Molecular brightness determined from a generalized form of Mandel's Q-parameter.
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 tau. 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(tau) into account. Q(0), which corresponds to the original Q-parameter, is severely affected by dead time and afterpulsing. Q(tau) for tau>0, on the other hand, is quite robust with respect to nonideal detector effects. Thus, analysis of Q(tau) 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(tau) 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(tau) analysis.
Sanchez-Andres
Alvaro
A
School of Physics and Astronomy, University of Minnesota, 116 Church Street SE, Minneapolis, MN 55455, USA.
Chen
Yan
Y
Müller
Joachim D
JD
D000077556:Alitretinoin; D000818:Animals; D001703:Biophysics / Q000379:methods*; D002460:Cell Line; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D006834:Hydrazines / Q000494:pharmacology; D008024:Ligands; D008027:Light; D015233:Models, Statistical; D008962:Models, Theoretical; D016011:Normal Distribution; D017785:Photons; D011336:Probability; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D047488:Retinoid X Receptors / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014158:Transcription, Genetic; D014212:Tretinoin / Q000737:chemistry
C417561:Alexa 488 hydrazide; D005456:Fluorescent Dyes; D006834:Hydrazines; D008024:Ligands; D047488:Retinoid X Receptors; D049452:Green Fluorescent Proteins; D000077556:Alitretinoin; D014212:Tretinoin