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Microscopy and Imaging Core Facility
- James T. Russell, DVM, PhD, Director
- Vincent Schram, PhD, Staff Scientist
- Louis (Chip) Dye, BS, Staff Scientist
- Lynne A. Holtzclaw, BS, Senior Research Assistant (Biologist)
The mission of the NICHD Microscopy and Imaging Core (MIC) is to provide state-of-the-art light microscopy and electron microscopy services to all NICHD scientists. The Core is designed as a multi-user facility where investigators can, with a minimum of time and effort, prepare, image, and analyze their samples. The facility is staffed by three full-time microscopists working under the supervision of James Russell. Vincent Schram oversees the light-microscopy operations and IT infrastructure, Lynne Holtzclaw supports light microscopy and immunohistochemistry, and Louis (Chip) Dye manages the electron microscopy arm of the facility.
The Division of Intramural Research (DIR) plans to relocate the MIC to the new facility currently under construction, the Porter Neuroscience Research Center–Phase II (PNRC II). The move should be completed by January, 2014. Within the PNRC, the MIC is expected to provide microscopy and imaging resources to eight other Institutes (NINDS, NIMH, NIDCD, NIDA, NIA, NEI, NIAAA, and NIDCR) in addition to NICHD Division of Intramural Research.
NICHD DIR is working to widen resources to meet the challenge of providing them to many more laboratories.
Mode of operation
The equipment and staff of the MIC are available to all laboratories within NICHD DIR, as well as to all laboratories housed in the PNRC II. The philosophy of the MIC is to ensure that only reliable, high-quality data are recorded on its instruments. For every new project, MIC staff meet with the Principal Investigator and the postdoctoral scientists involved in the study to discuss details of the experimental design. The background of the project and imaging goals are discussed at that time, and the most appropriate techniques and instrumentation are determined. Users are asked to sign a document outlining the policies to follow when using the Core equipment (https://science.nichd.nih.gov/confluence/display/mic/Policies). Positive feedback from long-term users suggests that this high level of interaction greatly improves the quality of data obtained and efficiency of each imaging project. The facility is accessible 24/7 using the NIH ID system, and users can reserve time on each instrument by using an online calendar (https://facility-dev.ninds.nih.gov/secure/scheduler/admin/adminHomePage.aspx).
In the fall of 2010, the MIC implemented a fee structure. The fee-for-service model serves to reduce frivolous use of the facility and makes it available to scientists who require microscopy services as a crucial tool in their research. The rates charged are nominal—between $20 and $40 per hour for confocal imaging for all researchers.
The light microscopy arm of the MIC operates in four different areas:
Equipment and maintenance:
The MIC operated several confocal laser scanning microscopes optimized for different applications:
- Zeiss LSM 510 inverted for high-resolution confocal imaging of fixed specimen;
- Zeiss Live DuoScan for high-speed imaging of live cells;
- Zeiss LSM 510 NLO for two-photon imaging of live tissue sections and live animals;
- Zeiss LSM 780 high sensitivity point scanning confocal microscope;
- Olympus platform dual-channel total internal reflection fluorescence (TIRF) (sub-resolution instrument);
- Photo-activation localization microscope (PALM) built in house (sub-resolution instrument);
- Nikon DSTORM microscope (sub-resolution instrument) with a spinning-disk confocal optical bench; the instrument was added during this year, and is functioning superbly;
- Zeiss Lumar Stereo microscope;
- High-end wide-field fluorescence instrument
Live-cell, tissue and live-animal imaging are supported with temperature, CO2, and humidity control and heated perfusion on most microscopes. Instrument downtime is kept to a minimum by providing full-time support to end users (phone and pager). For problems that require extensive repairs, most instruments are covered by manufacturers' service contracts and are usually serviced within a few days.
User training and support:
After counseling on specimen preparation and staining, each user receives hands-on training on the light microscope required for the project. The training covers the principles of fluorescence microscopy, confocal imaging, and optimum operation of the hardware platform and is followed by periodic refreshers at the user’s request or when MIC staff feel the equipment is not being used optimally.
The MIC operates a data analysis center with three high-end workstations and image-processing software environments (Metamorph, Volocity, Imaris, and Zeiss AIM). At the users’ request, we provide training and support for each software package and, when required, custom macros and high-throughput image analysis solutions. The facility offers extensive data storage services with an enterprise-level file server and a data backup system. The infrastructure is used to safeguard images and move data from the facility to each user's location on campus.
The MIC is involved in the development of methods for novel microscopy and image-processing techniques when the need arises. Recently, Vincent Schram implemented a single-molecule imaging platform and developed software required for imaging and analysis. Similar methods-development efforts will be undertaken according to Institute needs and availability of funds. We actively entertain such requests.
Because of the complex nature of electron microscopy (EM) sample preparation, the EM arm of the MIC operates in a different manner from the light microscopy imaging arm of the MIC.
Typically, all EM processing (fixation, embedding, tissue sectioning, and staining) is undertaken in-house by Chip Dye. Lynne Holtzclaw is now also proficient in EM sample preparation techniques and assists Mr. Dye in this effort. The MIC maintains and operates a fully equipped EM laboratory with a LKB Pyramitome, a Leica CM3050-S Cryostat, and a Reichert Ultracut-E Ultramicrotome. Given the labor involved, fewer projects are undertaken. By providing consistent and controlled incubation parameters, the PELCO Biowave Pro programmable incubator (Ted Pella, Inc.) has been instrumental in improving the preservation of ultrastructure and the quality of immunolabeling.
EM imaging is undertaken mostly by the microscopist, except in cases where the user has the inclination and necessary training. Imaging is carried out on a JEOL 1400 series electron microscope. This is a relatively new instrument, having been installed in the Spring of 2011. It offers two new imaging methodologies: cryo-EM and tomography. The main advantage of cryo-EM is that it preserves a high level of immunoreactivity and permits specimens to be imaged in a near-native state. Cryo-EM thus improves the quality of studies relying on immunogold labeling. Mr. Dye is slated to participate in a specialized course on cryo-EM techniques offered by Leica Micro Systems. Tomography provides three-dimensional imaging of structures at the EM level.
Chip Dye recently set up the required techniques for EM immunohistochemistry and dual immunolabeling to simultaneously image two separate antigens at EM resolutions and is using LUXFilm EM grids, which provide a view of the entire specimen and are crucial for imaging large structures, tracing features, searching for special details, and tomography imaging. The MIC also implemented a digital archive of all EM images and parameters, which is available online to investigators.
Given that the NICHD's Division of Intramural Research laboratories are scattered throughout the NIH campus, the MIC provides all necessary resources and facilities, such as tissue culture space, 5% and 10% CO2 incubators, animal holding and preparative space, and vibratomes for live and fixed tissues. Lynne Holtzclaw provides outstanding technical expertise on advanced cell and tissue-sample preparation for immunohistochemistry experiments. The MIC will move to a new facility within PNRC Phase II, which is nearing completion. The move will begin on December 15th of 2013.
The MIC is committed to promoting light and electron microscopy in the NICHD, DIR research community. We are making efforts to educate investigators on the benefits and pitfalls of advanced imaging techniques. These initiatives include: (i) coaching users on the principles of confocal microscopy during training and by publishing comprehensive operating protocols for each microscope; (ii) on-campus demonstrations of new instruments and software by vendors such as Zeiss, Olympus, Photometrics, Nikon, and Perkin-Elmer; and (iii) on-site assistance to investigators in their laboratories operating their own imaging equipment to optimize the quality of the data recorded. Furthermore, the MIC website (http://mic.nichd.nih.gov) is an important resource for tutorials and protocols for both fixed and live cell microscopy.
In addition, an educational outreach program was implemented within the MIC, with the goal of educating individual NICHD DIR senior scientists and post-doctoral scientists on microscopy techniques from the ground up. Instruction includes lectures as well as hands-on experimental work organized within the MIC facilities. We plan to offer this education curriculum annually.
Parallel to these efforts, the staff have continued to collaborate with other Institutes to promote the exchange of information and bring new imaging technologies to this Institute. Ongoing collaborations with the NIBIB on sub-resolution light microscopy (F-PALM) continue, and we are actively promoting the use of the trans-NIH Imaging consortium, and software development efforts with the Center for Information Technology.
The MIC currently serves more than 100 scientists associated with 41 NICHD principal investigators (PIs) and three PIs of sister Institutes within the campus. On any given week, approximately 10 different users spend half a day or more on MIC microscopes. Since its creation in 2004, usage has resulted in more than 120 publications, many of which were co-authored by Core personnel. For a full list of publications made possible through use of MIC facilities, go to https://science.nichd.nih.gov/confluence/display/mic/Publications.
Maintaining existing imaging rigs at peak operational status and replacing aging equipment are critical for the success of the MIC. To this end, a modern high-resolution and high-sensitivity point-scanning-confocal microscope (a Zeiss LSM 780) was recently purchased. During the fall of 2012, a new hybrid spinning disc confocal microscope and TIRF microscopy was purchased from Nikon. This microscope can also perform single-molecule imaging using the D-STORM technique. The MIC would require additional equipment (high-pressure freezing unit, cryo-ultra-microtome, and a freeze substitution unit) in order to make CyroEM technology routinely available within the MIC. The MIC continues its efforts towards technology development.
The portfolio of instruments within the MIC needs to be maintained at peak levels of performance at all times to serve the needs of NICHD scientists adequately. The quality of data acquired is heavily influenced by the quality of the optics. This can be achieved only with timely upgrades and replacement of aging instruments. If the MIC is expected to provide high-end imaging solutions to the DIR, particularly when the MIC moves into space within the PNRC and offers microscopy resources to scientists from eight additional Institutes, a new support model that allows for timely equipment upgrades is critical. Plans and processes need to be developed in order to reach this critical goal. The MIC is actively working to replace the aging LSM 510, the work horse instrument that has been with the facility for nearly 10 years. We hope this can be achieved during 2013–2014 fiscal year.
- Subramanian J, Dye L, Morozov A. Rap1 signaling prevents L type calcium channel dependent neurotransmitter release. J Neurosci 33;2013:7245-7252.
- James Pickel, PhD, Laboratory of Genetics, NIMH, Bethesda, MD
- Afonso Silva, PhD, Cerebral Microcirculation Unit, NINDS, Bethesda, MD