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: 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: 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

  •  FigureFigure 4.24.1 Sketch of the optical setting for an FCS measurement.
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  •  FigureFigure 4.24.2 Autocorrelation on cytosolic GFP. Autocorrelation function is collected from a confocal volume chosen in the cell cytoplasm where soluble GFP is being expressed (A). Note how the baseline for the autocorrelation function is flat (between 100 msec and 1 sec) yielding a good fit and diffusion estimate (B). The count rate is stable indicating the necessary lack of photobleaching (Inset C).
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  •  FigureFigure 4.24.3 Typical FCS curve and relevant parameters.
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  •  FigureFigure 4.24.4 Autocorrelation on plasma membrane–associated GPI-GFP. Autocorrelation function is collected from in a confocal volume chosen on the plasma membrane, where GFP is anchored via a GPI domain (A). Note how the baseline for the autocorrelation function is not flat (between 100 msec and 1 sec) because of photobleaching of the plasma membrane–anchored GFP (B). Photobleaching is apparent as an exponential decay in the countrate (Inset C).
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  •  FigureFigure 4.24.5 Autocorrelation function (circle) and associated fit (continuous line) for a solution of FITC-labeled IgG. The fit corresponds to a single component two-dimensional diffusion with a triplet relaxation equation (Equation (5)). The two relaxations can easily be distinguished: the slow one is associated with diffusion (diffusion = 500 µsec) and the fast one with triplet relaxation (triplet = 0.6 µsec). Their respective amplitudes are diffusion = 0.01 (hence there are 1/diffusion = 100 fluorescent objects per confocal volume) and triplet = 0.82 (hence, ptriplet = 0.45 as triplet = ptriplet/(1 – ptriplet).
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Videos

Literature Cited

Literature Cited
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    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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    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.
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