title: Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.
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.
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Palayret
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom; Genome Damage and Stability Centre, University of Sussex, Falmer, Sussex, BN1 9RQ, United Kingdom.
authors.forename: Helen
authors.initials: H
authors.lastname: Armes
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, United Kingdom.
authors.forename: Srinjan
authors.initials: S
authors.lastname: Basu
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Sussex, BN1 9RQ, United Kingdom.
authors.forename: Adam T
authors.initials: AT
authors.lastname: Watson
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Sussex, BN1 9RQ, United Kingdom.
authors.forename: Alex
authors.initials: A
authors.lastname: Herbert
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Lando
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Sussex, BN1 9RQ, United Kingdom.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Etheridge
authors.affiliation: Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 7, 60438, Frankfurt, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Endesfelder
authors.affiliation: Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 7, 60438, Frankfurt, Germany.
authors.forename: Mike
authors.initials: M
authors.lastname: Heilemann
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, United Kingdom.
authors.forename: Ernest
authors.initials: E
authors.lastname: Laue
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Sussex, BN1 9RQ, United Kingdom.
authors.forename: Antony M
authors.initials: AM
authors.lastname: Carr
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Klenerman
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.
authors.forename: Steven F
authors.initials: SF
authors.lastname: Lee
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells; D021621:Imaging, Three-Dimensional / Q000379:methods; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000295:instrumentation / Q000379:methods*; D029702:Schizosaccharomyces pombe Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics / Q000378:metabolism
chemical_list: D001324:Autoantigens; D018797:Cell Cycle Proteins; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C492013:Mbd3 protein, mouse; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C080926:cdc22 protein, S pombe