Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach

Nihal Altan‐Bonnet1, Grégoire Altan‐Bonnet2

1 Department of Biology, Rutgers University, Newark, New Jersey, 2 Program in Computational Biology and Immunology, Memorial Sloan Kettering Cancer Center, New York, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.24
DOI:  10.1002/0471143030.cb0424s45
Online Posting Date:  December, 2009
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Abstract

Fluorescence correlation spectroscopy (FCS) is a single‐molecule fluorescence technique used to monitor molecular dynamics. It can be applied to living cells expressing fluorescently labeled proteins and lipids to determine the diffusion timescales and the total number of diffusing fluorescent molecules in the cell. A practical step‐by‐step approach to performing FCS with a commercial spectroscopy/microscopy system, the Zeiss Confocor 3, how to set up live‐cell FCS experiments, acquire reliable data, and finally how to analyze the data acquired, are all described in this unit. Curr. Protoc. Cell Biol. 45:4.24.1‐4.24.14. © 2009 by John Wiley & Sons, Inc.

Keywords: fluorescence correlation spectroscopy; FCS; live‐cell imaging; green fluorescent protein; GFP; diffusion

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Setting Up Fluorescence Correlation Spectroscopy for Living‐Cell Measurements
  • Support Protocol 1: Checking the Confocal and FCS Alignment
  • Basic Protocol 2: Analyzing FCS Data
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Setting Up Fluorescence Correlation Spectroscopy for Living‐Cell Measurements

  Materials
  • Cells growing on a glass‐bottomed culture dish (Mat‐Tek) or chambered slide (Lab‐Tek, Nunc) with lid at 50% confluency
  • Medium for incubation on microscope stage (culture medium with serum but without phenol red, supplemented with 25 to 50 mM HEPES)
  • Petroleum jelly (e.g., Vaseline, Cheeseborough)
  • 100 mM Oregon Green in PBS or other fluorescently labeled molecule for calibration
  • FCS machine, e.g., Zeiss LSM 510 ConfoCor 3 FCS system
  • 8‐well Nunc‐Lab Tek chambers

Support Protocol 1: Checking the Confocal and FCS Alignment

  Materials
  • 1 µmol FITC solution in DMSO
  • No. 1 coverslips
  • ConfoCor FCS system (Zeiss LSM 510)
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Figures

Videos

Literature Cited

Literature Cited
   Altan‐Bonnet, G., Libchaber, A., and Krichevsky, O. 2003. Bubble dynamics in double‐stranded DNA. Phys. Rev. Lett. 90:138101.
   Altan‐Bonnet, N., Sougrat, R., Liu, W., Snapp, E.L., Ward, T., and Lippincott‐Schwartz, J. 2006. Golgi inheritance in mammalian cells is mediated through endoplasmic reticulum export activities. Mol. Biol. Cell 17:990‐1005.
   Bacia, K. and Schwille, P. 2003. A dynamic view of cellular processes by in vivo fluorescence auto‐ and cross‐correlation spectroscopy. Methods 29:74‐85.
   Bonnet, G., Krichevsky, O., and Libchaber, A. 1998. Kinetics of conformational fluctuations in DNA hairpin‐loops. Proc. Natl. Acad. Sci. U.S.A. 95:8602‐8606.
   Carl Zeiss Microimaging, 2006. ConfoCor 3 Operating Manual, Release 4.0.
   Chen, Y., Müller, J.D., So, P.T., and Gratton, E. 1999. The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys. J. 77:553‐567.
   Chen, Y., Müller, J.D., Ruan, Q., and Gratton, E. 2002. Molecular brightness characterization of EGFP in vivo by fluorescence fluctuation spectroscopy. Biophys. J. 82:133‐144.
   Gosch, M., Blom, H., Holm, J., Heino, T., and Rigler, R. 2000. Hydrodynamic flow profiling in microchannel structures by single molecule fluorescence correlation spectroscopy. Anal. Chem. 72:3260‐3265.
   Haupts, U., Maiti, S., Schwille, P., and Webb, W.W. 1998. Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 95:13573‐13578.
   Hess, S.T., Huang, S., Heikal, A.A., and Webb, W.W. 2002. Biological and chemical applications of fluorescence correlation spectroscopy: A review. Biochemistry 41:697‐705.
   Koppel, D.E. 1974. Statistical accuracy in fluorescence correlation spectroscopy. Phys. Rev. A 10:1938‐1945.
   Krichevsky, O. and Bonnet, G. 2002. Fluorescence correlation spectroscopy: The technique and its applications. Rep. Progr. Physics 65:251‐297.
   Magde, D., Elson, E., and Webb, W.W. 1972. Thermodynamic fluctuations in a reacting system—Measurement by fluorescence correlation spectroscopy. Phys. Rev. Lett. 29:705‐708.
   Rabut, G. and Ellenberg, J. 2004. Automatic real‐time three‐dimensional cell tracking by fluorescence microscopy. J. Microsc. 216:131‐137.
   Rigler, R., Mets, Ü., Widengren, J., and Kask, P. 1993. Fluorescence correlation spectroscopy with high count rate and low background: Analysis of translational diffusion. Eur. J. Biophys. 22:169‐175.
   Schwille, P. 2001. Fluorescence correlation spectroscopy and its potential for intracellular applications. Cell Biochem. Biophys. 34:383‐408.
   Wachsmuth, M., Waldeck, W., and Langowski, J. 2000. Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially‐resolved fluorescence correlation spectroscopy. J. Mol. Biol. 298:677‐689.
   Weiss, M., Hashimoto, H., and Nilsson, T. 2003. Anomalous protein diffusion in living cells as seen by fluorescence correlation spectroscopy. Biophys. J. 84:4043‐4052.
   Widengren, J. and Rigler, R. 1998. Fluorescence correlation spectroscopy as a tool to investigate chemical reactions in solutions and on cell surfaces. Cell Mol. Biol. 44:857‐879.
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