f luorescence correlation spectroscopy

FLUORESCENCE CORRELATION SPECTROSCOPY

MC and Christine

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

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

What Can You Get Out of It?

Diffusion Coefficients Hydrodynamic Radii Average Concentrations Kinetic Chemical Reaction Rates

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

FCS General PrinciplesFCS 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]

Brownian Motion This concept dictates the appearance and

disappearance of fluorescent molecules in small observation volume.

http://www.realinnovation.com/commentary/archive/organizational_brownian_motion.html [2]

Technique optimization1. 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.

Why low concentration? Magnitude of number fluctuations =

Mean square deviations

Typical Set up

http://en.wikipedia.org/wiki/File:Fluorescence_correlation_spectroscopy_instrument_diagram.png [2]

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

Autocorrelation Function Goal: to make sense out of data.

OR

Recall: Intensity autocorrelation g(t) = <I(t) I(t + t)>t

For small t

t

For larger tt

g(t)

ttc

Before and After Correlation

• = is the average time it takes for a molecule to diffuse through radial cavity.

𝝉𝒂𝒗𝒆

1/N

2D & 3D Correlation Function3D

r and l are radial and axial dimensions of volume.

For large r/l, same as 2D.

Diffusion Coefficient From autocorrelation plot we get N and The relationship between diffusion time

and coefficient is;

r is the radius of cavity – can be measured.

Therefore, we can find D!

Different Mobility Modes

Model autocorrelation curves for different kinds of particle motion [2]

Dual-Color Fluorecense Cross-Correlation Spectroscopy

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.

References1. Methods in Biomolecular Physics, Serdyuk and SZ²2. http://www.biophysics.org/Portals/1/PDFs/

Education/schwille.pdf3. http://research.stowers-institute.org/microscopy/ex

ternal/Technology/FCS/index.htm4. http://www.invitrogen.com/site/us/en/home/Refere

nces/Molecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlights/Fluorescence-Correlation-Spectroscopy-FCS.html

5. http://vohweb.chem.ucla.edu/voh/classes%5Cwinter09%5C221AID232%5CFCS.pdf

6. http://www.biophysics.org/Portals/1/PDFs/Education/schwille.pdf