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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 快速冷冻的生物样本显示出近乎自然的结构保存，但这一优点是有代价的；冷冻水合样本的对比度不大，而且样本对电子束非常敏感，因此很难使用电子显微镜来识别这类样本中感兴趣的区域。因此，我们正试图通过光学显微镜对冷冻样品进行预筛选，并将感兴趣的区域绘制在瞬变显微镜-查找器网格上。我们的方法旨在利用细胞生物学中常见的工具，例如GFP标记的感兴趣蛋白质和传统的荧光显微镜。然而，为了在筛选过程中保持样品质量，必须将样品温度保持在低于或等于-130摄氏度。在莫斯科一家生物物理实验室的帮助下，我们为蔡司荧光显微镜设计并建造了一个低温工作台(“光学显微镜低温工作台”，或LM-CS)，它可以容纳用于研究和记录的电子显微镜网格。该仪器由三部分组成：LM-CS本身、带有杜瓦的液氮蒸发系统和温度控制单元。低温工作台配备了一个夹具来固定电子显微镜栅格，该栅格被固定在一个圆柱孔中，可以将干燥的物镜放入其中。这个凹槽可以用泡沫塑料环覆盖，以最大限度地减少冷凝。杜瓦瓶中的氮气蒸发系统通过隔热管连接到冷冻器上；杜瓦瓶中的液氮以受控的速度加热，因此温度约为150摄氏度的气相氮气被煮熟，通过电网提供。栅格温度可以在从室温到146摄氏度的范围内保持高精度(正负0.5摄氏度)。与工作台相连的还有一个温度传感器，它与温度控制器通信；然后通过氮气蒸发速度来调节样品温度。LM-CS现已组装完成，并将用不同的冷冻水化样品进行测试。

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.