TY  - JOUR
A1  - Palayret, Matthieu
A1  - Armes, Helen
A1  - Basu, Srinjan
A1  - Watson, Adam T.
A1  - Herbert, Alex
A1  - Lando, David
A1  - Etheridge, Thomas J.
A1  - Endesfelder, Ulrike
A1  - Heilemann, Mike
A1  - Laue, Ernest
A1  - Carr, Antony M.
A1  - Klenerman, David
A1  - Lee, Steven F.
T1  - Virtual-"light-sheet" single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging
T2  - PLoS one
N2  - 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.
Y1  - 2015
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/37382
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-373820
SN  - 1932-6203
N1  - 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
VL  - 10
IS  - (4): e0125438
PB  - PLoS
CY  - Lawrence, Kan.
ER  -