ShEEP Request for Nikon TIRF STORM microscope

超分辨率显微镜打开了前所未有的机会，以成像单个分子或基团 分辨率在50 nm以下的分子。然而，具有这些功能的显微镜如果从 准备组装的商业供应商，超出了大多数个人调查人员的预算。和切割 建造自己的房子需要对光学的理解，这超出了大多数人的知识 生物学家。与爱荷华市退伍军人医疗中心有关的多名调查人员已经达到了他们的 需要将超分辨率显微镜应用于活细胞以推动研究的研究 往前走。这项研究针对的是与美国退伍军人相关的疾病：肺病 肺气肿，在慢性感染中观察到的宿主防御改变，如丙型肝炎、巨细胞病毒或 利什曼病、青光眼和肥胖。目前在退伍军人管理局或爱荷华大学都没有显微镜 这将使这些类型的研究成为可能。因此，我们需要一台尼康日食Ti-e2 具有全内反射(TIRF)和随机光学重建的倒置显微镜 显微镜(STORM)模块。TIRF光学将使那些研究通过组装发出信号的受体的人受益 质膜上的适配蛋白(T细胞受体，Bishop和Stapleton博士)或保持活性 进入内体后发出信号(McGowan博士)TIRF和STORM的结合将使McGowan博士， Allen和Anderson研究足体的组装或皮质下细胞骨架的重塑。 STORM将使威尔逊、梅尔和斯台普顿博士能够观察到来自病原体的蛋白质与 从宿主细胞中了解病毒或寄生虫是如何逃避宿主防御的。梅尔博士将利用风暴 研究病毒或哺乳动物蛋白的靶向降解或病毒蛋白与染色质的相互作用。全 这些项目需要高分辨率的多色荧光成像，以探测和定位光子，在 快速的帧速率，并具有高的方向和时间精度。我们选择了尼康H-TIRF/N- Storm系统，因为它在一个包中满足了所有这些要求，该包在 很大程度上是自动格式化的，这对于多用户仪器来说是非常理想的。要启用活细胞成像， 显微镜将配备一个台式孵化器，以控制环境并允许试剂 在不改变视野的情况下介绍。我们还需要功能强大的PC工作站和尼康 获取和分析图像所需的软件。对该仪器的更完整的描述可以 可在具体目标部分找到。由于这套方案涉及新兴的尖端技术，我们 希望这也将加强新调查人员加入退伍军人管理局研究计划时的研究计划 并追求为美国退伍军人未来的健康需求量身定做的新项目。

Super-resolution microscopy has opened unprecedented opportunities to image single molecules or groups of molecules with resolution below 50 nm. However microscopes with these capabilities, if purchased from a commercial vendor as ready to assemble, are beyond the budgets of most individual investigators. And cutting expenses by building one's own requires an understanding of optics which is beyond the knowledge of most biologists. Multiple investigators associated with the Iowa City VA Medical Center have reached a point in their investigations where applying super-resolution microscopy to living cells is required to move their research forward. This research is targeted towards diseases which are relevant to US military veterans: pulmonary emphysema, alterations in host defense observed in chronic infections such as hepatitis C, cytomegalovirus, or Leishmaniasis, glaucoma and obesity. There is currently no microscope on the VA or University of Iowa campuses which will enable these types of studies. Therefore, we are requesting a Nikon Eclipse Ti-e2 inverted microscope equipped with total internal reflection (TIRF) and stochastic optical reconstruction microscopy (STORM) modules. TIRF optics will benefit those studying receptors which signal by assembling adapter proteins at the plasma membrane (T-cell receptor, Drs. Bishop and Stapleton) or retain active signaling after entering endosomes (Dr. McGowan). Combining TIRF and STORM will enable Drs. McGowan, Allen and Anderson to study the assembly of podosomes or remodeling of the sub-cortical cytoskeleton. STORM will enable Drs. Wilson, Meier, and Stapleton to observe the assembly of proteins from pathogens with those from host cells to learn how viruses or parasites evade host defenses. Dr. Meier will use STORM to study targeted degradation of viral or mammalian proteins or the interaction of viral proteins with chromatin. All of these projects require multicolor fluorescent imaging, at high resolution, to detect and localize photons, at rapid frame rates and with high directional and temporal precision. We have chosen the Nikon H-TIRF/N- STORM system because it meets all of these requirements in a package which provides these features in a largely automated format, which is highly desirable for a multi-user instrument. To enable live cell imaging the microscope will be equipped with a stage-top incubator to control the environment and allow reagents to be introduce without altering the field of view. We are also requesting powerful PC workstations and Nikon software which is required to acquire and analyze images. A more complete description of the instrument may be found in the Specific Aims section. Because this package involves emerging state-of the art technology, we hope that it will also enhance the research programs of new investigators as they join the VA research program and pursue novel projects tailored to the future health needs of US veterans.