Spectrally Focused Stimulated Raman Scattering (sf-SRS) Microscopy for Label-Free Investigations of Molecular Mechanisms in Living Organisms

Abstract

Stimulated Raman Scattering (SRS) microscopy is a light-based non-linear imaging method for visualizing a molecule based on its chemical properties, i.e., the vibrational energy states reflecting the molecule’s structure and its environment. This technique, relying on the specificity of the molecule’s spectral fingerprint, enables label-free, high-sensitivity, and high-resolution 3D reconstruction of the distribution and the properties of a molecule within a tissue. Despite its tremendous potentials, the application of SRS is still not frequent in the field of life science, where it could be applied over an extremely broad investigation range, from the study of the molecular interactions at subcellular level to the characterization of tissue alterations in clinical studies. Trying to fill this gap, here, after describing the general principles of SRS, we present the materials and the methods to integrate spectrally focused Stimulated Raman Spectroscopy (sf-SRS) on commercial multiphoton microscopes and highlight the critical aspects to consider.