Correlative cry-single molecule fluorescence and electron microscopy of bacteria

低温相关光学和电子显微镜(CRYO-CLEM)是一种创新的、潜在的数据丰富的技术，它将保存在近自然状态下的细胞的功能和结构信息联系起来。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)在分辨率上的差异。第二个限制是细胞样本在两台成像仪器之间传输时产生的共定位误差。在哈威尔研究中心的中央激光设备(CLF)，一种专有透镜技术已经被开发出来，以克服这些限制。独特的透镜技术将传统低温调频的分辨率提高了3倍。此外，应用单分子局域成像策略，分辨率可以提高到几十纳米(如图1所示)。这为使用光学手段对单个大分子成像铺平了道路。此外，新光学元件的小尺寸使得在聚焦离子束扫描电子显微镜(FIB)系统的真空室内构建微型荧光显微镜成为可能。通过在一个实验单元中集成FM和EM，当将FM和EM图像关联时，可以实现最大的共定位精度，范围在几纳米范围内。在低温(C.A 70开尔文)下，荧光过程的效率提高，线宽减少，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精确测量，然而，由于没有合适的透镜具有正确的数值孔径，因此不可能在低温下利用这一点，这种透镜可以在低温下用于石油浸没系统。因为这一技术问题现在已经解决(见上文)，这为将单分子测量与改进的低温信号特性相结合开辟了可能性。在给定的分辨率范围内，这使得测量细菌细胞内的过程是可行的。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)外流通道组装(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实验室中标记构建物可用的阶段。然后，每个项目都可以根据目前能够实现的技术发展水平，在整个博士培训期间进行部署。与LSS的项目将在光学、舞台定位和激光开发方面提供必要的技术发展，这将是提高技术分辨率所必需的。所有这些项目都包括荧光标记的成分，将提供必要的生物学背景来解释新的图像。与任何新的显微镜一样，一旦分辨率障碍被打破，系统的新特征就被阐明，以及先前的假设被证实或否认。因此，预计该项目将产生一套特别丰富的数据，从而产生很大的影响。

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.