Correlative cry-single molecule fluorescence and electron microscopy of bacteria

冷冻相关光电子显微镜（cryo-CLEM）是一种创新的和潜在的数据丰富的技术，以联系在近天然状态下保存的细胞的功能和结构信息。然而，有两个限制因素阻碍了这项技术的性能。第一个限制是cryo-EM（纳米尺度）和cryo-FM（几百个数字尺度）之间的分辨率差异。第二个限制是由两个成像仪器之间的细胞样品的运输引起的共定位误差。在哈威尔研究中心的中央激光设施（CLF），一种专有的透镜技术已经开发出来，以克服这些限制。独特的透镜技术将传统cryo-FM的分辨率性能提高了3倍。此外，应用单分子定位成像策略，分辨率可以提高到几十纳米（如图1所示）。这为使用光学手段成像单个大分子铺平了道路。此外，新的光学元件的小尺寸，使结构内的聚焦离子束扫描电子显微镜（FIB）系统的真空室内的小型荧光显微镜。通过将FM和EM集成在一个实验单元中，当FM和EM图像相关时，可以实现最大的共定位精度，范围为几纳米。在低温（约70开尔文）下，荧光过程的效率增加，线宽减小，使测量更容易和更准确。单分子荧光测量依赖于对单个荧光团的准确测量，然而，由于缺乏具有正确数值孔径的合适的微透镜，因此不可能在低温下利用这一点，该微透镜将在这些低温下在油浸系统中工作。由于这个技术问题现在已经得到解决（见上文），这开辟了将单分子测量与低温温度的改善的信号特性相结合的可能性。鉴于分辨率范围，这使得测量细菌细胞内的过程变得可行。我们有许多细菌功能项目，如系统将有助于：（a）细菌细胞分裂（B）外排通道组装（c）外膜蛋白组装（d）质粒分离（e）DNA分离（f）RNA聚合酶组装和转录这些项目中的每一个都已经处于PI实验室的一个阶段，其中标记的构建体可用。然后，每个项目都可以根据当前技术发展水平的要求在整个博士培训中部署。LSS项目将提供光学、载物台定位和激光发展方面的必要技术发展，这对于提高该技术的分辨率是必要的。这些项目都包括荧光标记的成分，将提供必要的生物背景，以解释新的图像。与任何新的显微镜一样，一旦分辨率障碍被打破，系统的新特征就会被阐明，之前的假设也会被证实或否定。因此，该项目预计将产生一组数据特别丰富的结果，因此具有很高的影响力。

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.