At Imperial College London, we have developed a range of instruments for FLIM, including microscopes, endoscopes and optical tomography systems, and we have developed open source software tools for FLIM data acquisition and analysis. For practical FLIM with rapid image acquisition and low phototoxicity that is compatible with live cell imaging, we have focussed on wide-field time gated FLIM using a gated optical intensifier (GOI). This approach provides robust FLIM data acquisition that is sufficiently rapid to enable automated multiwell plate FLIM for high content analysis, including FLIM/FRET assays and label-free readouts of cellular autofluorescence to report changes in cell metabolism. We have also implemented FLIM in laser scanning microscopes utilising time-correlated single photon counting (TCSPC), including microscopes combining TCSPC with polarisation-resolved detection for studies complementing FLIM/FRET data with time-resolved fluorescence anisotropy measurements and laser scanning confocal FLIM endomicroscopy for in vivo FLIM/FRET studies to quantify intracellular chemotherapeutic drug-target binding.
Our open source FLIM data analysis software, FLIMfit, is applicable to both wide-field time-gated FLIM and TCSPC data and provides global analysis capabilities.
For FLIM data acquisition, TCSPC FLIM is available with a range of commercial microscopes or can be implemented as an commercially available upgrade with proprietary software. Wide-field time-gated FLIM can be implemented using our open source FLIM software to control the instrument and the data acquisition. This is available as a MicroManager plug-in for manual FLIM microscopy, as a module for our new MicroManager-based HCA platform or FLIM HCA can be implemented using our standalone FLIM HCA MicroManager plug-in that is supported by detailed instructions and hardware component lists in this JoVE paper.