Light microscopy The light microscopy branch of the MIC operates in 4 different areas: Equipment maintenance and upgrade: The MIC operates 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, and a Perkin-Elmer spinning disk for low-light imaging of photo-sensitive specimens. In addition, a dual-channel Olympus Total Internal Reflection Fluorescence (TIRF) platform and a high-end conventional fluorescence microscope are available. Live imaging is supported with temperature, CO2, 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, all instruments are covered by a service contracts provided by the manufacturers, 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 on 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 users request, or when the MIC staff feels 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 users location on campus. Method development During the past year, the MIC has dedicated a significant amount of time and effort in the custom development of a Photo-Activatable Localization Microscopy (FPALM) platform. The image acquisition side of this instrument is now complete for red fluorescent proteins. Funding allowing, we anticipate having a functional FPALM instrument for both green and red proteins by mid-spring 2010. FPALM also requires a complex image analysis module, which is still under development and will be available by Spring 2010. 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 done in-house by Mr. Dye. The MIC has a full-fledged EM laboratory with an LKB Pyramitome, a Leica CM3050-S Cryostat and a Reichert Ultracut-E Ultramicrotome. Due to the labor involved, the number of projects undertaken is comparatively fewer. 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 done most of the time by the microscopist except in cases where the user has the necessary inclination and training. The MIC operates an aging JEOL 1010 transmission electron microscope, which is slated to be replaced by Spring 2010 with a 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 immuno-reactivity and allows specimens to be imaged in a near native state. Cryo-EM will improve the quality of studies relying on immuno gold labeling. Tomography provides the ability to image three-dimensional structures at the EM level. This feature offers the ability to create 3D views of identified structures of interest which have been functionally characterized by other methods (for example, electrophysiology). Method development Mr. Dye has set up techniques for EM-level immuno-histochemistry, and double immunolabeling to mark two separate antigens at the same time. 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 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 Since the NICHD, DIR laboratories are scattered all over 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, vibratomes for live and fixed tissues. We wish to acknowledge the contribution of Mrs Lynne Holtzclaw, currently part 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 via publication of 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 the data recorded. Furthermore, the MIC web site (http://mic.nichd.nih.gov/) is an important resource for tutorials and protocols for both fixed and live cell microscopy. In parallel to these efforts, the staff have developed collaborations with other Institutes to promote the exchange of information and bring new imaging technologies to Childs Health. 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 PIs and 3 PIs of sister Institutes within the campus. On any given week, approximately 20 different users spend half a day or more on an MIC microscope. As of August 2009, users have logged more than 29,000 hours on the Cores equipment. Since its creation in 2004, usage has resulted in more than 65 publications, 6 of which have been co-authored by the Core personnel (see http://mic.nichd.nih.gov/publications.htm for a complete list). Looking ahead Several technology developments, such as sub-resolution fluorescence imaging (FPALM) and multiple immuno-labelling are currently underway. The new JEOL 1400 instrument will significantly improve the EM capabilities of the Core by providing cryo-EM and tomography. With continuing support from the Institute, the Microscopy &Imaging Core will continue to provide the NICHD community the competitive edge that is so important in todays research environment.
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