Fluorescence Lifetime Correlation Spectroscopy for Metal Ion Quantitation

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The late transition-metal ions have an ambiguous role in the life science. On the one hand, some of these are essential constituents of proteins. On the other hand, excess supply of the same elements is often associated with serious ailments like Alzheimer's disease. A real-time monitoring of the ion concentration in any living organism is challenging as most established, metal selective methods entail sample destruction. However, sensitive methods are mandatory for studying metal ion homeostasis, and new methods have to distinguish between the disposable ion pool and the strongly bound enzyme components.Recently, we could show in two publications that bivalent copper ions lead to a reduction of the fluorescence lifetime of fluorescent proteins. This approach could also be used to map the uptake of copper ions in plant root cells by fluorescence lifetime imaging microscopy. The observed reversible change obeys a titration behavior with a midpoint at micromolarity. Despite these promising achievements, the investigation of ion homeostasis demands further improvements: firstly, the sensitivity of the probe must be increased; secondly and more importantly, the concentration of the indicator cannot longer be neglected and must be quantified, e.g. by fluorescence correlation spectroscopy (FCS). We therefore propose to combine lifetime measurements and FCS in the so-called fluorescence lifetime correlation spectroscopy (FLCS) for accessing naturally occurring ion concentration in living systems.During the grant period, we will establish at the beginning FLCS in our laboratory with the already described copper-sensitive system and an existing setup. Afterwards, we'll head for nanomolarity by using synthetic fluorophores with a higher affinity for metal ions. Once FLCS will have been established for lower ion concentrations than micromolarity, we'll screen several described, copper-binding peptides for their compatibility with FLCS. These investigations are aimed at proposing a detection scheme as well as a peptide sequence for studying copper homeostasis. If time is left, we will address the question how copper can be quantified at these concentration levels among other, more abundant transition-metal ions.