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Proceedings Paper

Quantification of fluorescent samples by photon-antibunching
Author(s): Anton Kurz; Michael Schwering; Dirk-Peter Herten
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Paper Abstract

Typical problems in molecular biology, like oligomerization of proteins, appear on non-resolvable length scales. Therefore a method which allows counting numbers of fluorescent emitters beyond this barrier can help to unveil these questions. One approach engaging this task makes use of the photon antibunching (PAB) effect. Most fluorophores are single photon emitters. Therefore upon a narrow excitation pulse they might only run through one excitation cycle and emit one photon at a time. This behavior is known as PAB. By analyzing coincident events of photon detections (Coincidence Analysis, CCA ) over many excitation cycles the number of fluorophores residing in the confocal volume can be estimated. Simulations have shown that up to 40 fluorophores can be distinguished with a reasonable error. In follow-up experiments five fluorophores could be distinguished by CCA. In this work the method is applied to a whole sample set and statistical variance and robustness are determined. CCA is critical to several parameters like photo stability, background noise, label efficiency and photopysical properties of the dye, like brightness and blinking. Therefore a reasonable scheme for analysis is introduced and setup parameters are optimized. To proof the superiority of CCA, it has been applied to estimate the number of dyes for a well-defined probe and the results have been compared with bleach step analysis (BS analysis), a method based on the ability to observe single bleach-steps.

Paper Details

Date Published: 13 February 2012
PDF: 8 pages
Proc. SPIE 8228, Single Molecule Spectroscopy and Superresolution Imaging V, 82280K (13 February 2012); doi: 10.1117/12.909099
Show Author Affiliations
Anton Kurz, Univ. Heidelberg (Germany)
Michael Schwering, Univ. Heidelberg (Germany)
Dirk-Peter Herten, Univ. Heidelberg (Germany)

Published in SPIE Proceedings Vol. 8228:
Single Molecule Spectroscopy and Superresolution Imaging V
Jörg Enderlein; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling; Ingo Gregor, Editor(s)

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