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Analysis of Spatial Photon Counting Distributions in Fluorescence Fluctuation Spectroscopy
Abstract
The proposed method for analyzing photon counting distributions (PCD), calculated over the space (pixels) of a stack of fluorescent images obtained in the process of scanning measurement in fluorescence fluctuation spectroscopy, makes it possible to determine the characteristic brightness and number of molecules of the investigated substance. The method is valid for ergodic systems and is based on the theory of analysis of photon count histograms PCH (Photon Counting Histogram), developed for the analysis of single-point measurements. The method has been tested on the experimentally obtained images of green fluorescent protein. The results are compared with the results of a single-point experiment and the N&B (Number and Brightness) method, which is most often used for numerical analysis of a stack of fluorescent images obtained in scanning experiments. The resulting estimates of the characteristic brightness and number of molecules of the calculated substance are in good agreement with the estimates obtained using analysis methods in this area, which allows us to conclude that it is possible to use the theory of the PCH method for the analysis of spatial PCD calculated based on a stack of images. The developed method makes it possible to obtain estimates of the observed parameters based on a selected subregion of one image frame.
About the Authors
V. V. Skakun
Belarusian State University
Belarus
Belarus
Minsk
M. A. Hink
University of Amsterdam, dept Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy
Netherlands
Netherlands
Amsterdam
V. V. Apanasovich
Belarusian State University
Belarus
Belarus
Minsk
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Review
For citations:
Skakun V.V., Hink M.A., Apanasovich V.V. Analysis of Spatial Photon Counting Distributions in Fluorescence Fluctuation Spectroscopy. Zhurnal Prikladnoii Spektroskopii. 2024;91(4):571-580. (In Russ.)
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