High Pressure Freezing Of Cultured Neurons

保存细胞结构和分子在其天然状态是重要的科学家谁使用显微镜来探测细胞功能的机制。大多数细胞和生物样品的化学固定技术显著改变了制备中元素的分布。到目前为止，唯一的技术，保留在自然状态下的元素分布是冷冻保存。该技术涉及以接近每秒一百万摄氏度的冷却速率快速冷冻样品。较慢的冷冻速率允许冰晶的形成，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，通过插入冷冻或猛烈冷冻的可接受的冷冻深度限于几微米。最近引进了一种新技术，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）来延迟样品内的晶体形成，同时通过液氮喷射去除热量。本研究的目的是利用高压冷冻技术对培养的神经元进行冷冻保存。随后将对一些样本进行冷冻置换、包埋和薄切片，并在电子显微镜下进行断层扫描评价。其他样品将进行冷冻切片，并进行X射线微量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.