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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。快速冷冻的生物样品显示出接近天然的结构保存，但这种优点是有代价的;冷冻水合样品的对比度适中，并且样品对电子束非常敏感，因此很难使用电子显微镜来识别这些样品中的感兴趣区域。因此，我们正试图通过光学显微镜对冷冻样品进行预筛选，在TEM取景器网格上绘制感兴趣的区域。我们的方法旨在利用细胞生物学中常用的工具，例如，GFP标记的感兴趣的蛋白质和常规的荧光显微镜。然而，为了在筛选期间保持样品质量，必须将样品温度保持在小于或等于-130 ℃。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台（，或LM-CS），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，带杜瓦的液氮蒸发系统和温度控制器单元。冷冻台配备有一个夹具来固定TEM网格，TEM网格固定在一个圆柱形孔中，干燥物镜可以下降到该孔中。该凹部可以用泡沫聚苯乙烯环覆盖以最小化冷凝。杜瓦瓶中的氮气蒸发系统通过隔热管连接到低温台上;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沸，并通过网格进料。栅极温度可以在室温至± 146 ℃的范围内保持高精度（± 0.5 ℃）。与工作台相连的还有一个温度传感器，它与温度控制器相连;然后通过氮气蒸发速率调节样品温度。LM-CS现已组装完毕，并将使用不同的冷冻水化样本进行测试。

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.