Final Activity Report Summary - ACDIQDOTS (Asymmetric Cell Division Imaging with Quantum Dots) In this proposal, we aimed at developing new single molecule imaging methodologies to label and track key proteins involved in the polarity and the division of neuroblast stem-like brain cells from Drosophila flies. In particular, we focused on qualitatively determining the spatial and temporal requirements for Miranda (Mira) and myosin VI (Myo VI) proteins during the asymmetric cell division of neuroblasts. In order to better understand the spatial organisation and the dynamics of these two proteins we have employed a multidisciplinary approach that combines (i) the development of a microscope for ultrahigh sensitivity fluorescent imaging of dividing neuroblasts, (ii) the development of fluorescent probes for specific intracellular targeting and long-term single protein tracking of Mira and Myo VI, (iii) the development of intracellular/cytoplasmic delivery strategies of these fluorescent probes in neuroblasts and other cells and (iv) the development of methods for multiparametric image and single trajectory analysis. During the length of the project, we built a fully functional single molecule microscope allowing wide field and total internal reflection fluorescence (TIRF) imaging of single quantum dot-labeled proteins in live and diving neuroblasts. Using methodologies such as pinocytic uptake, Soft Lipid Assisted Microinjection and photochemical internalisation, we also optimised the cytoplasmic delivery of quantum probes in neuroblast cells as well as in other cell types. Importantly we conjugated quantum dots to recombinant proteins (e.g. Mira) and to antibodies, and successfully reintroduced these conjugates in living cells to image and track single Mira proteins as well as other key proteins participating to the asymmetric cell division of neuroblasts. To image single cytoplasmic proteins that cannot easily be produced in vitro (e.g. Myo VI), we developed a highly original and fully generic approach for in vivo single molecule imaging based on the re-complementation of a GFP split in two non-fluorescent fragments. This methodology allows specific, covalent and addressable targeting of any protein fused to the split-GFP and permit background free single molecule tracking in living cells. This approach permits the imaging of single molecule proteins even for very high protein expression levels. It also provides a unique way to verify the proper targeting of molecules introduced in living cells, and thus complement the toolbox of intracellular targeting strategies. Split-GFP based single molecule imaging and tracking was successfully demonstrated for a variety of mammalian protein fusions. It will also be applied to the imaging of Myo VI as originally proposed.