MC and Christine
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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
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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
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Diffusion Coefficients
Hydrodynamic Radii
Average Concentrations
Kinetic Chemical Reaction Rates
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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
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]

This concept dictates the appearance and
disappearance of fluorescent molecules in
small observation volume.
http://www.realinnovation.com/commentary/archive/organizational_brownian_mo
tion.html
[2]
1.
2.
3.
4.
5.
Small number of molecules.
Large number of molecules suppress effect of
fluctuations.
Low concentration is implied by 1.
Small area or cavity. One or less molecule.
Number of fluctuations is inversely related to
number of molecules.
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Magnitude of number fluctuations = Mean
square deviations
< ∆𝑁 2 > = < 𝑁 >
∆𝑁
𝑁
2
=
𝑁
𝑁
2
=
1
𝑁
2
1
=
𝑁
http://en.wikipedia.org/wiki/File:Fluorescence_correlation_spectroscopy_instrument_diagram.
png
[2]
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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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Goal: to make sense out of data.
𝐺𝐹
𝐹 𝑡 𝐹(𝑡 + 𝜏)
𝜏 =
𝐹 𝑡 2
OR
𝐺 𝛿𝐹
𝛿𝐹 𝑡 𝛿𝐹(𝑡 + 𝜏)
𝜏 =
𝐹 𝑡 2
𝐺 𝛿𝐹 = 𝐺 𝐹 − 1
g(t) = <I(t) I(t + t)>t
t
For small t
For larger t
t
g(t)
tc
t
Before and After Correlation
1/N
• 𝜏𝑎𝑣𝑒 = is the average
time it takes for a
molecule to diffuse
through radial cavity.
𝝉𝒂𝒗𝒆
3D:
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
𝐺 𝜏 =
1
1
𝜏
𝑁 1+
𝜏𝑎𝑣𝑒
1
1+
1
2
𝑟 2 𝜏
𝑙 𝜏𝑎𝑣𝑒
r and l are radial and axial dimensions of
volume.
For large r/l, same as 2D.
1
1
𝐺 𝜏 =
𝑁 1+ 𝜏
𝜏𝑎𝑣𝑒
1
2
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From autocorrelation plot we get N and 𝜏𝑎𝑣𝑒
The relationship between diffusion time and
coefficient is;
𝑟2
𝜏𝑎𝑣𝑒 =
4𝐷
r is the radius of cavity – can be measured.
Therefore, we can find D!
Model autocorrelation curves for different kinds of particle motion [2]
𝐺𝐹
𝐹 𝑡 𝑟𝐹𝑔(𝑡 + 𝜏)
𝜏 =
𝐹 𝑡 2
G+R
GR
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.
1.
2.
3.
4.
5.
6.
Methods in Biomolecular Physics, Serdyuk and SZ²
http://www.biophysics.org/Portals/1/PDFs/Education
/schwille.pdf
http://research.stowersinstitute.org/microscopy/external/Technology/FCS/ind
ex.htm
http://www.invitrogen.com/site/us/en/home/Referenc
es/Molecular-Probes-The-Handbook/Technical-Notesand-Product-Highlights/Fluorescence-CorrelationSpectroscopy-FCS.html
http://vohweb.chem.ucla.edu/voh/classes%5Cwinter09
%5C221AID232%5CFCS.pdf
http://www.biophysics.org/Portals/1/PDFs/Education
/schwille.pdf