Ultrafast photonics for fluorescence imaging and time-resolved assays
Drug discovery is becoming increasingly expensive as many drug candidates identified with conventional assays fail in animal or patient trials. High Content Analysis (HCA), which utilises high resolution imaging of cells, has begun to answer the urgent need therefore, for more specific readouts of drug actions in more realistic physiological contexts, identified earlier in the drug development process. However, it remains challenging to monitor the biomolecular interactions that control disease pathways. Fluorescence lifetime imaging (FLIM) can provide opportunities to quantitatively study such signalling processes within cells, tissue samples and small organisms.
By combining the time-gated imaging technology developed by Kentech Instruments Ltd with GE Healthcare multiwell plate imaging technology, we have demonstrated practical instrumentation for automated HCA based on fluorescence lifetime imaging (FLIM), which maps the rate at which excited fluorescence molecules lose energy. This fluorescence lifetime is a function of the local molecular environment and so FLIM can map changes in physical or chemical properties. In particular, the fluorescence lifetime can change when fluorescently labelled biomolecules are in close proximity (i.e. within ~10 nm of each other) and can exchange energy through an interaction called “Förster resonant energy transfer” (FRET). FLIM can therefore map the spatio-temporal behaviours of “protein-protein” and other biomolecular interactions, including those associated with disease processes. This provides much enhanced opportunities to study the efficacy of drug candidates and permit systematic investigations for drug discovery. Typical changes in fluorescence lifetime are on picosecond timescales and so FLIM requires ultrafast laser and measurement technology. This has impeded its take-up for drug discovery to date but we have developed practical prototype technology for FLIM assays that incorporates a novel time-gated imaging system and also provides 3-D and automated image acquisition, requiring only~15 minutes to read a 96 well plate, which makes it suitable for medium throughput applications in drug screening. We are evaluating this technology for commercial production and we are developing software tools that could be integrated with existing HCA software platforms. We have also investigated exemplar assays to illustrate the potential of FLIM readouts for HCA.
Translation along drug discovery pipeline: The superior quantitative readouts of biomolecular interactions that FLIM provides can be applied to elucidate signalling networks in cell based assays and also translated to live disease models such as zebrafish. This would permit direct comparison of drug candidates’ efficacy across different physiological contexts, permitting efficacy, side-effects and toxicology to be better established before expensive clinical trials.