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IDDRC

Cellular Morphology Core

Core services ending June 30th, 2014

Please see the Clinical Translational Core

    Cellular Morphology images

Dr. Garden

Gwenn Garden, M.D., Ph.D., Director, Cellular Morphology

gagarden@uw.edu

Overview

The Cellular Morphology Core is focused on providing CHDD Research Affiliates with access to state-of-the-art equipment and technical expertise needed to observe the pathology and pathogenesis of neurodevelopmental and neurodegenerative disorders at the cellular level. In doing so, the Cellular Morphology Core also aims to provide a comprehensive array of services by providing affiliated investigators with assistance from a very early point in the experimental design stage, and continuing that support even after data collection with assistance in image processing and quantitative image analysis. Moreover, the Cellular Morphology Core provides investigators with access to all of the equipment and technical assistance needed to study models of neurodevelopmental and neurodegenerative disorders at the cellular level by providing access to histology equipment and services, electron microscopy specimen preparation and analysis services, as well as access to and assistance with light, fluorescent, deconvolution, and confocal microscopy.

More specifically, this is accomplished by providing access to specific services within the following three facilities: (1) the Neuropathology/Histology Facility, (2) the Electron Microscopy Facility, and (3) the Digital Microscopy Center (DMC). The Neuropathology/Histology facility aims to provide IDDRC Research Affiliates with all of the equipment, training, and expertise needed to perform histological analysis of tissues and immunolabeling of both tissues and cells in culture. The Electron Microscopy Facility aims to provide a full range of specimen preparation, including cutting both semi-thin and ultra-thin sections, tissue staining, and technical assistance in viewing and photographing specimens on the electron microscope. Finally, the DMC aims to provide Research Affiliates with access to state-of-the-art technologies in digital microscopy including both conventional and multi-photon confocal imaging, 3-D deconvolution and reconstruction capabilities, and quantitative morphometric analyses

The Neuropathology/Histology Facility specific objectives are:

  • Provide technical services to Research Affiliates for the performance of routine neurohistological stains, standard immunohistochemistry and immunofluorescent labeling, and double immunofluorescent labeling for the concurrent identification of specific neural cell types expressing the protein of interest
  • Provide problem solving advice for Research Affiliates aiming to perform complex or novel labeling methodologies on CNS tissues
  • Train affiliate investigators as well as their students, post-doctoral fellows or staff in histological methods

The Electron Microscopy Facility specific objectives are:

  • Provide advice and technical services to Research Affiliates for the generation of appropriately preserved material for use in transmission electron microscopy (TEM), including assistance with fixation, embedding, sectioning, grid preparation and staining of ultrathin sections
  • Train affiliate investigators as well as their students, post-doctoral fellows or staff in the use of the TEM

The Digital Microscopy Center specific objectives are:

  • Provide affiliates and their laboratory members with access to digital image acquisition using widefield transmitted light and epifluorescence microscopes
  • Provide affiliates with the equipment and technical expertise needed to perform deconvolution microscopy or generate high resolution large field montage images using the Marianas system
  • Ensure that affiliates have reasonable access to both single and multi-photon confocal microscopy and receive expert training and technical assistance
  • Train and assist in the development of methodologies for microscopy of living cells, tissues, and transparent organisms
  • Provide the software, equipment, advice, and training needed to perform post-acquisition processing of digital microscopic images (including digitized TEM images) to enable the use of quantitative analysis and three-dimensional image transformations

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Location


south campus map Cellular Morphology location

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Faculty & Staff


Dr. Garden
Gwenn Garden, M.D., Ph.D.
Director,
Cellular Morphology Core

Dr. Deng
Mei Deng, M.D.
Neuropathology/ Histology Facility
Research Scientist
 Mr. Macdonald
Glen MacDonald
Digital Microscopy Center
Research Scientist

Ms. Gill
Nishi Gill, M.S.
Electron Microscopy Facility
Research Scientist

Mr. Eng
Jeremiah Eng
Digital Microscopy Center
Research Scientist

To Use Our Services

Investigators who are interested in using brain imaging in their projects are invited to read about the equipment and services we provide. Next send Dr. Garden a one-paragraph description of your project before beginning your experiments (gagarden@uw.edu, 206-616-9402).


Services & Equipment

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Neuropathology/Histology Facility

The neuropathology/histology facility contains all of the equipment needed for Research Affiliates to generate tissue sections for anatomical analysis. This facility is located in the RR wing of the CHDD Medical Research Tower and includes equipment for embedding tissue in paraffin, a paraffin microtome, a frozen sliding microtome and a cryostat, in addition to all of the small equipment needed for these histological methods such as water baths and warming plates. There are also several racks available for processing tissue following sectioning and performing routine histological stains or preparing tissue for immunolabeling.

Dr. Deng processing cells

Dr. Deng completes immunohistochemical procedure.

Dr. Mei Deng (dengm@uw.edu, 206 543-8769), Research Scientist, with more than 10 years of experience with the Cellular Morphology Core component can provide:

  • Assistance to affiliates and their lab members in developing the skills needed to perform these neuropathology and histology research methods described in more detail below
  • Assistance in the development of new immunohistochemical labeling protocols
  • Assistance in the development of double fluorescent immunolabeling methods, even for human tissue where the need for autofluorescence quenching often makes the development of these protocols very difficult

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Electron Microscopy Facility

CHDD Research Affiliates are provided with the equipment and support services which are located in two sites in the CHDD Medical Research Tower. The tissue and grid preparation area is located in RR737B/RR739, with the microtomy and light microscopic area located next door in RR737A. The preparation rooms contain a chemical hood, sinks, refrigerators, an evaporator for coating grids, balances, a glass knife maker and storage space. The adjacent microtomy/microscope area houses four Reichert ultramicrotomes (two older OMU2 instruments, an Ultracut E, and a newer Ultracut S), two dissecting microscopes, and a high quality histology light microscope with phase and dark field capabilities. The preparation and microtomy areas are next door to a general histology laboratory, and in close proximity to a suite in which to carry out surgical procedures; these facilities are available to users of the EM component of the Core as needed for their studies. The other principal part of the Electron Microscopy Facility consists of a Philips CM10 transmission electron microscope, provided through a cooperative agreement with the Vision Research Center, installed and available in Room RR017. This facility consists of the transmission microscope itself and a support darkroom needed for micrograph processing.

Nishi Gill (nishig@uw.edu, 206 543-3244), research scientist and neuroanatomist with extensive training in electron microscopy of the nervous system, aids investigators in the planning and execution of TEM projects. She can train affiliates and/or their lab members on appropriate methods of specimen preparation for TEM.

Phillips CM10 TEM

The Philips CM 10 is a transmission electron microscope with an accelerating voltage of between forty and one hundred kilovolts. The magnification range is from 18x to 450,000x, with a point to point resolution of 0.3 nm, using a tungsten filament. The images are recorded on 3 1/4"x4" Kodak 4489 plate film, and specimen tilt is between -60 and +60 degrees

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Digital Microscopy Center

DMC image DMC image DMC image DMC image DMC image

The Digital Microscopy Center (DMC) is housed in the lower level of the CHDD Clinic Building. Glen MacDonald (glenmac@uw.edu, 206 616-4156 or 206 543-8360), is a Research Scientist who manages the Digital Microscopy Center (DMC). He assists research affiliates in developing imaging modalities and quantitative imaging protocols using both fixed and living preparations. He also provides training sessions for the Olympus and confocal microscopes. Jeremiah Eng (eng683@uw.edu, 206 543-3380) provides training sessions on the use of the Nikon Optiphot, Marianas system and Slidebook software for Research Affiliates and their lab members. Jeremiah is also available on a limited basis to assist Research Affiliates with quantitative analysis projects and assists Glen MacDonald on improvement projects in the DMC.

This facility consists of 4 major tools providing a wide range of imaging and analytical capabilities to CHDD affiliates.

(1) A Fluoview-1000 (FV-1000) laser scanning confocal microscope from Olympus based on an IX-81 inverted microscope. Capabilities include four photomultiplier tube detectors for fluorescence and one for transmitted light detection. Two fluorescent detectors use diffraction grating filters as variable detection filters to allow spectral unmixing. A total of six lines (405 nm, 458 nm, 488 nm, 515 nm, 561 nm and 633 nm) from four lasers enable us to excite a wide range of fluorophores. Rapid line switching allows rapidly acquiring sequential channels, restriction of scanning to selected regions of interest, and new capabilities for photobleaching, photoconversion, and photoactivation. A Prior Instruments motorized stage allows multi-point positioning which is very useful in conjunction with timelapse imaging as well as for acquiring montages. The instrument has been equipped with a range of high performance dry objectives at 4X/.16, 10X/.4, 20X/.75 and oil immersion objectives at 40X/1.3, 60X/1.35 and 100X/1.4. Also available for special applications are a 20X/.45 long working distance objective for exceptionally thick materials and a 60X/1.2 water immersion objective for imaging live cells with minimal spherical aberration.

Olympus DMC / Confocal Microscope: Olympus Fluoview-1000

The Olympus FV-1000 confocal uses an Olympus IX-81 inverted microscope capable of brightfield, epifluorescence and DIC. The microscope is equipped with a motorized specimen stage and focus, plus automated control of lenses, condenser settings and filters. This confocal system can collect up to 4 channels of fluorescence and a transmitted light channel, as well as provide sequential image capture, spectral unmixing, photobleaching/photoactivation, 3D montage acquisition and complex 4D image capture.

This was acquired in 2007 by a shared equipment grant awarded by the National Center for Research Resources, NIH. It offers many functions not available on the MRC-1024 including the following:

  • Software controlled sample positioning - allows multi-point timelapse imaging and montages.
  • AOTF excitation control – Provides nearly instantaneous selection and independent attenuation of each laser line, which also allows minimizing bleedthrough by sequential capture of each image channel.
  • Rotatable scans – This feature allows 360° rotation of the field of view.
  • User defined regions of interest – The user may define up to 256 specific regions to be scanned or subjected to photoactivation or photobleaching operations.
  • Laser Intensity Feedback System – Allows correction of fluctuations in laser output to provide even illumination during timelapse imaging.
  • Multiple laser lines –6 laser lines at 405 nm, 458 nm, 488 nm, 514 nm, 561 nm and 633 nm.
  • Multiple Channels – 4 photomultipliers for fluorescent labels and 1 for brightfield/DIC signal capture.
  • Bi-directional scanning – 2 lasers can excite the sample in both directions along the x-axis for rapid acquisition of 2 labels during dynamic events.
  • Rapid line speed – Up to 4096 lines/second in conventional scanning.
  • 12-bit digitization – the signals are divided into 4096 intensity intervals for higher dynamic range, less saturation and more accurate deconvolution.
  • Spectral unmixing – Samples may be recorded and processed to separate overlapping emission spectra or to remove autofluorescence.

An additional component of the NCRR equipment is an SZX-ZB7 stereo zoom dissecting microscope mounted on a dual-arm boom stand.  This can be mounted on the airtable with the FV-1000 and used to place electrodes for experiments combining confocal imaging and electrophysiology.  This dissecting microscope routinely occupies space on a bench adjacent to the confocal and used for preparing samples involving in vivo imaging, but can be moved as needed to the confocal air table to facilitate simultaneous electrophysiology and confocal microscopy.


(2) The Marianas system is primarily intended for live cell imaging. This imaging system consists of a fully automated Zeiss Axiovert 200M equipped with an ASI motorized stage and CoolSnapHQ CCD camera (Princeton Instruments).  A shuttered xenon source coupled by a liquid lightguide and a pair of 10-position filterwheels (Sutter Instruments) provides a uniform field illumination and rapid capture of multiple wavelengths without pixel shift from filter cube misalignments.  Slidebook software from 3i controls routine image capture, as well as offers capabilities for montage imaging, timelapse, deconvolution, and stereology.  An incubation enclosure provides environmental control for living tissues during imaging, utilizing an air recirculating temperature controller.  In addition to standard selection of objectives from 5X to 100X oil immersion, this system also offers a 63X/1.2 water immersion objective and a 25X/.8 multi-immersion objective for live imaging and control of spherical aberration in critical samples.  The imaging software includes Slidebook 4.2 for Windows, which provided improved stage control, analytical capabilities, and an efficient interface for routine image capture. An adjacent dual processor G5 Macintosh provides additional deconvolution capability with Slidebook. For additional information email question here.

Axiovert microscope DMC/ Marianas Live Cell Workstation

Deconvolution Microscopy - Automated Live Cell Imaging

The Marianas imaging system is a fully automated microscope controlled by Slidebook software from Intelligent Imaging Innovations, which also provides deconvolution, advanced image analysis tools and stereology. It incorporates a Zeiss Axiovert 200M microscope with an X,Y motorized stage (ASI), shuttered 175 W xenon lamp (ASI) coupled with a liquid light guide, and a Roper CoolSnap HQ digital camera. Wavelength selection by filter wheels (Sutter) provides flexibility for viewing different fluorescent light wavelengths. This system is enclosed within a heated enclosure to provide tissue viability and thermal stability in live cell imaging experiments.


(3) The Nikon Optiphot-2 research microscope has been upgraded over the years and is currently equipped for brightfield, darkfield, phase contrast and epifluorescence. It is fitted with a Spot IIe cooled CCD camera with filters for RGB color images in addition to monochrome image captures for fluorescent samples. It is used for cellular morphometry and densitometry analysis, providing analytical capabilities on a wide range of samples (e.g., densitometry of material processed for in situ hybridization, autoradiography and immunocytochemistry as well as quantitative morphometry on images from the light microscope, the confocal microscope, and electron microscope). It is popular for tract tracing and ell counting due to its 1X and 2X objectives. A Macintosh G4 computer provides the software platform for scanning, image capture and analysis, and preparation of figures for publication. The primary analytical tool is the public domain application ImageJ. An Agfa T2500 flatbed scanner attached to this computer is also employed for digitization of gels and electron microscopy negatives.
Optiphot microscope DMC / Nikon Optiphot-2 widefield digital microscope

A Spot II digital camera is attached to a Nikon Optiphot upright microscope equipped for fluorescence, bright-field, dark-field and phase contrast imaging. This workstation also incorporates an Agfa T2500 flatbed scanner with a transparency drawer for scan resolutions up to 2500 dpi from prints and negatives.

Supporting Equipment

Image Processing Workstation

Image Processing Workstation, a Macintosh G5 provides a workstation for additional deconvolution of images obtained from the Marianas. This computer offers Slidebook, ImageJ and Photoshop for use with images from any microscope.

Deconvolution Workstation

Deconvolution Workstation Images requiring processor intensive operations or which are exceptionally large may be handled on a 4-processor server running 64-bit Linux, with 8 Gb of RAM. A mirrored RAID offers file security. Huygens Essential software from Scientific Volume Imaging provides deconvolution capability with several options for image rendering and 3D analysis. ImageJ is also available for large files.

Incubator

A CO2 incubator with an automatic tank switch is available for short term culture by investigators imaging live mammalian cells and tissues. It allows specimens to recover from transport between the investigator's laboratory and the DMC, as well as to maintain viability of samples waiting for imaging. The computer sitting to the right of the incubator is the 2Tb server.

Post-Imaging Processing and Analysis

We provide image processing and analysis to assist in the interpretation of changes that accompany neural development, neurodegeneration, and/or acute or chronic neural injury:

  • Computer-assisted quantitative morphometry
  • Automated grain counting from slide autoradiographs
  • High-resolution scanning for light and electron micrographs and export for slides or illustrations
  • Morphometric and densitometric measurements of digital images
  • Quantitative analysis of area, length, volume, number, etc., using video or microscope image input
  • 3-D cell reconstruction
  • Deconvolution of 3-D fluorescent images
  • Digital movies from optical volumes, reconstructions, or Quick-Time VR
  • Data archiving on CD and DVD media
  • Multi-channel fluorescence and RGB DIC image capture
  • Time-lapse imaging
  • 3-D segmentation and co-localization
  • 3-D and 4-D imaging
  • Montage images
  • Stereology

Multiphoton Confocal Microscope: Zeiss LSM510

Through an arrangement with the UW Department of Ophthalmology, CHDD Research Affiliates also have access to a Zeiss LSM510 Multi-photon confocal microscope system and associated technical support that became operational in September of 2001. This system is installed in Room RR019 of the CHDD Medical Research Tower. It consists basically of a Zeiss Axiovert inverted microscope with LSM 510 scanning head, three visible light lasers, and a Coherent Mira/Verdi IR femto-pulse laser with pulse compression, mounted on a Coherent modified FMC vibration isolation table. A Seimens computer system is used to control the microscope by means of Zeiss AIM image acquisition software. The system has six imaging detectors: three descanned PMTs for emitted or reflected light collection, one PMT for transmitted light detection, and two non-descanned detectors for ultra-high sensitivity multiphoton image capture. This system can be used for viewing both conventionally fixed fluorescent specimens and live tissues or cultured cells. A live cell stage is available plus other equipment needed to support living specimens during image acquisition. The room has been modified to control temperature and dust as required for the adequate operation of the Mira/Verdi laser. It is maintained by means of service contracts with Zeiss, Inc. and Coherent, Inc. Users receive assistance and computer access in the DMC for post- processing of images obtained from this imaging system

multi-photon microscope

Multiphoton Confocal Microscope: Zeiss LSM510

This microscope is equipped with a Coherent Mira 900 Infrared Femto-second Pulse laser capable of stimulating fluorescence from endogenous biochemical agents within living cells by 2- and 3-photon fluorescence and digitally capturing the resulting signal with ultra-sensitive non-descanned detectors.

Research Affiliates seeking additional information regarding the capabilities and specifications of this multi-photon microscope should contact Dan Possin (danpossn@uw.edu, 206 221-3845 or 206 543-3884). Affiliates wishing to initiate projects using this equipment should contact Dr. Garden (gagarden@uw.edu, 206-616-9402) for information regarding cost and access prior to using this instrument. Training and access will only be arranged after projects are proposed in writing and approved by Dr. Garden.

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University of Washington • Center on Human Development and Disability Box 357920 • Seattle WA 98195-7920 USA • 206-543-7701 • chdd@uw.edu