Matthieu Palayret, Helen Armes, Srinjan Basu, Adam T. Watson, Alex Herbert, David Lando, Thomas J. Etheridge, Ulrike Endesfelder, Mike Heilemann, Ernest Laue, Antony M. Carr, David Klenerman, Steven F. Lee
- Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.
MetadatenAuthor: | Matthieu Palayret, Helen Armes, Srinjan Basu, Adam T. Watson, Alex Herbert, David Lando, Thomas J. Etheridge, Ulrike EndesfelderORCiDGND, Mike HeilemannORCiDGND, Ernest Laue, Antony M. Carr, David Klenerman, Steven F. Lee |
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URN: | urn:nbn:de:hebis:30:3-373820 |
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DOI: | https://doi.org/10.1371/journal.pone.0125438 |
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ISSN: | 1932-6203 |
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Parent Title (English): | PLoS one |
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Publisher: | PLoS |
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Place of publication: | Lawrence, Kan. |
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Document Type: | Article |
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Language: | English |
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Date of Publication (online): | 2015/04/17 |
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Date of first Publication: | 2015/04/17 |
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Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
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Release Date: | 2015/04/22 |
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Volume: | 10 |
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Issue: | (4): e0125438 |
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Page Number: | 15 |
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Note: | Copyright: © 2015 Palayret et al. This is an open access article distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/ , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited |
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HeBIS-PPN: | 369177096 |
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Institutes: | Biochemie, Chemie und Pharmazie / Biochemie und Chemie |
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Dewey Decimal Classification: | 5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften |
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Sammlungen: | Universitätspublikationen |
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Licence (German): | Creative Commons - Namensnennung 4.0 |
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