1 May 1990 What determines the uncertainty of phase and modulation measurements in frequency-domain fluorometry?
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Abstract
The basic theory of a frequency domain fluorometer has been known since the original work of Gaviola (1)• With the introduction of multifrequency capabilities and the use of ultra high speed detectors, the basic understanding of the details of the instrumentation has become essential. The ultimate time resolution and separation capabilities for complex decay species critically depends on the noise characteristics of the instrument's various components. In frequency domain fluorometry, it is customary to measure the standard deviation of the phase and modulation at each individual frequency point. In the correlated single photon counting method, it is assumed that the standard deviation is given by '[i, where N is the number of counts in a time channel. In single photon counting techniques, the fluctuation of the number of counts is assumed to be intrinsic to the statistical properties of light, therefore, the only way to decrease the standard deviation of an individual channel is to increase the light intensity at the detector. A reduction of the standard deviation results in lower integration time and better instrument resolvability. The major feature emerging from the analysis of the residues and the standard deviation in the frequency domain technique is that, although they appear randomly distributed and uncorrelated from one frequency point to the next, there is a correlation between different experiments. The pattern of the residues is often reproducible. This observation questions the basic assumption used for statistical data analysis, i.e., that there is a gaussian distribution of each data point around the mean value and that the standard deviation of each point is significant for the statistical analysis of the frequency response.
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Beniamino B. Barbieri, Beniamino B. Barbieri, Fabio De Piccoli, Fabio De Piccoli, Martin J. vandeVen, Martin J. vandeVen, Enrico Gratton, Enrico Gratton, } "What determines the uncertainty of phase and modulation measurements in frequency-domain fluorometry?", Proc. SPIE 1204, Time-Resolved Laser Spectroscopy in Biochemistry II, (1 May 1990); doi: 10.1117/12.17723; https://doi.org/10.1117/12.17723
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