Principles and applications of expansion stimulated Raman scattering microscopy

Abstract Stimulated Raman Scattering (SRS) microscopy provides rapid, label-free chemical contrast based on molecular vibrations, but diffraction limits its resolution to approximately 300 nm – insufficient for nanoscale cellular architectures. This reviews Expansion Stimulated Raman Scattering (ExSRS), which couples SRS with Expansion Microscopy (ExM) to physically magnify specimens within a swellable hydrogel, bringing vibrational imaging below the diffraction limit using conventional optics. We frame ExSRS as an integrated pipeline in which sample chemistry and optical readout are interdependent: the hydrogel must preserve the biomolecules that generate Raman contrast, driving the development of protein-retentive protocols such as VISTA and the broadly compatible anchoring chemistry of MAGNIFIERS. A central trade-off governs the platform – resolution improves linearly with expansion while signal dilutes cubically – making sensitivity engineering and rigorous deformation metrology essential for quantitative nanoscale claims. Current implementations achieve effective lateral resolutions from approximately 78 nm to roughly 40 nm, and hyperspectral acquisition combined with silent-region Raman tags enables multiplexed molecular mapping. Looking ahead, we identify fingerprint-region access, standardized distortion calibration, and scalable multiplexing as key frontiers that will determine whether ExSRS matures into a broadly deployed tool for three-dimensional nanoscale chemical imaging in biology and translational research.