Assays
Figure 10
Berthold Detection Systems’ LF 502 NanoScan multimode fluorescence microplate
reader, with lifetime capability
product. The complex data from each well meas- ured in the NanoTaurus can be converted to a con- venient, single parameter readout of average life- time, , given by:
= (B1τ1)2 + (B2τ2)2 / (B1τ1 + B2τ2) where B1 and B2 are the amplitudes and τ1 and
τ2 are the lifetimes of the two components. The NanoTaurus uses proprietary, fast data harvesting
acquisition electronics and fluorescence decay analysis software to obtain high quality measure- ments in a 384-well plate, with sample volumes less than 10µl, in less than five minutes. In Almac FLEXYTE® assays (see below), a typical variation in average lifetime between substrate and product of 5ns is obtained, compared to the measurement precision of the NanoTaurus of better than 0.1ns. Using a long lifetime fluorophore as reporter, a high level of separation from interfering com- pounds, typically with a lifetime of a few nanosec- onds, can be routinely achieved. More sophisticat- ed expert software can be used to indicate, and in some cases to automatically compensate for, com- pound interference, hence dramatically reducing the incidence of false hits (Figure 9).
The LF 502 NanoScan multimode fluorescence microplate reader from Berthold Detection Systems (
www.berthold-ds.com) (formerly IOM GmbH) is probably the most versatile microplate
78
reader on the market among the instruments dedi- cated to fluorescence lifetime analysis. Technically, the basis is given by a nanosecond-pulsed laser with a low repetition rate and across the UV and visible range variable wavelength. These features lead directly to the unrivalled capability of seam- lessly measuring fluorescence lifetimes between nanoseconds and milliseconds, ie over more than six orders of magnitude. Nearly every fluorophore can be analysed. Detection is accomplished by two ultrafast PMTs in two separate channels enabling the simultaneous measurement at two different flu- orescence wavelengths. The NanoScan is capable of reading not only FLT assays over the whole span of lifetimes but also TRF and HTRF, Nano-TRF or fluorescence intensity assays. Due to its flexibility the NanoScan opens up a whole new world of applications. The availability of blue and green excitation wavelengths enables the use of novel quantum dot labels with outstanding long nanosecond lifetimes accompanied by large stokes shifts while avoiding compound fluorescence due to UV excitation. Furthermore, quantum dots are considered as basis for the development of multi- plexed assays. Short and intense UV laser pulses enable the efficient excitation of Terbium and Europium labels thus extending their use not only for TRF applications but rather in FLT mode with- in FRET or quenching assays. The microsecond- order lifetimes of transition metal complexes (eg Ruthenium) can easily be exploited by the NanoScan either in Nano-TRF assays (eg in a com- mercial cAMP assay) or directly in FLT mode. Recently progress was made to extend the span of detection methods to nanosecond time-resolved polarisation assays as recently demonstrated with a red fluorescing label in an enzymatic assay4. The LF502 NanoScan is ready to be integrated with stackers, dispensers and assay processors for appli- cations in 96 to 1536 well microplates (Figure 10).
Fluorescence Innovations (FI2) (www.fluorescen-
ceinnovations.com) and Montana Molecular (
www.montanamolecular.com) have teamed up to develop new live cell assays for FI2’s fluorescence lifetime microplate reader, which is based on microchip laser excitation sources and proprietary digitisers that record the complete fluorescence decay waveform on every excitation pulse. In addi- tion to lifetime, the reader (see Figure 11) is com- patible with fluorescence intensity, polarisation, and time-resolved polarisation formats in 96-, 384, and 1536 formats. The reading can be conducted for continuous scanning across the central 50% of the well or in stop-and-go mode. Read time for a
Drug Discovery World Summer 2010
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