Light microscopy The light microscopy arm of the MIC operates on 4 different aspects of imaging: Equipment maintenance and upgrade The MIC operates several confocal laser scanning microscopes optimized for different applications: The most recent acquisition is a microscope capable of multiple mods of imaging manufactured by Nikon Microscopes Inc. This images in TIRF (total internal reflection fluorescence), spinning disc confocal, and DSTORM ( ) modes of imaging, and is now made available for both live cell imaging and for fixed specimens. In addition the MIC maintains a Zeiss LSM 510 inverted for high-resolution point-scanning confocal imaging of fixed specimen, and a Zeiss LSM 510 NLO for two-photon imaging of live tissue sections and live animals. The MIC operates a home-built Photo-Activation Localization (PALM) Microscope for sub-resolution microscopy. This microscope and the necessary software were implemented by Dr. Schram in the MIC. Two high-end wide-field fluorescence microscopes, one on an upright platform and another on an inverted platform are also now functional. In addition, a fluorescence stereo microscope is available for imaging large specimens such as whole embryos and large tissue sections in low magnifications. Finally, an upright Zeiss fixed stage microscope is installed in wide-field configuration, which can image live cells under photo-conductive stimulation mode. Live cell imaging is supported with temperature, CO2, humidity control and heated perfusion on most microscopes. The newly acquired microscopes including the ones currently under procurement processes are equipped with computer-controlled constant focus capabilities which allows for long term imaging of live cell preparations. 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 service contracts provided by the manufacturers, and are usually serviced within a one to two days. User training and Support Each user then 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. During the Spring of 2012, and 2013 MC staff offered a series of classroom lessons as well as hands-on laboratory courses on various aspects of microscopy and practical microscopy techniques.These courses were attended by over 30 individual postdoctoral scientists and technical staff in each year. The courses were so well received by all the participants and their PIs, that it will be repeated at least annually. Image analysis The MIC operates a data analysis center with three high-end workstations and image analysis, and image processing software (ImageJ/Fiji, Metamorph, Volocity, Imaris and Zeiss AIM, MatLab, LabView). 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 offers extensive data storage and archival services with an enterprise-level file server and a data backup system. Method development MIC is actively involved in the development of newer microscopy and imaging methodologies de novo, and in collaborative effort with scientists within the NIH IRP. Electron microscopy Because of the complex and delicate nature of Electron Microscopy (EM) sample preparation, the EM operations of the MIC function differently from the light microscopy imaging side. 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 a 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. Imaging EM imaging is done most of the time by the microscopist, except in cases where the user has the necessary training and experience. The newly acquired JEM 1400 transmission electron microscope is currently fully operational. This scope was installed during the Spring of 2011 and it offers two new imaging methodologies, cryo-EM and tomography. The main advantage of cryo-EM is preservation of a high level of immuno-reactivity and imaging of specimen in a near native state. Cryo-EM will improve the quality of studies relying on immunogold 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 for tomography. The MIC has implemented a digital archival system for all EM images and parameters. More recently Mr. Dye has been involved in the development of the use of the different methods to achieve correlative light microscopy and electron microscopy of the same specimen. Use of the Minisog, and other approaches such as nano-particle based fliuorescent probes, which can be detected using fluorescence microscopy and also electron microscopy are being developed to do correlative LM-EM in the same specimen. 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, Olympus, 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 (https://science.nichd.nih.gov/confluence/display/mic/Home) is an important resource for tutorials and protocols for both fixed and live cell microscopy. Looking ahead Over the last two years, a Nikon multimode microscope and a Zeiss LSM 780, point scanning confocal microscope were acquired. The Nikon instrument allows for TIRF, DSTORM and spinning disc confocal microscopy modalities. This hybrid instrument is capable of live-cell imaging as well as single molecule fluorescence imaging and localization. The LSM 780 is a high-sensitivity, high-resolution point scanning confocal microscope.The legacy instrument from Carl Zeiss, the LSM 510 point scanner will be upgraded to a LSM 710 instrument during 2014. The portfolio of instruments within the MIC needs to be maintained at peak levels of performance at all times to adequately serve the needs of NICHD scientists. Due to lack of funds we plan not to maintain the LSM 510 confocal microscope under a service agreement starting this year, and when funds are available acquire a new confocal microscope on an inverted microscope platform within the next 12 to 18 months. The quality of the acquired data is heavily influenced by the quality of the optics and therefore the MIC strives to keep the instruments upgraded periodically.
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