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


Type

Article

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Authors

Palayret, Matthieu 
Armes, Helen 
Watson, Adam T 
Herbert, Alex 

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.

Description

Keywords

Animals, Autoantigens, Cell Cycle Proteins, Cells, Cultured, Centromere Protein A, Chromosomal Proteins, Non-Histone, DNA-Binding Proteins, Embryonic Stem Cells, Imaging, Three-Dimensional, Mice, Microscopy, Fluorescence, Molecular Imaging, Schizosaccharomyces pombe Proteins, Transcription Factors

Journal Title

PLoS One

Conference Name

Journal ISSN

1932-6203
1932-6203

Volume Title

10

Publisher

Public Library of Science (PLoS)
Sponsorship
Wellcome Trust (082010/Z/07/Z)
Biotechnology and Biological Sciences Research Council (BB/K013726/1)
The Royal Society (uf120277)
Medical Research Council (MR/K015850/1)
Wellcome Trust (093756/Z/10/Z)
We thank the Wellcome Trust for the PhD studentship of MP (093756/B/10/Z), and the Royal Society for the University Research Fellowship of SFL (UF120277). The work by SB and DL was also funded by the Wellcome Trust (082010/Z/07/Z). UE and MH acknowledge funding by the German Science Foundation (grants EXC 115 and SFB 902). SB is funded by a BBSRC grant (BB/K013726/1). AMC acknowledges ERC Award 268788-SMI-DDR. We also thank the European Commision for support through the 4DCellFate project (EC FP7 CP 277899).