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

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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.