Label-free Imaging of Single-Biomolecule Structure and Interaction by Stimulated Raman Photothermal Encoded...

Current single molecule methods either rely on fluorescence or lack chemical information. Here we report stimulated Raman photothermal encoded scattering (SRPSCAT) microscopy for quantitative bond-selective imaging of single-biomolecule structures and interactions in native environments. In this approach, scattering of the target molecule is modulated by the deposited energy from stimulated Raman gain and loss processes, thereby encoding vibrational spectroscopic information. Leveraging single-molecule sensitivity of interferometric scattering, SRPSCAT can map single proteins with chemical specificity, determine their mass, and distinguish protein secondary structures based on their Raman fingerprints. Furthermore, single protein binding kinetics are quantified and the conformational dynamics of single de novo designed allosteric proteins are observed. Together, these results highlight the potential of SRPSCAT for label-free structural, functional and dynamic analysis at the single-molecule level.