High Pressure Freezing Of Cultured Neurons

保存细胞结构和分子的原始状态对于使用显微镜探索细胞功能机制的科学家来说是很重要的。大多数用于细胞和生物样品的化学固定技术显著地改变了制备中元素的分布。迄今为止，唯一能保持自然状态下元素分布的技术是低温保存。这项技术包括以接近每秒100万摄氏度的冷却速度快速冷冻样品。较慢的冻结速度允许冰晶的形成，从而破坏细胞结构。不幸的是，由于通过生物样品的相对缓慢的热传递，可接受的冷冻深度，无论是跳水冷冻或猛击冷冻限制在几微米。最近引进了一项新技术，可以将含水样品冷冻到几百微米的深度。这种技术被称为高压冷冻，利用显著升高的压力（约2300巴）来延缓样品内的晶体形成，同时通过液氮喷射除去热量。本项目的目的是利用高压冷冻对培养的神经元进行冷冻保存。一些样品随后将被冷冻取代、包埋、切片并在电子显微镜下进行层析评估。其他样品将被冷冻切片并进行x射线微分析或可能的电子能量损失光谱分析。

Preservation of cellular structures and molecules in their native state is important for scientists who use microscopy to probe the mechanisms of cellular function. Most chemical fixation techniques for cells and biological samples significantly alter the distribution of elements in the preparation. To date the only technique that retains the elemental distribution in the native state is cryo preservation. This technique involves rapidly freezing the sample at a cooling rate approaching one million degrees centigrade per second. Slower freezing rates permit the formation of ice crystals that disrupt cellular structures. Unfortunately, because of the relatively slow heat transfer through biological samples, the depth of acceptable freezing by either plunge freezing or slam freezing is limited to a few microns. A new technique has been recently introduced that permits freezing of aqueous samples to depths of several hundred microns. This technique referred to as high pressure freezing, utilizes significantly elevated pressure, approximately 2300 bar, to retard crystal formation within the sample while heat is removed via liquid nitrogen jets. The purpose of this project is to use high pressure freezing to cryo-preserve cultured neurons. Some samples will subsequently be freeze-substituted, embedded, and thin sectioned and evaluated tomographically in the electron microscope. Other samples will be cryo sectioned and undergo x-ray microanalysis or possibly electron energy loss spectroscopy.