Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Abstract

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.

Raman micro-spectroscopy reveals the spatial distribution of fumarate in cells and tissues

“…Future studies that require lower LODs or faster scanning could adopt coherent Raman techniques such as stimulated (photothermal) Raman spectroscopy (SRS) 48 , coherent anti-Stokes Raman spectroscopy (CARS) 49 or surface-enhanced Raman spectroscopy (SERS) [50][51][52][53] , which provide resonant signal-enhancements and fluorescencebackground suppression 54 . Although coherent Raman techniques require more specialist microscopy equipment, they also afford substantially greater sensitivity and imaging speed compared to conventional spontaneous Raman microscopy, offering a promising route to enable broader metabolite imaging and quantification using the Raman scattering process.…”