Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.
Bibliographical noteKAUST Repository Item: Exported on 2022-09-14
Acknowledged KAUST grant number(s): OSR-2016-CRG5-3017-01
Acknowledgements: D. P. acknowledges funding from the European Union project CRIMSON under Grant Agreement No. 101016923 and the Regione Lombardia project NEWMED under Grant Agreement No. POR FESR 2014-2020. G. C. acknowledges funding from the European Union project GRAPHENE Core3 under grant agreement number 881603. G. C. also acknowledges funding from King Abdullah University of Science and Technology, Grant Award Number: OSR-2016-CRG5-3017-01.
This publication acknowledges KAUST support, but has no KAUST affiliated authors.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Immunology and Microbiology(all)
- Chemical Engineering(all)