Wide-field subdiffraction imaging by accumulated binding of diffusing probes

doi:10.1073/pnas.0609643104

Wide-field subdiffraction imaging by accumulated binding of diffusing probes

Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323

Contributed by Robin M. Hochstrasser, October 30, 2006 (received for review October 11, 2006)

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 ≈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.

Footnotes

*To whom correspondence should be addressed. E-mail: hochstra{at}sas.upenn.edu
Author contributions: A.S. and R.M.H. designed research; A.S. and R.M.H. performed research; A.S. and R.M.H. analyzed data; and A.S. and R.M.H. wrote the paper.
The authors declare no conflict of interest.
Abbreviations:

LUV,

large unilamellar vesicle;

GUV,

giant unilamellar vesicle;

PSF,

point-spread function;

POPC,

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine;

PAINT,

points accumulation for imaging in nanoscale topography.