1. MC and Christine

2.  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.  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.  Diffusion Coefficients  Hydrodynamic Radii  Average Concentrations  Kinetic Chemical Reaction Rates

5.  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

6. 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]

7.  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]

8. 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.

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

11.  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

13. g(  ) = t For small   For larger   g(  )  cc

14. Before and After Correlation 1/N

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

20. 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.

21. 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/external/Technology/FCS/ind ex.htm http://research.stowers- institute.org/microscopy/external/Technology/FCS/ind ex.htm 4. http://www.invitrogen.com/site/us/en/home/Referenc es/Molecular-Probes-The-Handbook/Technical-Notes- and-Product-Highlights/Fluorescence-Correlation- Spectroscopy-FCS.html http://www.invitrogen.com/site/us/en/home/Referenc es/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 http://vohweb.chem.ucla.edu/voh/classes%5Cwinter09 %5C221AID232%5CFCS.pdf 6. http://www.biophysics.org/Portals/1/PDFs/Education /schwille.pdf http://www.biophysics.org/Portals/1/PDFs/Education /schwille.pdf