Overview

The Cellular Imaging and Immunology Core provides cost-effective access to crucial instruments, reagents, training, and education and in support of morphological and cell biological studies, immunological assays, and single-cell and spatial transcriptomics approahces.

Core Personnel

Andrew Luster, MD, PhD
Co-Director

Dennis Brown, PhD
Co-Director

Shiv Pillai, MD, PhD
Co-Director

Richard Bouley
Core Manager

Shannon Bromley
Core Manager

Diane Capen
Core Research Technologist

Objectives

  1. Provide access to sophisticated cellular imaging, immunological, histopathological, and single-cell and spatial transcriptomics approaches that would otherwise be prohibitive to individual researchers
  2. Provide an environment for training key personnel from Center laboratories
  3. Evolve to meet the needs of the CSIBD community by introducing new equipment and techniques
  4. Maintain the highest level of integrity in generating, analyzing, and reporting data by focusing on quality control, rigor and reproducibility parameters in research, and data management
  5. Promote the development of junior investigators and foster collaborations by providing a connection point between investigators of varying research approaches

Services

Imaging

  • 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. The Nikon A1R confocal microscope is dedicated to live-cell imaging and associated techniques, including FRET and FRAP, and spectral imaging.
  • Zeiss LSM800 Airyscan confocal imaging. This system allows image capture at close to double the resolution and much greater sensitivity compared to conventional confocal systems.
  • Nikon AXR confocal platform. This microscope uses advanced hardware and AI assistance to perform all functions from image capture to final analysis more rapidly and with high sensitivity due to its high-speed resonance imaging capability.
  • Airyscan imaging and Huygens deconvolution. Application of Huygens deconvolution software to Airyscan images results in greatly increased clarity and resolution.
  • 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 adapted our TIRF microscope to perform Ca++ and pH ratio imaging.
  • Super-resolution STORM 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.
  • Imaging flow cytometry. Amnis technology fully distinguishes and ­­visualizes appropriate information from undesired background debris by a combination of image analysis and brightfield examination.
  • Central antibody bank. We have a bank of over 2,000 antibody reagents, many of which were developed in-house.

Routine immunology

  • Flow cytometry for both basic (up to 21 color parameters with the Cytoflex LX) and more complex (up to 33 color parameters with the Astrios sorter) analysis
  • Cell sorting and leukocyte isolation using high-speed cell sorting
  • Dendrimer-based in situ hybridization and immunofluorescence on both FFPE and frozen sections
  • Phosphoproteomics in activated T and B cells
  • Histopathology on mouse or human paraffin embedded or frozen tissue sections

Specialized immunology

  • Next-generation sequencing to identify clonal expansion of T and B cells in human blood and tissues
  • Single-cell cloning of activated B cells to match Ig H and L sequences and create disease-related human monoclonal antibodies
  • MHC class II tetramers to identify, purify, and characterize disease-causing antigen-specific T cells
  • Antigen multimers to identify, purify, and characterize disease-causing antigen-specific B cells
  • Novel pathway-based computational approaches and CRISPR on primary immune cells
  • Identification of causal variants underlying genetic associations of relevance to digestive diseases
  • GPCR-seq, a multiplexed screening platform that allows simultaneous screening of hundreds of GPCR-ligand interactions
  • Toolbox of base editors

Personnel training. We provide support for CSIBD investigators through (1) formal and informal end-to-end consultations, (2) short-term technical training, and (3) intensive training of selected personnel in specific activities that will be used extensively by participating laboratories.