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Estimation of the number of single-photon emitters for multiple fluorophores with the same spectral signature

Fluorescence microscopy is of vital importance for understanding biological function. However, most fluorescence experiments are only qualitative inasmuch as the absolute number of fluorescent particles can often not be determined. Additionally, conventional approaches to measuring fluorescence inte...

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Autores principales: Li, Wenchao, Li, Shuo, Brown, Timothy C., Sun, Qiang, Wang, Xuezhi, Yakovlev, Vladislav V., Kealy, Allison, Moran, Bill, Greentree, Andrew D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Vacuum Society 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10694824/
http://dx.doi.org/10.1116/5.0162501
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author Li, Wenchao
Li, Shuo
Brown, Timothy C.
Sun, Qiang
Wang, Xuezhi
Yakovlev, Vladislav V.
Kealy, Allison
Moran, Bill
Greentree, Andrew D.
author_facet Li, Wenchao
Li, Shuo
Brown, Timothy C.
Sun, Qiang
Wang, Xuezhi
Yakovlev, Vladislav V.
Kealy, Allison
Moran, Bill
Greentree, Andrew D.
author_sort Li, Wenchao
collection PubMed
description Fluorescence microscopy is of vital importance for understanding biological function. However, most fluorescence experiments are only qualitative inasmuch as the absolute number of fluorescent particles can often not be determined. Additionally, conventional approaches to measuring fluorescence intensity cannot distinguish between two or more fluorophores that are excited and emit in the same spectral window, as only the total intensity in a spectral window can be obtained. Here we show that, by using photon number resolving experiments, we are able to determine the number of emitters and their probability of emission for a number of different species, all with the same measured spectral signature. We illustrate our ideas by showing the determination of the number of emitters per species and the probability of photon collection from that species, for one, two and three otherwise unresolvable fluorophores. The convolution binomial model is presented to represent the counted photons emitted by multiple species. Then, the expectation-maximization (EM) algorithm is used to match the measured photon counts to the expected convolution binomial distribution function. In applying the EM algorithm, to leverage the problem of being trapped in a sub-optimal solution, the moment method is introduced to yield an initial guess for the EM algorithm. Additionally, the associated Cramér–Rao lower bound is derived and compared with the simulation results.
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spelling pubmed-106948242023-12-05 Estimation of the number of single-photon emitters for multiple fluorophores with the same spectral signature Li, Wenchao Li, Shuo Brown, Timothy C. Sun, Qiang Wang, Xuezhi Yakovlev, Vladislav V. Kealy, Allison Moran, Bill Greentree, Andrew D. AVS Quantum Sci Quantum Photonics Fluorescence microscopy is of vital importance for understanding biological function. However, most fluorescence experiments are only qualitative inasmuch as the absolute number of fluorescent particles can often not be determined. Additionally, conventional approaches to measuring fluorescence intensity cannot distinguish between two or more fluorophores that are excited and emit in the same spectral window, as only the total intensity in a spectral window can be obtained. Here we show that, by using photon number resolving experiments, we are able to determine the number of emitters and their probability of emission for a number of different species, all with the same measured spectral signature. We illustrate our ideas by showing the determination of the number of emitters per species and the probability of photon collection from that species, for one, two and three otherwise unresolvable fluorophores. The convolution binomial model is presented to represent the counted photons emitted by multiple species. Then, the expectation-maximization (EM) algorithm is used to match the measured photon counts to the expected convolution binomial distribution function. In applying the EM algorithm, to leverage the problem of being trapped in a sub-optimal solution, the moment method is introduced to yield an initial guess for the EM algorithm. Additionally, the associated Cramér–Rao lower bound is derived and compared with the simulation results. American Vacuum Society 2023-12 2023-11-14 /pmc/articles/PMC10694824/ http://dx.doi.org/10.1116/5.0162501 Text en © 2023 Author(s). https://creativecommons.org/licenses/by/4.0/All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ).
spellingShingle Quantum Photonics
Li, Wenchao
Li, Shuo
Brown, Timothy C.
Sun, Qiang
Wang, Xuezhi
Yakovlev, Vladislav V.
Kealy, Allison
Moran, Bill
Greentree, Andrew D.
Estimation of the number of single-photon emitters for multiple fluorophores with the same spectral signature
title Estimation of the number of single-photon emitters for multiple fluorophores with the same spectral signature
title_full Estimation of the number of single-photon emitters for multiple fluorophores with the same spectral signature
title_fullStr Estimation of the number of single-photon emitters for multiple fluorophores with the same spectral signature
title_full_unstemmed Estimation of the number of single-photon emitters for multiple fluorophores with the same spectral signature
title_short Estimation of the number of single-photon emitters for multiple fluorophores with the same spectral signature
title_sort estimation of the number of single-photon emitters for multiple fluorophores with the same spectral signature
topic Quantum Photonics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10694824/
http://dx.doi.org/10.1116/5.0162501
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