Abstract
Fluorescence correlation spectroscopy (FCS) is a powerful technique used to measure diffusion, fluctuations, and other transport processes in biomolecular systems. It is, however, prone to artifacts and subject to considerable experimental difficulties when applied to living cells. In this chapter, we provide protocols to conduct quantitative FCS measurements on DNA inside living eukaryotic and prokaryotic cells. We discuss sample preparation, dye selection and characterization, FCS data acquisition, and data analysis, including a method to com pensate for photobleaching to obtain quantitatively meaningful spectra.
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Acknowledgments
This work was funded through internal resources of the University of Michigan. The FCS measurements were carried out at the Single-Molecule Analysis in Real Time (SMART) Center with the help of J. D. Hoff on equipment acquired through NSF MRI-ID grant DBI-0959823 to Nils G. Walter. Michael Jones contributed to the dye characterization measurements. The authors would like to thank Jörg Enderlein from the University of Göttingen for generously sharing computer code for the calculation of correlation functions from photon arrival times with us.
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Hodges, C., Meiners, JC. (2018). Fluorescence Correlation Spectroscopy on Genomic DNA in Living Cells. In: Lyubchenko, Y. (eds) Nanoscale Imaging. Methods in Molecular Biology, vol 1814. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8591-3_25
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DOI: https://doi.org/10.1007/978-1-4939-8591-3_25
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