Abstract
This chapter presents an overview of quantitative fluorescence brightness experiments with special emphasis on single-color measurements of protein homo-interactions inside living cells. We discuss practical considerations in the choice of the fluorescent labels and the calibration measurements necessary for quantitative interpretation of brightness experiments. Methods to identify and avoid common pitfalls, such as bleaching and saturation, are addressed. We examine the interpretation of brightness data with moment analysis. In particular, we focus on how to avoid or correct for undersampling, as well as how to characterize and adjust for photon detector effects. We conclude by describing brightness titration experiments which determine the binding curve and stoichiometry of a protein from apparent brightness data.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Chen Y, Mueller JD, So PT et al (1999) The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 77:553–567
Chen Y, Wei L-N, Mueller JD (2003) Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy. Proc Natl Acad Sci USA 100:15492–15497
Wu B, Chen Y, Mueller JD (2009) Fluorescence fluctuation spectroscopy of mCherry in living cells. Biophys J 96:2391–2404
Day RN, Davidson MW (2009) The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev 38:2887
Day RN, Davidson MW (2009) Aequorea victoria fluorescent proteins. Zeiss Online Campus. http://zeiss-campus.magnet.fsu.edu/articles/probes/jellyfishfps.html. Accessed 01 September 2012
Piston DW, Robertson RE, Day R.N., et al. (2009) Anthozoa fluorescent proteins. Zeiss Online Campus. http://zeiss-campus.magnet.fsu.edu/articles/probes/anthozoafps.html. Accessed 01 September 2012
Macdonald PJ, Chen Y, Wang X et al (2010) Brightness analysis by z-scan fluorescence fluctuation spectroscopy for the study of protein interactions within living cells. Biophys J 99:979–988
Chen Y, Mueller JD, Ruan Q et al (2002) Molecular brightness characterization of EGFP in vivo by fluorescence fluctuation spectroscopy. Biophys J 82:133–144
Wu B, Mueller JD (2005) Time-integrated fluorescence cumulant analysis in fluorescence fluctuation spectroscopy. Biophys J 89:2721–2735
Hillesheim LN, Mueller JD (2003) The photon counting histogram in fluorescence fluctuation spectroscopy with non-ideal photodetectors. Biophys J 85:1948–1958
Sanchez-Andres A, Chen Y, Mueller JD (2005) Molecular brightness determined from a generalized form of Mandel’s Q-parameter. Biophys J 89:3531–3547
Acknowledgments
This work was supported by grants from the National Institutes of Health (GM64589 & GM091743) and the National Science Foundation (PHY-0346782).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Macdonald, P.J., Johnson, J., Chen, Y., Mueller, J.D. (2014). Brightness Experiments. 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_32
Download citation
DOI: https://doi.org/10.1007/978-1-62703-649-8_32
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-648-1
Online ISBN: 978-1-62703-649-8
eBook Packages: Springer Protocols