Repository logo

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



Change log


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


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.



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


Volume Title



Public Library of Science (PLoS)
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).