Analysis of Spatial Distributions of the Number of Photon Counts in Fluorescence Fluctuation Spectroscopy

IF 0.8 4区 化学 Q4 SPECTROSCOPY Journal of Applied Spectroscopy Pub Date : 2024-09-14 DOI:10.1007/s10812-024-01788-8
V. V. Skakun, M. A. Hink, V. V. Apanasovich
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Abstract

A method is proposed for analysis of the photon-count number distributions (PCD) calculated over the space (pixels) of a stack of fluorescent images obtained in the process of a scanning measurement in fluorescence fluctuation spectroscopy which makes it possible to determine the characteristic brightness and number of molecules of the material being studied. The method is applicable to ergodic systems and is based on a theory of the analysis of Photon Counting Histograms (PCH) developed for single-point measurements. The method is tested on experimentally obtained images of a green fluorescent protein. The obtained 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 estimates of the characteristic brightness and number of molecules of the studied substance are in good agreement with estimates obtained with the aid of the methods of analysis used in this region, which makes it possible to conclude that the theory of the PCH method can be used for analyzing spatial PCDs calculated on the basis of a stack of images. The developed method makes it possible to obtain estimates of the studied parameters based on a selected subregion of a single frame of the image.

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荧光波动光谱中光子计数的空间分布分析
本文提出了一种方法,用于分析在荧光波动光谱扫描测量过程中获得的荧光图像堆栈空间(像素)上计算出的光子计数数量分布(PCD),从而确定被研究材料的特征亮度和分子数量。该方法适用于遍历系统,基于为单点测量开发的光子计数直方图(PCH)分析理论。该方法在实验获得的绿色荧光蛋白图像上进行了测试。获得的结果与单点实验结果和 N&B(数量和亮度)方法进行了比较,后者最常用于对扫描实验中获得的一叠荧光图像进行数值分析。对所研究物质的特征亮度和分子数量的估计值与借助该领域使用的分析方法获得的估计值非常一致,因此可以得出结论:PCH 方法的理论可用于分析根据叠加图像计算出的空间 PCD。所开发的方法可以根据单帧图像的选定子区域获得所研究参数的估计值。
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来源期刊
CiteScore
1.30
自引率
14.30%
发文量
145
审稿时长
2.5 months
期刊介绍: Journal of Applied Spectroscopy reports on many key applications of spectroscopy in chemistry, physics, metallurgy, and biology. An increasing number of papers focus on the theory of lasers, as well as the tremendous potential for the practical applications of lasers in numerous fields and industries.
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