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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References
Chiantia S, Ries J, Schwille P (2009) Fluorescence correlation spectroscopy in membrane structure elucidation. Biochim Biophys Acta 1788:225–233
Michelman-Ribeiro A, Mazza D, Rosales T, Stasevich TJ, Boukari H, Rishi V, Vinson C, Knutson JR, McNally JG (2009) Direct measurement of association and disassociation rates of DNA binding in live cells by fluorescence correlation spectroscopy. Biophys J 97:337–346
Gröner N, Capoulade J, Cremer C, Wachsmuth M (2010) Measuring and imaging diffusion with multiple scan speed image correlation spectroscopy. Opt Express 18:21225–21236
Kafle RP, Liebeskind MR, Bourg JT, White E, Meiners JC (2015) Artifacts from photobleaching and dye dissociation in fluorescence correlation spectroscopy. Proc SPIE 9548:94581M
Windengreen J, Rigler R (1996) Mechanisms of photobleaching investigated by fluorescence correlation spectroscopy. Bioimaging 4:149–157
Eriksson M, Karlsson HJ, Westman G, Akerman B (2003) Groove-binding unsymmetrical cyanine dyes for staining of DNA: dissociation rates in free solution and electrophoresis gels. Nucleic Acids Res 31:6235–6242
Skinner S, Sepúlveda L, Xu H, Golding I (2013) Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization. Nat Protoc 8:1100–1113
Wahl M, Gregor I, Patting M, Enderlein J (2003) Fast calculation of fluorescence correlation data with asynchronous time-correlated single photon counting. Opt Express 11:3583–3591
Hodges C, Kafle RP, Hoff JD, Meiners JC (2018) Fluorescence correlation spectroscopy with photobleaching correction in slowly diffusing systems. J Fluoresc:1–7 https://doi.org/10.1007/s10895-018-2210-y
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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