Multicolor Fluorescence Nanoscopy by Photobleaching: Concept Verification and its Application to Resolve Selective Storage of Proteins in Platelets
2014 (English)In: ACS Nano, ISSN 1936-0851, Vol. 8, no 5, 4358-4365 p.Article in journal (Refereed) Published
Fluorescence nanoscopy provides means to discernthe finer details of protein localization and interaction in cells by offeringan order of magnitude higher resolution than conventional optical imagingtechniques. However, these super resolution techniques put higher demands onthe optical system as well as on the fluorescent probes, making multicolorfluorescence nanoscopy a challenging task. Here we present a new and simpleprocedure which exploits the photostability and excitation spectra of dyes toincrease the number of simultaneous recordable targets in STED nanoscopy. Weuse this procedure to demonstrate four color STED imaging of platelets with ≤40 nm resolution and low crosstalk. Platelets can selectively store, sequesterand release a multitude of different proteins, and in a manner specific fordifferent physiological and disease states. By applying multicolor nanoscopy tostudy platelets, we can achieve spatial mapping of the protein organizationwith a high resolution, for multiple proteins at the same time and in the samecell. This provides a means to identify specific platelet activation states fordiagnostic purposes and to understand the underlying protein storage andrelease mechanisms. We studied the organization of the pro- and anti-angiogenicproteins VEGF and PF-4 together with fibrinogen and filamentous actin, andfound distinct features in their respective protein localization. Further,colocalization analysis revealed only minor overlap between the proteins VEGFand PF-4 indicating that they have separate storage and release mechanisms,corresponding well with their opposite rules as pro- and anti-angiogenicproteins, respectively.
Place, publisher, year, edition, pages
2014. Vol. 8, no 5, 4358-4365 p.
multicolor, super resolution microscopy, photobleaching, STED, platelets, thrombocytes, α-granules
Research subject Biological Physics
IdentifiersURN: urn:nbn:se:kth:diva-141008DOI: 10.1021/nn406113mISI: 000336640600026ScopusID: 2-s2.0-84901649796OAI: oai:DiVA.org:kth-141008DiVA: diva2:693793
FunderEU, FP7, Seventh Framework Programme, 801237
Updated from "Submitted" to "Published". QC 201406302014-02-052014-02-052014-06-30Bibliographically approved