Abstract
Studies of lateral diffusion are used for the characterization of the dynamics of biological membranes. One of the techniques that can be used for this purpose is fluorescence correlation spectroscopy (FCS), which belongs to the single-molecule techniques. Unfortunately, FCS measurements, when performed in planar lipid systems, are associated with a few sources of inaccuracy in the determination of the lateral diffusion coefficient. The main problems are related to the imperfect positioning of the laser focus relative to the plane of the sample. Another source of inaccuracy is the requirement for external calibration of the detection volume size. This protocol introduces a calibration-free method called Z-scan fluorescence correlation spectroscopy (Z-scan FCS), which is based on the determination of the diffusion time and particle number in steps along the optical (z-) axis by sequential FCS measurements. Z-scan FCS could be employed for diffusion measurements in planar membrane model systems—supported phospholipid bilayers (SPBs) and giant unilamellar vesicles (GUVs) and also in biological membranes. A result from measurements in SPBs is also presented in the protocol as a principle example of the Z-scan technique.
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Acknowledgements
The authors thank Martin Štefl for his comments and suggestions and collaborators from Institute of Organic Chemistry and Biochemistry of ASCR for providing halictine peptide. This work was supported by Czech Science Foundation through Grant No. P208/10/0376 and by Ministry of Education, Youth and Sports of the Czech Republic via BIO-OPT-XUV Research Team Advancement at the Faculty of Biomedical Engineering, Czech Technical University in Prague, registration nr.: MEYS ESF Project CZ.1.07/2.3.00/20.0092. M.H. acknowledges financial support by AS CR via Praemium Academiae award.
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Steinberger, T., Macháň, R., Hof, M. (2014). Z-Scan Fluorescence Correlation Spectroscopy as a Tool for Diffusion Measurements in Planar Lipid Membranes. In: Engelborghs, Y., Visser, A. (eds) Fluorescence Spectroscopy and Microscopy. Methods in Molecular Biology, vol 1076. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-649-8_28
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DOI: https://doi.org/10.1007/978-1-62703-649-8_28
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