Virtual-'Light-Sheet' Single-Molecule Localisation Microscopy Enables Quantitative Optical Sectioning for Super-Resolution Imaging |
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Authors: | 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 |
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Institution: | 1. Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.; 2. Genome Damage and Stability Centre, University of Sussex, Falmer, Sussex, BN1 9RQ, United Kingdom.; 3. Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, United Kingdom.; 4. Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 7, 60438, Frankfurt, Germany.; Tufts University, UNITED STATES, |
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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. |
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