Fingerprint‐to‐CH stretch continuously tunable high spectral resolution stimulated Raman scattering microscope

• Published in: Journal of biophotonics 2019-09, Vol.12 (9), p.e201900028-n/a
• Main Authors: Laptenok, Sergey P., Rajamanickam, Vijayakumar P., Genchi, Luca, Monfort, Tual, Lee, Yeonwoo, Patel, Imran I., Bertoncini, Andrea, Liberale, Carlo
• Format: Article
• Language: English
• Subjects: Biological properties; Biological samples; Broadband; coherent Raman imaging; Fingerprints; Hyperspectral imaging; Image contrast; lipid droplets; Microscopy; Narrowband; Organic chemistry; Quantitative analysis; Raman spectra; Raman spectroscopy; Spectral resolution; Statistical analysis; Statistical methods; stimulated Raman scattering; Tunable filters
• ISSN: 1864-063X; 1864-0648
• DOI: 10.1002/jbio.201900028

Stimulated Raman scattering (SRS) microscopy is a label‐free method generating images based on chemical contrast within samples, and has already shown its great potential for high‐sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio‐samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub‐cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto‐optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon‐Hydrogen (CH)‐stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm−1, is demonstrated. Stimulated Raman Scattering (SRS) microscopy is a sensitive and labelfree method generating images based on chemical contrast within the samples. Here a novel configuration for a widely tunable SRS microscope is presented, which allows a continuous scan from the Fingerprint to the CH stretch regions, with high spectral resolution and without requiring any modification of the optical setup.