Wide-field subdiffraction imaging by accumulated binding of diffusing probes

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.

Fig. 1.

The total integrated intensity vs. time for a single vesicle probed by Nile red. The intermittency is caused by fluorescence bursts of Nile red molecules undergoing collisions with the lipid bilayer.

Fig. 2.

Imaging of vesicles. Fluorescence image (a) and synthetic high-resolution image (b) of vesicles attached to a glass surface. The synthetic image, obtained as described in the text, displays the average coordinates of single fluorophores. (c and d) Fluorescence and synthetic images of two vesicles with a center-to-center distance of ≈200 nm.

Fig. 3.

Spatial distribution of fluorescent spots. (a) Example of a density distribution of fluorescent spots around the center of a vesicle. The solid line represents a Gaussian fit with σ = 28 nm. (b) Histogram of distribution widths obtained as in a for 185 vesicles. (c) Distribution of points density obtained by simulations for 50-nm-radius disks without any diffusive motion of the probes (dots) and with diffusion (D = 3 μm²/sec) for different residence times of 1 (dash), 10 (solid), and 100 (dash-dot) msec.

Fig. 4.

Image of a supported bilayer on glass, probed by Nile red. (a) Conventional fluorescence image. (b) High-resolution synthetic image obtained by locating 2,778 single Nile red probes collected in 4,095 frames.

Fig. 5.

High-resolution synthetic image of the contour of a supported bilayer. Transferrin conjugated with Alexa Fluor 568 creates the image by targeting areas on the glass surface that are not covered by the bilayer (bright regions), whereas the bilayer appears to be unaffected by the flux of labeled proteins (dark area).

Fig. 6.

Stages in the determination of the locations of Nile red molecules on the surface of an LUV. (Left) Stage 1 (see text) determines the x,y coordinates to a precision of ≈50 nm. (Right) After fitting to 2D Gaussian (stage 2), the precision becomes ≈25 nm.