Electron Microscopy

Location: Thomas Building, DE-780
Contact phone: (206) 667-4289
Contact e-mail: emsr@fredhutch.org

Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is primarily useful for giving a three-dimensional image of the surface of the specimen and is for viewing large objects (e.g. Drosophila).

Scanning electron microscopy services include:

  • Fixation and dehydration
  • HMDS or critical point drying (Tousimis critical point dryer)
  • Sputter Coating (Denton desk IV sputter coater)
  • Scanning EM of whole cells, bulk specimens, i.e. embryos, Drosophila, etc.
  • Microscopy using the JEOL JSM 6610LV
  • Microscopy and Training

Loading SEM sample

Microscope operation on the scanning electron microscope (JEOL JSM 6610LV) may be conducted by individuals (either alone or with assistance), or may be provided by EM staff. Sign-up for microscope time or viewing time with resource staff using iLab.

Training

Training will be provided by the EM staff on the operation of the microscope. All self users will be required to demonstrate competency on the instrument to be considered qualified to use the instrument alone. For more in depth classes in various areas of electron microscopy please see training. Links to many EM training resources are provided in the EM Resources and Training LibGuide.

Obtaining your Data (Images)

Images resulting from microscopy can be saved in your Homelink account, on the server maintained by Center's IT department.

If you do not have a Homelink account, please submit see Data Storage and Archiving.

Transmission Electron Microscopy (TEM)

Transmission electron microscopy (TEM) provides information on internal structures in thin-sections (70-100nm) from your specimens. Cellular and sub-cellular structures can be viewed in great detail. TEM is also useful for viewing suspensions of microscopic particles, such as bacteria or virus using negative staining techniques. Very small amounts of sample are necessary for this technique.

TEM services provided by the EM facility include:

  • Conventional specimen fixation, dehydration and embedding
  • Specimen sectioning
  • 1 micron epoxy survey sections
  • Ultra thin sectioning (70-100nm) on ultra-microtomes (using Leica EMUC7 and UCT Ultra-microtomes), and Vibrating blade microtomy with Leica VT1200S
  • Immunoelectron microscopy
  • EM tomography for 3-D analysis of structures
  • Cryotechniques
  • High Pressure freezing
  • Freeze substitution
  • Cryo-ultramicrotomy (Leica EM FC6 cryochamber)
  • Negative Staining (Beckman airfuge)
  • Viral and Particle Quantification
  • Platinum shadowing and carbon coating (Denton desktop vacuum evaporator)
  • Microwave techniques (Ted Pella Research Microwave System)
  • Microscopy using JEOL 1400 transmission electron microscope

Microscope operation on the transmission electron microscope (JEOL JEM 1400) may be conducted by individuals (either alone or with assistance), or may be provided by EM staff. Sign-up for microscope time or viewing time with resource staff using iLab.

Training

Training will be provided by the EM staff on the operation of the microscope. All self users will be required to demonstrate competency on the instrument to be considered qualified to use the instrument alone. For more in depth classes in various areas of electron microscopy please see training. Links to many EM training resources are provided in the EM Resources and Training LibGuide.

Sample preparation

Every sample is different.Every sample is different. Please consult with the EM Staff before a project is started. You can save time and money by providing references or reprints relating to EM projects similar to the one being proposed.
The EM staff provides fixative (1/2 Karnovsky's) and will handle the specimen preparation after fixation has taken place. Samples for electron microscopy should not normally be prepared with fixatives used for light microscopy, such as formalin. See our overview of a conventional specimen protocol for TEM.

Samples of our EM Tomography work

Obtaining your Data

Images resulting from microscopy can be saved in your Homelink account, on the server maintained by Center's IT department. If you do not have a Homelink account, please submit see Data Storage and Archiving.

Immunoelectron Microscopy

This service is offered on a consultant basis. If you wish to explore this option, please contact emsr@fredhutch.org to discuss your needs.

This technique uses antibodies to detect the intracellular location of structures of particular proteins by electron microscopy. Ultra thin sections are labeled with antibodies against the required antigen and then labeled with gold particles. Gold particles of different diameters enable two or more proteins to be studied. (View chapter 4 in this excellent resource on Applications of Immunocytochemistry for more information.)

The EM Resource offers post-embedding immunogold labeling of samples in LRW resin. The investigator must supply the primary and secondary antibodies. The investigator should do immunolabeling at the fluorescent light microscopy level before attempting it at the EM level. (The Pelco low temperature UV polymerization system is also available for embedding of LRW resin.)

The EM Resource also offers cryo equipment for immunolabeling. This consists of the High Pressure Freezing (HPF) apparatus, freeze substitution system, and low temperature cryochamber for low temperature sectioning. The Leica EM PACT2 is a high pressure freezing machine that is designed to freeze samples up to 200-300 um into the specimen without significant ice crystal damage. HPF provides for better antigen retention for immunolabeling.

HPF is followed by freeze substitution or cryo-ultramicrotomy. The Leica EM AFS2 is capable of freeze substitution, progressive lowering of temperature techniques, and low temperature embedding and polymerization of resins for improved preservation of ultra structure and antigenicity. Cryo-ultramicrotomy is performed by the Leica EM FC6 cryochamber. It is designed for low temperature sectioning of samples at temperatures from -15 to -185 degrees C. The Tokuyasu technique for immunolabeling can then be carried out on the cut sections.

Cryotechniques (EM)

Electron microscopy owns several pieces of Leica cryo equipment including a High Pressure Freezing apparatus, freeze substitution system, and low temperature cryochamber. This equipment produces superior cellular preservation for morphological studies and better antigen retention for immunolabeling work.

Automatic Freeze substitution (AFS)

Automatic Freee Substitution (AFS) chamber

The Leica EM AFS2 is capable of freeze substitution, progressive lowering of temperature techniques, and low temperature embedding and polymerization of resins for improved preservation of ultra structure and antigenicity.

Cryo-microtomy

Cryo-ultramicrotomy is performed by the Leica EM FC6 cryochamber. It is designed for low temperature sectioning of samples at temperatures from -15 to -185 degrees C. More information on the equipment is found at the Leica web site.

Leica VT1200 S Fully automated vibrating blade microtome:

The Leica VT1200 S fully automated Vibratome uses a vibrating razor blade to cut sections from 1-999 microns under physiological conditions without freezing or embedding, and thus ultrastructure is well preserved. The tissue can be fixed or fresh when using a vibratome. The vibratome maintains cell morphology, enzyme activity and cell activity. The microtome is well used for immunocytochemistry and histoenzymology.

Ultimately, vibratome tissue sections can be trimmed down and used for high pressure freezing.

Located in the South Lake Union neighborhood of Seattle, the facility serves Fred Hutchinson, Cancer Consortium, and all other Seattle area researchers.