Advances in time-resolved fluorescence microscopy: Simultaneous FRAP, FLIM and tr-FAIM to image rotational and translation diffusion in living cells

Fluorescence imaging techniques are powerful tools in the biological and biomedical sciences, because they are minimally invasive and can be applied to live cells and tissues. It is advantageous to exploit the many properties of fluorescence in imaging experiments.[1–3] We demonstrate a novel experimental arrangement for measurements of intracellular dynamics by simultaneous acquisition of fluorescence recovery curves (FRAP), fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging (FAIM). We have used this set-up to obtain the translational and rotational diffusion properties of green fluorescent protein (GFP)-labelled proteins in living cells. This method allows extraction of fluorescence lifetimes, rotational correlation times and diffusion characteristics simultaneously and thus avoids excessive photobleaching or artefacts due to cell movement. It can also measure phenomena that each method on its own cannot measure, e.g. diffusing homo-dimers.