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research article

Time-resolved molecule counting by photon statistics across the visible spectrum

Grussmayer, Kristin Stefanie  
•
Herten, D. -P.
2017
Physical Chemistry Chemical Physics

In the past few years quantification of fluorescently labeled (bio-) molecules has become of increasing importance and several approaches have been developed to address this task. Counting by photon statistics measures the distribution of multiple photon detection events that carry information about the number and brightness of independently emitting fluorophores. The method enables absolute and non-destructive quantification, with the quality of estimates critically depending on the ability to accurately measure said photon statistics. Here, we present a combination of simulations and experiments that relate fundamental properties of fluorophores, i.e. their molecular brightness and photostability, to important experimental conditions, i.e. excitation power and acquisition time. Thereby, experimental settings and analysis parameters can be quantitatively evaluated, making counting by photon statistics a robust method for absolute counting of the number of emitters in a diffraction limited observation volume. We show that the time-resolution of counting varies with the fluorophore brightness and can be as fast as 10-100 ms. At the same time, the range of suitable fluorophores can be easily assessed. We evaluated the brightness and photostability of 16 organic dyes across the visible spectrum, providing information crucial for a range of single-molecule spectroscopy applications. This opens up exciting possibilities to analyze absolute stoichiometries in dynamic multi-component complexes.

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Type
research article
DOI
10.1039/c7cp00363c
Web of Science ID

WOS:000399004700036

Author(s)
Grussmayer, Kristin Stefanie  
Herten, D. -P.
Date Issued

2017

Publisher

Royal Society of Chemistry

Published in
Physical Chemistry Chemical Physics
Volume

19

Issue

13

Start page

8962

End page

8969

Editorial or Peer reviewed

REVIEWED

Written at

OTHER

EPFL units
IMT  
Available on Infoscience
May 30, 2017
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/137974
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