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

电子显微镜是确定细胞内部结构细节的唯一方法。由于细胞很小，但包含数千种成分，这对于了解这些成分在细胞环境中如何工作以及它们如何受到发育、疾病、环境或突变的影响是至关重要的。电子显微镜的问题是必须对细胞进行处理，因为它们必须被切成薄片并放入真空中。加工可能会带来结构性变化。处理从固定开始，这涉及到化学交联剂将所有细胞成分彼此连接以保持它们在适当的位置。这使得水分可以被去除，这是必要的，因为水会在显微镜的真空中蒸发，然后被固化的液体树脂取代，从而进行切片。这有几个问题：(1)与我们研究的过程相比，固定速度很慢；(2)一些细胞成分因固定而改变：(3)一些用作模型系统的生物，如植物、真菌、蠕虫和苍蝇，对固定剂是不可渗透的。这意味着它们死亡的速度非常慢，因为它们被修复了，导致保存不佳。这些问题可以通过将样品冷却到-100℃，然后用在这个温度下是液体的含有修复物质的溶剂来代替固体水来解决。低温将细胞组件保持在适当的位置，而固定分子将所有东西粘合在一起。然后用设定好的液体树脂代替。样品被加热，我们切下非常薄的切片和图像。这里最困难的部分是结冰，因为如果形成冰晶，就会破坏结构。我们必须使用一种“玻璃化”的过程，即样品冷却得如此之快，以至于冰无法形成。这可以通过将样品放入非常冷的液体中来实现，但这种方法只能在大约单个细胞的深度起作用。为了获得有用的玻璃化深度，样品必须在冷却时加压，从而抑制冰的生长。为此，我们需要一种复杂的仪器，称为高压冷冻机，它在应用液体冷却剂时同步对样品加压。由于这一步骤是即时的，因此可以准确地捕获快速过程。此外，不透水的样品(例如植物和蠕虫)可以有效地固定，因为结构在低温缓慢固定期间保持不变。这种方法允许我们使用带有小金色标记的抗体(可以在显微镜下识别)，在切片中定位和识别复杂结构中的特定细胞成分。尽管冷冻后固定是一个缓慢的过程，但它可以在一种名为冷冻替代装置的自动化仪器中完成。我们花了数年时间开发利用高压冷冻和冷冻替代技术处理模型生物和其他系统的方法。我们开发了抗体标记技术，使我们能够定位蛋白质，并将结果与新的光学显微镜方法相关联。我们的仪器陈旧、过时、不可靠，不能再修理了，需要更换。现代高压冷冻机处理样品的功能更加多样，使我们能够扩展我们的样品类型。它们还具有冷冻前的光刺激等设施，使我们能够在电子显微镜水平上开发光遗传学的新领域。这使得生物过程可以通过光刺激来控制。例如，神经细胞可以被激活，然后在设定的毫秒数后，高压冻结，以便以受控的方式捕捉变化。我们开发了这些方法，用于我们的广泛研究，着眼于细胞骨架在人类组织(如晶状体和皮肤)中对植物抗病和衰老的作用。我们用它们来研究分子穿梭的过程，并将它们移入和移出细胞。我们能够研究细菌、毒素和突变是如何影响肠道的。我们将开发光遗传学方法来研究自闭症的发展，并将其扩展到其他细胞过程。

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.