Alan D. Elder

A white light confocal microscope for spectrally resolved multidimensional imaging.

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.

A quantitative protocol for dynamic measurements of protein interactions by Förster resonance energy transfer-sensitized fluorescence emission

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.

The application of frequency-domain Fluorescence Lifetime Imaging Microscopy as a quantitative analytical tool for microfluidic devices

We describe the application of wide-field frequency domain Fluorescence Lifetime Imaging Microscopy (FLIM) to imaging in microfluidic devices. FLIM is performed using low cost, intensity modulated Light Emitting Diodes (LEDs) for illumination. The use of lifetime imaging for quantitative analysis within such devices is demonstrated by mapping the molecular diffusion of iodide ions across a microchannel.

Theoretical investigation of the photon efficiency in frequency-domain fluorescence lifetime imaging microscopy

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.

mhFLIM: resolution of heterogeneous fluorescence decays in widefield lifetime microscopy.

Frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate way of measuring fluorescence lifetimes in widefield microscopy. However, the resolution of multiple exponential fluorescence decays has remained beyond the reach of most practical FD-FLIM systems. In this paper we describe the implementation of FD-FLIM using a 40 MHz pulse train derived from a supercontinuum source for excitation. The technique, which we term multi-harmonic FLIM (mhFLIM), makes it possible to accurately resolve biexponential decays of fluorophores without any a priori information. The systems performance is demonstrated using a mixture of spectrally similar dyes of known composition and also on a multiply-labeled biological sample. The results are compared to those obtained from time correlated single photon counting (TCSPC) microscopy and a good level of agreement is achieved. We also demonstrate the first practical application of an algorithm derived by G. Weber [1] for analysing mhFLIM data. Because it does not require nonlinear minimisation, it offers potential for realtime analysis during acquisition.

ϕ 2 FLIM: a technique for alias-free frequency domain fluorescence lifetime imaging

A new approach to alias-free wide-field fluorescence lifetime imaging in the frequency domain is demonstrated using a supercontinuum source for fluorescence excitation and a phase-modulated image intensifier for detection. This technique is referred to as phi-squared fluorescence lifetime imaging (phi(2)FLIM). The phase modulation and square-wave gating of the image intensifier eliminate aliasing by the effective suppression of higher harmonics. The ability to use picosecond excitation pulses without aliasing expands the range of excitation sources available for frequency-domain fluorescence lifetime imaging (fd-FLIM) and improves the modulation depth of conventional homodyne fd-FLIM measurements, which use sinusoidal intensity modulation of the excitation source. The phi(2)FLIM results are analyzed using AB-plots, which facilitate the identification of mono-exponential and multi-exponential fluorescence decays and provide measurements of the fluorophore fractions in two component mixtures. The rapid acquisition speed of the technique enables lifetime measurements in dynamic systems, such as temporally evolving samples and samples that are sensitive to photo-bleaching. Rapid phi(2)FLIM measurements are demonstrated by imaging the dynamic mixing of two different dye solutions at 5.5 Hz. The tunability of supercontinuum radiation enables excitation wavelength resolved FLIM measurements, which facilitates analysis of samples containing multiple fluorophores with different absorption spectra.

Quantitative fluorescence microscopy techniques.

Fluorescence microscopy is a non-invasive technique that allows high resolution imaging of cytoskeletal structures. Advances in the field of fluorescent labelling (e.g., fluorescent proteins, quantum dots, tetracystein domains) and optics (e.g., super-resolution techniques and quantitative methods) not only provide better images of the cytoskeleton, but also offer an opportunity to quantify the complex of molecular events that populate this highly organised, yet dynamic, structure.For instance, fluorescence lifetime imaging microscopy and Förster resonance energy transfer imaging allow mapping of protein-protein interactions; furthermore, techniques based on the measurement of photobleaching kinetics (e.g., fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and fluorescence localisation after photobleaching) permit the characterisation of axonal transport and, more generally, diffusion of relevant biomolecules.Quantitative fluorescence microscopy techniques offer powerful tools for understanding the physiological and pathological roles of molecular machineries in the living cell.