JEOL Freeze Fracture/Freeze Etch Device

使用冷冻断裂、深蚀刻和分子复制的方法目前正在发现一个巨大的 现代电子显微镜中实用工具的复兴。不管低温技术的复杂程度如何- 方法和其他先进的电磁技术以及光分辨率的相应改进 显微镜，传统的EM显微镜仍然有令人难以置信的用途。此外，由于许多设施已经被 随着高分辨率光学显微镜的出现，剩下的那些已经变得越来越多，并且 值得注意的是，在我们的案例中，非常活跃，与全国各地的研究小组合作。这个 复制方法的使用重新出现的原因是需要在更高的位置定位分子 膜中的分辨率。传统上，我们在这项技术上拥有丰富的专业知识，虽然专业知识 仍然存在，我们设施中的设备已经不存在了。我们的冷冻折断机，翻新了~20年 Cressington CFE50，两年前因真空、枪支和电气问题而退役 由于这种特定的仪器已不再生产，该公司进行了补救。正因为如此，我们 申请资金购买JEOL JFD-V-TP冷冻-断裂/冷冻-蚀刻/旋转阴影机 匹兹堡大学医学院生物成像中心(CBI)。我们需要的原因是 通过购买新仪器继续使用这些协议有四个方面：1.最近招募了 几名新的教员对这台仪器提供的技术提出了关键要求， 包括不能作为独立部件购买的机器的旋转遮挡部件。 2.长期合作者对获得高分辨率结构-功能成像的持续请求 膜和分子的分子复制品。3.将光学显微镜模式与 膜上的高分辨率分析，特别是利用FRIL(冷冻-骨折复制免疫- 标签)。4.匹兹堡大学没有其他类似的设备，也没有 大匹兹堡地区，就这一点而言。CBI的任务是提供全方位的光线和 向所有研究小组(超过250个NIH资助)提供电子光学、图像分析和形态测量方法 匹兹堡大学内部以及许多外部机构的用户)。收购这台机器 将极大地帮助我们的主要用户，并为所有其他研究人员提供访问重要的高分辨率 技术，以促进他们的研究。

The use of freeze-fracture, deep-etch and molecular replica methods are currently finding a tremendous resurgence of utility in modern electron microscopy. Regardless of the increased sophistication of cryo- methods and other advanced EM technologies and coincident improvements in the resolution of light microscopy, traditional EM microscopies still have incredible utility. Moreover, as many facilities have been closed with the advent of high-resolution light microscopy, those that remain have become increasingly, and notably in our case, phenomenally active, collaborating with research groups from all over the country. The cause of the re-emergence in the use of replica methods springs from the need to localize molecules at higher resolution in membranes. Traditionally, we have significant expertise in the technique, and while the expertise still exists, the equipment in our facility does not. Our freeze-fracture machine, a refurbished ~20 year old Cressington CFE50, was retired two years ago due to vacuum, gun and electrical problems that could not be remedied by the company since this specific instrument is no longer manufactured. Because of this, we are requesting funds to purchase a JEOL JFD-V-TP freeze-fracture/freeze-etch/rotary shadowing machine for the Center for Biologic Imaging (CBI) at the University of Pittsburgh Medical School. The reason for our need to continue the use of these protocols by purchasing a new instrument is four-fold: 1. The recent recruitment of several new faculty members that have critical requirements for technology afforded by this instrument, including the rotary shadowing component of the machine that cannot be purchased as a stand alone feature. 2. Ongoing requests from long time collaborators for access to high-resolution structure-function imaging of membranes and molecular replicas of molecules. 3. Integration of light microscopy modalities coupled with high-resolution analysis at the membrane, especially utilizing FRIL (Freeze-Fracture Replica Immuno- Labeling). 4. There are no other devices of this nature anywhere at the University of Pittsburgh, or in the greater Pittsburgh area, for that matter. The mandate of the CBI is to provide access to a full range of light and electron optical, image analysis, and morphometric methods to all research groups (over 250 NIH-funded users) within the University of Pittsburgh as well as many from outside institutions. Acquisition of this machine will assist our major users enormously and provide all other researchers access to important high-resolution technologies to further their research.