Development of a super-resolving STED FLIM microscope for biological applications

We aim to develop a fluorescence microscope to image molecular processes with significantly improved spatial resolution in order to study the mechanisms of disease at a molecular level. By using fluorescent molecules ('fluorophores') to label proteins associated with signalling, e.g. at the interface ('immune synapse') between white blood cells and suspect cells being interrogated by the immune system, it is possible learn about the spatial and temporal organisation of specific proteins and their interactions. Unfortunately the resolution of fluorescence microscopes is limited by diffraction to ~300 nm and the molecules of interest are much smaller. One way to probe their interactions is to use spectroscopic techniques because the properties of fluorescence emission can vary according to the local environment of the fluorophore and can also be used to distinguish different molecular species. By labelling different proteins with fluorophores emitting at different wavelengths and then comparing the different 'colour' images, one can obtain information about co-localisation, from which interaction can be inferred - albeit limited by the spatial resolution. More can be learned from the fluorescence lifetime - the time over which a fluorescence signal decays - which can be sensitive to its local physical or chemical environment and to the presence of other molecules. Fluorescence lifetime imaging (FLIM) can be used to map changes in molecular environment and to detect protein-protein interactions by exploiting Förster Resonant Energy transfer (FRET), where the emission of one fluorophore-labelled protein is quenched by direct energy transfer to nearby suitable fluorophores that can be labelling a second molecule. This energy transfer, which can only occur if the fluorophores are within ~10 nm, also reduces the fluorescence lifetime and so FLIM provides a means to map when and where pairs of proteins interact - to a precision limited by the spatial resolution of the microscope. In a study of inter-cell signalling, we used FLIM-FRET to image the phosphorylation (i.e. the 'activation') of a key molecule (the KIR receptor) involved in determining the response of a white blood cell when interrogating a suspect cell. Unexpectedly, we observed that the phosphorylation of the KIR receptor occurred in small microclusters - but the confocal microscope did not have sufficient spatial resolution to do more than detect their presence. This is an exemplar application for which we are developing a new microscope capable of resolving structures below the diffraction limit. Others include the colocalisation and segregation of signalling molecules, the assembly of signalling complexes and the use of FLIM to elucidate the state and distribution of actomyosin cross bridges in muscle fibres. We aim to build a super-resolving fluorescence microscope using the technique pioneered by S. Hell called stimulated emission depletion (STED). A regular confocal microscope scans a focussed excitation beam across a sample and detects the resulting fluorescence to acquire an image, the resolution of which depends on the size of the focussed spot on the sample. In STED, the sample is scanned by two collinear beams: the first excites fluorescence in the usual way but the second 'STED' beam suppresses it by depleting the excited state population through stimulated emission. This second beam has a 'doughnut' profile with a hole in the middle such that the outside of the excitation spot is 'switched off' while the centre remains, thereby realising a smaller effective spot and resolution beyond the diffraction limit. Having demonstrated a STED prototype microscope incorporating FLIM and adaptive compensation of aberrations in the microscope, we now aim to build a system suitable for use by biologists to study the organisation of cell signalling and other molecules with resolution beyond the diffraction limit using techniques like FLIM and FRET.

Microscopes offering resolution beyond the diffraction limit can probe biological structures and process on scales below ~300 nm. We have developed interdisciplinary research programmes to study cell signalling, many of which exploit FLIM to study molecular environments and interactions, including FLIM of membrane lipid microdomain, or 'lipid raft', activity, FLIM-FRET imaging of phosphorylation of signalling molecules and FLIM of actomyosin cross bridges in muscle sarcomeres. All these experiments were limited by the spatial resolution of our confocal microscope and our biology collaborators are keen to progress to experiments with superior resolution. As a first step, we demonstrated a novel STED FLIM microscope implemented in a standard laser scanning confocal microscope (LSCM) and utilising, for the first time, an ultrafast laser supercontinuum source to provide spectral versatility at relatively low cost. This novel instrument also included spatial light modulator technology to adaptively control the spatial profile of the depleting beam and to compensate for aberrations in the microscope. FLIM was implemented using time correlated single photon counting, making this the first super-resolved FLIM microscope, as well as the first STED microscope in the UK. Here we aim to develop a 'biology-friendly' instrument offering the full capabilities of a LSCM with the ability to 'zoom in' and obtain super-resolved (intensity and FLIM) STED images anywhere in the field of view. In our prototype STED microscope, we established that instabilities in the galvanometric scanners of our commercial LCSM limited our achievable resolution, as did vibrations from the pump laser. We will build a new, stable, instrument with stage and resonant scanning to be applied to studies of signalling molecule interactions in microclusters, the assembly of signalling complexes, kinetic segregation model studies, membrane nanotubes and actomyosin states in mammalian muscle sarcomeres.