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Molecular Brightness Determined from a Generalized Form of Mandel's Q-Parameter

School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota 55455

Correspondence: Address reprint requests to Joachim Mueller, University of Minnesota, School of Physics and Astronomy, 116 Church St. SE, Minneapolis, MN 55455. Tel.: 612-625-4369; Fax: 612-624-4578; E-mail: mueller{at}physics.umn.edu.

Mandel's Q-parameter, which is determined from the first two photon count moments, provides an alternative to PCH analysis for determining the brightness of fluorophores. Here, the definition of the Q-parameter is generalized to include correlations between photon counts that are separated by a time . We develop and experimentally verify a theory that takes the effects of dead time, afterpulsing, and the finite sampling time on the generalized parameter into account. which corresponds to the original Q-parameter, is severely affected by dead time and afterpulsing. for on the other hand, is quite robust with respect to nonideal detector effects. Thus, analysis of provides a robust method for determining the brightness of fluorophores. We extend the theory to a mixture of species, which is characterized by an apparent brightness. The brightness of EGFP in CV-1 cells is measured as a function of protein concentration to demonstrate the feasibility of analysis in cells. In addition, we monitor protein association of the ligand-binding domain of retinoid X receptor in the presence and absence of 9-cis-retinoic acid by analysis.

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