|Atul K. Bhan, MD
|Dennis Brown, PhD
|Fred Preffer, PhD
|Gregory Lauwers, MD
|Patricia Della Pelle
Core Research Technologist
Core Research Technologist
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.
The objectives of the Morphology Core are as follows:
- 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
- Generate an environment appropriate for the training of key personnel from participating laboratories
- 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
- Provide a central location for imaging activities in which different investigators can interact and share ideas for pushing IBD research forward
- 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
- Laser scanning confocal microscopy. The new Nikon A1R confocal microscope is an invaluable tool for pursuing studies on cells and tissues with sophisticated techniques such as FRET and FRAP as well as real-time imaging. Our older Zeiss Radiance 2000 series confocal continues to be a valuable asset for studies on basic protein localization in cultured cells and tissue sections.
- 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.
- 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.
- Helium ion microscopy (HIM). Over the past two years, Dr. Brown and colleagues have spent a great deal of time exploring the use of helium ion scanning electron microscopy for imaging cells. This technique provides a dramatic increase in clarity and resolution compared to regular electron scanning microscopy. While not part of the Core per se, Dr. Brown can introduce interested users to the MIT NanoStructures Laboratory.
- 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.