NICHD Microscopy and Imaging Core
- Vincent Schram, PhD, Biologist
- Louis (Chip) Dye, BS, Research Assistant
- Lynne A. Holtzclaw, BS, Research Assistant
- Sara Felsen, BS, MS, Postbaccalaureate Fellow
The mission of the NICHD Microscopy and Imaging Core (MIC) is to provide service in four different areas: (1) sample preparation for light and electron microscopy studies; (2) wide-field and confocal light microscopy; (3) transmission electron microscopy (TEM); and (4) image analysis and data extraction. The Facility is operated as a 'one-stop shop,' where investigators can, with minimum effort, go from their scientific question to the final data.
Mode of Operation
Located on the ground floor of the Porter Building (building 35A), the MIC is accessible 24/7, and users can reserve time on each microscope by using an online calendar. The Facility is available free of charge to all NICHD investigators and, resources allowing, to anyone within the Porter building. The Facility is supported by the Office of the Scientific Director.
Vincent Schram is the point person for light microscopy and data analysis. The EM branch of the Facility is staffed by Chip Dye, and Lynne Holtzclaw is in charge of sample preparation (histology). Chip Dye and Lynne Holtzclaw report to Vincent Schram, who serves as interim director under the management of Chris McBain (NICHD). Tamás Balla (NICHD) acts as scientific advisor for the Facility.
The MIC has an open-door policy with the NINDS Light Imaging Facility (LIF, Building 35), where the two cores freely exchange users, equipment, and support. Although not officially sanctioned, this mode of operation provides extended support hours, wider expertise, and access to more equipment than each Institute could afford on its own.
The MIC serves over 300 registered users in 68 laboratories. NICHD uses 80% of the Facility resources, NINDS 15%, and other Institutes (NIBIB, NIA, and NIMH) the remaining 5%.
Light microscopy
The MIC is equipped with six modern confocal microscopes, each optimized for certain applications:
- Zeiss LSM 710 for high-resolution confocal imaging of fixed specimen and live cells;
- Zeiss LSM 780 for challenging specimens that require both high resolution and high sensitivity;
- Nikon Spinning Disk/Total Internal Reflection Fluorescence (TIRF) hybrid microscope for high-speed confocal imaging and selective recording of membrane-bound events in live cells;
- Zeiss LSM 880 2-photon confocal for thick tissues and live animals;
- Zeiss 800 optimized for confocal imaging of large specimens (tiling);
- Zeiss 880 Airy, which offers near superresolution without the need for special dyes or protocols.
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 differential interference contrast (DIC), and large specimens.
After an initial orientation, during which the staff research the project and decide upon the best approach, users receive hands-on training on the equipment and/or for the software best suited to their goals, followed by continuous support, when required. Once image acquisition is complete, the staff devise solutions and train users in how to extract usable data from their images. Additional training and support is offered to the community in different ways:
- on-site assistance and training on equipment owned by individual investigators;
- an extensive yearly workshop covering light and electron microscopy, image analysis, and sample processing;
- MIC staff volunteer time to teach FAES (NIH's Foundation for Advanced Education in the Sciences) classes; and
- the Facility organizes frequent on-campus demonstrations of new imaging equipment, technology, and software by vendors in a dedicated space. The equipment demonstrations are open to the entire NIH community.
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 for the light microscopy section, where end users do their own processing. In the past 12 months, Chip Dye processed a total of 196 samples; 6 of them were immuno–EM studies. John Heuser, part of Joshua Zimmerberg's unit (NICHD), continued to spend a significant amount of time on the JEOL electron microscope.
Tissue preparation
Lynne Holtzclaw continues to provide sample processing, training, and services to the Facility’s users, both for light and electron microscopy applications. She dedicates a significant amount of time to training users in various techniques, such as rodent perfusion, cryopreservation, cryosectioning, immunofluorescence, and tissue clearing. Drs. Dever, Dufau, Fields, Hoffman, Klein, Le Pichon, Loh, Marini, McBain, Pfeifer, Sackett, Stojilkovic, and Stopfer (NICHD), Mankodi, Roche, Youle, and Ward (NINDS), and Chen (NIBIB) received support and training from Mrs. Holtzclaw.
In a collaborative endeavor with David Klein (NICHD), we characterizing pineal cell types for which genes of interest had been documented by RNA-seq [Reference 1]. A collaborative effort with the NINDS laboratory of Katherine Roche (NINDS) also resulted in a publication [Reference 2]. The continuing collaboration with the laboratory of Richard Youle (NINDS) to study the accumulation of ubiquitinated protein aggregates in brain and liver of a TAX1BP1 knock-out mouse continued during the year and is expected to be completed by year’s end.
Image analysis
Extracting usable information from the collected images is an essential part of the Facility's services. Vincent Schram provides training and support for image analysis on two high-end computers running advanced image analysis packages: Imaris from Bitplane (3D rendering, segmentation and analysis), Huygens from Scientific Volume Imaging for deconvolution of regular confocal images, and ImageJ, Zeiss Zen and Nikon Elements for regular image processing. The latter now includes Denoise.AI, a unique denoising module for point scanner images based on machine learning.
Publications
- Mays JC, Kelly MC, Coon SL, Holtzclaw L, Rath MF, Kelley MW, Klein DC. Single-cell RNA sequencing of the mammalian pineal gland identifies two pinealocyte subtypes and cell type-specific daily patterns of gene expression. PLoS One 2018;13(10):e0205883.
- Lomash RM, Petralia RS, Holtzclaw LA, Tsuda MC, Wang YX, Badger JD 2nd, Cameron HA, Youle RJ, Roche KW. Neurolastin, a dynamin family GTPase, translocates to mitochondria upon neuronal stress and alters mitochondrial morphology in vivo. J Biol Chem 2019;294(30):11498-11512.
Collaborators
- Tamás Balla, PhD, Section on Molecular Signal Transduction, NICHD, Bethesda, MD
- David C. Klein, PhD, Scientist Emeritus, NICHD, Bethesda, MD
- Katherine W. Roche, PhD, Receptor Biology Section, NINDS, Bethesda, MD
- Carolyn L. Smith, PhD, Light Imaging Facility, NINDS, Bethesda, MD
- Richard Youle, PhD, Neurogenetics Branch, NINDS, Bethesda, MD
Contact
For more information, email schramv@mail.nih.gov or visit http://mic.nichd.nih.gov.
