Correlative cry-single molecule fluorescence and electron microscopy of bacteria

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Cryo- correlative light and electron microscopy (cryo-CLEM) is an innovative and potentially data-rich technique to link the functional and structural information in cells preserved in a near-native state. However there are two limiting factors hindering the performance of this technique. The first limitation is the discrepancy in resolving power between cryo-EM (nanometre scale) and cryo-FM (a few hundred number scale). And the second limitation is the co-localisation errors arising from the transport of cell samples between two imaging instrument. At the Central Laser Facility (CLF) at the Research Complex at Harwell, a proprietary lens technology has been developed to conquer these limitations. The unique lens technology enhances the resolution performance of conventional cryo-FM by a factor of 3. Furthermore, the resolution can be improved to a few tens of nanometers applying single molecule localisation imaging strategy (as demonstrated in Fig. 1). This paves the way of imaging single macro-molecules using optical means. In addition, the small size of the new optical components has enabled the construction a mini-type fluorescence microscope inside the vacuum chamber of focused ion beam-scanning electron microscopy (FIB) system. By integrating FM and EM in one experimental unit, the maximum co-localisation precision, in the range of a few nanometers, can be achieved when correlating FM and EM images.At cryo temperatures(c.a 70 Kelvin), the efficiency of fluorescence processes increases and linewidths are reduced, making measurements easier and more accurate. Single molecule fluorescence measurements rely on accurate measurement of a single fluorophore, however it has been impossible to exploit this at cryo temperatures due to the absence of a suitable lense with the correct numerical aperture that will work in an oil immersion system at these low temperatures. As this technical problem has now been solved (see above) and this opens up the possibility of combining single molecule measurements with the improved signal properties of cryo-temperatures. Given the resolution range, this makes the measurement of processes inside the bacterial cell feasible. We have a number of bacterial functional projects that such as system would help with:(a) Bacterial cell division(b) Efflux channel assembly(c) Outer membrane protein assembly(d) Plasmid segregation(e) DNA segregation(f) RNA polymerase assembly and transcriptionEach of these projects are already at a stage in the PI's lab where labeled constructs are available. Each project can then be deployed throughout the PhD training as required by the level of technological development currently enabled. The project with LSS will provide the necessary technological developments in optics, stage positioning and laser development that will be necessary in increasing the resolution of the technique. The projects, all of which include fluorescently labeled components, will provide the necessary biological context with which to interpret the new images. As with any new microscopy, once a resolution barrier has been broken, new features of systems are elucidated, as well as previous hypothesis confirmed or denied. As such this project is expected to produce a particularly data rich set of results and hence be of high impact.