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). Several conventional (wide-field) light microscopes provide 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) MIC staff are 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 As of this writing, the MIC has a total of 217 registered users in 66 laboratories. Since last fiscal year, the overall usage of the facility has increased significantly due to a higher usage by the Institute. The three confocal instruments were used a total of 5,843 hours in the last twelve months (37 hours per week per confocal microscope). All three confocals were used almost equally. 54% of this usage comes from NICHD investigators, 27% from NINDS, and the remaining hours are mostly pilot projects ran by the facilitys staff. If usage continues to increase at the present pace, we may have to manage access and orient certain projects toward regular wide-field or legacy confocal microscopes owned by NINDS. Usage of conventional (wide-field) light microscopes was significant but not heavy. 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 21 specimens for ultra-structural imaging projects by 12 NICHD investigators. Cryo-tomography was used extensively as well, in particular by Dr. Zimmerberg (Andrea Farra). Sample preparation Mrs Lynne 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, Chen, Chou, Crouch, Danner, Dufau, Fields, Kaler, Loh, Maraia, McBain, Ozata, Pfeiffer, Porter and Stratakis. At the same time, she is pursuing two collaborative endeavors: 1) An immunohistochemical study of the expression of a mitochondrial protein (Complex V) in Ataxic mice with Dr. Crouch, NICHD. The project includes perfusion, cryo-sectioning of prepared cerebella, immuno-fluorescence and imaging. 2) A project with Dr. McBain involving perfusion-fixation, immuno-histochemical and immuno-fluorescence of thick sections and dual immuno-EM. Future direction The facility continues to diversify and offer new services to the community. A modern two-photon point scanning confocal system, slated for delivery in the next few months, will fill a hole in the facilitys capabilities by allowing deep tissue and live animal imaging. Once Mrs Holtzclaw receives additional training to refresh her skills with the surgery involved, we plan to offer trans-cranial imaging on live rodents. The two-photon will also allow for high-resolution imaging of cleared tissue specimens, albeit at a lower speed than a dedicated light sheet microscope. A cryo ultra-microtome has also been purchased recently. This device will allow us to expand cryo-electron microscopy techniques as required by several investigators. Publications Since its inception in 2004, the work carried out in the MIC has been included in more than 180 publications. For a complete list, head to: https://science.nichd.nih.gov/confluence/display/mic/Publications
Showing the most recent 10 out of 23 publications
