Assays
Figure 12: Fluorescence lifetimes of three FRET pairs obtained with direct waveform recording using FI2’s lifetime reader. The samples consisted of Cerulean and Venus fluorescent proteins separated by 5, 17, and 32 amino acid linkers. See Koushik et al, Biophysical Journal, 91, L99-L101 (2006) for details on construct generation and comparison data obtained via two-photon excited time-correlated single photon counting. Each protein was expressed in a total of 22 wells on a 96-well plate and the entire plate was scanned four times. Scan time per plate was 29 seconds. Fluorescence decays for the Cerulean donor undergoing FRET were fit to a single exponential decay. Error bars represent one standard deviation and are substantially smaller than in the Koushik et al work
1536 plate in continuous scanning mode is less than two minutes. The FI2 direct recording approach is extremely fast, accurate and precise. The response of thousands of photoevents is recorded on every excitation pulse rather than one count for 100 exci- tation pulses as in time-correlated single photon counting (TCSPC). Signal-to-noise ratio (SNR) at the peak of the decay waveform can easily exceed 500:1 even at 10 wells per second read speed, equivalent to 250,000 counts in the peak channel by TCSPC. The excellent precision makes it possi- ble to reliably monitor extremely small and subtle changes in the fluorescence decay characteristics even when operating in high throughput screening (HTS) mode. FI2 plans to bring to market a next- generation plate reader designed to accommodate a wider variety of laser excitation sources by the end of 2010. Fluorescence lifetime imaging microscopy (FLIM) is the usual form in which live cells express- ing genetically encoded sensors are combined with fluorescence lifetime, but FLIM is unsuitable for HTS. FI2 and Montana Molecular have demon- strated the feasibility of simply averaging the fluo- rescence emitted by a collection of cells in the well. Illustrative data is shown in Figure 12 for three Cerulean::Venus FRET pairs that have a short link-
Drug Discovery World Summer 2010
Figure 11: Current version of the two-channel FI2 plate reader equipped with a microchip laser excitation source. Filters are used to isolate either the 532nm or 355nm laser wavelengths for excitation. Each channel has a six-position filter wheel for fluorescence wavelength selection
er consisting of 5, 17, or 32 amino acids between the chromophores. The FRET pairs were transient- ly expressed in HEK293 cells. As expected, the shorter the linker, the higher the FRET efficiency and the shorter the Cerulean donor lifetime. Twenty-two wells for each of the three pairs were studied in a 96-well plate. The figure shows that lifetime differences of just 0.1ns or 0.2ns are reli- ably distinguished.
AssayMetrics (
www.assaymetrics.com) has devel- oped a range of fluorescence labels specifically suit- able for assays using fluorescence lifetime (FLT) as their readout signal. The first distinctive feature of these labels is their very long lifetime compared to, for example, fluorescein or rhodamine, providing a much enhanced signal window. The second is that their fluorescence can be dynamically quenched by two natural amino acids, tyrosine and tryptophan. Further, the labels are extremely photo stable. Together, these characteristics allow the researcher in drug discovery to rapidly develop cost-effective, customised, robust assays. The assay signal is pro- duced by a single label attached to, for example, a peptidic enzyme substrate. No secondary compo- nents such as antibodies, beads, etc are necessary.
79
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92