title: The dual-color photon counting histogram with non-ideal photodetectors.
abstract: Dual-color photon counting histogram (PCH) analysis utilizes the photon counts in two detection channels to distinguish species by differences in brightness and color. Here we modify the existing dual-color PCH theory, which assumes ideal detectors, to include the non-ideal nature of the detector. Specifically, we address the effects of deadtime and afterpulsing. Both effects modify the shape of the dual-color PCH and thus potentially lead to incorrect values for the brightness and number of molecules if an ideal model is assumed. We use the modified theory to predict the effects of detector non-idealities on dual-color PCH as a function of concentration and brightness. In addition, we introduce a method based on moment analysis to determine the error in brightness due to non-ideal detector effects. We verify our theory experimentally by measuring a dye solution as a function of concentration and brightness. We determine the deadtime and afterpulse probability of our detectors and show that both effects play an important role in the analysis of dual-color PCH experiments. We demonstrate that resolving a mixture of CFP and YFP requires taking non-ideal detector effects into account. These corrections are also crucial for cellular measurements, as shown for GFP and RFP in mammalian cells.
authors.affiliation: School of Physics and Astronomy, University of Minnesota, 116 Church Street SE, Minneapolis, MN 55455, USA.
authors.forename: Lindsey N
authors.initials: LN
authors.lastname: Hillesheim
authors.forename: Joachim D
authors.initials: JD
authors.lastname: Müller
mesh_terms: D000465:Algorithms; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D055592:Biophysical Phenomena; D001703:Biophysics; D019556:COS Cells; D002522:Chlorocebus aethiops; D003116:Color; D005456:Fluorescent Dyes / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006834:Hydrazines / Q000494:pharmacology; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D015233:Models, Statistical; D016011:Normal Distribution; D017785:Photons; D010957:Plasmids / Q000378:metabolism; D016012:Poisson Distribution; D013997:Time Factors; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006834:Hydrazines; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C417561:Alexa 488 hydrazide; C485182:Cyan Fluorescent Protein; C054562:yellow fluorescent protein, Bacteria