Journal
PLOS ONE
Volume 10, Issue 4, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0125438
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Categories
Funding
- Wellcome Trust [093756/B/10/Z, 082010/Z/07/Z]
- Royal Society [UF120277]
- German Science Foundation [EXC 115, SFB 902]
- BBSRC [BB/K013726/1]
- European Commission [EC FP7 CP 277899]
- ERC [268788-SMI-DDR]
- Biotechnology and Biological Sciences Research Council [BB/K013726/1] Funding Source: researchfish
- Medical Research Council [MR/K015850/1] Funding Source: researchfish
- BBSRC [BB/K013726/1] Funding Source: UKRI
- MRC [MR/K015850/1] Funding Source: UKRI
- Wellcome Trust [082010/Z/07/Z] Funding Source: Wellcome Trust
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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.
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