Counting by Photon Statistics - Fluorescence Quantification based on Photon Antibunching
Event details
| Date | 18.03.2016 |
| Hour | 14:00 |
| Speaker | Dr. Kristin S. Grußmayer, Heidelberg University (D) |
| Location | |
| Category | Conferences - Seminars |
BIOENGINEERING SEMINAR
Abstract:
Quantitative information is key to unravel molecular processes in all fields of research. Counting by Photon Statistics (CoPS) is a single molecule technique that provides such quantification for fluorescent species. CoPS exploits the photon antibunching effect, i.e. a dye as a quantum system may emit at most one photon per laser excitation cycle. The simultaneous detection of multiple photons in a four-detector confocal microscope enables the estimation of the number of independent emitters and their molecular brightness [1,2]. Since the method is directly based on the measured photon statistics, it does not require calibration.
Using both simulations and experiments with defined, DNA-based probes, we analyzed the critical interplay of fluorophore properties and analysis parameters to achieve reliable quantification. We discovered that the time resolution can be up to ~10ms at high molecular brightness, which opens new possibilities for time resolved measurements. The findings stress that the choice of analysis parameters is vital and provide a practical guideline to choose suitable dyes.
We show that CoPS is feasible with many fluorophores in different applications. In the biological context, we investigated the label number distribution of several commonly used fluorescent markers, e.g., SNAP-tag and nanobodies, a prerequisite for quantitative investigations in cells [3]. Moreover, we show that CoPS offers new perspectives for characterization of photophysical processes in photoluminescent materials. We studied the behavior of conjugated polymers, materials important for organic semiconductor devices like polymer solar cells. Time-resolved CoPS measurements revealed heterogeneous dynamics in polymer chain photophysics [4].
[1] H. Ta, J. Wolfrum, and D.-P. Herten, Laser Physics 2010, 20, 119–124
[2] K. S. Grußmayer and D.–P. Herten, in Advanced Photon Counting, (Eds.: P. Kapusta, M. Wahl, R. Erdmann), Springer International Publishing, Cham, Apr. 2015, pp. 159–190
[3] K. S. Grußmayer, A. Kurz, D.–P. Herten, ChemPhysChem Feb. 2014, 15, 734–742
[4] K. S. Grußmayer, F. Steiner, J. M. Lupton, D.–P. Herten, and J. Vogelsang, ChemPhysChem Dec. 2015, 16, 3578–3583
Abstract:
Quantitative information is key to unravel molecular processes in all fields of research. Counting by Photon Statistics (CoPS) is a single molecule technique that provides such quantification for fluorescent species. CoPS exploits the photon antibunching effect, i.e. a dye as a quantum system may emit at most one photon per laser excitation cycle. The simultaneous detection of multiple photons in a four-detector confocal microscope enables the estimation of the number of independent emitters and their molecular brightness [1,2]. Since the method is directly based on the measured photon statistics, it does not require calibration.
Using both simulations and experiments with defined, DNA-based probes, we analyzed the critical interplay of fluorophore properties and analysis parameters to achieve reliable quantification. We discovered that the time resolution can be up to ~10ms at high molecular brightness, which opens new possibilities for time resolved measurements. The findings stress that the choice of analysis parameters is vital and provide a practical guideline to choose suitable dyes.
We show that CoPS is feasible with many fluorophores in different applications. In the biological context, we investigated the label number distribution of several commonly used fluorescent markers, e.g., SNAP-tag and nanobodies, a prerequisite for quantitative investigations in cells [3]. Moreover, we show that CoPS offers new perspectives for characterization of photophysical processes in photoluminescent materials. We studied the behavior of conjugated polymers, materials important for organic semiconductor devices like polymer solar cells. Time-resolved CoPS measurements revealed heterogeneous dynamics in polymer chain photophysics [4].
[1] H. Ta, J. Wolfrum, and D.-P. Herten, Laser Physics 2010, 20, 119–124
[2] K. S. Grußmayer and D.–P. Herten, in Advanced Photon Counting, (Eds.: P. Kapusta, M. Wahl, R. Erdmann), Springer International Publishing, Cham, Apr. 2015, pp. 159–190
[3] K. S. Grußmayer, A. Kurz, D.–P. Herten, ChemPhysChem Feb. 2014, 15, 734–742
[4] K. S. Grußmayer, F. Steiner, J. M. Lupton, D.–P. Herten, and J. Vogelsang, ChemPhysChem Dec. 2015, 16, 3578–3583
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