Automated multiwell fluorescence lifetime imaging for Förster resonance energy transfer assays and high content analysis

Abstract

Fluorescence lifetime measurements can provide quantitative assays of the local fluorophore environment and can be applied to read out biomolecular interactions via Förster resonance energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can be automated for high content analysis (HCA) to map protein–protein interactions with applications in drug discovery, systems biology and basic research. The automated acquisition of FLIM data over 100's of fields of view provides statistical power to overcome noise in instrumentation and biological systems and thus exploit relatively small changes in mean lifetime to provide useful readouts that would not be practically achievable in manual microscopy experiments. We present here an automated HCA system with the ability to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential through exemplar applications of different FRET readouts.