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.
Bibliographical noteFunding Information:
L.N.H. thanks Bin Wu for his helpful comments on the non-ideal dual-color PCH theory, and Yan Chen for her advice on cellular measurements and protein purification, and for assistance with the GFP and RFP plasmids.This work was supported by grants from the National Institutes of Health (No. GM64589) and National Science Foundation (No. MCB-0110831) and by the Doctoral Dissertation Fellowship from the University of Minnesota.