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

  • 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

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

Literature Cited
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   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.
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   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.
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   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.
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