Ragon Institute Immunology Core-Flow Cytometry and Microscopy
The mission of the Ragon Institute Immunology Core is to bring the latest imaging modalities to bear on fundamental molecular and cell biological questions pertaining to human disease. As an MGH core facility, it serves the greater MGH community in all aspects of flow cytometry, cell sorting, advanced microscopy and image analysis. The Microscopy division of the Ragon Institute Imaging Core is located on the 8th floor of 400 Technology Square in Cambridge (Main Street). The core offers training, assistance and access to a wide variety of imaging systems including slide scanners (TissueFAXS and Zeiss MIRAX MIDI) for rapid imaging of tissues and adhered cell populations, high resolution confocal imaging (Zeiss LSM510), and advanced live cell imaging systems. There is also high-speed, flow-based imaging of cell populations in suspension using the ImagingStreamX MkII by Amnis. Our facility is unique in that our confocal and live cell imaging systems are situated in a BL2S facility. In addition, consultation is provided on sample preparation, experimental design, and advanced image analysis, in addition to assistance with all aspects of the imaging process.
Director: Michael Waring
Co-Director (Microscopy): Thomas Diefenbach, Ph.D.
Phone: (857) 268-7046
Facilities and Equipment:
Microscopy (400 Technology Square, Rooms 810 and 812, Cambridge, MA 02139):
- Zeiss LSM510 Laser Scannning Confocal Microscope based on an inverted Axio Observer Z1 with environmental control (heat and CO 2), 405/458/477/488/514/561/633 nm laser lines, and a motorized stage. Imaging is controlled via Zen software (Zeiss).
- Zeiss Axio Observer Z1 inverted fluorescence microscope with Orca-R2 12/16 bit digital camera, environmental control (heat and CO 2), motorized stage, PIFOC piezoelectric objective stepping motor with 5 nm step resolution, and a Sutter Instruments DG-4 ultra-high speed wavelength switcher. Imaging is controlled via Metamorph software (Molecular Devices).
- Zeiss Axio Observer A1 manual microscope with AxioCam MRc 12 bit color and black and white CCD camera. Controlled through Axiovision image capture software (Zeiss).
- Zeiss MIRAX MIDI whole slide scanner with Zeiss 20x 0.8NA Plan-Apochromat objective. Capable of scanning up to 12 slides in one session. Utilizes two 12 bit cameras for either fluorescence or brightfield imaging of slides. Images are tiled to provide a global view of the entire section for offline “virtual” microscopy. Controlled by Zeiss MIRAX imaging and image presentation software.
- TissueFAXS by Tissue Gnostics is a unique slide scanning system based on a Zeiss Axio Imager Z2 automated microscope with motorized Maerzhaeuser stage with an eight-slide tray. Controlled by Tissue Gnostics imaging software. Images can be tiled for a global view of a slide. Associated TissueQuest and HistoQuest software provide a means to rapidly identify cell populations based on fluorescent or cytological markers, display 2D plots of colocalized staining, and permit gating of cell populations similar to flow cytometric analysis.
- High-end image analysis workstation equipped with Metamorph (Molecular Devices), Imaris (Bitplane), in addition to TissueQuest and HistoQuest (Tissue Gnostics) image analysis software. Viewing software for the MIRAX MIDI and Zeiss confocal microscope (Zen) are also provided and are available without license for offline viewing.
Flow Cytometers (400 Technology Square, room 910, 911, and 912, Cambridge MA 02139):
- Two 5 Laser LSR Fortessas (BD Biosciences) with 355, 405, 488, 561, and 640 excitation lines, up to 18 colors, and the HTS option for acquiring from 96 well plates.
- 4 Laser LSR2 (BD Biosciences) with 355, 405, 488, and 633 excitation lines, 16 colors.
- 4 Laser LSR2 (BD Biosciences) with 355, 405, 488, and 640 excitation lines, 16 colors.
- 3 Laser LSR2 (BD Biosciences) with 405, 488, and 633 excitation, 16 colors, and the HTS option for acquiring from 96 well plates.
- Imagestream X Mark II (Amnis) imaging cytometer,
- 5 laser FACSAria cell sorter (BD Biosciences) with 350, 405, 488, 561 and 633 excitation, 18 colors, housed within a biosafety cabinet for sorting of human and infectious samples requiring up to BL2+ containment. The sorter is run by core personnel only, and no training is provided in its operation.
- 4 laser FACSAria Fusion cell sorter (BD Biosciences) with 405, 488, 561, and 640 excitation, housed in a biosafety cabinet for BL2+ sorting.
- 3 laser SONY SH800 cell sorter (Sony Biotech) with 405, 488, and 640 excitation and 6 colors.
- BioPlex 3D (BioRad) XMap reader
- AutoMACS Procell separator(Miltenyi) magnetic column cell separation.
- Computer workstations for data analysis and access to platform-specific software.
- An introductory course is provided for new users in the fundamentals of microscopy, including confocal microscopy and image analysis.
- Consultation in terms of the imaging system best suited to the needs of the investigator is provided.
- Individual training in the use of any of the imaging systems in the core is provided so that users are able to work independently with the systems.
- For more sophisticated applications, assisted use of the systems is provided.
- Imaging core staff are available to answer questions or to assist in troubleshooting.
- Training in image analysis applications is also provided for core users.
- The core continues to evolve to suit the needs of the Ragon Institute and the greater MGH community.
To access the Imaging Core please email Michael and Thomas with a description of what resources you were interested in utilizing.
Departmental Web Links:
Ragon Institute Core Services home page: https://www.ragoninstitute.org/resources/cores/