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.

在其本地状态下保存细胞结构和分子对于使用显微镜探测细胞功能机制的科学家很重要。大多数用于细胞和生物样品的化学固定技术都显着改变了制剂中元素的分布。迄今为止，保留本地状态元素分布的唯一技术是冷冻保存。该技术涉及以每秒100万度的冷却速率快速冷冻样品。较慢的冻结速率允许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，因此可以通过陷入冻结或猛烈冻结的可接受冻结深度仅限于几微米。最近引入了一种新技术，该技术允许将水性样品冷冻到数百微米的深度。该技术称为高压冻结，利用了大约2300 bar的压力升高，以在样品内延迟晶体形成，同时通过液氮射流去除热量。该项目的目的是使用高压冻结来进行冷冻培养的神经元。随后将在电子显微镜中通过冻结，嵌入和薄的切片和评估的冻结，嵌入和薄的分段和评估。其他样品将进行冷冻切片并进行X射线微分析或可能的电子能量损失光谱。