Quantification of the oligomerisation state and conformational changes of the epidermal growth factor receptor in its
constitutive and active states are critical to fully understand their signal transduction pathway. We show the ability of
single-molecule fluorescence microscopy to determine this information and its implementation by combining
multidimensional emission optics with total internal reflection fluorescence microscopy. Importantly, the signalling
process is analysed in live cells under physiological conditions.
The fluorescence decay in fluorescence lifetime imaging (FLIM) is typically fitted to a multi-exponential model with discrete lifetimes. The interaction between fluorophores in heterogeneous samples (e.g. biological tissue) can, however, produce complex decay characteristics that do not correspond to such models. Although they appear to provide a better fit to fluorescence decay data than the assumption of a mono-exponential decay, the assumption of multiple discrete components is essentially arbitrary and often erroneous. The stretched exponential function (StrEF) describes fluorescence decay profiles using a continuous lifetime distribution as has been reported for tryptophan, being one of the main fluorophores in tissue. We have demonstrated that this model represents our time-domain FLIM data better than multi-exponential discrete decay components, yielding excellent contrast in tissue discrimination without compromising the goodness of fit, and it significantly decreases the required processing time. In addition, the stretched exponential decay model can provide a direct measure of the sample heterogeneity and the resulting heterogeneity map can reveal subtle tissue differences that other models fail to show.
We report a whole-field fluorescence imaging microscope that combines 3-D spatial resolution by optical sectioning, using structured illumination, with fluorescence lifetime imaging and spectrally-resolved imaging. We show the potential of this technique in the elimination of common artefacts in fluorescence lifetime imaging and apply it to study the dependence of the lifetime on the emission wavelength in biological tissue.
The Expectation Maximisation (EM) algorithm for computing the Maximum Likelihood image has
been applied to both simulated and experimental cone-beam SPECT projection data. Reconstructions of
simulated data show the superiority of the EM algorithm over a convolution and backprojection (CBP)
algorithm with respect to image uniformity across the field of view and tolerance to noise. Phantom
studies demonstrate the need for an accurate attenuation correction routine. A method of correcting for
photon attenuation has been applied to the EM algorithm assuming the object to be a uniform
attenuator. A method of extending this to allow for non uniform photon attenuation is also proposed.
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