Lifetime Characterization of Cerulean::Venus FRET Standards in Live Cells Using the NovaFluor PR Fluorescence Lifetime Plate Reader
Fluorescence Innovations and Montana Molecular have teamed up to develop new live cell assays for the NovaFluor PR Fluorescence Lifetime Microplate Reader, which utilizes Direct Waveform Recording™ based on microchip laser excitation sources and proprietary digitizers that record the complete fluorescence decay waveform on every excitation pulse. In addition to lifetime, the reader is compatible with intensity, polarization, and time-resolved polarization applications in 96-, 384-, and 1536 formats. The reading can be conducted using continuous scanning across the central 50% of the well or in stop-and-go mode. Read time for a 1536 plate in continuous scanning mode is less than 2 minutes. The Direct Waveform 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 excitation 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 ten well per second read speeds, equivalent to 250,000 counts in the peak channel by TCSPC.
The excellent precision makes it possible to reliably monitor extremely small and subtle changes in the fluorescence decay characteristics even when operating in high throughput screening (HTS) mode. Fluorescence lifetime imaging > microscopy (FLIM) is the usual form in which live cells expressing genetically encoded sensors are combined with fluorescence lifetime, but FLIM is unsuitable for HTS. Fluorescence Innovations and Montana Molecular have demonstrated the feasibility of simply averaging the fluorescence emitted by a collection of cells in the well. Illustrative data is shown in the accompanying figure for three Cerulean::Venus FRET pairs that have a short linker consisting of 5, 17, or 32 amino acids between the chromophores (Koushik SV et.al. Biophys J 2006). The FRET pairs were transiently expressed in HEK293 cells. As expected, the shorter the linker, the higher the FRET efficiency and the shorter is the Cerulean donor lifetime. Twenty-two wells for each of the three pairs were studied in a 96-well plate at a total plate read time of 29 seconds. The figure shows that lifetime differences as small as 0.1 ns are reliably distinguished.
High Throughput Fluorescence Lifetime Measurements of FRET-Based Protein Sensors in Live Cells
Although widely used for basic research applications, genetically encoded fluorescent biosensors have had little impact on drug discovery because of difficulties in measuring and interpreting fluorescence intensity read-outs, including poor signal to noise ratios, variability in cell expression, and interference from fluorescence emitted by compounds. Combining the specificity of genetically encoded fluorescent sensors with fluorescence lifetime measurements overcomes many of these issues; however traditional methods of measuring fluorescence lifetimes are relatively slow and thus unsuitable for high throughput screening. In this application note a NovaFluor PR Plate Reader equipped with a pulsed Nd:YAG laser with a 532 nm line and ultra high speed Direct Waveform Recording™ enables us to measure fluorescence decay from transiently expressed red fluorescent fusion proteins in living HEK 293 cells with unprecedented precision and speed. We show accurate fluorescence lifetime measurements from living cells in 96-well plates in less than 30 seconds per plate.
The transfection was done using the standard lipofectamine 2000 protocol (Invitrogen) with little optimization.
Lifetime measurements can readily differentiate between different fluorescent proteins. To test the accuracy of the lifetime measurements, we expressed different fluorescent fusion proteins in HEK 293 cells in different wells of the same 96 well plate. A 29 second scan of the entire plate was sufficient to show that we can readily resolve the difference between lifetimes of a tagRFP::PKC-delta fusion, an mKOK::CiVSP fusion, and a membrane targeted mCherry protein.
The data plotted to the right is normalized for comparison and shows lifetime decay waveforms from 22 different wells demonstrating exceptional reproducibility.
Casper3-GR, a genetically encoded sensor for apoptosis: Comparing fluorescence intensity to lifetime for HTS
Plotted above in figure 3 are intensity vs. lifetime measurements from living HEK 293 cells in a 96 well plate transiently expressing a FRET-based caspase sensor, Casper3. Intensity measurements vary from well-to-well due to differences in cell density and expression levels; the staurosporin triggered apoptosis in wells 13 to 24 is undetectable in intensity mode. Fluorescence lifetime is less sensitive to this variability, providing an unambiguous readout of caspase activation by staurosporin. The waveforms used to produce this data are shown below.
Casper3 carries the donor tagGFP and the acceptor tagRFP. Staurosporin triggers apoptosis, which in turn causes a caspase to cleave a linker between the FRET pair. Plotted in red are the tagGFP lifetimes from 12 different wells, and in green is the change in donor lifetimes, from 12 wells, caused by triggering the apoptotic pathway. This data was collected in under 30 seconds using the NovaFluor Plate Reader and demonstrates that FRET changes in living cells can be detected reliably in HTS mode.
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