Wide-field subdiffraction imaging by accumulated binding of diffusing probes

TLDR

The method exploits the constant flux of diffusing probes to enable examination of an essentially unlimited number of individual probe molecules. It introduces a subdiffraction imaging technique that accumulates points through collisional flux. The approach targets object surfaces with fluorescent probes that diffuse in solution, immobilizes each probe upon collision, and localizes it precisely by replacing its point‑spread function with its centroid. The technique produced high‑resolution images of lipid bilayers and vesicles with ~25 nm spatial resolution, demonstrating rapid nanoscale imaging below the Rayleigh limit without the need for fluorescent labeling.

Abstract

A method is introduced for subdiffraction imaging that accumulates points by collisional flux. It is based on targeting the surface of objects by fluorescent probes diffusing in the solution. Because the flux of probes at the object is essentially constant over long time periods, the examination of an almost unlimited number of individual probe molecules becomes possible. Each probe that hits the object and that becomes immobilized is located with high precision by replacing its point-spread function by a point at its centroid. Images of lipid bilayers, contours of these bilayers, and large unilamellar vesicles are shown. A spatial resolution of approximately 25 nm is readily achieved. The ability of the method to effect rapid nanoscale imaging and spatial resolution below Rayleigh criterion and without the necessity for labeling with fluorescent probes is proven.