Abstract
Fluorescence recovery after photobleaching (FRAP) is one of the most useful microscopy techniques for studying the mobility of molecules in terms of a diffusion coefficient. Here, we describe a FRAP method that allows such measurements, relying on the photobleaching of a rectangular region of any size and aspect ratio. We start with a brief overview of the rectangle FRAP theory, and next we provide guidelines for performing FRAP measurements, including a discussion of the experimental setup and the data analysis. Finally, we discuss how to verify correct use of the rectangle FRAP method using test solutions.
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Acknowledgements
Financial support by the Ghent University Special Research Fund (Centre for Nano- and Biophotonics) is acknowledged with gratitude. Ranhua Xiong gratefully acknowledges the financial support from China Scholarship Council (CSC). Hendrik Deschout is a doctoral fellow of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT), Belgium.
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Xiong, R., Deschout, H., Demeester, J., De Smedt, S.C., Braeckmans, K. (2014). Rectangle FRAP for Measuring Diffusion with a Laser Scanning Microscope. 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_18
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DOI: https://doi.org/10.1007/978-1-62703-649-8_18
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