J Fluoresc (2007) 17:43–48 DOI 10.1007/s10895-006-0145-1 ORIGINAL PAPER Fluorescence Lifetime Correlation Spectroscopy Peter Kapusta · Michael Wahl · Aleˇ s Benda · Martin Hof · J¨ org Enderlein Received: 19 July 2006 / Accepted: 13 October 2006 / Published online: 17 November 2006 C  Springer Science+Business Media, LLC 2007 Abstract This article explains the basic principles of FLCS, a genuine fusion of Time-Correlated Single Photon Counting (TCSPC) and Fluorescence Correlation Spectroscopy (FCS), using common terms and minimum mathematics. The use- fulness of the method is demonstrated on simple FCS ex- periments. The method makes possible to separate the auto- correlation function of individual components of a mixture of fluorophores, as well as purging the result from parasitic contributions like scattered light or detector afterpulsing. Keywords FCS . TCSPC . Lifetime . Correlation . Multichannel detection . Scattering . Afterpulsing Introduction The combination of Time-Correlated Single Photon Count- ing (TCSPC) and Fluorescence Correlation Spectroscopy (FCS), called Fluorescence Lifetime Correlation Spec- troscopy (FLCS), is a method that uses picosecond time- resolved detection for separating different FCS contribu- P. Kapusta () · M. Wahl PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany e-mail: photonics@picoquant.com A. Benda · M. Hof J. Heyrovsk´ y Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejˇ skova 3, 18223 Prague, Czech Republic J. Enderlein IBI-1, Forschungszentrum J¨ ulich in der Helmholtzgesellschaft, 52425 J¨ ulich, Germany tions. The emphasis is on the word separating. FLCS does not involve fitting of a multiple-parameter model to a com- plex autocorrelation function. Instead, a separate autocorre- lation function is calculated for each fluorescence lifetime component, emitted for example by various species in the sample. The only assumption is that the various components have distinct and non-changing lifetime signatures. The core of the method is a statistical separation of different fluores- cence contributions on a single photon level. An essential requirement for FLCS is a sub-nanosecond pulsed excitation instead of continuous wave (CW) illumina- tion. The second requirement is the ability to simultaneously measure the fluorescence photon arrival time on two different time scales: relative to the excitation pulse with picosecond resolution (TCSPC) and relative to the start of the experiment with nanosecond precision. The experimental and analysis technique outlined is an important extension of standard FCS. The core idea ap- peared in 2001 [1] but received only a little attention until now, although recent publications [2, 3] present exciting new applications of this method. The principle FLCS is best understood in comparison with conventional FCS using CW excitation and single channel detection. The standard result of an FCS experiment is the autocorrelation function (ACF) of the fluorescence intensity fluctuations. This can be calculated by a hardware autocorrelator, but the recent state-of-the-art is to use more versatile software pro- cessing of recorded individual photon arrival times [9]. The core problem of FCS is that if the detected signal contains more than one component, the resulting ACF is a linear com- bination of the contributions from the different components. A trivial example is a sample with two kinds of diffusing Springer