A LIQUID NITROGEN-COOLED LIGHT MICROSCOPE STAGE TO SCREEN SAMPLES FOR CRYOEM

用于筛选 CRYOEM 样品的液氮冷却光学显微镜台

• 批准号: 7722838
• 负责人: CINDY L SCHWARTZ
• 金额: $ 0.92万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722838
• 关键词: Area; Biological; Biological Preservation; Biophysics; Cellular biology; Computer Retrieval of Information on Scientific Projects Database; Cryoelectron Microscopy; Electron Beam; Electron Microscopy; Fluorescence; Freezing; Funding; Furuncles; Gases; Grant; Heating; Institution; Label; Laboratories; Light Microscope; Liquid substance; Maps; Moscow; Nitrogen; Phase; Physical condensation; Price; Proteins; Range; Rate; Research; Research Personnel; Resources; Sampling; Screening procedure; Source; Specimen; Staging; Structure; System; Temperature; Testing; United States National Institutes of Health; design; evaporation; feeding; fluorescence microscope; instrument; interest; light microscopy; sensor; styrofoam; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Rapidly frozen biological samples show near-to-native preservation of structure, but this virtue comes at a price; contrast in frozen-hydrated specimens is modest, and the samples are very sensitive to the electron beam, so it is difficult to use electron microscopy to identify areas of interest in such specimens. We are therefore trying to pre-screen frozen-samples by light microscopy, mapping areas of interest on a TEM-finder grid. Our approach has been designed to make use of tools that are commonly available in cell biology, e.g., GFP-labeled proteins of interest and a conventional fluorescence light microscope. To maintain sample quality during screening, however, one must maintain sample temperature less than or equal to -130 degrees C. With the help of a biophysics laboratory in Moscow we have designed and built a cryostage for our Zeiss fluorescence microscope ("light-microscope cryo-stage", or LM-CS) that can hold a TEM-grid for study and recording. This instrument is made in three parts: the LM-CS itself, a liquid nitrogen evaporation system with Dewar, and a temperature controller unit. The cryostage is equipped with a clamp to hold a TEM-grid, which is held in a cylindrical hole into which a dry objective can be lowered. This recess can be covered with a styrofoam-ring to minimize condensation. The nitrogen evaporation system in the Dewar is attached to the cryotable via thermo-isolated tubing; the liquid nitrogen in the Dewar is heated at a controlled rate, so gas phase nitrogen with temperature of about 150 degrees C is boiled up to be fed over the grid. The grid temperature can be maintained with high precision (plus or minus 0.5 degrees C) in the range from room temperature down to 146 degrees C. Also connected to the table is a temperature sensor that communicates with the temperature controller; sample temperature is then regulated by the rate of nitrogen evaporation. The LM-CS is now assembled and will be tested with different frozen-hydrated specimen.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 快速冷冻的生物样品显示出结构的近乎原始保存，但这种美德是有代价的。冷冻水分标本中的对比度适中，样品对电子束非常敏感，因此很难使用电子显微镜来识别此类标本中感兴趣的区域。 因此，我们试图通过光学显微镜预屏幕冷冻样本，并在Tem-Finder网格上映射感兴趣的区域。我们的方法旨在利用在细胞生物学中常用的工具，例如，感兴趣的GFP标记的蛋白质和常规的荧光光学显微镜。但是，要在筛选过程中保持样品质量，必须将样品温度保持小于或等于-130度C。在莫斯科的生物物理学实验室的帮助下，我们为Zeiss荧光显微镜设计并建造了一个低温量（“轻型微镜阶段”，或者可以容纳用于研究和记录的Tem-grid和记录。该仪器分为三个部分：LM-CS本身，带有Dewar的液氮蒸发系统和温度控制器单元。 低温量配备了夹具以容纳TEM-GRID，该夹子固定在一个圆柱孔中，可以将干燥的物镜降低。该凹槽可以用泡沫聚苯乙烯环覆盖，以最大程度地减少凝结。露水中的氮蒸发系统通过热透明管连接到冷冻的氮。露水中的液氮以受控的速率加热，因此温度约为150摄氏度的气相氮气被煮沸以在网格上馈送。网格温度可以保持高精度（加上或负0.5度C），范围从室温降低到146摄氏度。也连接到桌子的是温度传感器，该温度传感器与温度控制器通信。然后，样品温度受氮蒸发率调节。现在组装了LM-CS，并将使用不同的冷冻水合试样进行测试。