Assays
Fluorescence lifetime assays – the basics
When a light photon is absorbed by a molecule, the energy is used to lift an outer electron from its ground state into an excited state. Upon its return to the ground state, the energy can be dissipated through various channels; one possibility is to re-emit a photon, usually with a slight- ly reduced energy. This process is called fluorescence. The time interval between absorbing a photon and re-emitting one, is called the fluores- cence lifetime. For each individual molecule the length of time spent in the excited state will vary. As an ensemble of many molecules, the decay of the proportion of molecules in the excited state is similar to a radioactive decay, and the mathematical description is an exponential func- tion. The fluorescence lifetime is the time after which the exponential curve reaches a value of 1/e, or about 37%. Most fluorescent samples do not convert 100% of the absorbed photons into fluorescence photons. The relative probabilities between the
radiative process and those channels where no photon is emitted (thus: non-radiative), govern the fluorescence lifetime of the sample. The bal- ance between the radiative and non-radiative channels can be altered, eg by bringing a quencher into the vicinity of the fluorescent molecule: the quencher adds a non-radiative channel, thereby reducing the fluorescence lifetime. Typical values for the fluorescence lifetime are in the order of one to five nanoseconds (10-9s). Fluorescein, rhodamine and similar dyes fall into this class. Some better known exceptions are rare earth metal ions in a chelate or kryptate complex; these have fluorescence lifetimes of up to one millisecond (10-3s). Similarly, a complex with the transition metal ruthenium displays fluorescence with lifetimes of several hundreds of nanoseconds (10-5 to 10-6s). Measuring the fluorescence lifetime has been done in several ways. The most intuitive method is to excite the sample with a very short light pulse and record the fluorescence intensity decay with sub-nanosecond resolution. The resulting decay curve can then be analysed by a mathe- matical curve fit, assuming a mono-exponential function in the easiest case. The best known technique for recording these extremely fast time courses is time-correlated single photon counting (TCSPC). Alternatively, one can analyse the sample's response to excitation light modulated at a high-frequency (phase-modulation method). Lesser known methods employ, for instance, boxcar integration with varying delay and gate widths, and fast digitising of the entire decay curve. Note that the term ‘time-resolved fluorescence’ is often used to describe measurements in which the fluorescence intensity is integrated after a fixed delay time. This technique can help suppress background fluorescence if a label with suitably long lifetime is used (see the above mentioned rare-earth and ruthenium complexes). No real time resolution is currently used in these assays but may provide further information and assay stability.
Utilising fluorescence lifetime as a readout signal in drug screen-
ing assays requires a sufficiently large change of the lifetime. With current measurement techniques a lifetime value can be recorded with a precision of a few tens of picoseconds, while keeping record- ing times at about one second per sample. This necessitates that the assay signal changes by several nanoseconds. Standard fluorophores with lifetimes between 1ns and 5ns are therefore relatively imprac- tical starting points. More recent developments (see Almac, Assaymetrics below) have produced labels with lifetimes between 10 and 25 nanoseconds, making fluorescence lifetime available for assay development in drug discovery.
Top panel: Typical FLT data recorded by TCSPC. The fluorescence intensity is plotted semi-logarithmically versus a nanosecond timescale. Two sets of data are shown, corresponding to samples with 10ns (blue) and 14ns lifetimes (red). Each curve is made up of approximately 2,500 points, with a time resolution below 50ps between points. A total of 200,000 photons were collected per curve. The continuous lines are the fitted mono-exponential curves, yielding the lifetime values, at a typical relative error of 0.5%. Lower panel: Displays the weighted residuals for the fits, which are the normalised deviations of the fit from the data. They are useful as a quality control tool during assay development.
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Drug Discovery World Summer 2010
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