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Imaging Facility (Wistar)

Directors: Hayden, James, B.A., R.B.P., F.B.C.A.; Janicki, Susan M, Ph.D.

Summary:

The Imaging Facility is a shared resource with the primary goal of providing exceptional microscopy and imaging services, as well as individual access to a variety of state-of-the-art imaging resources for members of the Wistar research community. The imaging systems have been designed to be extremely flexible to reflect a broad range of challenging scientific questions and specimens. Each system provides a combination of illumination, optics and image capture options. Diverse subjects ranging from fluorescently tagged live cell cultures and stained tissue sections, to 3D tumor spheroids and low magnification explanted tissues, can be accommodated with available systems.

Current equipment includes standard upright and inverted fluorescence microscopes, a customized live-cell time lapse microscope capable of 6D imaging, a laser scanning confocal microscope, a 2-photon microscope designed for in vivo imaging, a small animal, whole body luminescence and fluorescence imager, special low magnification (photomacrography) systems as well as a variety of traditional photographic cameras, lenses and lighting equipment. Users of the facility may be trained for unassisted use of all core assets, or they may elect assisted service with the facility staff performing the imaging.

The Imaging staff also provides assistance to researchers with additional aspects of their imaging requirements. Ideal approaches to specimen documentation are often unique to the experiment and the staff can help design the most effective imaging protocols to answer a particular question. On-site assistance is available to help investigators get the most out of their own systems. Image analysis and specialized Photoshop training, creative imaging for journal covers, and guidance on digital imaging ethics help to round out the services available from the facility.

Affiliations:

People:

Resources:

Instruments

  • Leica DMRE upright microscope (for fluorescence) ( Upright microscope )

    The Leica DMRE microscope has the following features:

    Lenses:
    2.5X, 10X, 20X, 40X and 100X oil
    Illumination options:
    Brightfield and Fluorescence
    Fluorescence Filter Cubes:
    DAPI, GFP/Aqua, HQ FITC, Piston GFP, HQ R, CY5.5, G/R (for viewing only), GFP/DsRed FRET
    Image Capture:
    Hammamatsu ORCA digital camera w/ ImagePro Plus software for fluorescence monochrome capture – 1344 x 1024 or 672 x 512 pixel resolution.

    Workstation
    Pentium 4 Dell PC with ImagePro v.6.2, 48X CD reader/writer, Zip 250 drive, front USB port

  • Leica TCS SP5 II scanning confocal microscope ( Laser scanning confocal microscope )

    The Leica TCS SP5 II scanning confocal microscope has the following features:

    Microscope:
    Leica DMRBE Inverted system
    Lenses:
    10X dry, 25X oil, 40X oil, 100X oil
    Illumination options:
    Brightfield, Fluorescence, DIC
    Fluorescence Filter Cubes (for visible inspection):
    DAPI, FITC, TRITC, Dual FITC/TRITC
    Fluorescence Excitation Wavelengths:
    458nm, 488nm, 568nm, 633nm
    Image Capture:
    Leica PMT detection system – adjustable resolution, usually 1024 x 1024 pixels

  • Nikon D200 digital SLR camera ( Digital camera )

  • Nikon E600 upright widefield microscope ( Upright microscope )

    The Nikon E600 has the following features:

    Lenses:
    2X, 4X, 10X, 20X, 40X, 60X and 100X oil
    Illumination options:
    Brightfield, Darkfield, Fluorescence, Polarization
    Fluorescence Filter Cubes:
    DAPI, FITC, TRITC, CFP, YFP, (triple cube available for viewing only)

    Image Capture:
    Spot RT Slider digital camera w/ ImagePro Plus software for fluorescence monochrome capture - 1600 x 1200 pixel resolution.
    Nikon DXM 1200F digital camera w/ Nikon ACT1 software for color capture –
    Variable pixel resolution, 1280 x 1024 pixels standard

    Workstation
    Pentium 4 Dell PC with ImagePro v.4.5, 48X CD reader/writer, Zip 250 drive, front USB port

  • Nikon SMZ800 stereo microscope ( Stereo microscope )

    The Nikon SMZ800 stereo microscope has the following features:

    Magnification Range:
    1X objective with 1X – 6.3X variable zoom
    Illumination options:
    Brightfield, Darkfield, Polarization, Oblique Illumination, Episcopic Lighting, Fiber optic light tubes
    Fluorescence:
    GFP possible with Illumatool 9900 system
    Image Capture:
    Nikon DXM 1200F digital camera w/ Nikon ACT1 software for color capture – Variable pixel resolution, 1280 x 1024 pixels standard

    Workstation
    Pentium 4 Dell PC with 48X CD reader/writer, Zip 250 drive, front USB port (share with Zeiss Axioplan back-up upright microscope)

  • Nikon TE2000 inverted microscope ( Inverted microscope )

    The Nikon TE2000 inverted microscope has the following features:

    Lenses:
    4X, 10X, 20X, 40X (all Long Working Distance)
    2X (non-phase), 60X (non-phase, close working distance) and 100X oil (non-phase) available
    Illumination options:
    Brightfield, Phase, Oblique, Fluorescence
    Fluorescence Filter Cubes:
    DAPI, CFP, FITC, TRITC, Texas Red
    Image Capture:
    • Q-Imaging Retiga EX digital camera, high sensitivity monochrome camera
    1360 x 1036 pixel resolution
    • Nikon DXM 1200F digital camera w/ Nikon ACT1 software for color capture – Variable pixel resolution, 1280 x 1024 pixels standard
    • Single-shot color imaging available through front camera port with Nikon D200 digital SLR camera

    Workstation
    Pentium 4 Dell PC with ImagePro v.6.2, CD reader/writer, DVD reader/writer, USB and Firewire connections

  • Nikon TE300 inverted microscope ( Inverted microscope )

    The Live-Cell Time-Lapse System is comprised of a Nikon TE300 inverted microscope with the following features:

    Lenses:
    • 4X, 10X, 20X, 40X (all Long Working Distance)
    • 2X (non-phase), 60X (non-phase, close working distance) and 100X oil (non-phase) available
    Illumination options:
    Brightfield, Phase, Oblique, Fluorescence
    Fluorescence Filter Cubes:
    DAPI, FITC, TRITC, CFP, YFP, (triple cube available for viewing only)
    Fluorescence Filter Wheel:
    DAPI, Pacific Blue, CFP, GFP, TRITC, CY5

    Motorized XY Stage
    Multiple inserts for well plates, 35mm dishes, standard glass slides. Positions can be saved for return sessions.

    Environmental Chamber
    Temperature and CO2 control surrounding entire microscope
    Image Capture:
    • Q-Imaging Retiga EX digital camera, high sensitivity monochrome camera 1360 x 1036 pixel resolution
    • Single-shot color imaging through front camera port with Nikon D200 digital SLR camera

    Workstation
    Dell Core 2 Duo with ImagePro v.6.2, CD reader/writer, DVD reader/writer, USB and Firewire connections, Dual 19” Flat Screens

    Additional
    Z-axis focusing controller with encoder, Automatic fluorescence shutter

    Time-Lapse Capabilities
    6D imaging (X, Y, Z, time, wavelength, positions)

  • Prairie Instruments Ultima two-photon microscope ( Microscope )

    The Prairie Instruments Ultima two-photon microscope has the following features:

    Microscope:
    Fixed stage, upright system based on Olympus BX81
    Lenses:
    10X, 20X, 40X and 60X dipping lenses
    Laser:
    Coherent Chameleon XR Ti:Sapphire laser, 710-980nm wavelengths (variable)
    Image Capture:
    4 detectors for Second Harmonic Generation and DAPI (457-487nm), GFP (503-537nm), YFP (525-570nm) and RFP (580-652nm)
    Time Lapse Capabilities:
    5D imaging (X, Y, Z, time, wavelength)

  • Xenogen IVIS 200 Whole Body Imager ( In vivo bioluminescence imaging system )

    "The Perkin-Elmer IVIS 200 whole body imager is designed for detection of weak fluorescent or bioluminescent signals deep in living mice for non-invasive longitudinal studies of growing (or regressing) tumors in mouse models. The system is housed in the Institute’s barrier mouse facility and is only available to those researchers with approved protocols and animals housed in the colony. The integrated photon counting software can normalize the data for direct comparison to future studies or to similar instruments anywhere in the world."

  • Zeiss Axioplan upright microscope (for Brightfield) ( Upright microscope )

    The Zeiss Axioplan has the following features:

    Lenses:
    10X, 20X, 40X and 100X oil
    Illumination options:
    Brightfield
    Image Capture:
    Nikon DXM 1200F digital camera w/ Nikon ACT1 software for color capture – Variable pixel resolution, 1280 x 1024 pixels standard

    Workstation
    Pentium 4 Dell PC with 48X CD reader/writer, Zip 250 drive, front USB port (share with SMZ800 steromicroscope)

Services

  • 2, 3, 4, and 5D image analysis ( Access service )

    "Capturing images is only the first step in scientific documentation. The images also need to be analyzed to extract meaningful data. The facility maintains multiple workstations and a variety of software packages for
this purpose with internal support and training from the staff, or custom support available from the suppliers for special cases."

    Software:
    Nikon ACT-1
    Adobe Creative Suite 2
    Photoshop
    InDesign
    GoLive
    Illustrator
    Macromedia Flash
    Macromedia Freehand
    Microsoft Office XP
    PowerPoint
    Excel
    Word
    Kodak PhotoDesk

  • Confocal microscopy service ( Material analysis service )

    "A Leica TCS SP5 II scanning laser confocal system with resonant scanner and environmental chamber is also available for individual and assisted imaging. This instrument allows investigators to carry out high-resolution, single cell observations, spectral separations, thick specimen analysis and co-localization studies in both fixed and dynamically interacting cell populations. It supports studies designed for both single timepoint examinations as well as multi-dimensional time-lapse observations. This equipment will allow long-term, environmentally controlled, minimmaly photo-toxic, reduced noise, high resolution microscopy, and the ability to carry out FRET, FRAP, confocal reflectance, 3D and 4D tracking, and temporal, spectral and spatial analysis.

    This system has 9 laser lines and 3 spectral PMT detectors, as well as 2 HyD detectors for superior sensitivity and single photon capability. Sample preparation is generally done by the interested lab and analysis of the captured images can be done with the Leica analysis package or with 3rd party options available in the Core."

  • Imaging consultation service ( Support service )

    "All successful experiments begin with an understanding of the capabilities of the existing equipment and software. The facility staff constantly work with researchers to help design protocols that can be effectively accomplished with the resources at hand. Choice of fluorophores, freezing and fixation techniques, thickness of specimens, and other issues can all negatively affect results if the instrumentation is not considered in advance and, in the end, cannot support those results. The facility staff also takes great care to make users aware of the dangers of unacceptable image manipulations that can alter the results of their experiments."

  • Imaging data storage service ( Data storage service )

    "The Imaging Facility maintains storage space in the Center for Systems and Computational Biology, located in the Wistar Bioinformatics Facility. All Imaging systems are directly linked to the storage system and all images created in the Imaging Facility should be saved directly to this server. The system has redundant back-up protocols and is automatically archived. Files may be accessed by users and transferred to individual laboratory drop boxes for personal back-up and storage. Although the Imaging Facility will do all that it can to maintain the integrity of imaging files, the user is ultimately responsible for their own data."

  • Imaging equipment, image processing, and image analysis training ( Training service )

    "All facility equipment is available to be used unassisted or with assistance. Users must first be trained for each specific piece of equipment before they can be certified by the Facility for unassisted usage. Some equipment requires significant training to attain proficiency, so the staff can always work with a user directly to obtain the desired results. Training also includes image processing and analysis, as well as digital imaging ethics. Training is mandatory prior to unassisted use of all Facility equipment."

  • Imaging Facility instrument access ( Access service )

    "Access to all Imaging Facility equipment is coordinated through the main laboratory in room 287. Researchers may be authorized to work with the equipment unassisted or services can be provided by the facility staff. Please contact the facility manager with any questions or requests."

  • In-lab microscopy assistance and maintenance service ( Support service )

    "In addition to providing core instrumentation and services, the facility staff is also available to help support microscopy instrumentation elsewhere in the Institute. Common maintenance issues such as cleaning and bulb replacement in individual labs are easily supported and response time is more immediate from the Imaging Facility than from outside vendors. Consolidation of outdated and discarded microscopes by the Facility also creates a repository of used equipment, which can easily be redistributed as needed throughout the Institute."

  • Live-cell time-lapse microscopy ( Material analysis service )

    "Using the Nikon TE300 inverted microscope in room 287 with its ImagePro workstation, 5D time-lapse studies of cells grown in culture can be accomplished (X, Y, time, wavelength and location). Imaging combinations using phase contrast and fluorescence illumination are possible, taken at short or long intervals, from several minutes to several days. The environmental chamber has temperature and CO2 control and is monitored continuously. Cells are normally presented in multi-well plates or 35mm glass-bottom dishes, but other vessels can be accommodated as needed. Common uses for this system include scratch (wound) assays and cellular proliferation studies.

    For higher resolution options, including 3D timelapse, The Leica SP5 II confocal microscope (see below) is also capable of multi-day timelapse, with CO2 and temperature control."

  • Low magnification and photomacrography
 service ( Material analysis service )

    "Specimens that are too large for a standard microscope require flexible equipment configurations for illumination and imaging conditions that vary depending on the specimen. We have the ability to image anything from whole mount slides to entire Petri dishes, as well as gross specimen photography of excised biological tissues using our stereomicroscopes or digital SLR-based macro equipment. Transmitted or reflected light, as well as fluorescence can be accommodated with our systems."

  • Small animal whole body luminescence and fluorescence imaging
 ( Analysis service )

    "The Perkin-Elmer IVIS 200 whole body imager is designed for detection of weak fluorescent or bioluminescent signals deep in living mice for non-invasive longitudinal studies of growing (or regressing) tumors in mouse models. The system is housed in the Institute’s barrier mouse facility and is only 
available to those researchers with approved protocols and animals housed in the colony. The integrated photon counting software can normalize the data for direct comparison to future studies or to similar instruments anywhere in the world."

  • Specialty image capture service ( Analysis service )

    "The facility maintains equipment to provide “traditional” photographic support, such as photography of small animals (mice), experimental set-ups, copy stand specimens, or even human groups and portraits when needed, as well as special equipment for unusual situations such as GFP fluorescence in large specimens like adult mice. The Nikon D200 digital camera has a variety of interchangeable lenses, depending on the situation, and special portable studio lighting is also available as needed."

  • Two-photon microscopy ( Material analysis service )

    "2-photon microscopy continues where traditional confocal leaves off. With laser excitations in the longer wavelengths, 2P allows deeper imaging and reduced phototoxicity. This system was designed to be used for intravital imaging, with tissues such as inguinal lymph nodes, but it can also used with cell cultures in acceptable vessels, tissue slides, explanted specimens such as lymph nodes and brain slices, kept viable in heated media. This system has the ability to resolve non-labeled structural details with 2nd harmonics (SHG), as well as capture emission wavelengths simultaneously with three other detectors. Thick, fixed specimens, such as spheroids and skin reconstructs can benefit from the increased penetration and imaging capabilities of this system, as well as fixed cultures in deep collagen matrices."

  • Widefield microscopy ( Material analysis service )

    "Conventional microscopy, utilizing standard techniques in brightfield, darkfield, fluorescence, phase contrast and differential interference contrast, is available on a variety of instruments in the main Core Facility room 287. Multiple upright and inverted microscopes capable of low to high magnification documentation are available with individual image capture workstations networked to the Imaging server. Sensitive monochrome cameras and one-shot digital color cameras are available on each system. Real-time stitching software allows high resolution imaging of larger samples."

Software

  • Helicon Focus ( Software )

    "Helicon Focus is a program for focus stacking. Focus stacking is a post-processing technique that enables you to extend the depth of field beyond what is available in one shot. First take several shots of the same scene; then let the program blend them together into one image with an extended depth of field. For each shot, you focus your lens on a different part of the object. The resulting series of photographs is called a "stack," and the process of merging them into one image is called "focus stacking."

    If you do macro photography — close-ups of flowers, insects, or jewelry, for example — you're familiar with the problem created by a shallow depth of field. Shallow depth of field is an eternal problem in microscope photography, too, because the greater the magnification, the shallower the resulting depth of field. Sometimes this can also be an issue for landscape photography if you want both the foreground and the background to be in focus. Focus stacking with Helicon Focus can help you to overcome shallow depth of field.

    Focus stacking with Helicon Focus also allows you to create 3D models of your objects without extra effort, after which you can export them as stereo pairs, anaglyphs, and even videos of the rotating models."

  • Image-Pro Plus ( Software )

  • Volocity ( Software )

    Volocity is a sophisiticated software for visualization, deconvolution, and quantitative analysis of 3- and 4-dimensional image data.


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Last updated: 2016-03-10T14:16:00.930-05:00

Copyright © 2016 by the President and Fellows of Harvard College
The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016