The dead time of the detector significantly distorts the fluorescence correlation function of fluorescent particles in solution. This distortion of the correlation function is similar to the saturation effect of the correlation function in a high-power excitation region. The correlation amplitude is significantly reduced by the dead time. The deviations in the number of molecules and the diffusion time are empirically given by the deviation of the fluorescence intensity linearity. The empirical curves of the deviations can be applied to the systematic error estimation of the parameters. The proportionality of the number of molecules to the concentration of fluorophores is no longer maintained with a large dead time, although almost all of the proportionality of the diffusion time to the inverse diffusion constant remains. This fact makes the dead-time effect different from the saturation effect, which is due to photokinetics. In practice, these distortions can be reduced by use of a smaller excitation power in which the proportionality of the fluorescence intensity is maintained.
© 2005 Optical Society of America
(030.5290) Coherence and statistical optics : Photon statistics
(170.3890) Medical optics and biotechnology : Medical optics instrumentation
(180.1790) Microscopy : Confocal microscopy
(300.2530) Spectroscopy : Fluorescence, laser-induced
Goro Nishimura and Masataka Kinjo, "Dead-time distortion in fluorescence correlation measurements," Appl. Opt. 44, 3458-3467 (2005)