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M. Axmann, G. Schütz, J. Huppa:
"Single Molecule Fluorescence Microscopy on Planar Supported Bilayers";
Journal of Visualized Experiments, 104 (2015), 531581 - 5315816.

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.

Bioengineering, Issue 104, Small/Large Unilamellar Vesicles, Planar Glass-Supported Lipid Bilayer, Laser, Total Internal Reflection Microscopy, Fluorescence Recovery after Photo-Bleaching, Single Molecule Microscopy