Label-Free Imaging of Single Proteins and Binding Kinetics Using Total Internal Reflection-Based Evanescent Scattering Microscopy

Single-molecule detection can push beyond ensemble averages and reveal the statistical distributions of molecular properties. Measuring the binding kinetics of single proteins also represents one of the critical and challenging tasks in protein analysis. Here, we report total internal reflection-based evanescent scattering microscopy with label-free single-protein detection capability. Total internal reflection is employed to excite the evanescent field to enhance light–analyte interaction and reduce environmental noise. As a result, the system provides wide-field imaging capability and allows excitation and observation using one objective. In addition, this system quantifies protein binding kinetics by simultaneously counting the binding of individual molecules and recording their binding sites with nanometer precision, providing a digital method to measure binding kinetics with high spatiotemporal resolution. This approach does not employ specially designed microspheres or nanomaterials and may pave a way for label-free single-protein analysis in conventional microscopy.