Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging

Palayret, Matthieu, Armes, Helen, Basu, Srinjan, Watson, Adam T, Herbert, Alex, Lando, David, Etheridge, Thomas J, Endesfelder, Ulrike, Heilemann, Mike, Laue, Ernest, Carr, Antony M, Klenerman, David, Lee, Steven F and Unset (2015) Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging. PLoS ONE, 10 (4). e0125438. ISSN 1932-6203

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Abstract

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.

Item Type: Article
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Depositing User: Antony Carr
Date Deposited: 15 Jan 2016 14:56
Last Modified: 07 Mar 2017 05:31
URI: http://sro.sussex.ac.uk/id/eprint/59224

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Project NameSussex Project NumberFunderFunder Ref
Single Molecule Imaging of the DNA Damage Response in Live CellsG0250EUROPEAN UNION268788