National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2015 Annual Report of the Division of Intramural Research

The Microscopy and Imaging Core Facility

Ben Feldman
  • James T. Russell, DVM, PhD, Director, Microscopy and Imaging Core Facility
  • 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. Since the move to the Porter Neuroscience Research Center (PNRC II), building 35-A, in February, 2014, the MIC has been functioning smoothly in the new location. The PNRC facility is effective, efficient, and accessible to all MIC's clients. The MIC 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 component of the facility.

Within the PNRC II, 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's Division of Intramural Research (DIR). Since the move to PNRC, MIC has totally abandoned its fee-for-service policy, and services are now free of charge.

Mode of operation

The equipment and staff of the MIC are available to everyone within the NICHD DIR, as well as to all occupants of the PNRC. 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 experimental design, the background of the project, and its imaging goals and the most appropriate techniques and instrumentation are determined. Users are asked to sign a document outlining the policies to be followed when using the Core equipment. 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. The MIC also offers periodic training courses on the principles of and detailed techniques in microscopy and specimen preparation techniques. Periodic invited lectures and new equipment demonstrations offered by microscope manufacturers are also offered.

Light microscopy

The light microscopy arm of the MIC operates in four different areas:

Equipment maintenance and upgrade

The MIC operates several confocal laser scanning microscopes optimized for various applications: a Zeiss LSM 780 inverted for high-resolution confocal microscopy of both live and fixed fluorescently labelled cells and tissues; a Zeiss 510 inverted for high-resolution confocal imaging of fixed specimens; during 2015, the aging LSM 510 point-scanning confocal microscope was replaced by a new-generation point-scanning confocal, the LSM 710 for two-photon imaging of live tissue sections and live animals. During the summer of 2013, the MIC acquired a Nikon inverted microscope platform with all the required accessories to implement a high-resolution, ultra-fast imaging platform using spinning disc confocal scanning, and a high sensitivity camera. This high-end instrument is also capable of providing Total Internal Reflection (TIRF) microscopy as well as direct Stochastic Optical Reconstruction Microscopy or dSTORM. A sub-resolution microscope with a home-built Photo-Activation Localization Microscope (PALM) is also available at the MIC. In addition, two high-end wide-field fluorescence microscopes are available, as is a fluorescence stereo microscope.

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 (by 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 the MIC's staff feel that the equipment is not being used optimally. When necessary, hands-on training in sample preparation is provided in the MIC’s labs so that postdoctoral fellows are adequately trained in optimal sample preparation. Individual training in tissue handling and sample preparation is offered by MIC staff upon request.

Image analysis

The MIC operates a data-analysis center with three high-end workstations and image-processing software environments (Metamorph, Volocity, Imaris, and Zeiss AIM, Image J, and Fiji). At the user's 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 the NIH campus.

Methods development

The MIC develops methods for novel microscopy and image-processing techniques when the need arises. For example, a single-molecule imaging platform was implemented, and Vincent Schram developed software required for imaging and analysis. Similar methods-development efforts will be undertaken according to Institute needs and availability of funds. The MIC actively entertains such requests.

Electron microscopy

Because of the complex nature of electron microscopy (EM) sample preparation, the EM component of the MIC (MIC-EM) functions differently, and all sample preparation and microscopy are carried out by the staff microscopist. This is necessary because, to achieve optimal image quality, the procedures involved are critical and exacting.

Sample processing

Typically, all EM processing (fixation, embedding, tissue sectioning, and staining) is undertaken in-house by Chip Dye. Lynne Holtzclaw is also now proficient in EM sample-preparation techniques and assists Chip 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. 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 immuno-labeling.

Imaging

EM imaging is done mostly by the microscopist, except in cases where the user has the necessary inclination and training. Imaging is carried out on a JEOL JEM 1400 series electron microscope. The instrument has become the work-horse for transmission electron microscopy within the MIC. It offers two new imaging methodologies: cryo-EM and tomography. The main advantages of cryo-EM are that it preserves a high level of immuno-reactivity and allows specimens to be imaged in a near-native state. Cryo-EM improves the quality of studies that rely on immuno-gold labeling. Tomography provides three-dimensional imaging of structures at the EM level. We are exploring the possibility of expanding the EM component by including specimen preparation equipment for high-pressure freezing and freeze substitution. The techniques should provide better immune detection at EM–level resolutions with well-preserved cellular architectures.

Methods development

Chip Dye recently set up the required techniques for EM immuno-histochemistry and dual immuno-labeling to simultaneously image two separate antigens at EM resolution; he 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. Dye is expected to incorporate cryo-EM techniques and tomography in his repertoire.

Ancillary support

Given that the NICHD's Division of Intramural Research laboratories are scattered over 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 in advanced cell and tissue-sample preparation for immuno-histochemistry experiments. The recently completed move to PNRC facilities requires us to provide microscopy support to all occupants of the PNRC, which has somewhat increased our user base.

Facility usage

The MIC currently serves over 100 scientists associated with 41 NICHD principal investigators (PIs) and three PIs of sister Institutes within the campus. On any given week, approximately 10 separate users spend half a day or more on the MIC's 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 the MIC's facilities, go to https://science.nichd.nih.gov/confluence/display/mic/Publications.

2014 and 2015 publications emanating from work carried out in the MIC facility

Bojjireddy N, Botyanszki J, Hammond G, Creech D, Peterson R, Kemp DC, Snead M, Brown R, Morrison A, Wilson S, Harrison S, Moore C, Balla T. Pharmacological and genetic targeting of the PI4KA enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate levels. J Biol Chem 2014;289:6120-6132.

Farber CR, Reich A, Barnes AM, Becerra P, Rauch F, Cabral WA, Bae A, Quinlan A, Glorieux FH, Clemens TL, Marini JC. A novel IFITM5 mutation in severe atypical osteogenesis imperfecta type VI impairs osteoblast production of pigment epithelium-derived factor. J Bone Miner Res 2014;29:1402-1411.

Guzmán-Hernández ML, Potter G, Egervári K, Kiss JZ, Balla T. Secretion of VEGF-165 has unique characteristics, including shedding from the plasma membrane. Mol Biol Cell 2014;25:1061-1072.

Hammond GR, Machner MP, Balla T. A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi. J Cell Biol 2014;205:113-126.

Jovic M, Kean MJ, Dubankova A, Boura E, Gingras AC, Brill JA, Balla T. Endosomal sorting of VAMP3 is regulated by PI4K2A. J Cell Sci 2014;127:3745-3756.

Lin L, Long LK, Hatch MM, Hoffman DA. DPP6 domains responsible for its localization and function. J Biol Chem 2014;289:32153-32165.

Swift MR, Pham VN, Castranova D, Bell K, Poole RJ, Weinstein BM. SoxF factors and Notch regulate nr2f2 gene expression during venous differentiation in zebrafish. Dev Biol 2014;390:116-125.

Wang H, Kane AW, Lee C, Ahn S. Gli3 repressor controls cell fates and cell adhesion for proper establishment of neurogenic niche. Cell Rep 2014;8:1093-1104.

Chandra G, Bagh MB, Peng S, Saha A, Sarkar C, Moralle M, Zhang Z, Mukherjee AB. Cln1 gene disruption in mice reveals a common pathogenic link between two of the most lethal childhood neurodegenerative lysosomal storage disorders. Hum Mol Genet 2015;24:5416-5432.

Cheng Y, Rodriguiz RM, Murthy SRK, Senatorov V, Thouennon E, Cawley NX, Aryal DK, Ahn S, Lecka-Czernik B, Wetsel WC, Loh YP. Neurotrophic factor-a1 prevents stress-induced depression through enhancement of neurogenesis and is activated by rosiglitazone. Mol Psychiatry 2015;20:744-54.

Choi EY, Patel K, Haddad MR, Yi L, Holmes C, Goldstein DS, Dutra A, Pak E, Kaler SG. Tandem duplication of exons 1-7 neither impairs ATP7A expression nor causes a Menkes disease phenotype. JIMD Rep 2015;20:57-63.

Kim YJ, Guzman-Hernandez ML, Wisniewski E, Balla T. Phosphatidylinositol-phosphatidic acid exchange by Nir2 at ER-PM contact sites maintains phosphoinositide signaling competence. Dev Cell 2015;33:549-561.

London E, Wassif CA, Horvath A, Tatsi C, Angelousi A, Karageorgiadis AS, Porter FD, Stratakis CA. Cholesterol biosynthesis and trafficking in cortisol-producing lesions of the adrenal cortex. J Clin Endocrinol Metab 2015;100:3660-3667.

Pelkey KA, Barksdale E, Craig MT, Yuan X, Sukumaran M, Vargish GA, Mitchell RM, Wyeth MS, Petralia RS, Chittajallu R, Karlsson RM, Cameron HA, Murata Y, Colonnese MT, Worley PF, McBain CJ. Pentraxins coordinate excitatory synapse maturation and circuit integration of parvalbumin interneurons. Neuron 2015;85:1257-1272.

Vullhorst D, Mitchell RM, Keating C, Roychowdhury S, Karavanova I, Tao-Cheng JH, Buonanno A. 2015. A negative feedback loop controls NMDA receptor function in cortical interneurons via neuregulin 2/ErbB4 signalling. Nat Commun 2015;6:7222.

Xie Y, Zhou B, Lin MY, Wang S, Foust KD, Sheng ZH. Endolysosomal deficits augment mitochondria pathology in spinal motor neurons of asymptomatic fALS mice. Neuron 2015;87:355-370.

Yi L, Kaler SG. Direct interactions of adaptor protein complexes 1 and 2 with the copper transporter ATP7A mediate its anterograde and retrograde trafficking. Hum Mol Genet 2015;24:2411-2425.

Community outreach

The MIC is committed to promoting light and electron microscopy in the PNRC as well as for NICHD's Division of Intramural Research. We are making efforts to educate investigators on the benefits and pitfalls of advanced imaging techniques. Our initiatives include: (1) coaching users on the principles of confocal microscopy, during training and by publishing comprehensive operating protocols for each microscope; (2) on-campus demonstrations of new instruments and software by vendors such as Zeiss, Olympus, Photometrics, Nikon, and Perkin-Elmer; and (3) on-site assistance to investigators in their laboratories operating their own imaging equipment to optimize the quality of the data recorded. Furthermore, the MIC's website (http://mic.nichd.nih.gov) is an important resource for tutorials and protocols for both fixed and live cell microscopy.

In addition, during 2011, 2013, and 2015, we conducted educational outreach programs. The goal was to educate individual NICHD DIR senior scientists and post-doctoral scientists on microscopy techniques from the ground up. Instruction included lectures as well as hands-on experimental work organized within the MIC's facilities. More than 50 participants took part in a month-long series of lessons and lab work. The program was very well received by our user-base both at senior level and by postdoctoral scientists. We plan to offer this education curriculum whenever there is demand, and plan to query the community of their wishes. In addition, we have offered individual training when such requests are received; Ms. Holtzclaw provides such training in our laboratories.

Parallel to these efforts, the staff have continued to collaborate with other Institutes to promote an exchange of information and bring new imaging technologies to this Institute. Ongoing collaborations include: imaging of live animals (NINDS, NIMH); and correlative light and electron microscopy in the same specimen. We are also initiating discussions on implementing some form of light-sheet microscopy within the MIC.

NICHD has contributed to a jointly funded Stimulated Emission Depletion (STED) microscopy platform, which is hosted by the NHLBI Imaging core facility headed by Chris Combs.

Publications

  1. Subramanian J, Dye L, Morozov A. Rap1 signaling prevents L type calcium channel dependent neurotransmitter release. J Neurosci 2013;33:7245-7252.

Collaborators

  • James Pickel, PhD, Laboratory of Genetics, NIMH, Bethesda, MD
  • Afonso Silva, PhD, Laboratory of Functional and Molecular Imaging, NINDS, Bethesda, MD

Contact

For more information, email james@helix.nih.gov or visit http://mic.nichd.nih.gov.

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