We report what to our knowledge is a novel approach for simultaneous imaging of two different Förster resonance energy transfer (FRET) sensors in the same cell with minimal spectral cross talk. Previous methods based on spectral ratiometric imaging of the two FRET sensors have been limited by the availability of suitably bright acceptors for the second FRET pair and the spectral cross talk incurred when measuring in four spectral windows. In contrast to spectral ratiometric imaging, fluorescence lifetime imaging (FLIM) requires measurement of the donor fluorescence only and is independent of emission from the acceptor. By combining FLIM-FRET of the novel red-shifted TagRFP/mPlum FRET pair with spectral ratiometric imaging of an ECFP/Venus pair we were thus able to maximize the spectral separation between our chosen fluorophores while at the same time overcoming the low quantum yield of the far red acceptor mPlum. Using this technique, we could read out a TagRFP/mPlum intermolecular FRET sensor for reporting on small Ras GTP-ase activation in live cells after epidermal growth factor stimulation and an ECFP/Venus Cameleon FRET sensor for monitoring calcium transients within the same cells. The combination of spectral ratiometric imaging of ECFP/Venus and high-speed FLIM-FRET of TagRFP/mPlum can thus increase the spectral bandwidth available and provide robust imaging of multiple FRET sensors within the same cell. Furthermore, since FLIM does not require equal stoichiometries of donor and acceptor, this approach can be used to report on both unimolecular FRET biosensors and protein-protein interactions with the same cell.

We report a novel, compact and automated multidimensional spectrofluorometer that exploits a fibre‐laser‐pumped ultrafast supercontinuum source to provide resolution with respect to intensity, excitation and emission wavelength, decay time and polarisation. This instrument has been applied to study the photophysics of the phase‐sensitive membrane probe di‐4‐ANEPPDHQ and to characterise protein‐protein interactions via Förster resonance energy transfer. It can be applied to in situ measurements via a fibre‐optic probe in medical and other contexts and is demonstrated here to provide a comprehensive characterisation of tissue autofluorescence. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

We report an automated optically sectioning fluorescence lifetime imaging (FLIM) multiwell plate reader for high content analysis (HCA) in drug discovery and accelerated research in cell biology. The system utilizes a Nipkow disc confocal microscope and performs unsupervised FLIM with autofocus, automatic setting of acquisition parameters and automated localisation of cells in the field of view. We demonstrate its applications to test dye solutions, fixed and live cells and FLIM‐FRET. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

We present a time domain optically sectioned fluorescence lifetime imaging (FLIM) microscope developed for high-speed live cell imaging. This single photon excited system combines wide field parallel pixel detection with confocal sectioning utilizing spinning Nipkow disc microscopy. It can acquire fluorescence lifetime images of live cells at up to 10 frames per second (fps), permitting high-speed FLIM of cell dynamics and protein interactions with potential for high throughput cell imaging and screening applications. We demonstrate the application of this FLIM microscope to real-time monitoring of changes in lipid order in cell membranes following cholesterol depletion using cyclodextrin and to the activation of the small GTP-ase Ras in live cells using FRET.