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Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging
journal contribution
posted on 2023-06-09, 00:01 authored by Matthieu Palayret, Helen Armes, Srinjan Basu, Adam WatsonAdam Watson, Alex Herbert, David Lando, Thomas Etheridge, Ulrike Endesfelder, Mike Heilemann, Ernest Laue, Antony M Carr, David Klenerman, Steven F LeeSingle-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.
Funding
Single Molecule Imaging of the DNA Damage Response in Live Cells; G0250; EUROPEAN UNION; 268788
History
Publication status
- Published
File Version
- Published version
Journal
PLoS ONEISSN
1932-6203Publisher
Public Library of ScienceExternal DOI
Issue
4Volume
10Article number
e0125438Department affiliated with
- Sussex Centre for Genome Damage Stability Publications
Full text available
- Yes
Peer reviewed?
- Yes
Legacy Posted Date
2016-01-15First Open Access (FOA) Date
2016-01-15First Compliant Deposit (FCD) Date
2016-01-15Usage metrics
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