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Proceedings Paper

Modulated or alternating excitation in fluorescence correlation spectroscopy
Author(s): Gustav Persson; Tor Sandén; Per Thyberg; Jerker Widengren
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Paper Abstract

We have previously shown that formation of triplet states and other photo-induced states can be controlled by modulating the excitation with pulse widths and periods in the range of the transition times of the involved states. However, modulating the excitation in fluorescence correlation spectroscopy (FCS) measurements normally destroys correlation information and induces ringing in the correlation curve. We have introduced and experimentally verified a method to retrieve the full correlation curves from FCS measurements with modulated excitation and arbitrarily low fraction of active excitation. Modulated excitation applied to FCS experiments was shown to suppress the triplet build-up more efficiently than reducing excitation power with continuous wave excitation. The usefulness of the method was demonstrated by measurements done on fluorescein at different pH, where suppression of the triplet significantly facilitates the analysis of the protonation kinetics. Using a fluorophore where the protonation-coupled fluorescence intensity fluctuations are due to spectral shifts, introduction of two-color alternating excitation and spectral crosscorrelation can turn the protonation component of the correlation curve into an anti-correlation and further facilitate the distinction of this component from those of other processes.

Paper Details

Date Published: 24 February 2009
PDF: 10 pages
Proc. SPIE 7185, Single Molecule Spectroscopy and Imaging II, 718509 (24 February 2009); doi: 10.1117/12.808912
Show Author Affiliations
Gustav Persson, Royal Institute of Technology (Sweden)
Tor Sandén, Royal Institute of Technology (Sweden)
Per Thyberg, Royal Institute of Technology (Sweden)
Jerker Widengren, Royal Institute of Technology (Sweden)

Published in SPIE Proceedings Vol. 7185:
Single Molecule Spectroscopy and Imaging II
Jörg Enderlein; Zygmunt Karol Gryczynski; Rainer Erdmann, Editor(s)

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