Estimation of molecular concentration and brightness based on controlled attenuation of fluorescence intensity

Authors

  • Viktor V. Skakun Belarusian State University, 4 Niezaliezhnasci Avenue, Minsk 220030, Belarus
  • Vladimir V. Apanasovich Independent researcher, Minsk, Belarus

Keywords:

fluorescence fluctuation spectroscopy, generating functional, photon counting distribution, PCD, probability of zero photon counts within the registration interval, fluorescence intensity distribution analysis, FIDA, photon counting histogram, PCH
Supporting Agencies
The authors express their gratitude to the department of parasitology at the Leiden University Medical Centre (Netherlands), and in particular to doctor O. Maiboroda, for providing the results of measuring the photon counting distribution of the Alexa-488 solution in buffer.

Abstract

A new method for estimating the concentration and characteristic brightness of complex molecular assemblies in fluorescence fluctuation spectroscopy is proposed. This method measures the probability of zero photon counts within the registration interval for different excitation intensities. The excitation intensity can be varied by changing the laser power, neutral density filters that attenuate the laser radiation intensity, and independent photon count thinning. The method is based on an expression relating the probability of zero photon counts within the registration interval to the excitation intensity attenuation coefficient. Expressions are derived with and without correction for out-of-focus emission. Estimates of the parameters of the test substance are found by analysing the obtained non-linear dependence using the least-squares method. The developed method is simple to implement, does not require measuring complex characteristics of the recorded photon count flow, and, as a result, allows the use of a detector with significant dead time and afterpulse probability. The performance of the method was verified by analysing simulated and experimentally measured data and comparing its results with the results obtained using the photon counting histogram method.

Author Biographies

  • Viktor V. Skakun, Belarusian State University, 4 Niezaliezhnasci Avenue, Minsk 220030, Belarus

    PhD (physics and mathematics), docent; head of the department of system analysis and computer simulation, faculty of radiophysics and computer technologies

  • Vladimir V. Apanasovich, Independent researcher, Minsk, Belarus

    doctor of science (physics and mathematics), full professor; independent researcher

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Published

2026-04-10

Issue

No. 1 (2026): Journal of the Belarusian State University. Physics

Section

Optics and Spectroscopy

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How to Cite

(1)
Skakun, V. V.; Apanasovich, V. V. Estimation of Molecular Concentration and Brightness Based on Controlled Attenuation of Fluorescence Intensity. Журнал Белорусского государственного университета. Физика 2026, No. 1, 13-25. https://doi.org/10.33581/2520-2243-2026-1-%p.
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