Correction for out-of-focus emission in fluorescence fluctuation spectroscopy; generalization of the algorithms

Abstract

Workshop abstracts. One of the challenges of FFS is an adequate approximation of brightness profile. The key feature of the Fluorescence Intensity Distribution Analysis (FIDA) [1] is, for example, a polynomial model used to approximate broad spectrum of brightness profile functions. On the other hand Perroud [2] and Huang [3] have suggested an approach for brightness profile correction, as an extension of Photon Counting Histogram (PCH) analysis. In this case the additional fitting parameters are defined as a relative difference between integrals of the actual brightness profile and its Gaussian approximation. In current work we show that this approach can be derived straightforward from the FFS theory based on generation functions (GF). One of the advantages of GF is better performance of the algorithm; mainly because the applications of successive convolutions for total distribution calculation are not necessary anymore. As a logical consequence of the developed theory we apply corrections for out-of-focus emission in Fluorescence Cumulants Analysis (FCA) and show that applied corrections improve results of analysis that makes FCA an appropriate method for the analysis of FFS data alongside with PCH and FIDA.

[1] Kask, P., K. Palo, D. Ullmann, and K. Gall. Fluorescence-intensity distribution analysis and its application in biomolecular detection technology. Proc. Natl. Acad. Set USA. 96:13756-13761 (1999). [2] Perroud T.D., B. Huang, M.I. Wallace, and R.N. Zare. Photon Counting Histogram for One-Photon Excitation. ChemPhysChem. 4:1121-1123 (2003). [3] Huang B., T.D. Perroud, and R.N. Zare. Photon Counting Histogram: One-Photon Excitation. ChemPhysChem. 5:1523-1531 (2004).