Challenges
Datasets
Workspaces
Papers
Discussions
Leaderboard
Log inSign up
Challenges
Datasets
Workspaces
Papers
Discussions
Leaderboard
Blog
Job Board
Q3AS

© 2026 Aqora Quantum S.A.S.

TermsPrivacyLegal Notice

Laser & Optoelectronics Progress

Identifying and Distinguishing Single-molecule Based on the Fluorescence Photon Statistic Measurement

Peng Shuang-Yan, Huang Tao

Published Jan 1, 2006• 0 citations
OpenAlex

Abstract

Identifying and distinguishing single-molecule by fluorescence photon statistic measuring have been widely applied in physics, biology, chemistry and information processing et al.. Two kinds of methods of identifying and distinguishing single-molecule are analyzed and reviewed. Firstly, two identification methods to identify singlemolecule photon source are introduced: one by measuring laser stimulated single-molecule fluorescence field twoorder autocorrelation function with start-stop method, the other based on single photon counting statistic measurement. Secondly, the other kind of methods are to distinguish which species molecules the single-molecule detected belongs to when a couple of species molecules are mixed. Two methods, based on property-correlated single-photon counting statistic measurement and probability calculation and analysis, are chiefly introduced: the maximum likelihood method and the lookup table method.

Related papers

Mapping molecules quantitatively in confocal fluorescence microscopy (Conference Presentation)

Marcelle Koenig, Caroline Berlage, Paja Reisch +4 more

Single-molecule fluorescence microscopy has been established in life science as an essential tool for studying the characteristics and dynamics of individual fluorescent emitters both in vitro as well as in vivo. Acquiring quantitative information from the confocal observation volume is still a challenging task and knowing the absolute number or concentration of proteins in, e.g., cellular structures can significantly improve our understanding of cell biology, which is an important step towards quantitative microscopy. In this talk, a new quantitative analytical tool called Counting by Photon Statistics (CoPS) will be presented. The approach relies on a statistical analysis of detected photon coincidences to estimate the number of independent fluorescent labels in the observation volume [1]. CoPS does exploit the photon antibunching effect: a single photon emitter can only generate one photon at a time. Originally developed for point measurements, CoPS has recently been extended to an imaging scheme [2]. Using a confocal fluorescence microscopy setup with pulsed excitation, four single-photon, detectors and parallellized time-correlated single photon counting electronics (MicroTime 200, PicoQuant), we prove the applicability of the method with artificial model systems (immobilized DNA origami) and present first steps towards biological samples. [1] Ta, H., Wolfrum, J., Herten, D.-P., An extended scheme for counting fluorescent molecules by photon-antibunching Laser Phys. 20:119 (2010). [2] Ta, H. et al., Mapping molecules in scanning far-field fluorescence nanoscopy. Nat. Commun. 6:7977 (2015).

Single Molecule Spectroscopy and Superresolution Imaging•Mar 4, 2019•0 citations

Identifying single molecule based on the photon statistics

Peng Shuang-Yan, Huang Tao, Xiaobo Wang +4 more

We theoretically study the photon counting statistics characterizations of the real single molecular photon source with a Poisson statistics background and that of an ideal double molecular system based on the standard HBT detection. Mandel parameter Q of each source with the same mean photons n—was analyzed. We have obtained an efficient method to identify single molecule by the two-photon statistics probability P(2), which shows the relation P(2)2. Additionally, we studied the effect of single-to-background ratio of the single molecular photon source on the criterion.

Acta Physica Sinica•Jan 1, 2005•0 citations

An extended scheme for counting fluorescent molecules by photon-antibunching

Haisen Ta, J. Wolfrum, Dirk‐Peter Herten

Acquisition of quantitative information from microscopic biological samples is highly desirable in the context of the emerging field of systems biology. We derive a statistical approach to estimate the number of fluorescent molecules in the observation volume based on a confocal microscope for single-molecule detection. The method employs ps-pulsed laser sources for excitation and time-correlated single-photon counting with 4 avalanche photon diodes (APDs) for detection of individual photons. The feasibility for estimating the number of molecules is shown based on simultaneous emission and detection of multiple photons (photon-antibunching) under realistic experimental conditions. In theory, it should be possible to estimate the number of molecules with errors of less than 1% by using novel photo-stabilizing agents. The proposed method puts into perspective its application for high-resolution microscopy without the need for photo-switching or photo-activation of fluorescence dyes.

Laser Physics•Jul 29, 2009•25 citations

Distribution analysis of the photon correlation spectroscopy of discrete numbers of dye molecules conjugated to DNA

Samantha Fore, Ted A. Laurence, Yi‐Cheun Yeh +4 more

The formation of protein complexes with other proteins and nucleic acids is critical to biological function. Although it is relatively easy to identify the components present in these complexes, it is often difficult to determine their exact stoichiometry and obtain information about the homogeneity of the sample from bulk measurements. We demonstrate the use of single molecule photon-pair correlation spectroscopy to distinguish between discrete numbers of molecules in biological complexes. Fluorescence photon antibunching is observed from a single molecule by employing time-correlated single photon counting in combination with a Hanbury-Brown and Twiss coincidence setup. In addition, pulsed laser excitation and time-tagged time-resolved data collection allow for the measurement of photon arrival times with nanosecond time resolution. The interphoton time distribution between consecutively arriving photons can be calculated and provides a measure of the second-order temporal correlation function. Analysis of this function yields an absolute measure of the number of molecules, N, present in a given complex. It is this ability to measure N that renders this technique powerful for determining stoichiometries in complex biological systems at the single molecule level. We investigate the counting efficiency and statistics of photon antibunching of specifically designed biological samples labeled with multiple copies of the same fluorescent dye and derive conclusions about its use in the analytical evaluation of complex biological samples.

IEEE Journal of Selected Topics in Quantum Electronics•Jul 1, 2005•10 citations

Characterization and Application of Photon-Statistics in Single-Molecule Measurements for Quantitative Studies of Fluorescently Labeled Samples

Anton Kurz

The lack of reliable quantitative methods in fluorescence microscopy prevents the detailed investigation of basic molecular processes. Single-molecule fluorescence spectroscopy offers several techniques to determine the stoichiometry of fluorescent probes. Among them, an approach using the single-photon emission of an individual quantum system, also known as photon antibunching, yields access to both the emitter number and their molecular brightness. Previous work demonstrated the feasibility of the method to estimate up to five dyes without further physical characterization. In this work, I investigated and improved the reliability and robustness of this method. I developed different fluorescent probes with a defined label stoichiometry to explore the performance of our approach \nin experiments. I could demonstrate, in combination with simulations, that it can provide reliable estimates of up to 18 fluorescent emitters at a time resolution of approximately 200 ms. Furthermore, I inspected the dependency of the estimation bias and precision on the laser intensity, the analysis time and the number of expected molecules in detail. My results designate the scope for future stoichiometry determinations. In initial experiments, I analyzed the labeling distributions of different fluorescent probes. In addition, I could combine our counting approach with super-resolution STED-microscopy and identify critical parameters for possible applications

•Jan 1, 2013•0 citations

Optics detection of single molecules

Jia Suo

The principle of single photon source based on three-levels single molecule is introduced. By use of Hanbury-Brown-Twiss method, the photon statistics P(n, n=0、1、2) and the Mandel parameter are analyzed by recording of every photo-electrical event from the single photon source. When signal to background ratio SBR2.4, an efficient method to distinguish single molecules by the double-photon statistics probability P(2), which shows P(2)12(2-4-2n-)~2, is presented. Here, n- is the mean photon number.

•Jan 1, 2006•0 citations
Identifying and distinguishing single-molecule by fluorescence photon statistic measuring have been widely applied in physics, biology, chemistry and information processing et al.. Two kinds of methods of identifying and distinguishing single-molecule are analyzed and reviewed. Firstly, two identification methods to identify singlemolecule photon source are introduced: one by measuring laser stimulated single-molecule fluorescence field twoorder autocorrelation function with start-stop method, the other based on single photon counting statistic measurement. Secondly, the other kind of methods are to distinguish which species molecules the single-molecule detected belongs to when a couple of species molecules are mixed. Two methods, based on property-correlated single-photon counting statistic measurement and probability calculation and analysis, are chiefly introduced: the maximum likelihood method and the lookup table method.
Single-molecule fluorescence microscopy has been established in life science as an essential tool for studying the characteristics and dynamics of individual fluorescent emitters both in vitro as well as in vivo. Acquiring quantitative information from the confocal observation volume is still a challenging task and knowing the absolute number or concentration of proteins in, e.g., cellular structures can significantly improve our understanding of cell biology, which is an important step towards quantitative microscopy. In this talk, a new quantitative analytical tool called Counting by Photon Statistics (CoPS) will be presented. The approach relies on a statistical analysis of detected photon coincidences to estimate the number of independent fluorescent labels in the observation volume [1]. CoPS does exploit the photon antibunching effect: a single photon emitter can only generate one photon at a time. Originally developed for point measurements, CoPS has recently been extended to an imaging scheme [2]. Using a confocal fluorescence microscopy setup with pulsed excitation, four single-photon, detectors and parallellized time-correlated single photon counting electronics (MicroTime 200, PicoQuant), we prove the applicability of the method with artificial model systems (immobilized DNA origami) and present first steps towards biological samples. [1] Ta, H., Wolfrum, J., Herten, D.-P., An extended scheme for counting fluorescent molecules by photon-antibunching Laser Phys. 20:119 (2010). [2] Ta, H. et al., Mapping molecules in scanning far-field fluorescence nanoscopy. Nat. Commun. 6:7977 (2015).
We theoretically study the photon counting statistics characterizations of the real single molecular photon source with a Poisson statistics background and that of an ideal double molecular system based on the standard HBT detection. Mandel parameter Q of each source with the same mean photons n—was analyzed. We have obtained an efficient method to identify single molecule by the two-photon statistics probability P(2), which shows the relation P(2)2. Additionally, we studied the effect of single-to-background ratio of the single molecular photon source on the criterion.
Acquisition of quantitative information from microscopic biological samples is highly desirable in the context of the emerging field of systems biology. We derive a statistical approach to estimate the number of fluorescent molecules in the observation volume based on a confocal microscope for single-molecule detection. The method employs ps-pulsed laser sources for excitation and time-correlated single-photon counting with 4 avalanche photon diodes (APDs) for detection of individual photons. The feasibility for estimating the number of molecules is shown based on simultaneous emission and detection of multiple photons (photon-antibunching) under realistic experimental conditions. In theory, it should be possible to estimate the number of molecules with errors of less than 1% by using novel photo-stabilizing agents. The proposed method puts into perspective its application for high-resolution microscopy without the need for photo-switching or photo-activation of fluorescence dyes.
The formation of protein complexes with other proteins and nucleic acids is critical to biological function. Although it is relatively easy to identify the components present in these complexes, it is often difficult to determine their exact stoichiometry and obtain information about the homogeneity of the sample from bulk measurements. We demonstrate the use of single molecule photon-pair correlation spectroscopy to distinguish between discrete numbers of molecules in biological complexes. Fluorescence photon antibunching is observed from a single molecule by employing time-correlated single photon counting in combination with a Hanbury-Brown and Twiss coincidence setup. In addition, pulsed laser excitation and time-tagged time-resolved data collection allow for the measurement of photon arrival times with nanosecond time resolution. The interphoton time distribution between consecutively arriving photons can be calculated and provides a measure of the second-order temporal correlation function. Analysis of this function yields an absolute measure of the number of molecules, N, present in a given complex. It is this ability to measure N that renders this technique powerful for determining stoichiometries in complex biological systems at the single molecule level. We investigate the counting efficiency and statistics of photon antibunching of specifically designed biological samples labeled with multiple copies of the same fluorescent dye and derive conclusions about its use in the analytical evaluation of complex biological samples.
The lack of reliable quantitative methods in fluorescence microscopy prevents the detailed investigation of basic molecular processes. Single-molecule fluorescence spectroscopy offers several techniques to determine the stoichiometry of fluorescent probes. Among them, an approach using the single-photon emission of an individual quantum system, also known as photon antibunching, yields access to both the emitter number and their molecular brightness. Previous work demonstrated the feasibility of the method to estimate up to five dyes without further physical characterization. In this work, I investigated and improved the reliability and robustness of this method. I developed different fluorescent probes with a defined label stoichiometry to explore the performance of our approach \nin experiments. I could demonstrate, in combination with simulations, that it can provide reliable estimates of up to 18 fluorescent emitters at a time resolution of approximately 200 ms. Furthermore, I inspected the dependency of the estimation bias and precision on the laser intensity, the analysis time and the number of expected molecules in detail. My results designate the scope for future stoichiometry determinations. In initial experiments, I analyzed the labeling distributions of different fluorescent probes. In addition, I could combine our counting approach with super-resolution STED-microscopy and identify critical parameters for possible applications
The principle of single photon source based on three-levels single molecule is introduced. By use of Hanbury-Brown-Twiss method, the photon statistics P(n, n=0、1、2) and the Mandel parameter are analyzed by recording of every photo-electrical event from the single photon source. When signal to background ratio SBR2.4, an efficient method to distinguish single molecules by the double-photon statistics probability P(2), which shows P(2)12(2-4-2n-)~2, is presented. Here, n- is the mean photon number.