title: Dense small molecule labeling enables activator-dependent STORM by proximity mapping.
abstract: 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.
authors.affiliation: Immune Signalling Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia.|Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, VIC, Australia.
authors.forename: Ye
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, VIC, Australia.|Artificial-Intelligence Nanophotonics Laboratory, School of Science, RMIT University, Melbourne, VIC, 3001, Australia.
authors.forename: Min
authors.initials: M
authors.lastname: Gu
authors.affiliation: School of Medical Sciences, UNSW Australia, Sydney, NSW, 2052, Australia.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Gunning
authors.affiliation: Immune Signalling Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia. Sarah.russell@petermac.org.|Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, VIC, Australia. Sarah.russell@petermac.org.|Department of Pathology, The University of Melbourne, Parkville, VIC, Australia. Sarah.russell@petermac.org.|Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia. Sarah.russell@petermac.org.
authors.forename: Sarah M
authors.identifier: http://orcid.org/0000-0001-5826-9641
authors.initials: SM
authors.lastname: Russell
mesh_terms: D000199:Actins / Q000032:analysis / Q000737:chemistry*; D002478:Cells, Cultured; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D008856:Microscopy, Fluorescence; D010590:Phalloidine / Q000032:analysis / Q000737:chemistry*; D013194:Staining and Labeling; D013269:Stochastic Processes
chemical_list: D000199:Actins; D010590:Phalloidine
keywords: Actin; Multi-channel imaging; Single-molecule localization microscopy; Small molecule labels; Super-resolution imaging