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:	03 Jul 2019 01:24
URI:	http://sro.sussex.ac.uk/id/eprint/59224

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