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Microscopy and Imaging Core Facility

James T. Russell, DVM, PhD, Director
Louis (Chip) Dye, BS, Staff Scientist
Vincent Schram, PhD, Staff Scientist

The mission of the NICHD Microscopy and Imaging Core (MIC) is to provide high-end light 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. Located in the fifth and sixth floors of Building 49, the facility is staffed by two full-time microscopists working under the supervision of James Russell: Vincent Schram oversees the light microscopy operations and IT infrastructure, and Chip Dye manages the electron microscopy branch.

Mode of operation

The equipment and staff of the Core are available to everyone within the Institute free of charge, with the proviso that all investigators receive equal support. The mission 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 study’s Principal Investigator and postdoctoral scientists to discuss details of the experimental design. The background of the project and the imaging goals are discussed, and the most appropriate techniques and instrumentations are determined. Users are asked to sign a document outlining the policies governing use of the Core equipment (Rules of Use). Positive feedback from long-term users suggests that this high level of interaction greatly improves the efficiency of each imaging project. The facility is accessible 24 hours a day, 7 days a week, and users can reserve time at each instrument on an online calendar (https://next.cirklo.org/nichd).

Light microscopy

The light microscopy branch of the MIC operates in 4 different areas:

Equipment and maintenance: The MIC operates several confocal laser scanning microscopes optimized for various 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
Perkin-Elmer spinning disk for low-light imaging of photo-sensitive specimens
Dual-channel Olympus Total Internal Reflection Fluorescence (TIRF) platform
High-end conventional fluorescence microscope

Live imaging is fully supported, with temperature control, heated perfusion, and solution change available on every microscope; most instruments also have an environmental chamber with temperature, CO₂, and humidity control. Instrument downtime is kept to a minimum by providing full-time support to end users (phone and pager). For problems that require extensive repairs, all instruments are covered by a manufacturer's service contract and are usually serviced within the next business day.

User training and support: After counseling on specimen preparation and staining, each user receives hands-on training for the light microscope required by the project. The training covers the principles of fluorescence microscopy, confocal imaging, and optimum operation of the hardware platform. The initial training is followed by periodic refreshers at the user’s request, or when MIC staff feel the equipment is not being used properly.

Image analysis: The MIC operates a data analysis center with three high-end workstations and imaging software (Metamorph, Volocity, Imaris, and Zeiss AIM). At the users’ request, training and support are provided for each software package. When required, custom macros and high-throughput image analysis solutions are also provided. The facility also offers extensive data storage services with an enterprise-strength file server and a data backup system. This infrastructure is used to safeguard images and move data from the facility to each user’s location on campus.

Method development: During the past year, the MIC dedicated a significant amount of time and effort to the custom development of a subresolution imaging device—the Fluorescent Photo-Activatable Localization Microscopy (F-PALM) platform, which is being developed under the trans–NIH Imaging Initiative. The image acquisition side of this instrument is now complete for red fluorescent proteins. Funding permitting, we anticipate having a fully functional FPALM instrument for both green and red proteins by mid-spring, 2010. FPALM requires a complex image analysis module, which is still under development and will also be available by Spring, 2010.

Upgrades: To ensure that it addresses users’ needs optimally, the MIC continuously upgrades its equipment portfolio with new capabilities. A recently completed instrument upgrade is the replacement of an older wide-field Leica fluorescence microscope with a more modern, fully automated Olympus wide-field microscope. The data analysis software has also been significantly upgraded during the past 12 months with the addition of Bitplane’s Tmaris software, which offers advanced three-dimensional rendering, quantitation, and filament tracing.

Electron microscopy

Because sample processing for Electron Microscopy (EM) is more sophisticated and requires close attention to detail, the EM component of MIC operates differently from the light microscopy projects.

Sample processing: Typically, all EM processing (fixation, embedding, cutting, and staining) is performed in-house by Mr. Dye. The MIC has a fully equiped EM laboratory with an LKB Pyramitome, a Leica CM3050-S Cryostat, and a Reichert Ultracut-E Ultramicrotome. Given to the labor involved, fewer projects are undertaken than for light microscopy. The PELCO Biowave Pro programmable incubator (Ted Pella, Inc.) has been instrumental in improving the quality of both ultrastructure and immunolabeling for immunohistochemistry by providing consistent and controlled incubation parameters.

Imaging: EM imaging is performed mostly by the microscopist except when the user has the necessary inclination and training. The MIC operates an aging JEOL 1010 transmission electron microscope, which will be replaced by Spring 2010 with a more modern JEOL-1400. This upgrade will provide two new methodologies: cryo-electron microscopy and tomography imaging. The main advantage of cryo-EM is to preserve a high level of immunoreactivity, and it allows specimens to be imaged in a near-native state. Cryo-EM will improve the quality of studies relying on immunogold labeling. Tomography permits the imaging of three-dimensional structures at the EM level.

Method development: Mr. Dye has instituted techniques for EM-level immuno-histochemistry and double immunolabeling to simultaneously mark two separate antigens. The use of specialized grids (LUXFilm) has also been developed. LUXFilm EM grids allow a view of the entire specimen and are crucial for imaging large structures, tracing features, searching for special details, and for tomography imaging. The MIC has also implemented a digital archive of all EM images and parameters, which will be available online to investigators in the near future.

Ancillary support

Given that the DIR laboratories of the NICHD are scattered around the NIH campus, the MIC provides all necessary techniques and facilities within building 49: tissue culture hood, 5 and 10% CO₂ incubators, animal holding and preparative space, and vibratomes for live and fixed tissues. We wish to acknowledge the contribution of Mrs. Lynne Holtzclaw, currently a member of the Section on Cell Biology and Signal Transduction, for providing outstanding technical expertise on advanced cell and tissue sample preparation.

Community outreach

The MIC is committed to promoting light and electron microscopy in the NICHD, DIR, research community. Efforts are being made to educate investigators on the benefits and pitfalls of advanced imaging techniques. These initiatives include a) coaching users on the principles of confocal microscopy, during training and by publishing comprehensive operating protocols for each microscope; b) on-campus demonstrations of new instruments and software by vendors such as Zeiss, Photometrics, Nikon and Perkin-Elmer; and c) on-site assistance to investigators in their own laboratories operating their imaging equipment to optimize the quality of recorded data. Furthermore, the MIC web site (mic.nichd.nih.gov) is an important resource for tutorials and protocols for both fixed and live-cell microscopy.

In parallel with these efforts, the staff have developed collaborations with other Institutes to promote the exchange of information and bring new imaging technologies to NICHD. Ongoing collaborations include imaging of live animals (Dr. Afonso Silva, NINDS) and sub-resolution light microscopy (F-PALM, trans-NIH Imaging initiative).

Facility usage

The MIC currently serves a total of 107 registered users associated with 36 NICHD principal investigators (PIs) and three PIs of sister Institutes within the campus. During any given week, approximately 20 different users spend half a day or more at an MIC microscope. As of August 2009, users had logged more than 29,000 hours on the Core’s equipment. Since its creation in 2004, usage has resulted in more than 65 publications, six of which were co-authored by the Core personnel (see www.nichd.nih.gov/about/org/dir/other-facilities/cores/microscopyandimaging/publications for a complete list).

Looking ahead

Several technology developments, such as sub-resolution fluorescence imaging (FPALM) and multiple immuno-labelling are currently under way. The new JEOL 1400 instrument will significantly improve the EM capabilities of the Core by providing cryo-EM and tomography. With support from the Institute, the Microscopy and Imaging Core will continue to provide the NICHD community the competitive edge that is so important in today’s research environment.

Publications

Atkin SD, Patel S, Kocharyan A, Holtzclaw LA, Weerth SH, Schram V, Pickel J, Russell JT. Transgenic mice expressing a cameleon fluorescent Ca2+ indicator in astrocytes and Schwann cells allow study of glial cell Ca2+ signals in situ and in vivo. J Neurosci Meth 2009 181:212-226.
Balla T, Várnai P. Visualization of cellular phosphoinositide pools with GFP-fused protein-domains. Curr Protoc Cell Biol 2009 24:24-44.
Besser L, Chorin E, Sekler I, Silverman WF, Atkin S, Russell JT, Hershfinkel M. Synaptically released zinc triggers metabotropic signaling via a zinc-sensing receptor in the hippocampus. J Neurosci 2009 29:2890-2901.
Fisahn A, Neddens J, Yan L, Buonanno A. Neuregulin-1 modulates hippocampal gamma oscillations: implications for schizophrenia. Cereb Cortex 2009 19:612-618.
Koshimizu H, Senatorov V, Loh YP, Gozes I. Neuroprotective protein and carboxypeptidase E. J Mol Neurosci 2009 39:1-8.
Park JJ, Koshimizu H, Loh YP. Biogenesis and transport of secretory granules to release site in neuroendocrine cells. J Mol Neurosci 2009 37:151-159.
Rafikova E, Melikov K, Ramos C, Dye L, Chernomordik L. Transmembrane protein-free membranes fuse into Xenopus nuclear envelope and promote assembly of functional pores. J Biol Chem 2009 284:29847-29859.
Tanaka N, Ito K, Stopfer M. Odor-evoked neural oscillations in Drosophila are mediated by widely branching interneurons. J Neurosci 2009 29:8595-8603.
Samoshkin S, Arnaoutov A, Jansen L, Ouspenski I, Dye L, Karpova T, McNally J, Dasso M, Cleveland D, Strunnikov A. Human condensin function is essential for centromeric chromatin assembly and proper sister kinetochore orientation. PLoS One 2009 4:e6831.
Uveges TE, Kozloff KM, Ty JM, Ledgard F, Raggio CL, Gronowicz G, Goldstein S, Marini JC. Alendronate treatment of Brtl osteogenesis imperfecta mouse improves femoral geometry and load response before fracture but decreases predicted material properties and has detrimental effects on osteoblasts and bone formation. J Bone Miner Res 2009 24:849-859.
Wollert T, Wunder C, Lippincott-Schwartz J, Hurley JH. Membrane scission by the ESCRT-III complex. Nature 2009 458:172-177.

Contact

For more information, visit mic.nichd.nih.gov.

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