Discrete frequency chemical imaging with stimulated Raman scattering microscopy

Chemical imaging, the process of using chemically-specific label-free light-matter interactions as a contrast mechanism for imaging or microscopy, is a powerful set of tools for performing investigations where the distribution of chemical constituents within a specimen is of importance. This can include the locations of distinct cell types within a tissue biopsy, the distribution of oriented molecules within a polymer sample, or the concentration of a dissolved analyte in a fluidic system. Coherent Raman scattering (CRS) spectroscopies have gained increasing attention in recent years, as they represent a class of techniques which affords high-resolution, z-stack capable, not-perturbative, rapid chemical imaging. Stimulated Raman scattering (SRS) microscopy is particularly attractive because a linear response to analyte concentration allows for quantitative investigation. Unlike more traditional vibrational spectroscopic techniques such as Fourier-transform infrared (FT-IR) absorption and spontaneous Raman scattering, CRS instruments are often operated in a single-frequency or limited bandwidth fashion and investigate only one small piece of the specimen’s vibrational spectrum at any given time. This difference has implications for experimental design, imaging protocols, and subsequent data analysis. Nevertheless, the increasing interest in and apparent utility of these tools is driving many implementations of chemical imaging towards this ‘discrete-frequency’ approach. Here, we describe the construction and deployment of an SRS microscope, followed by the evaluation of this technology as a tool for the label-free classification of tissue biopsies. Additionally, we explore applications which are better-suited to the specific strengths of this imaging modality, namely those which benefit from 3D volumetric imaging or the investigation of aqueous systems, both of which are not achievable with most implementations of infrared absorption measurements