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

¹ University of Minnesota

^* To whom correspondence should be addressed. E-mail: mueller{at}physics.umn.edu.

Submitted on May 20, 2005
Revised on June 17, 2005
Accepted on 15 August 2005

	Abstract

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 t. We develop and experimentally verify a theory that takes the effects of dead-time, afterpulsing, and the finite sampling time on the generalized parameter Q(t) into account. Q(0), which corresponds to the original Q-parameter, is severely affected by dead-time and afterpulsing. Q(t) for t>0, on the other hand, is quite robust with respect to non-ideal detector effects. Thus, analysis of Q(t)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 Q(t) 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 Q(t) analysis.

Key Words: Afterpulsing, Dead-Time, Fluorescence Correlation Spectroscopy, Fluorescence Fluctuation Spectroscopy, Photon Statistics, Protein-protein interactions

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