Northwestern University Cell Imaging Facility

Table of Contents

  1. Description of the facility
    1. Organizational Structure
    2. Services provided
    3. Services Available
    4. Instruments Available
    5. Administration of the facility
  2. Users of the Facility
The Cell Imaging Facility is a core facility available to all Northwestern University investigators. The facility provides state of the art instruments, technical support, training and service work for most aspects of light, confocal and electron microscopy. The operations of the facility are funded primarily through charge-backs to grants and contributions from the Medical School and Dept. of Cell and Molecular Biology.

I. Description of the facility

The Cell Imaging Center is a Medical School Core Facility located on the 7th floor of the Ward Building. Its purpose is to provide access to equipment, support, service work and training in the fundamental techniques required for analysis of structure and function at the tissue, cellular and subcellular levels. It seeks to maintain technologically advanced equipment that will provide investigators a resource for furthering their research. Techniques currently employed in the Cell Imaging Facility include light, fluorescence, confocal and electron microscopy; computerized image analysis and digital image manipulation; and the use of laser based techniques for the manipulation of cells such as optical trapping, laser ablation and fluorescence recovery after photobleaching techniques. The Center is available for use by all Medical School investigators who wish to take advantage of its instrumentation or services. These range from the preparation of samples for microscopy and the use of the microscopes, sophisticated computer enhanced imaging and digital processing to help with the production of prints, slides, and posters for publication or presentation.

A. Organizational Structure

The Cell Imaging Facility grew out of a microscopy facility established by Dr. Robert Goldman when he became Chair of the Cell Biology and Anatomy department in 1981. Initially the facility focused on providing electron microscopy services to members of the Department. Over time the facility has added instrumentation allowing it to offer light, fluorescent and confocal microscopy. During the past two years the facility has been generally opened up to the Northwestern University research community. This was made possible by a significant contribution toward salary support for the facility staff from the Medical School's Dean's Office. The facility now exists as a free standing core facility managed through the Dept. of Cell and Molecular Biology as outlined below in section C below.

B. Services provided

The Cell Imaging Facility has several goals as part of its mission:

A. To provide access to a variety of state of the art microscopic instrumentation at a single location. Because of its high cost and the widespread need of many investigators for only occassional use it is not feasible for every investigator who needs microscopy in their research to have their own instruments. Providing access to these instruments through a core facility makes these instruments available to investigators who might otherwise not have access to them.

B. To provide service for each step in EM sample preparation, light and electron microscopy imaging and photography. Investigators can arrange to do part or all of the techniques necessary for their research, with the facility providing the service for the remainder.

C. Provides skilled training and technical advice for a broad range of microscopic equipment and techniques.

Services Available

Currently, a number of microscopy services are available through the facility, as well as access to and training on a wide variety of research equipment. Services are provided on a per hour fee basis with additional charges for consumable supplies provided. The following is a list of the services currently available through the facility.

Standard transmission EM processing and embedding. The facility provides complete or partial processing and embedding of samples for TEM. A normal process for samples will include fixation of tissues in cacodylate or phosphate buffered glutaraldehyde and/or paraformaldehyde, buffer rinses, post-fixation in osmium tetroxide, en-block staining with uranyl acetate acetate, step-wise dehydration in ethanol or acetone and step-wise infiltration and embedding in an epoxy resin. Techniques such as re-embedding for orientation can also be performed.

Ultramicrotomy. The critical step for TEM of ultramicrtomy can be performed by the facility staff, or the microtomy equipment can be used by investigators, The ultramicrotomy facilities are located in a 95 square foot room containing a Reichert Ultracut E Ultramicrotome, a Reichert FC 4D Low-Temperature Sectioning System, tow LKB Nova Ultotomes, and two LKB 7800 Knifemakers. A wide range of grids of various materials are available. Microtomy can be done both on glass and diamond knives. In addition to standard microtomy, semi-thin sectioning, enface and serial sectioning are available. Cryo-ultramicrotomy can also be arranged on frozen samples.

Formvar, Parlodion, and carbon grid coating. Support films for negative staining, delicate sections, or other applications can be prepared by the facility. Both parlodion and formvar grids can be prepared, and these can be carbon coated and glow discharged before use. A wide variety of grids are available. Coating and glow discharge can be performed in a Denton DV-502 Vacuum Evaporator.

Grid staining. Staining of grids with ultrathin sections is also available. Standard staining is with uranyl acetate and lead citrate, but other special techniques can also be arranged.

Negative staining. Negative staining of protein, macromolecule, virus or other types of samples is available in the facility. Standard negative staining, the pseudo-replica technique, land other techniques are available. A spray apparatus can be used for even distribution of the sample.

Rotary shadowing. Rotary shadowing can be utilized for preparing spread samples such as DNA preps, isolated structural proteins, land other types of specimens for TEM examination. For optimal distribution of sample for TEM a protein spray apparatus produces a fine mist that spreads micro-droplets across a surface. A Balzers BAF 400 Freeze Etching System can produce a high vacuum low angle metal coating with platinum, as well as carbon coating for stabilization.

Instruments Available

The facility contains a large number of state-of-the-art instruments that are available for use by Northwestern University investigators:

1. Sample preparation is performed in the 550 square foot main laboratory supplied with ample work benches and the following specialized equipment.

2. Facilities for chemical processing of EM and LM samples are carried out with the following additional equipment:

3. Ultramicrotomy facilities are located in a 95 square foot room equipped with:

4. The center supports all aspects of conventional photography as they relate to light and electron microscopy. A 50 square foot negative darkroom with a Leedal wet bench and nitrogen burst system for controlled processing of photographic negatives. A printing area 144 square foot darkroom is equipped with the following items:

5. Zeiss LSM 10 Laser Scanning/Confocal Microscope with various scanning modes include laser and scanning modes with or without confocal imaging. It can also be used as a conventional light microscope. Options for both modes are bright field, phase contrast, differential interference contrast, epifluorescence, and reflectance microscopy. The microscope is driven by a 486/50 MS-DOS/Windows based computer system which includes a magneto-optical drive for storage of digital images aquired by the confocal microscope. An extensive image processing system is built into the LSM which allows for a wide variety of manipulations of the digitally recorded image.

6. Zeiss Axiovert 135 with an attached Cell Robotics Laser Tweezers 2000 LT-2000 with C Stage/Laser Tweezers Optical Trapping System and Photometrics CH250 CCD Camera System and Supporting Image 200 Image Acquisition Software. This system can be used for manipulation of cells and subcellular organelles in living cells. In addition it can be used for in vitro motility assays of purified molecular motor molecules such as dynein, kinesin and myosin. Thus the manipulation of microscopic particles and high resolution measurements of cell properties can be obtained.

7. Nikon Diaphot 200 inverted microscope with an attached Cell Robotics Laser Scissors laser ablation system. This system can be used for laser microsurgery on cells or for laser mediated photobleaching experiments.

8. JEM 100CX Transmission Electron Microscope supplies a means of viewing ultra-thin sections of a wide range of samples embedded in plastic resins for ultrastructual analysis. This instrument produces high resolution micrographs. The Jeol 100CX is a high resolving power transmission electron microscope with 0.2nm resolving power. It has a KV range of 20 to 100 in steps of 20KV. For ease of sample exchange it employs a side-entry eccentric goniometer stage capable of holding two grids at a time for faster and more efficient viewing. Arrangements can be made for the microscope to be critically aligned in whatever KV and aperture configuration best fits the investigator's needs. We encourage investigators to use a variety of settings on the microscope to find the optimal results for their samples.

9. Zeiss Axioskop used for transmitted light and epi-fluorescence imaging. This microscope is heavily used for standard immunofluorescence analsis. It has brightfield, phase, differential interference contrast, and fluorescence capabilities. This microscope is equiped with two 35mm cameras with automatic exposure control, complete filter sets for rhodamine, fluorescein, and DAPI fluorescence. This instrument is also equipped with a Photometrics Sensys cooled CCD camera purchased through a previous grant from the Committee on Shared Facilities.

10. In addition to conventional film based images, digital images can be aquired from all of the light microscopes in the facility. To facilitate acquisition and manipulation of digital images the facility includes a pentium computer with 48 MB RAM and a 1.3 GB optical drive which runs the Metamorph image acquisition and analysis software package.

11. To facilitate the production of figures for publication from digital images the facility houses a second pentium and a PentiumPro computer which has a variety of graphics software packages including Adobe Photoshop. This computer has direct interfaces to a 300 dpi Tektronics Phaser 2 SDX dye sublimation printer, a LaserMaster film recorder capable of recording 24 bit, 4000 line color slides, and a 24 bit HP-ScanJet IVc color scanner for the conversion of conventional photographs into digital images.

12. To provide high end manipulation and analysis of data acquired from the instruments in the facility, a Silicon Graphics Indigo2 200 MHz R4400 microprocessor based graphics workstation with 200 MB of RAM, 4 GB harddrive storage and 1.3 GB optical drive is also housed in the facility. Available software includes the Voxel View suite, with Voxel Math and Voxel Animator for the production of 3D reconstructions of data acquired from the confocal microscope. This computer is also equipped with video input and output capabilities allowing investigators to record the output of three dimensional reconstructions directly onto videotape.

13. The facility, with the support of the Howard Hughes Medical Research Institute has recently acquired a state-of-the-art JEOL 1220 transmission electron microscope. This instrument is the first of its kind to be installed in the United States. This instrument utilizes a pole piece optimized for improved contrast of biological specimens. The microscope is outfitted with a Kodak digital camera allowing digital acquisition of electron micrographs.

C. Administration of the facility

The day to day operations of the facility are managed by the Director of the Cell Imaging Facility, a research track faculty position which has been recently established. A nationwide search is currently underway to fill this position. The successful candidate for this position will have experience with light, fluorescence and confocal microscopy, as well as digital acquisition of images and image processing. This individual will be responsible for the day to day operation of the facility, including equipment maintenance, training, billing ordering of supplies and budget preparation. The Director of the Facility will report to Dr. Rex Chisholm and the Cell Imaging Facility Advisory Committee (see below). The facility has also recently hired Ms. Maya Moody, an electron microscopist with five years of experience in the laboratory of Professor Hewson Swift at the University of Chicago. She is responsible for providing electron microscopy services.

Facility policies are set by the Cell Imaging Facility Advisory Committee, which is chaired by Dr. Jonathan Jones. The current membership of the advisory committee is:

Cell Imaging Facility Advisory Committee

Stephen Adam, Ph.D., CM Biology

Lester Binder, PhD., Cell and Molecular Biology

Kathleen Green, PhD., Pathology

Philip Hockberger, PhD., Physiology

Robert D. Goldman, CM Biology

Sam Rao, M.D., Pathology

Jonathan Jones, PhD., CM Biology, Chairman

II. Users of the Facility

The facility is available for use by Northwestern University researches who wish to take advantage of a variety of microscopy and ancillary instruments and services. These range from the preparation of samples for microscopy and the use of the microscopes, to the production of prints, slides, and posters for publication or presentation. Authorized users have access to the instruments in the facility based on their level of experience. Access to the facility is via a keycard system. Authorized users can have their keycards validated for 24 hour access once they have demonstrated proficiency with the instruments to be used. Access to instruments is on a first come first served basis with users able to reserve instrument time up to one week in advance.