Assays
Repetitive Excitation
Many Cycles w/o photon
t Start-Stop Time 1 Start-Stop Time 2 Start-Stop Time 3 Start-Stop Time 4
cts t
Figure 8: Principles of TCSPC measurements on Tecan’s Ultra Evolution. A pulsed laser repetitively excites the sample. The intensity of the excitation pulses is adjusted such that for any one pulse no more than one photon counting event is generated on the detector. According to the time measured between the laser pulse and the detector event, this count is added to a histogram collecting count numbers. The channel numbers are sorted to reflect the arrival time of the single photons at the detector. One to ten million repetitions of this elementary experiment lead to smooth histogram data that represent the fluorescence decay curve
differences plotted on a histogram. Between one and ten million repetitions of these elementary events produces a smooth histogram representing the fluo- rescence decay curve. As fluorescence intensity-based readouts are background limited, they are susceptible to numerous assay interferences. Consequently the drug discovery community has investigated more
robust assay principles, including fluorescence polar- isation (FP) and, more recently, time-resolved fluo- rescence (TRF) and time-resolved fluorescence reso- nance energy transfer (TR-FRET) assays. The chal- lenge is to reduce the number of short-lived autoflu- orescence signals from plates, buffers and com- pounds. When it comes to data robustness and reduction of fluorescence-based interferences, Tecan’s customers find fluorescence lifetime method- ology a powerful and robust alternative to TR- FRET-based measurements. FLT is highly susceptible to changes in the micro-environment surrounding the label, an aspect which could be exploited to allow the development of assay platforms targeting a variety of enzyme classes. In addition to a suitable microplate reader, researchers also need access to appropriate reagents which are compatible with FLT applications, and previously there has been a lack of reagent providers offering ready-made FLT kits suit- able for specific drug discovery applications. In addi- tion, the complexity of FLT readout demands that researchers become familiar with the principles and practice of FLT-based applications. These prerequi- sites currently limit the broad use of FLT. The latest drug discovery related conferences show interesting new activities and offerings around FLT, striving for robust, complementary and synergistic screening solutions. Tecan is working closely with its cus- tomers, exploring this promising technique and iden- tifying opportunities where it can offer the technolo- gy to a broader industrial base (Figure 8).
Figure 9: Edinburgh Instruments’ NanoTaurus, the first dedicated FLT plate reader 76
Edinburgh Instruments (
www.edinst.com), with more than 30 years’ experience in pioneering devel- opments in FLT, has developed the NanoTaurus, the first dedicated FLT plate reader. The NanoTaurus is based on the FLT method of choice, ie Time Correlated Single Photon Counting (TCSPC). The principle of TCSPC is analogous to using a fast stop watch to repetitively measure the time difference between each excitation pulse and the detection of the first fluorescence photon. Each measurement is added to form a histogram which depicts the time resolved sample emission. TCSPC outperforms all other methods for time resolved fluorescence in terms of sensitivity (detection at the quantum limit of single photons), dynamic range, accuracy and precision. Importantly, as a digital process, the data noise in TCSPC is Poissonian in nature (the noise of each data point is given by the square root of the number of counts) meaning that decays can be analysed further into the tail, helping precision. The measured fluorescence time response in an assay is made up by a linear combination of the emissions from the dye labelled substrate and its
Drug Discovery World Summer 2010
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