High Pressure Freezing Of Cultured Neurons

高压冷冻培养的神经元

• 批准号: 6548708
• 负责人: Richard D Leapman
• 金额: --
• 依托单位: OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6548708
• 关键词: X ray microscopy; cryopreservation; freezing; neurons; spectrometry; tissue /cell culture; transmission electron microscopy

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.

对于使用显微镜探索细胞功能机制的科学家来说，保持细胞结构和分子的天然状态非常重要。大多数细胞和生物样品的化学固定技术显着改变了制剂中元素的分布。迄今为止，保留自然状态元素分布的唯一技术是冷冻保存。该技术涉及以每秒接近一百万摄氏度的冷却速率快速冷冻样品。较慢的冷冻速度会形成破坏细胞结构的冰晶。不幸的是，由于通过生物样品的热传递相对较慢，通过骤冷或骤冷的可接受的冷冻深度仅限于几微米。最近推出了一项新技术，可以将水样冷冻至数百微米的深度。该技术称为高压冷冻，利用显着升高的压力（约 2300 巴）来延迟样品内的晶体形成，同时通过液氮喷射除去热量。该项目的目的是使用高压冷冻来冷冻保存培养的神经元。一些样品随后将被冷冻替代、包埋、切成薄片并在电子显微镜下进行断层扫描评估。其他样品将进行冷冻切片并进行 X 射线微量分析或可能进行电子能量损失光谱分析。