Abstract
Our paper presents the first theoretical and experimental study using single-molecule Metal-Induced Energy Transfer (smMIET) for localizing single fluorescent molecules in three dimensions. Metal-Induced Energy Transfer describes the resonant energy transfer from the excited state of a fluorescent emitter to surface plasmons in a metal nanostructure. This energy transfer is strongly distance-dependent and can be used to localize an emitter along one dimension. We have used Metal-Induced Energy Transfer in the past for localizing fluorescent emitters with nanometer accuracy along the optical axis of a microscope. The combination of smMIET with single-molecule localization based super-resolution microscopy that provides nanometer lateral localization accuracy offers the prospect of achieving isotropic nanometer localization accuracy in all three spatial dimensions. We give a thorough theoretical explanation and analysis of smMIET, describe its experimental requirements, also in its combination with lateral single-molecule localization techniques, and present first proof-of-principle experiments using dye molecules immobilized on top of a silica spacer, and of dye molecules embedded in thin polymer films.
Highlights
Single-molecule localization based super-resolution microscopy has become an important tool in biological imaging
The combination of single-molecule Metal-Induced Energy Transfer (smMIET) with single-molecule localization based super-resolution microscopy that provides nanometer lateral localization accuracy offers the prospect of achieving isotropic nanometer localization accuracy in all three spatial dimensions
Stochastic Optical Reconstruction Microscopy (STORM)/direct STORM (dSTORM) primarily improves the resolution in the lateral direction, techniques such as astigmatism-based imaging,3 biplane imaging,4 or helical wavefront shaping5 enabled the generation of images with full three-dimensional resolution
Summary
Single-molecule localization based super-resolution microscopy has become an important tool in biological imaging. STORM/dSTORM primarily improves the resolution in the lateral direction (xy-plane), techniques such as astigmatism-based imaging, biplane imaging, or helical wavefront shaping enabled the generation of images with full three-dimensional resolution. For all these techniques, the achievable axial resolution (50 nm) is still approximately one order of magnitude less than typical intramolecular distances. Until now, they were not much used for structural biology on the single macromolecule level. The most prominent members of this class are interferometric Photoactivated Localization Microscopy (iPALM) and 4pi-STORM. As only one remarkable example of their capability, we cite
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