3464/JN - Galvo-Scanning TIRF Microscope System for Single Molecule Imaging

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This is a contract awarded via mini-competition under Lot 4 (Super Resolution and Enhanced Resolution Microscopes) of the APUC Framework Agreement for: Microscopes and Imaging Equipment including Associated Servicing, Maintenance and Repairs LAB1028 AP The Twelvetrees Lab work on understanding the molecular mechanisms or slow axonal transport so we can develop a deeper understanding of how this process supports neuronal function and longevity. The group uses a combination of biochemistry, cell biology and single molecule methods to probe transport mechanisms, through generous funding from a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society. The lab is based in the Sheffield Institute for Translational Neuroscience and collaborates with many groups in the building. Our work studies how microtubule motor proteins interact differentially with a variety of cargo to regulate distinct rates of transport. Many of our experiments are built around fluorescence microscopy assays; we rely on self labelling enzymes (SNAPf, CLIPf and HaloTag) in combination with their fluorescent ligands (e.g. TMR, 647-SiR, JF549 etc) for single molecule imaging, and also use fluorescent protein markers (EGFP, mTurquoise2, mScarlet-I etc). We use two broad classes of assay system: 1. primary neuronal cultures for live cell imaging and; 2. in vitro motile systems reconstituted from purified proteins. Typical microscopy applications of the lab include, but are not limited to: live-cell imaging of primary neurons over time scales from minutes to hours; single-molecule imaging in live cells; single molecule imaging of in vitro reconstituted motile systems; FRET of single molecules in vitro and populations of molecules within cells; photoablation and activation; super resolution microscopy. The University now requires a microscopy system to support the research in this area. In order to image live neurons and in vitro reconstituted systems, the system must be capable of a number of distinct microscopy applications, including but not limited to: single molecule TIRF microscopy, instantaneous FRAP and TIRF, live-cell imaging, FRET, DNA-PAINT, widefield fluorescence microscopy. To facilitate single molecule imaging in demanding conditions, the system must have an automated focus drift compensation system to maintain z-position. To preserve the activity and integrity of live neurons, the system will be housed in a temperature controlled enclosure that can be operated at a range of temperatures, from room temperature up to 37C. The system must be mounted on an anti-vibration table to minimise environmental vibrations interfering with imaging. Computer control of the system must facilitate both sub second time lapse imaging of single molecules as well as automated time lapse imaging of several points in the sample over many hours.

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