CORE C: IMAGING CORE

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。自上一报告所述期间以来，主要人员的支助情况没有变化。在下一个预算期内，任何关键人员的工作水平都不会有重大变化。预计未支配余额将不超过本年度预算的25%。a.具体目标：项目目标没有改变。我们正在构建一个共享使用的双光子显微镜系统，并制定一个培训计划，为UTSA的用户准备利用这一重要的新实验技术。b.今年的进展：过去一年，成像核心的主要工作是对两台光子显微镜的最终配置进行技术决策，并购买设备。由于该领域的技术变化如此之快，许多关于设置细节的决定必须在最后一刻做出。此外，价格的变化，特别是钛蓝宝石激光器，提供了在最初预算制定时并不明显的机会。提出的配置是一组显微镜围绕一个单一的激光器。这是因为激光是整个显微镜的主要成本决定因素。审查委员会指出了这个设计中的一些缺陷。审稿人特别指出的一个问题是不能容忍激光操作中的故障。如果激光不工作，所有4个工作站的实验都将停止。审查委员会建议购买一台以上的激光器。在这一年中，激光器成本的降低使这项建议成为可能。我们能够购买两个Ti：蓝宝石激光器，围绕它建立我们的显微镜装置。激光器已被购买，但在撰写本文时，它们尚未到达UTSA。我们面临的第二个主要问题是激光扫描设备的选择。在我们提出建议的时候，在我们的价格范围内有两种可能的配置。一种是购买一个扫描头，作为一个内部探测器和软件的包装。这基本上意味着购买共聚焦显微镜的扫描头，如奥林巴斯Fluoview，并修改它用于我们的激光器。在这种情况下，我们还需要修改显微镜以添加外部检测器（pmt），并修改电子设备以使软件读取来自这些检测器的信号并正确解释它们。另一种方法是将扫描头、软件和探测器的部件组装在一起。这种显微镜很常见。它们中的大多数都是基于最初的贝尔实验室小组的设计，并且可以从开发它们的科学家那里获得信息和软件。像这样的显微镜正在准备作为萨特仪器的商业包装，尽管它还没有出现在他们的目录中。这是非常划算的，这本来就是我们的顾问推荐的。这种类型的显微镜存在于圣安东尼奥的UT健康科学中心附近。然而，自从我们提交赠款以来，出现了第三种选择。Prairie Technologies是一家生产许多特殊用途成像产品的公司，现在提供一种带有软件和对现有显微镜进行修改的扫描头。我们的顾问提出了这个解决方案，我们遵循了这个建议。该系统的一个主要优点是，它是为同时膜片钳记录和成像而设计的，这是我们在UTSA设施的主要用途。该系统还具有一定的安全优势，因为激光束可以完全封闭。包装系统的成本与共聚焦扫描头的成本相当，需要修改才能使用。我们已经购买了两个这样的系统。我们打算建立两个更自制类型的显微镜。我们的配置是两张桌子，每一张都比最初提议的要小，每一张都有自己的激光器。一个Prairie Technologies装置将放置在每个桌子上，同时提供成像和贴片夹紧。激光束通常会射向这台显微镜。此外，还将制定一项规定，将光束引导到第二台显微镜上，这台显微镜将更多地由家庭建造，并将为独特和特殊目的的实验进行重新配置。这些工作站将更具可扩展性，并且将依赖于实验者提供的特殊部件。使用这些工作站将需要更广泛的设备知识和更详细的安全培训和安全程序。过去一年，在我们的监督下，UTSA成像设施的旧部分一直很有成效。电子显微镜实验室有两个主要用户。一位是Laura Mery博士，她是Brenda Claiborne博士实验室的博士后研究员。Drs。梅里和克莱本正在研究齿状回神经元树突棘的变化。实验室的第二个主要用户是詹姆斯·鲍尔博士的实验室。鲍尔博士正在研究由颗粒细胞轴突与小脑浦肯野神经元的树突和树突棘形成的突触连接。我们还接管了UTSA现有的Bio-Rad 1024共聚焦显微镜的维护。这是生物系唯一共享使用的共聚焦显微镜，它被大量用于各种各样的项目。我们已经开始努力扩大UTSA社区对活的完整组织的多光子成像的当地熟悉程度。UTSA的与会者参加了两次由外部发言人举办的关于双光子显微技术的研讨会。一篇是由加州大学的Colleen Witt博士完成的，另一篇是由斯坦福大学的Mark Schnitzer博士完成的，研究的是哺乳动物大脑的显微内窥镜。意义：在过去的一年里，双光子显微技术在生物学的各个领域得到了极大的发展。此外，利用显微内窥镜更深入地成像大器官（如大脑）的方法已经开发出来，这些依赖于双光子成像的可用性。这项技术在UTSA的发展比以往任何时候都更加重要。d.来年计划：分配给影像实验室的空间尚未可用。这座大楼的建造进度有些超出计划。根据我们最初的计划，我们将在写这篇文章的时候（2005年5月）搬进来。在这一点上，我们几乎所有的设备已经到达，我们准备搬进去。我推迟了购买大型光学桌的时间，因为我们没有地方存放它们，而且它们又大又重。如果该建筑在未来2个月内完工，就有可能在预算年度结束前购买这些最后的部件。这种情况预计不会发生。显然，我们要到今年年底才能搬进这栋楼。这是一个相对较小的延迟，只是由于表的原因而引起关注。我们希望有可能结转一些资金专门用于购买光学表。这并不奇怪，很难找到一个合适的经理来管理这个设施。这项工作需要难得的训练和才能的结合。我相信我们已经找到了一位合适的经理，但她不想在大楼准备好，真正的工作可以开始之前就开始。她目前受雇于一家制作成像软件的公司，是双光子显微镜方面的专家。假设在这段时间内她的情况没有任何变化，我们应该可以在大楼空置的时候雇佣她。在即将到来的一年里，当设施的空间可用时，我们打算为教师和学生开设一个2光子显微镜的短期课程，包括与该技术相关的理论，实践和安全问题。我们的顾问已经同意参加这个工作坊

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. ¿ There has been no change in the suppoort of key personnel since the last reporting period. ¿ There will not be any significant change in the level of effort for any key personnel in the next budget period. ¿ The estimated unobligated balance will not be greater than 25% of the current year's budget. a. Specific Aims: The aims of the project have not changed. We are constructing a 2-photon microscope system for shared use, and developing a training program to prepare users at UTSA to take advantage of this important new experimental technology. b. Progress this year: The main effort of the imaging core over the past year has been expended in making technical decisions regarding the final configuration of the two photon microscopes, and making purchases of the equipment. Because the technology in this field is changing so fast, many decisions about the particulars of the setup had to be made at the last possible moment. Also, changes in pricing, especially of Ti:Sapphire lasers, offered opportunities that were not apparent at the time the original budget was formulated. The configuration proposed was a set of microscopes surrounding a single laser. This was proposed because laser was the main cost determinant of the entire microscope. The review committee pointed out a number of flaws in this design. One, pointed out specifically by the reviewers, is intolerance to faults in laser operation. If the laser is inoperative, experiments at all 4 workstations is halted. The review committee recommended that more than one laser be purchased. Reductions in the cost of lasers during the year made it possible to follow this recommendation. We were able to purchase two Ti:Sapphire lasers around which to build our microscope installation. The lasers have been purchased, but at the time of this writing they have not yet arrived at UTSA. The second major question for us was the choice of laser scanning devices. At the time of our proposal, two possible configurations were possible in our price range. One is the purchase of a scan head as a package with internal detectors and software. This basically means buying the scan head for a confocal microscope, such as the Olympus Fluoview, and modifying it for our laser. In this case we would also need to modify the microscope to add external detectors (PMTs) and modify the electronics to get the software to read the signals from these detectors and interpret them correctly. The alternative was to put together a scan head, software and detectors from parts. This kind of microscope is common. Most of them are based on designs from the original Bell Labs group, and the information and software are available from the scientists who developed them. A microscope like this is being prepared as a commercial package from Sutter Instruments, although it does not yet appear in their catalog. This is very cost-effective, and it is originally what our consultants recommended. Microscopes of this type exist nearby at the UT Health Science Center in San Antonio. Since the submission of our grant, however, a third option has arisen. Prairie Technologies, a company that makes a number of special purpose imaging products, now offers a scan head with software and modifications to an existing microscope as a package. Our consultants suggested this solution, and we followed this advice. A major advantage of this system is that it was designed for simultaneous patch-clamp recording and imaging, which is the main use intended for our facility at UTSA. The system also has some safety advantages, in that the laser beam can be completely enclosed. The cost of the packaged system was comparable to that of the confocal scan heads that would require modification for our use. We have purchased two of these systems. We intend to set up two of the more homebrew type of microscopes as well. Our configuration will be two tables, each smaller than originally proposed, and each with its own laser. One Prairie Technologies setup will be placed on each table, with provision for simultaneous imaging and patch clamping. The laser beam will ordinarily be directed to this microscope. A provision will be made to direct the beam to the second microscope as well, which will be more home-built, and will be reconfigurable for unique and special purpose experiments. These workstations will be more expandable, and will rely on special parts supplied by the experimenter. Use of these workstations will require more extensive knowledge of the apparatus and more elaborate safety training and safety procedures. The older parts of the imaging facility at UTSA have been productive under our supervision for the past year. The electron microscopy laboratory has two major users. One is Dr. Laura Mery, who is a postdoctoral fellow in the laboratory of Dr. Brenda Claiborne. Drs. Mery and Claiborne are studying changes in dendritic spines of neurons in the dentate gyrus. The second major user of the laboratory is Dr. James Bower's laboratory. Dr. Bower is studying the synaptic connections formed by granule cell axons onto the dendrites and dendritic spines of Purkinje neurons in the cerebellum. We have also taken over the maintenance of the existing Bio-Rad 1024 Confocal microscope at UTSA. This is the only shared-use confocal microscope in the Biology Department, and it is heavily used on a wide variety of projects. We have begun our effort to expand local familiarity of the UTSA community to multiphoton imaging inl iving, intact tissues. Two seminars by outside speakers on 2-photon microscopic techniques were attended by UTSA participants. One was by Dr. Colleen Witt from Univ. California, and one (at the Health Sciences Center) by Dr. Mark Schnitzer (Stanford, University) on microendoscopy in the mammalian brain.c. Significance: During the past year, the use of 2-photon microscopy of living material has expanded dramatically in all areas of biology. In addition, methods for imaging more deeply into large organs (like the brain) using microendoscopy have been developed, and these depend upon the availability of 2-photon imaging. The development of this technology at UTSA is more essential than ever. d. Plans for the Upcoming Year: The space allocated for the imaging laboratory has not yet become available. Construction of the building is somewhat over schedule. According to our original plan, we were to move in at about the time of this writing (May 2005). At this point, almost all of our equipment has arrived and we are ready to move in. I have delayed buying the large optical tables, because we have no place to store these, and they are very large and heavy. If the building were to be completed in the next 2 months, it would be possible to buy these last parts before the end of the budget year. This is not expected to happen. Apparently, we will not be able to move into the building until the end of this calendar year. This is a relatively small delay, and is only a concern because of the tables. We hope it may be possible to carry forward some funds specifically for purchase of the optical tables.It is not surprising that it was difficult to find a suitable manager for this facility. The job requires a rare combination of training and talent. I believe we have identified a suitable manager, but she does not want to begin until the building is ready and the real job can begin. She is currently employed by a company that makes imaging software, and is an expert on 2 photon microscopy. Assuming nothing changes about her situation during this delay, we should be able to hire her about the time that the building becomes available.In the upcoming year, when the facility's space becomes available, we intend to institute a short course in 2 photon microscopy for faculty and students, that will include theoretical, practical and safety issues associated with the technique. Our consultants have agreed to participate in this workshop