Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.
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 us...
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doaj-c2c023d829814008afe5d2b304949d1b2020-11-25T01:22:06ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01104e012543810.1371/journal.pone.0125438Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.Matthieu PalayretHelen ArmesSrinjan BasuAdam T WatsonAlex HerbertDavid LandoThomas J EtheridgeUlrike EndesfelderMike HeilemannErnest LaueAntony M CarrDavid KlenermanSteven 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.http://europepmc.org/articles/PMC4401716?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
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 |
spellingShingle |
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 Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging. PLoS ONE |
author_facet |
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 |
author_sort |
Matthieu Palayret |
title |
Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging. |
title_short |
Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging. |
title_full |
Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging. |
title_fullStr |
Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging. |
title_full_unstemmed |
Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging. |
title_sort |
virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2015-01-01 |
description |
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. |
url |
http://europepmc.org/articles/PMC4401716?pdf=render |
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