Single Molecule Fluorescence Microscopy on Planar Supported Bilayers.

M Axmann, GJ Schütz, JB Huppa

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cellcell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile proteinfunctionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Original languageEnglish
Article numbere53158
Pages (from-to)e53158
JournalJournal of visualized experiments : JoVE
Volume2015
Issue number104
DOIs
Publication statusPublished - 31 Oct 2015

Keywords

  • Bioengineering
  • Fluorescence recovery after photo-bleaching
  • Issue 104
  • Laser
  • Planar glass-supported lipid bilayer
  • Single molecule microscopy
  • Small/large unilamellar vesicles
  • Total internal reflection microscopy
  • Proteins/analysis
  • Glass/chemistry
  • Exocytosis
  • Molecular Imaging/methods
  • Lipid Bilayers/chemistry
  • Cell Membrane/chemistry
  • Microscopy, Fluorescence/methods

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