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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。快速冷冻的生物样本显示出近乎天然的结构保存，但这种优点是有代价的；对比在冷冻水合标本是适度的，样品是非常敏感的电子束，所以很难使用电子显微镜来确定感兴趣的领域在这样的标本。因此，我们正试图通过光学显微镜对冷冻样品进行预筛选，在TEM-finder网格上绘制感兴趣的区域。我们的方法被设计为利用细胞生物学中常用的工具，例如，感兴趣的gfp标记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，必须保持样品温度小于或等于-130摄氏度。在莫斯科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冷冻级（光显微镜冷冻级），可以容纳tem网格进行研究和记录。该仪器由三部分组成：LM-CS本身，带有杜瓦瓶的液氮蒸发系统和温度控制器单元。低温级配备了一个夹子来固定tem栅格，栅格被固定在一个圆柱形孔中，干燥的物镜可以放进去。这个凹槽可以用泡沫聚苯乙烯环覆盖，以减少冷凝。杜瓦瓶中的氮气蒸发系统通过热隔离管与低温容器相连；杜瓦瓶中的液氮以受控的速率加热，因此温度约为150摄氏度的气相氮被煮沸并通过电网输送。网格温度可以保持高精度（±0.5摄氏度），范围从室温到¿146摄氏度。还连接到表是一个温度传感器，与温度控制器通信；然后通过氮的蒸发速率来调节样品温度。LM-CS现已组装完毕，将用不同的冷冻水合试样进行测试。

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.