MICROSCOPY AND IMAGING CORE FACILITY
, Director
Vincent Schram, PhD, Staff Scientist
Louis (Chip) Dye, BS, Senior Research Assistant
Mode of operation
The facility’s equipment and staff are available to everyone within the Institute free of charge, provided that existing resources are distributed equally among investigators requesting MIC services. 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 project’s background, and the project’s imaging goals in order to determine the most appropriate techniques and instrumentation. Users must sign a document outlining the policies that must be followed when using the Core’s equipment (http://mic.nichd.nih.gov/pdf/mic_rules_of_use.pdf). Long-term user feedback indicates that the high level of interaction between Core staff and researchers has greatly improved the efficiency of each imaging project.
Light microscopy
After counseling on specimen preparation and staining, each user undergoes hands-on training on the light microscope required by the project. Once staff are confident that the user can safely collect high-quality data, the user may proceed without supervision but with continuous support and guidance. The facility is accessible 24 hours a day/seven days a week, and users may reserve time on each instrument by using an online calendar (https://dircal.nichd.nih.gov/calendar/login.html).
The MIC operates several confocal laser scanning microscopes that address different needs.
(1) Zeiss LSM 510 inverted with UV and spectral detection capabilities, optimal for high-resolution imaging.
(2) Zeiss LSM 510 upright with two-photon excitation and an integrated electrophysiology platform for combined physiology/confocal imaging on live tissue sections and whole animals.
(3) Perkin-Elmer spinning disk for low-light imaging of photosensitive specimens.
In addition, an Olympus Total Internal Reflection Fluorescence (TIRF) microscope and a wide-angle fluorescence microscope from Leica are available. Live imaging is fully supported on every microscope, with temperature control, heated perfusion, and solution change available. Most instruments have an environmental chamber with CO2 and humidity control.
To address users’ needs optimally, the MIC continuously upgrades its equipment portfolio with new capabilities. During the past year, we added a Zeiss Live DuoScan confocal microscope specifically designed for ultra–high-speed, high-sensitivity imaging. Ideally suited for fluorescent protein constructs and fully equipped for live cell imaging, the Live DuoScan has an auxiliary attachment for high-speed photobleaching and photoactivation. High-speed volume acquisition and calcium imaging are fully supported. Over the last year, we made three significant upgrades to existing instruments. The excitation and emission paths of the Zeiss LSM 510 upright microscope were modified for a significant (4 to 6×) increase in detection efficiency. The Olympus TIRF microscope was outfitted with a dual-channel exciter for high-resolution imaging of fine membrane-bound features in dual colors. We are replacing the Leica wide-field microscope with a more modern, fully automated Olympus upright microscope.
Electron microscopy
Given that sample processing for electron microscopy (EM) is more sophisticated than for light microscopy, the EM division of the Core operates differently than the light microscopy branch. Typically, Mr. Dye carries out all EM processing (fixation, embedding, cutting, and staining). The Core has a full-fledged EM laboratory with an LKB Pyramitome, a Leica CM3050-S Cryostat, and a Reichert Ultracut-E Ultramicrotome. Imaging on the facility’s JEOL 1010 transmission electron microscope is performed by either the microscopist or, for users with the inclination and proper training, the end user. Due to the labor involved, the number of projects undertaken is necessarily small. In an effort to alleviate any backlog, the MIC has acquired a microwave-assisted programmable incubator (PELCO BioWave Pro, Ted Pella, Inc.). The device has been instrumental in reducing processing times and allowing Mr. Dye to support more investigators. Consistent and controlled incubation parameters have also significantly improved the quality of both ultrastructure and immunolabeling studies. Mr. Dye recently set up techniques for EM-level immunohistochemistry, and the use of the new microwave technology has enabled excellent preservation of morphology as well as of immunogenicity.
Ancillary support
Given that the NICHD’s Division of Intramural Research (DIR) laboratories are scattered across the NIH campus, we have provided necessary techniques and facilities within building 49: tissue culture hood, 5 and 10 percent CO2 incubators, animal holding and preparative space, and vibratomes for live and fixed tissues. The MIC also operates a data analysis center with three high-end workstations and imaging software (Metamorph, Volocity, Imaris, and Zeiss LSM, with 3D rendering), an enterprise-strength file server, and a data backup server. We offer training and support on all imaging and analysis software as well as custom macros or stand-alone batch analysis programs.
Community outreach
The MIC is committed to promoting light and electron microscopy in the NICHD’s DIR research community. We are working to educate investigators on the benefits and pitfalls of advanced imaging techniques by (1) coaching users on the principles of confocal microscopy during training and publication of comprehensive operating protocols for each microscope; (2) providing on-campus demonstrations of new instruments and software by vendors such as Zeiss, Olympus, Nikon, and Bitplane; and (3) delivering on-site assistance to investigators operating their own imaging equipment to optimize the quality of the data recorded. Moreover, the MIC Web site (http://mic.nichd.nih.gov/) contains an extensive list of protocols for both fixed and live tissue microscopy and is an important resource for end users. In parallel to these efforts, staff have developed collaborations with other Institutes to promote the exchange of information and bring new imaging technologies to NICHD. We are currently collaborating with Dr. Afonso Silva (NINDS), Dr. Richard Saunders (NIMH), Dr. Alexei Morozov (NIMH), and Dr. Indu Ambudkar (NIDCR). One of the most significant outcomes has been the acquisition of the procedural expertise required to image live animals.
Facility use
The MIC currently serves 140 registered users associated with 36 NICHD principal investigators and 4 principal investigators of sister Institutes on campus. Approximately 15 users spend half a day or more on an MIC microscope during any given week. This level of use has resulted in more than 50 publications since creation of the MIC in early 2004. During 2007, an estimated 20 new publications contained data acquired at the MIC.
Looking ahead
Several technology developments, such as multiple immunolabeling for EM and improved sensitivity in infrared imaging, are currently in progress. We have to overcome some challenges to expand the facility beyond its current capacity, but, with continuing support from the Institute, the Microscopy and Imaging Core will continue to provide the NIH community with the competitive edge that is essential in today’s research environment.
For further information, contact james@helix.nih.gov.
