Abstract
Single-molecule measurements provide statistical distributions of molecular properties, in addition to the ensemble averages. Evanescent detection approaches have been widely used for single-molecule detection because the evanescent field can significantly enhance the light-analyte interaction and reduce the background noise. However, current evanescent single-molecule detection systems mostly require specially designed sensing components. Here, we show that single proteins can be imaged on a plain cover glass surface by detecting the evanescent waves scattered by the target molecules. This allows us to quantify the protein-antibody interactions at the single-molecule level. This protocol describes a label-free single-molecule imaging approach with conventional consumables and may pave the road for detecting single molecules with commercial optical microscopy.