear Raman microscopy method, based on coherent Stokes Raman scattering, has never been demonstrated because researchers thought its larger fluorescence background made it impractical. Sandro Heuke and Hervé Rigneault, researchers with the French National Center for Scientific Research at the Fresnel Institute, have now achieved coherent Stokes Raman scattering microscopy (CSRS, pronounced “scissors”).

Heuke and Rigneault combine two approaches to suppress the fluorescence in CSRS (Nat. Commun. 2023, DOI: 10.1038/s41467-023-38942-4). First, they use two tilted narrow-bandpass filters that transmit sharp signal bands and reject the fluorescence. Second, they modulate the two excitation laser beams and demodulate the signal beam to discriminate between the CSRS signal and the fluorescence background. They used CSRS to image various samples, including olive oil, human skin, onion, avocado, and a fruit fly larva’s wing disc.

The researchers also calculated, but have not yet experimentally demonstrated, that conservation of momentum enables CSRS to work in a configuration that collects the signal in what the researchers call the backward direction. Other nonlinear Raman microscopy approaches lose much of their signal when collected in a similar way. Such a setup might enable easier analysis of thick samples and the development of more efficient endoscopy based on CSRS.

“There is a lot of interest in label-free imaging using the vibrations of molecules,” Zachary D. Schultz, an expert on Raman imaging at Ohio State University, writes in an email. “This is a nice addition to this area of research.”



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