CORE C: IMAGING CORE

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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