Dense small molecule labeling enables activator-dependent STORM by proximity mapping
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Stochastic optical reconstruction microscopy (STORM) enables high-resolution imaging, but multi-channel 3D imaging is problematic because of chromatic aberrations and alignment errors. The use of activator-dependent STORM in which spectrally distinct activators can be coupled with a single reporter can circumvent such issues. However, the standard approach of linking activators and reporters to a single antibody molecule is hampered by low labeling density and the large size of the antibody. We proposed that small molecule labels might enable activator-dependent STORM if the reporter or activator were linked to separate small molecules that bound within 3.5 nm of each other. This would greatly increase the labeling density and therefore improve resolution. We tested various mixtures of phalloidin- or mCling-conjugated fluorophore to demonstrate this feasibility. The specific activation was dependent on the choice of activator, its density, a matching activating laser and its power. In addition to providing an effective means of multi-channel 3D STORM imaging, this method also provides information about the local proximity between labels, potentially enabling super-resolved mapping of the conformation of the labeled structures.
KeywordsSuper-resolution imaging Single-molecule localization microscopy Actin Small molecule labels Multi-channel imaging
We thank Sara Jones (Harvard University) for technical advice on STORM and Andrew Clayton (Swinburne University of Technology) for helpful discussions. The work was supported by a project grant from the Australian National Health and Medical Research Foundation (APP1061647), the Kids Cancer Project to PWG, and fellowships to SMR from the Australian Research Council (Future Fellowship) and the Australian National Health and Medical Research Foundation (Senior Research Fellowship).
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Conflict of interest
None of the authors has a conflict of interest related to this work.
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