Enabling Nanoscale Dynamic Imaging of Vesicles and Organelles

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 DESCRIPTION (provided by applicant): The long-term aim of this project is to enable the imaging of vesicle and organelle dynamics inside living cells with unprecedented spatial and temporal resolution. The most compelling advantage of new super-resolution techniques such as single molecule switching (SMS) nanoscopy is the potential to image dynamic processes in living cells with 10-20 nm resolution and hence solve the many open questions in cell biology which need both high structural and temporal resolution. One such problem is how the Golgi is dynamically organized, a major and highly debated enigma. Although SMS imaging in fixed cells is already yielding impressive new biological discoveries, the potential to resolve dynamics is far from fully realized. Key limiting factors include: (i) lack of instrumentation capable of both attaining the highest resolutions and doing so in an environment and at a speed which are compatible with extended imaging in living cells, (ii) lack of good probes which can non-toxically label and switch inside a live cell with high specificity, density and brightness, and (iii) uncertainty about how to deal with the potentially incomplete data that high-speed super-resolution microscopy delivers. Motivated by a long-standing biological problem, namely the mechanism by which proteins are trafficked through the Golgi complex, we propose to address these major current limitations and develop the microscope hardware, probes and algorithms to make dynamic nanoscopy a reality. Our specific aims are: 1) To implement a 4Pi-SMS instrument which will deliver the best possible 3D resolution in living cells with minimal photodamage, 2) To develop a new genre of blinkable high-density live-cell SMS probes to image the Golgi, and 3) To develop new image processing tools which leverage prior biophysical knowledge to improve the reconstruction and quantification of Golgi morphology. These three methodological developments will be applied to a novel synthetic biology system that 'landlocks' Golgi cisternae to mitochondria and will facilitate favorable geometries to monito Golgi function. Although targeted at the Golgi, our methodological developments will be broadly applicable to live-cell super-resolution dynamic imaging of nearly every organelle within the cell.