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dc.contributor.authorYoung, Laurenceen
dc.contributor.authorStröhl, Florianen
dc.contributor.authorKaminski, Clemensen
dc.date.accessioned2015-11-16T12:04:14Z
dc.date.available2015-11-16T12:04:14Z
dc.date.issued2016-05-30en
dc.identifier.citationJournal of Visualized Experiments: JoVE (2016) Issue 111. doi:10.3791/53988.en
dc.identifier.issn1940-087X
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/252608
dc.description.abstractOptical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.
dc.languageEnglishen
dc.language.isoenen
dc.publisherMYJoVE Corporation
dc.rightsAttribution 4.0 International
dc.rightsAttribution 4.0 Internationalen
dc.rightsAttribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectColoren
dc.subjectMicroscopy, Fluorescenceen
dc.titleA Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors.en
dc.typeArticle
prism.issueIdentifier111en
prism.publicationDate2016en
prism.publicationNameJournal of Visualized Experimentsen
dc.rioxxterms.funderEPSRC
dc.rioxxterms.funderWellcome Trust
dc.rioxxterms.funderMRC
dc.rioxxterms.projectidEP/H018301/1
dc.rioxxterms.projectidEP/G037221/1
dc.rioxxterms.projectid089703/Z/09/Z
dc.rioxxterms.projectidMR/K015850/1
dc.rioxxterms.projectidMR/K02292X/1
dcterms.dateAccepted2015-10-26en
rioxxterms.versionofrecord10.3791/53988en
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserveden
rioxxterms.licenseref.startdate2016-05-30en
dc.contributor.orcidKaminski, Clemens [0000-0002-5194-0962]
dc.identifier.eissn1940-087X
rioxxterms.typeJournal Article/Reviewen
pubs.funder-project-idEPSRC (EP/H018301/1)
pubs.funder-project-idMRC (MR/K02292X/1)
pubs.funder-project-idMRC (MR/K015850/1)
pubs.funder-project-idWellcome Trust (089703/Z/09/Z)
pubs.funder-project-idEPSRC (EP/G037221/1)


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International