Explore This Lab

Overview

The Morphology Core serves the CSIBD community by providing Center investigators with the technical support, expertise and access to instrumentation necessary for their morphological and cell biological studies related to IBD. These techniques are centered around examining the localization of proteins that are implicated in pathogenesis of various forms of bowel disease. The experimental approaches facilitated by the Core are essential to many investigations performed by Center members, and often involve sophisticated equipment and technical skill.

Core Personnel

Atul K. Bhan, MD
Co-Director

Dennis Brown, PhD
Co-Director

Kate Jeffrey, PhD
Associate Director

Omer Yilmaz, MD, PhD
Associate Director

Diane Capen
Core Research Technologist

Objectives

  1. Provide a cost-effective and efficient solution for the morphology and imaging needs of the CSIBD user base via sophisticated instrumentation and outstanding intellectual and technical support
  2. Generate an environment appropriate for the training of key personnel from participating laboratories
  3. Strive to continually upgrade equipment and available techniques to allow the user base to stay at the cutting edge of cell and tissue imaging procedures
  4. Provide a central location for imaging activities in which different investigators can interact and share ideas for pushing IBD research forward

Services

  • Routine tissue fixation and processing for light microscopy. The Core also performs special stains (e.g., trichrome, PAS, Sirius Red).
  • Perfusion-fixation of organs. The type of fixative depends on the experiments to be carried out and sensitivity of antigens to fixation; it is determined empirically based on our experience.
  • Conventional electron microscopy. Images are captured digitally using our JEOL 1011 electron microscope and AMT CCD camera. They are stored on a central file server.
  • Low temperature embedding in Lowicryl HM20. Embedding tissues in hydrophilic resin preserves antigenicity better than conventional embedding and allows the post-embedding detection of antigens on thin sections of tissues, using immunogold labeling techniques.
  • Immunocytochemistry. The Core performs and instructs users on a variety of immunocytochemical procedures, including the following:
    • Light microscope immunocytochemistry on cells and tissue sections with 3D reconstruction
    • Light microscopy of intact cells and tissues
    • Electron microscopy on intact cells and tissues (pre-embedding labeling)
    • Immunogold electron microscopy on thin sections
    • Ultracryomicrotomy for immunogold labeling of thin, frozen sections
  • Nikon A1R laser scanning confocal microscopy. This is an invaluable tool for pursuing a variety of studies on cells and tissues with sophisticated techniques such as FRET and FRAP, real time and spectral imaging
  • Zeiss LSM800 Airyscan confocal imaging. Our new confocal system allows image capture at close to double the resolution and much greater sensitivity compared to conventional confocal systems.
  • Opal multiplexed imaging (Perkin Elmer). This is a new technique that allows up to seven antibodies (even if they are all raised in the same species) to be used for labeling. Each step results in covalent crosslinking of a specific fluorophore with the target antigen, followed by stripping of the antibodies, and re-labeling with the next antibody – up to seven times. It requires a confocal, such as our Nikon A1R system, that is capable of precise spectral imaging to distinguish the seven probes with no overlap.
  • Spinning disk confocal microscopy. The Perkin Elmer spinning disk confocal microscope is designed for close to real-time acquisition of images from live cells or other small specimens.
  • Total internal reflection fluorescence (TIRF) microscopy. TIRF is a powerful technique for visualizing fluorescently labeled molecules that are in or near the cell membrane.
  • Calcium/pH ratio imaging. We have adapted our TIRF microscope to perform Ca++ and pH ratio imaging.
  • Nikon STORM (super-resolution) microscopy. This system is a form of single molecule light microscopy that enables the visualization of interactions, such as between proteins or between proteins and nucleic acids, at the nanoscopic level. STORM capabilities include the use of three-color and three-dimensional imaging.
  • Multiphoton confocal microscopy via the MGH Center for Systems Biology (CSB). The Program in Membrane Biology is within the MGH Center for Systems Biology. Interested users from the CSIBD may gain access to these Olympus systems by approaching Dr. Brown, who will refer them to the CSB Director (and CSIBD member) Ralph Weissleder for further consultation and discussions.
  • Imaging flow cytometry. As examples of work done in the Simches facility, members of the flow laboratory studied the internalization of DsRed-labeled bacteria and colocalization to the autophagosome marker light chain 3 (LC3, labeled with GFP) using WT HeLa cells alongside HeLa cells in which LC3 expression was knocked down. Debris, single cells, and cellular aggregates can be distinguished by comparison of the cell population’s area vs. aspect ratio measurements (not shown) and subsequently analyzed for staining at the single-cell level (Fig. 1). This type of cell analysis typifies the power of the Amnis technology to fully distinguish and visualize appropriate information from undesired background debris by a combination of image analysis and brightfield examination.
  • Central antibody bank. We have a bank of close to 2,000 antibody reagents, many of which were developed in-house.
  • Personnel training. We provided support for CSIBD investigators by (1) consultation activities, (2) short-term technical training, and (3) intensive training of selected personnel in specific activities that will be used extensively by participating laboratories.