Mandel's Q-parameter, which is determined from the first two photon count moments, providesan 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 τ. 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(τ) into account. Q(0), which corresponds to the original Q-parameter, is severely affected by dead time and afterpulsing. Q(τ) for τ > 0, on the other hand, is quite robust with respect to nonideal detector effects. Thus, analysis of Q(τ) 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(τ) 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(τ) analysis.
Bibliographical noteFunding Information:
This work was supported by grants from the National Institutes of Health (GM64589) and the National Science Foundation (PHY-0346782). A.S.A. acknowledges support by a “La Caixa” Foundation Fellowship.