Convex lens-induced confinement for imaging single molecules.

Fluorescence imaging is used to study the dynamics of a wide variety of single molecules in solution or attached to a surface. Two key challenges in this pursuit are (1) to image immobilized single molecules in the presence of a high level of fluorescent background and (2) to image freely diffusing single molecules for long times. Strategies that perform well by one measure often perform poorly by the other. Here, we present a simple modification to a wide-field fluorescence microscope that addresses both challenges and dramatically improves single-molecule imaging. The technique of convex lens-induced confinement (CLIC) restricts molecules to a wedge-shaped gap of nanoscale depth, formed between a plano-convex lens and a planar coverslip. The shallow depth of the imaging volume leads to 20-fold greater rejection of background fluorescence than is achieved with total internal reflection fluorescence (TIRF) imaging. Elimination of out-of-plane diffusion leads to an approximately 10,000-fold longer diffusion-limited observation time per molecule than is achieved with confocal fluorescence correlation spectroscopy. The CLIC system also provides a new means to determine molecular size. The CLIC system does not require any nanofabrication, nor any custom optics, electronics, or computer control.