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Biophys. J. BioFAST: First Published August 26, 2005. doi:10.1529/biophysj.105.066951
© 2005 by the Biophysical Society.

A more recent version of this article appeared on November 1, 2005.
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

The Dual-color Photon Counting Histogram with Non-ideal Photodetectors

Lindsey N. Hillesheim 1* and Joachim D. Mueller 1

1 University of Minnesota

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

Submitted on May 19, 2005
Revised on July 11, 2005
Accepted on 9 August 2005


  Abstract
Dual-color photon counting histogram (PCH) analysis utilizes the photon counts in two detection channels and distinguishes 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 dead-time 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 dead-time 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.

Key Words: Afterpulsing, Dead-time, Fluorescence Correlation Spectroscopy, Fluorescence Microscopy, Photon Statistics, Single Molecule Spectroscopy




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L. N. Hillesheim, Y. Chen, and J. D. Muller
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Copyright © 2005 by the Biophysical Society.