Cryo-confocal microscopy imaging of host-pathogen interactions - an essential pipeline for infectious disease research

Advancing our knowledge of disease-causing pathogens is fundamental to improving human health. Recent technological advances now enable us to visualise biological processes in unprecedented detail deep inside cells, at close to the level of individual atoms. To do this, special techniques are needed to cryogenically preserve biological material in a near-native state, by rapid freezing at ultra-low temperatures. These cryo-preserved biological samples can then be subjected to ultra-high-resolution imaging, using techniques such as electron microscopy and tomography, to investigate fundamental biological questions that have hitherto been inaccessible. An important area of research that these cutting-edge techniques are needed for is investigating how infectious organisms (including viruses, parasites and bacteria) interact with and exploit our cells. This proposal seeks to establish a multi-user facility within Cambridge University to support a wide range of scientists to prepare cryo-preserved samples containing infectious organisms and obtain high-quality images of the infected cells using a specially designed microscope that enables imaging of these samples under cryogenic conditions. This is a vital pipeline to produce samples that are of high enough quality for the application of cutting-edge ultra-high-resolution cryo-imaging techniques such as cryo-electron tomography. Establishing capacity for cryo-preservation and cryo-microscopy imaging under biosafety containment will open up new fields of discovery for the vibrant infection biology research community within the University of Cambridge and beyond. This research will have immediate and long-lasting impact on research into human diseases and the pathogens that cause them.

The focus of this proposal is to establish a new "Pathogen Cryo-Microscopy Facility" within the Department of Pathology. The requested cryo-confocal microscope and plunge freezer will be installed within dedicated Containment Level 2 (CL2) laboratory space to provide an integrated user-friendly University-wide facility supported by the School of Biological Sciences. This facility will enable preparation of vitrified samples and high-resolution fluorescence microscopy imaging of these samples by confocal microscopy under cryogenic conditions, all at CL2 containment. This will be a vital pipeline to prepare high quality samples to exploit new technologies for near-atomic resolution analysis of biological processes deep within cells, for example focused ion beam milling coupled to scanning electron microscopy (FiB-SEM). For access to these techniques, particularly those based at national facilities such as the Electron Bio-Imaging Centre (eBIC), evidence of high-quality sample availability and accurate high-quality 3-dimensional subcellular positioning data for regions or proteins of interest is vital. This is precisely what the Pathogen Cryo-Microscopy Facility will provide.
Establishing capacity for cryo-preservation and cryo-fluorescence microscopy imaging under containment will open up new fields of discovery to the vibrant infection and immunity research community within Cambridge University and beyond. This research will have immediate and long-lasting impact in pathogen biology, host-pathogen interactions, cell biological process, and immune cell interactions.