Enhanced resolution in vivo time-lapse imaging system for subcellular CLEM

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Correlated light and electron microscopy (CLEM) is a powerful approach to study the subcellular and molecular basis of cellular processes in physiology and pathology. Especially in neural and other complex tissues, where dynamic remodeling at spatial scales of the limit of classical light microscopy– e.g. synaptic plasticity, neuron-glia interactions or organelle trafficking – play key roles in development and disease, such approaches have proven especially necessary and informative. This necessity is underscored by the fact that true superresolution techniques do not routinely achieve full performance inside an intricate tissue environment, and due to sparse or molecular labelling often still require electron microscopy (EM) as a dense structural background. Here, we apply for an enhanced resolution in vivo time-lapse microscopy system that is capable of routinely achieving xy-resolution of ≈ 150 nm at relatively high speed (>1 HZ at full frame/ >10Hz at 512x512 pixel) across a variety of complex tissues, including brain, spinal cord, peripheral nerve and muscle. This system is to be seamlessly embedded into a CLEM workflow starting with time-lapse recordings of subcellular dynamics in situ (e.g. synaptic or glial process dynamics or calcium signaling, cytoskeletal remodeling, organelle transport, vesicle recycling etc.) ending at semi-automated three-dimensional (3D) scanning EM (3D-SEM), including automated tape ultramicrotome-SEM (ATUM-SEM), focussed ion beam SEM (FIB-SEM) and the hybrid, that we recently developed, ATUM-FIB. The acquired instrument will be incorporated into an imaging unit that is used by a range of DFG-funded consortia (including a FOR, CRC/TRR and an Excellence Cluster) and by a core of six research groups, as well as an MSc program that they run, but which will also be made accessible to the larger Munich neuroscience community. This imaging unit comprises a unique set of custom-built and custom-adapted in vivo imaging systems (ranging from whole-brain imaging in larval zebrafish to in vivo imaging in the cortex, spinal cord and peripheral nervous system of murine disease models) and will be tethered to the SyNergy Excellence Cluster’s EM facility via a range of in-house developed correlation approaches – all of these applications supported by permanent expert staff. The aim of this proposal is hence to expand the available scope of high end in vivo imaging approaches to include the routine time-lapse imaging of subcellular dynamics at enhanced resolution followed by further analyses using a range of 3D CLEM techniques.