senior research associate

Áron SIPOS research associate
Ferenc SARLÓS scientific administrator


The fluorescence kinetics of a chromophore highly depends on its interactions with the molecular micro-environment. While the excited-state lifetime of an isolated chromophore typically falls into the ns timescale, upon environmental interactions it could reduce even to several ps. Hence, fluorescence kinetics can sensitively monitor the structural states of heterogeneous biological materials.

In our laboratory we recently constructed a measuring apparatus for detecting fluorescence kinetics in the 100 fs – 10 ns time range and the whole visible spectral region. This device combines the advantages of two measuring methods: the high sensitivity of time-correlated single photon counting and the high time resolution of fluorescence upconversion.

For working with biological samples a key requirement is the successful analysis of multicomponent fluorescence kinetics data. To that end we developed a novel method based on the principle of compressed sensing. This approach seeks for representations of complex kinetic data with a large set of lifetimes, but prefers the sparsest one, that is which has the minimum number of nonzero amplitudes. We have found that the Basis Pursuit Denoising (BPDN) optimization technique is an excellent method for the analysis of complex fluorescence decay kinetics1.

Figure 1: Fluorescence kinetics from FAD in aqueous solution at different wavelengths (red indicates high intensity).

A typical fluorescence kinetics dataset obtained from flavin adenine dinucleotide (FAD) coenzyme by the above methods is presented in Fig. 1. In solvents the FAD molecules can exist in both open and stacked conformations, characterized by ns and ps fluorescence lifetime(s), respectively.

Figure 2: Results obtained by BPDN analysis of the kinetics presented in Fig. 1. Left panel: Amplitude distribution over the space of time constants and wavelengths (red: positive, green: zero, blue: negative). Right panel: Decay associated difference spectra corresponding to the traces shown in the left panel.

Our analysis resulted in five distinct wavelength-independent lifetimes (Fig. 2). A ns component can be attributed to the open conformation, and three other ones falling to the 1-100 ps range could correspond to different stacked states. The fifth, subpicosecond component, which has positive amplitude in the low and negative one in the high spectral region, is the manifestation of an excited-state spectral shift, related to the solvation dynamics of water molecules surrounding the chromophore. In flavocytochrome C sulfide dehydrogenase enzyme, which contains a covalently bound FAD molecule, a marked downshift of the lifetimes was observed, indicating further interactions with the amino acid residues.

Selected publications

Groma, G.I., Heiner, Z., Makai, A. and Sarlós, F. (2012). Estimation of kinetic parameters from time-resolved fluorescence data: A compressed sensing approach. RSC Advances 2: 11481-11490.