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FCS (Fluorescence Correlation Spectroscopy) and PCH (Photon Counting Histogram) are the two most basic measurement and analysis techniques worked on and developed in our lab. FCS is a method by which the autocorrelation function of the fluorescence from a sample observed over a period of time is calculated. The autocorrelation curve is characterized by the diffusion dynamics and the concentration of the molecule under study. It is therefore analyzed to determine the molecule's diffusion coefficient as well as the number of particles in the sample volume. PCH involves binning the photon counts collected from the fluorescence experiments into a histogram. The curve obtained from this histogram is used to quantify the number of molecules in the sample volume and the brightness of the fluorescent molecule. |
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In the standard FCS measurements the sample is excited by focusing a stationary beam in order to monitor fluctuations in time. Scanning FCS introduces spatial fluctuations through the use of galvonometer scan mirrors. The spatial fluctuations changes the shape of the autocorrelation curve giving it more structure for fitting. |
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The idea that there are more information in fluorescence fluctuation spectroscopy (FFS) data than what FCS or PCH use is not new. We incorporate the brightness in PCH and correlation time in FCS into a same model: Factorial Cumulant Analysis (FCA). So we can combine the advantages of FCS and PCH to describe the real system. We rebin the FFS data to get the equivalent data for different binning time. We then develop a theory to describe the factorial cumulant of photon counts for arbitrary binning time as well as an error model to describe the uncertainty in these cumulants. By fitting the experimental cumulant data to the theoretical model, we can recover the brightness and correlation time of the fluorophore. |