Fluorescence Correlation Spectroscopy

1
Fluorescence Correlation Spectroscopy

• MC and Christine

2
An Introduction

• FCS is a high-resolution spatial and temporal
  analysis of very low concentrations of
  biomolecules
• This is done by measuring the spontaneous
  intensity fluctuations caused by the minute
  deviations of the system from equilibrium

3
Uses

• Developed in the early seventies as a way to
  analyze relaxation
• Study the Behavior of Individual Molecules
• Study Serum Biomarkers
• Monitoring biological molecular association and
  disassociation processes

4
What Can You Get Out of It?

• Diffusion Coefficients
• Hydrodynamic Radii
• Average Concentrations
• Kinetic Chemical Reaction Rates

5
History

• 1916 Smoluchowski gave the first description of
  amplitude and temporal decay of number
  fluctuations in diffusion system
• 1972-1974 Magde, Elson, Webb published book on
  potential of FCS and first developed the
  technique at Cornell University
• 1990 Rigler reached single molecule detection
  limit on FCS
• 1994 Eigen and Rigler proposed dual color cross
  correlation for FCS
• 2000 FCS evolved and also dual color cross
  correlation made and used

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FCS General Principles

• FCS is a method in which the florescence
  intensity arising from a very small volume
  containing fluorescent molecules is
  correlated/analyzed to obtain information about
  the processes that give rise to fluctuations in
  the fluorescence. 1

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Brownian Motion

• This concept dictates the appearance and
  disappearance of fluorescent molecules in small
  observation volume.

2
http//www.realinnovation.com/commentary/archive/o
rganizational_brownian_motion.html
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Technique optimization

1. Small number of molecules.
2. Large number of molecules suppress effect of
   fluctuations.
3. Low concentration is implied by 1.
4. Small area or cavity. One or less molecule.
5. Number of fluctuations is inversely related to
   number of molecules.

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Why low concentration?

•  

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Typical Set up
http//en.wikipedia.org/wiki/FileFluorescence_cor
relation_spectroscopy_instrument_diagram.png
2
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Measurements

• During FCS you measure the Fluorescent Intensity.
• Data not very useful yet!
• Notice that these fluctuations are caused by
  diffusion of fluorescent molecules through the
  cavity or just changes in fluorescence over time

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Autocorrelation Function

•  

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Recall Intensity autocorrelation

• g(t) ltI(t) I(t t)gtt

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Before and After Correlation
1/N

•  

 
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2D 3D Correlation Function

•  

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Diffusion Coefficient

•  

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Different Mobility Modes

• Model autocorrelation curves for different kinds
  of particle motion 2

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Dual-Color Fluorecense Cross-Correlation
Spectroscopy
19

•  

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Cross-correlation curves at different time points
during an endonucleolytic cleavage reaction.
Dotted lines are the original data. Fitted curves
are given in solid lines. During the reaction the
cross-correlation amplitude, which is a measure
of the reaction progress, gradually decreases.
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References

1. Methods in Biomolecular Physics, Serdyuk and SZ²
2. http//www.biophysics.org/Portals/1/PDFs/Education
   /schwille.pdf
3. http//research.stowers-institute.org/microscopy/e
   xternal/Technology/FCS/index.htm
4. http//www.invitrogen.com/site/us/en/home/Referenc
   es/Molecular-Probes-The-Handbook/Technical-Notes-a
   nd-Product-Highlights/Fluorescence-Correlation-Spe
   ctroscopy-FCS.html
5. http//vohweb.chem.ucla.edu/voh/classes5Cwinter09
   5C221AID2325CFCS.pdf
6. http//www.biophysics.org/Portals/1/PDFs/Education
   /schwille.pdf