Imaging Single Molecules vs. Single-Molecule Imaging: Counting Single Molecules using Dextran-Streptavidin-Antibody Clusters (DSA)

Abstract

Single-molecule imaging using TIRF has become a routine technique in many biophysical applications. However, a complex instrument setup, cumbersome surface treatment procedures and stringent requirements for sample preparation pose a challenge. For the case of digital counting, some single-molecule imaging approaches can be achieved using tools tuned for single-molecule detection. Here, we demonstrate a simple method for imaging a single target molecule using an epi-fluorescence wide-field microscope. Polystyrene magnetic microparticles instead of a specially treated glass were used as the surface for target immobilization. Such antibody-coated particles are easy to manipulate and can be stored for an extended period of time. Conventionally, detection of target molecules is performed with fluorescently-labeled reporter antibody. However, the autofluorescence of many commercially available microparticles dominates over even the brightest fluorophores and interferes with imaging. To overcome this limitation, we enhance the brightness of the reporter by preparing biotinylated antibodies complexed with a dextran-streptavidin scaffold. Subsequently, we detect this antibody assembly using multiple streptavidin-phycoerythrin (PE) conjugates. The resulting signal increase, driven by numerous PE molecules, overcomes the background and allows for direct detection of a single target molecule. Thus, imaging of a single antibody sandwich can be performed on microparticles using multi-molecular fluorescent conjugates.