Development of Cryo-Methods for Preparation of samples for Structural Analysis of Model Biological Systems and Optogenetics

Electron microscopy is the only method to determine fine details of internal cell structure. Because cells are small, but contain thousands of components, this is essential to understand how components work within the context of the cell and how they are affected by development, disease, environment or mutations. Electron microscopy has the problem that cells have to be processed because they have to be cut into thin sections and introduced into a vacuum. Processing can introduce structural changes. Processing starts with fixation, which involves chemical cross-linkers that attach all the cell components to each other to hold them in place. This allows water to be removed, necessary because water would evaporate in the vacuum of the microscope and then replaced by liquid resin that is solidified, allowing sectioning. This has several problems: (1) Fixation is slow compared to the processes that we study; (2) Some cell components are altered by fixation: (3) Some organisms used as model systems, such as plants, fungi, worms and flies, are impermeable to fixatives. This means they die very slowly as they are fixed leading to poor preservation. These problems can be circumvented by cooling the sample to -100'C, then replacing the solid water with solvent which is liquid at this temperature, containing fixes. Low temperature holds the cell components in place while fix molecules stick everything together. This is then replaced by liquid resin which is set. The sample is warmed and we cut very thin sections and image. The most difficult part here is freezing because if ice crystals form they damage the structure. We have to use a process of "vitrification" where the sample is cooled so rapidly that ice cannot form. This can be achieved by plunging the sample into a very cold liquid, but this only works to a depth of about single cell. To obtain useful depths of vitrification, the sample has to be pressurized while it is cooled, suppressing ice growth. For this we need a sophisticated instrument, called a high pressure freezer, which synchronously pressurizes the sample as liquid coolant is applied. Because this step is instant, rapid processes are capture accurately. Also impermeable samples (e.g. plants and worms) can be effectively fixed because the structure is maintained during slow fixation by low temperature. This method allows us to use antibodies with small gold markers attached (which can be identified in the microscope), to locate and identify specific cell components within the complex structure in sections. Although post-freezing fixation is a slow process, it can be done in an automated instrument, called a freeze substitution unit.
We spent years developing methods for processing model organisms and other systems using high pressure freezing and freeze substitution. We developed antibody labelling allowing us to locate proteins and correlate the results with new light microscopy methods. Our instruments are old, obsolete, unreliable, can no longer be repaired and need replacing. Modern high pressure freezers are more versatile for sample handling, allowing us to expand our types samples. They also have facilities such as light stimulation prior to freezing allowing us to develop the new field of optogenetics at the electron microscopy level. This allows biological process to be controlled by light stimulation. For instance nerve cells can be activated and then a set number of milliseconds later, high pressure frozen, in order to capture changes in a controlled manner.
We developed these methods for our broad research looking at the role of the cell skeleton in plant disease resistance and ageing in human tissues such as lens and skin. We use them to study processes that shuttle molecules around, and move them into and out of cells. We are able to study how bacteria, toxins and mutations affect the gut. We will develop optogenetic methods to study development of autism, and extend this to other cell processes.

Biological specimens are mostly water, which is incompatible with the vacuum of an electron microscope (EM). Water has to be vitrified, or removed and replaced, with hard resin, allowing ultrathin sectioning. Although cryo-EM is the gold standard for cellular ultrastructure, it is incompatible with immuno-localisation, demanding, and requiring specialised TEMs, limiting the data for any one experiment. Conventional fixation and embedding introduces artefacts, rapid processes are not captured and some impervious specimens, including important models (plants, fungi and invertebrates), can take hours to fix, introducing artefacts. The alternative is to vitrify the sample, then to replace the water with fix containing solvent at about -100'C, then acrylic resin at -50'C, which is polymerised by UV. Plunge freezing results in vitrification to only about one cell thickness, but pressurizing the sample as it is cooled, suppresses ice crystals and enables vitrification to at least 10X the depth. High Pressure Freezing (HPF) then freeze substitution (FS) is the method of choice for preparing EM samples. We have spent years developing HPF/FS for a range of samples, but current instrumentation is no longer fit for purpose. Newly developed instruments will enable us to capitalise on our experience, to broaden applications and develop new approaches. We focus on plants, yeast, C. elegans, Drosophila, zebrafish lens and engineered human skin. We study the cytoskeleton and its role in development, ageing and disease. Because the cytoskeleton is dynamic and prone to fixation induced aggregation, HPF/FS is essential to determine native organisation. Processes such as nucleo-cytoplasmic transport, endocytosis and vesicle transport, which we study in yeast and plants, are dynamic and rapid, requiring instant cryo-fixation. The latest HPF will also allow us to develop optogenetic methods in EM, initially to study development of autism, and then extend this to other cell processes.

The objectives of the impact plan are (1) to ensure that the UK plant cell biology and model system communities have high quality electron microscopy and immuno electron microscopy facilities available to them (2) to work with other UK cell biologists to optimize HPF/FS techniques for use with plants, crops and other model systems. (3) to provide training for HPF/FS and EM imaging (4) to share facilities with N8 partners and other collaborators (5) to protect potential applicable discoveries and raise awareness with industrial sponsors (6) to provide high level training to research associates interested in nanoscale imaging (7) to integrate with the local communities via outreach.
HPF/FS will be advertised to the N8 partnership and when methods are fully developed, workshops will be organized around the needs of interested parties. Co-investigators on this proposal are established leaders in animal and fungal as well as plant cell biology. Moreover to help advertise our EM capabilities, they will be included on the Department's Microscopy and Bioimaging Facility webpages and brochure, and the Durham Centre for Bioimaging technology (DCBT) website. An internet gateway to the microscopy facility and program of work will be hosted by DCBT. This will offer non-technical explanations of our activities and summarise key published discoveries for the general public, media and potential industrial collaborators.
All work and promotion of new capabilities will be communicated through publication in research journals and engagement at conferences open to both academic and industrial scientists.
Any exploitation of Durham based research will be discussed with the PI in association with the Durham University Technology Transfer Team who provide support to staff and research students whose research outputs have commercial significance. Groups will contact the Durham University Technology Transfer Office for example: when they may require patent protection; are interested in exploring the possibility of setting up a spin-off company or are looking to identify a commercial partner for a joint business venture. To this end, we will approach and engage with appropriate companies.
The permanent Senior Experimental Officers who run the Microscopy and Bioimaging and electron microscopy facilities will train research associates who are part of the research groups in this program. As a result RAs will gain experience in HPF/FS and an appreciation of all the technologies available in the facility. The Department is involved with numerous outreach activities throughout the academic year and the PI and SEOs in the Microscopy and Bioimaging and electron microscopy facilities give presentations and demonstrations of our advanced imaging capabilities. These outreach activities will continue.
The PI and Co-Is will be responsible for the overall management of impact. Durham University has active, well established managers and offices that give significant support to Departments for outreach activities, media relations and for interactions with industry.