Light microscopy The facility operates three modern confocal microscopes, each optimized for certain applications: 1) A Zeiss LSM 710 inverted for high-resolution confocal imaging of fixed specimen and live cells. 2) A Zeiss LSM 780 for challenging specimens that require both high resolution and high sensitivity. 3) A Nikon Spinning Disk / Total Internal Reflection Fluorescence (TIRF) hybrid microscope for high-speed confocal imaging or selective recording of membrane-bound events in live cells (TIRF). The facility just received a Zeiss LSM 880 2-photon confocal for thick specimen and live animal microscopy. Several conventional (wide-field) light microscopes provides imaging modalities such as transmission (visible stains), large-scale tiling of tissue slices, high-speed phase contrast and DIC, and large specimens. High-end computer workstations with imaging software (Zeiss Zen, Nikon Element, Bitplane Imaris, Metamorph and ImageJ) are also available. After an initial orientation where their project is discussed and the best approach is decided upon, users receives hands-on training on the equipment and / or software best suited to their goals, followed by continuous support when required. Additional training and support is offered to the community in different ways: 1) On-site assistance and training on equipment owned by individual investigators. 2) An extensive yearly workshop on microscopy covering light and electron microscopy, image analysis and sample processing. 3) The MIC staff is volunteering time to teach FAES classes. 4) The facility organizes frequent on-campus demonstrations of new instruments and software by vendors such as Zeiss, Olympus, Nikon, Leica, etc The MIC has a total of 210 registered users in 65 laboratories. At 7,582 instrument-hours over 12 months, overall usage has grown slightly since last fiscal year, although this figure includes wide-field microscopes which are now seeing significant usage. Half of the total usage is coming from NICHD investigators, most of them within the Porter building. The MIC used an additional 21% of these resources for user training and pilot projects, and other Institutes, predominantly NINDS, used the remaining 28%. All three confocal microscopes were used equally at an average of 43 hours per week. To alleviate this continuous high usage pattern, the facility is considering the acquisition of additional point scanning confocals. Electron microscopy The electron microscopy branch of the facility processes specimens from start to finish: fixation, embedding, cutting, ultra-fine sectioning, staining and imaging on the JEOL 1400 transmission electron microscope. Because of the labor involved, the volume is necessarily smaller than the light microscopy branch where end users do their own processing. In the past 12 months, Mr. Dye processed a total of 85 samples: 70 from NICHD investigators, 9 from MIC internal test projects, and 6 from other Institutes. Thanks to a generous endowment from Dr. Zimmerberg, the facility received an AMT Biosprint 29 camera with a wider field of view, enhanced resolution, and slightly lower pixel size. The JEOL 1400 electron microscope is now on the facilitys calendar and trained investigators have direct access to the instrument. Dr. Zimmerberg and Dr. Wu (NINDS) are the major users. Tissue preparation Mrs. Holtzclaw continues to provide sample processing training and services to the facilitys users, both for light and electron microscopy applications. She is spending a significant amount of time training users in different techniques such as rodent perfusion, cryopreservation, cryosectioning, immunofluorescence and tissue clearing. During the past year, she trained staff, processed samples and acquired images for Drs. Balla, Baron, Basser, Bezrukov, Bonifacino, Crouch, Dasso, Fields, Hoffman, Klein, Lilly, Loh, McBain, Mukherjee, Ozato, Penzo (NIMH), Porter, Stojilkovic, Stopher, and Stratakis. At the same time, she is pursuing a collaborative endeavor with Dr. Klein on rat pineal glands to characterize the cell types in which 3 genes of interest, identified by single cell RNA sequencing, are located. The project includes perfusion, cryosectioning of pineal glands, immunofluorescence and imaging. Publications Since its inception in 2004, the work carried out in the MIC has been included in more than 187 publications. For a complete list, head to: https://science.nichd.nih.gov/confluence/display/mic/Publications
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