Modulated or alternating excitation in fluorescence correlation spectroscopy Gustav Persson, Tor Sandén, Per Thyberg, Jerker Widengren* Royal Institute of Technology, Department of Applied Physics, Experimental Biomolecular Physics, SE-106 91 Stockholm, Sweden 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 cross- correlation 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. Keywords: fluorescence correlation spectroscopy, fluorescence cross-correlation spectroscopy, modulated excitation, alternating excitation, photobleaching, triplet state, protonation 1. INTRODUCTION Fluorescence correlation spectroscopy (FCS) is a technique that makes use of intrinsic fluctuations to gain information of the dynamics of a system at thermodynamic equilibrium 1-7 . Two of its greatest virtues are its non-invasiveness, i.e. the possibility to study the fluctuations of a system around its true unperturbed equilibrium, and its high sensitivity. Virtually any dynamic process affecting the fluorescence intensity can be studied by FCS. Fluctuations in the detected fluorescence intensity may arise from the diffusion of molecules in and out of the detection volume or by changes in fluorescence characteristics due to reaction kinetics or conformational changes. A common complication in FCS measurements is, however, that several processes take place in the same time regime. Thus, their contributions to the correlation curve will overlap, making them difficult to separate. We have previously shown that the amount of triplet buildup can be controlled by modulating the excitation with pulse widths and separations in the range of the triplet relaxation time 8 . We have also shown that modulated excitation can be applied to FCS measurements without loss of information by carefully designing the excitation pulse trains and filtering the correlation curve through division by a time-shifted correlation 9 . The use of modulated excitation in FCS makes it possible to selectively suppress a photoinduced process, thus favoring the study of other processes in the same time range. It has been shown that modulated excitation can be used to suppress triplet population build-up more efficiently than by reducing the CW excitation power 9 . This results in higher fluorescence intensity during excitation and reduced photobleaching. In a current project we introduce spectral cross-correlation as a tool to selectively highlight a process giving rise to a spectral shift of absorption and emission. We use a pH-sensitive so called ratiometric fluorophore and show that, by application of spectral cross-correlation, the protonation-deprotonation process will manifest itself as an anticorrelation, making the distinction from, e.g., diffusion or triplet kinetics substantially more robust. The fluorophore used in this study is the experimental compound NK 138 from ATTO-Tec GmbH, Siegen, Germany. Separating the protonated and * jerker@biomolphysics.kth.se; phone +46 8 5537 8030; fax +46 8 5537 8216; www.biomolphysics.kth.se Single Molecule Spectroscopy and Imaging II, edited by Jörg Enderlein, Zygmunt K. Gryczynski, Rainer Erdmann Proc. of SPIE Vol. 7185, 718509 · © 2009 SPIE · CCC code: 1605-7422/09/$18 · doi: 10.1117/12.808912 Proc. of SPIE Vol. 7185 718509-1