Fluorescence detection of acceptor molecules sensitized by Förster resonance energy transfer (FRET) is a powerful method to study protein interactions in living cells. The method requires correction for donor spectral bleed-through and acceptor cross-excitation as well as the correct normalization of signals to account for varying fluorophore concentrations and imaging parameters. In this paper, we review different methods for FRET signal normalization and then present a rigorous model for sensitized emission measurements, which is both intuitive to understand and practical to apply. The method is validated by comparison with the acceptor photobleaching and donor lifetime-imaging techniques in live cell samples containing EYFP and ECFP tandem constructs exhibiting known amounts of FRET. By varying the stoichiometry of interaction in a controlled fashion, we show that information on the fractions of interacting donors and acceptors can be recovered. Furthermore, the method is tested by performing measurements on different microscopy platforms in both widefield and confocal imaging modes to show that signals recovered under different imaging conditions are in quantitative agreement. Finally, the method is applied in the study of dynamic interactions in the cyclin-cdk family of proteins in live cells. By normalizing the obtained signals for both acceptor and donor concentrations and using a FRET exhibiting control construct for calibration, stoichiometric changes in these interactions could be visualized in real time. The paper is written to be of practical use to researchers interested in performing sensitized emission measurements. The correct interpretation of the retrieved signals in a biological context is emphasized, and guidelines are given for the practical application of the developed algorithms.

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Summary Spectrofluorometric imaging microscopy is demonstrated in a confocal microscope using a supercontinuum laser as an excitation source and a custom‐built prism spectrometer for detection. This microscope system provides confocal imaging with spectrally resolved fluorescence excitation and detection from 450 to 700 nm. The supercontinuum laser provides a broad spectrum light source and is coupled with an acousto‐optic tunable filter to provide continuously tunable fluorescence excitation with a 1‐nm bandwidth. Eight different excitation wavelengths can be simultaneously selected. The prism spectrometer provides spectrally resolved detection with sensitivity comparable to a standard confocal system. This new microscope system enables optimal access to a multitude of fluorophores and provides fluorescence excitation and emission spectra for each location in a 3D confocal image. The speed of the spectral scans is suitable for spectrofluorometric imaging of live cells. Effects of chromatic aberration are modest and do not significantly limit the spatial resolution of the confocal measurements.

SummaryHigh brightness light emitting diodes are an inexpensive and versatile light source for wide-field frequency-domain fluorescence lifetime imaging microscopy. In this paper a full calibration of an LED based fluorescence lifetime imaging microscopy system is presented for the first time. A radiofrequency generator was used for simultaneous modulation of light emitting diode (LED) intensity and the gain of an intensified charge coupled device (CCD) camera. A homodyne detection scheme was employed to measure the demodulation and phase shift of the emitted fluorescence, from which phase and modulation lifetimes were determined at each image pixel. The system was characterized both in terms of its sensitivity to measure short lifetimes (500 ps to 4 ns), and its capability to distinguish image features with small lifetime differences. Calibration measurements were performed in quenched solutions containing Rhodamine 6G dye and the results compared to several independent measurements performed with other measurement methodologies, including time correlated single photon counting, time gated detection, and acousto optical modulator (AOM) based modulation of excitation sources. Results are presented from measurements and simulations. The effects of limited signal-to-noise ratios, baseline drifts and calibration errors are discussed in detail. The implications of limited modulation bandwidth of high brightness, large area LED devices ( ∼ 40 MHz for devices used here) are presented. The results show that phase lifetime measurements are robust down to sub ns levels, whereas modulation lifetimes are prone to errors even at large signal-to-noise ratios. Strategies for optimizing measurement fidelity are discussed. Application of the fluorescence lifetime imaging microscopy system is illustrated with examples from studies of molecular mixing in microfluidic devices and targeted drug delivery research.

We investigate the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy, using both theoretical and Monte Carlo methods. Our analysis differs from previous work in that it incorporates the data fitting process used in real experiments, allows for the arbitrary choice of excitation and gain waveforms, and calculates lifetimes as well as associated F-values from higher harmonics in the data. Using our analysis, we found different photon efficiencies to those previously reported and were able to propose optimal excitation and gain waveforms. Additionally, we suggest measurement protocols that lead to further improvement in photon efficiency. We compare our results to other techniques for lifetime imaging and consider the implications of our higher-harmonic analysis for multi-exponential lifetime determination.