Abstract
We present a fundamentally new approach to 3D superresolution microscopy based on the principle of surface-generated fluorescence. This near-field fluorescence is strongly dependent on the distance of fluorophores from the coverslip and can therefore be used to estimate their axial positions. We established a robust and simple implementation of supercritical angle fluorescence detection for single-molecule localization microscopy, calibrated it using fluorescent bead samples, validated the method with DNA origami tetrahedra, and present proof-of-principle data on biological samples.
Highlights
Two general approaches allow for three-dimensional resolution in localization microscopy
The z position of single emitters can be determined from the shape of the point spread function (PSF) either by imaging two focal planes or after PSF engineering
The axial resolution is usually worse than the lateral one, which itself is compromised by the modification of the PSF
Summary
Two general approaches allow for three-dimensional resolution in localization microscopy. The z position of single emitters can be determined from the shape of the point spread function (PSF) either by imaging two focal planes (bi-plane) or after PSF engineering (e.g. astigmatic PSF, double helix PSF or self-bending PSF).
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