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An emerging theme in cell biology is that cell surface receptors need to be considered as part of supramolecular complexes of proteins and lipids facilitating specific receptor conformations and distinct distributions, e.g., at the immunological synapse. Thus, a new goal is to develop bioimaging that not only locates proteins in live cells but can also(More)
An understanding of cellular signalling from a systems-based approach has to be robust to assess the effects of point mutations in component proteins. Outcomes of these perturbations should be predictable in terms of downstream response, otherwise a holistic interpretation of biological processes or disease states cannot be obtained. Two single, proximal(More)
As T cells and natural killer (NK) cells survey the surface of other cells, cognate receptors and ligands are commonly organized into distinct micrometer-scale domains at the intercellular contact, creating an immune or immunological synapse (IS). We aim to address the still unanswered questions of how this organization of proteins aids immune surveillance(More)
In fluorescence microscopy, the fluorescence emission can be characterised not only by intensity and position, but also by lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, and time-resolved fluorescence anisotropy(More)
Fluorescence lifetime imaging (FLIM) is a functional imaging methodology that can provide information, not only concerning the localisation of specific fluorophores, but also about the local fluorophore environment. It may be implemented in scanning confocal or multi-photon microscopes, or in wide-field microscopes and endoscopes. When applied to tissue(More)
The fluorescence intensity and lifetime of the 4,4'-difluoro-4-bora-5-(p-oxoalkyl)phenyl-3a,4a-diaza-s-indacene (1) show a strong correlation with the viscosity of the medium due to the viscosity-dependent twisting of the 5-phenyl group, which gives access to the dark nonemissive excited state. We propose a sensitive and versatile method for measuring the(More)
Diffusion-mediated cellular processes, such as metabolism, signalling and transport, depend on the hydrodynamic properties of the intracellular matrix. Photodynamic therapy, used in the treatment of cancer, relies on the generation of short-lived cytotoxic agents within a cell on irradiation of a drug. The efficacy of this treatment depends on the viscosity(More)
We have developed a wide-field time-resolved imaging system to image quantitatively both the fluorescence lifetime and the rotational correlation time of a fluorophore. Using a polarization-resolved imager, we simultaneously image orthogonal polarization components of the fluorescence emission onto a time-gated intensified CCD. We demonstrate imaging of(More)
Fluorescence lifetime imaging (FLIM) and fluorescence polarization imaging are complementary techniques that can be used to extract information about macromolecules from biological samples. Owing to the sensitivity of fluorescence to the physicochemical environment, and nanometer-scale interactions via Förster resonance energy transfer (FRET), FLIM has been(More)