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Introduction        PALM       Single particle tracking PALM        FCS        FLIM        GSD Microscopy

Despite very high resolution, these single molecule imaging techniques lack the ability to report protein-protein interactions inside living cells. This is a very important measure - being able to report protein complex formation, with high spatial resolution in cells is extremely powerful, especially combined with all the other molecular imaging approaches. In order to achieve this, we use fluorescence lifetime imaging microscopy (FLIM) to

Quantifying FRET is difficult, or almost impossible, using intensity measurement. Measuring the excited state fluorescence lifetime of the donor in a FRET system provides a much more direct route to quantifying energy transfer, with the advantage of requiring low excitation light levels, being oblivious to donor concentration, and in addition providing information about the ratio of interacting versus non-interacting proteins in a system. The fluorescence lifetime of a fluorophore is a characteristic molecular property, describing the time spent between the absorption of an exciting photon and the return to the ground state (illustrated right and left.)

To measure this, we use a pulsed laser excitation source with a pulse-width substantially shorter than the lifetimes we are quantifying. In the ideal world, this would function as in the illustration, left. The laser pulse, shown as a dot in the upper panel, and a red spike in the graph, results in excitation of the fluorescent molecules in the cells. The emission of a photon is a good approximation of the return to the ground state, so the time period between the laser pulse and this event describes the fluorescence lifetime. If we could measure this time for every single photon, in every pixel of an image, then we would have a measure of the excited state lifetimes of the molecules in those pixels. As this is stochastic, the only way to quantify this is to make many thousands of measurements and estimate the half-time of the combined decays - as in the graph to the left.

Unfortunately, it isn't possible to do this in the way illustrated - instead we acquire the data pixel-by-pixel and build up histograms of emission photon arrival times, correlated to the laser pulse responsible for each excitation event (illustrated right). This is relatively easy with a laser-scanning microscope and suitable fast electronics and single photon detectors. The disadvantage is that it is slow - it takes at least 1 minute to acquire sufficient data to make accurate fits estimating the decay times.

Once the fluorescence lifetimes have been calculated, the challenge is to present these data in a meaningful way for cell biologists. To achieve this, the calculated lifetimes are displayed as a false-colour, with every pixel maintaining its spatial position (left). In these images, therefore, the colours in each compartment of the cell represent the donor fluorescence lifetime, with long lifetimes displayed in blue, and short lifetimes in red. If FRET occurs in the presence of a proximal interacting acceptor molecule, the donor lifetime is substantially shortened. This is seen as a change in colour in those locations from blue towards red - clearly visible in some of the lower panels, left. In combination with the other molecular imaging approaches it is possible to localise single molecules in cells, track their mobilities, diffusions, real time binding kinetics and interactions, all inside living samples.

report Foerster resonance energy transfer (FRET) between 2 fluorescent molecules. FRET is a physical effect, where energy is transferred from one molecule (the higher energy donor) to a proximal acceptor molecule, when the pair have overlapping emission and excitation spectra, respectively. Importantly, this effect is very distance-dependant and occurs only over distance scales (<6 nm or so) encompassing protein-protein interactions.