Multiplexed confocal FLIM for dynamic molecular imaging in live cells

Quantitative measurements of the dynamics of biomolecular interactions allow biologists to develop a better understanding of biological processes that are critical to new diagnostic tools, drug discovery, and personalized treatments of diseases. Such measurements require multidimensional (spatial, spectral, and temporal) imaging with a high frame rate. Conventional single point confocal microscopy can produce 3D images at video rate but faces difficulties in accurately measuring fluorescence lifetime images (FLIM) while maintaining low excitation power to avoid phototoxicity and photobleaching in live cells. Multipoint confocal fluorescence lifetime imaging offers access to microscopic dynamics at the subcellular resolution. We have designed a 32 × 32 point multiplexing time-resolved confocal microscope to address these problems and demonstrated the power of this system to measure live cell FLIM of Förester resonance energy transfer (FRET). Using a pinhole array simplifies the optical system design, allowing improved optical efficiency for imaging at faster frame rates with a temporally calibrated single photon avalanche detector (SPAD) array. These efficiency improvements are leveraged by redesigning the optomechanical system and software processing to achieve a frame rate 12 times faster than previously demonstrated. Through dilution series measurements, we demonstrate that a concentration as low as 10 µM Coumarin6 can be measured accurately at 4 Hz frame rates. The performance is also demonstrated with fixed, stained samples and FLIM-FRET constructs in live cells at a maximum imaging rate of 4 Hz with an image dimension of 960 × 960 pixels.