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

PLoS One. 2015 Apr 17;10(4):e0125438. doi: 10.1371/journal.pone.0125438. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autoantigens / analysis
  • Autoantigens / genetics
  • Autoantigens / metabolism
  • Cell Cycle Proteins / analysis
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cells, Cultured
  • Centromere Protein A
  • Chromosomal Proteins, Non-Histone / analysis
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism
  • DNA-Binding Proteins / analysis
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Embryonic Stem Cells
  • Imaging, Three-Dimensional / methods
  • Mice
  • Microscopy, Fluorescence / methods*
  • Molecular Imaging / instrumentation
  • Molecular Imaging / methods*
  • Schizosaccharomyces pombe Proteins / analysis
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism
  • Transcription Factors / analysis
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • Autoantigens
  • Cell Cycle Proteins
  • Centromere Protein A
  • Chromosomal Proteins, Non-Histone
  • DNA-Binding Proteins
  • Mbd3 protein, mouse
  • Schizosaccharomyces pombe Proteins
  • Transcription Factors
  • cdc22 protein, S pombe