Electron Microscope Research Core


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Director

Karen L. de Mesy Bentley, M.S.

Mission Statement 

The principal mission of this Electron Microscope Research Core (EM Core) is to provide University of Rochester researchers support in high magnification image analysis of cells and tissue in the fields of Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM) and combined Scanning/Transmission EM (STEM).  The EM Core operates as a “fee for service” support laboratory with a “wet lab” for specimen processing, embedding, sectioning and staining.  Educational seminars and workshops on the principles of EM and interpretation of cellular and tissue ultrastructure are given yearly.

Core Services

An initial consultation with the Director who has 27 years of teaching and diagnostic experience in Ultrastructural Pathology and Immunoelectron Microscopy is required prior to submitting research specimens for electron microscopy processing.  This consultation defines the best and most cost effective experimental strategy and methods to achieve a researcher’s goals.  Once the EM Core receives the fixed tissue or cells they are generally processed as follows:  post-fixed in osmium tetroxide, processed through a graded series of alcohols, infiltrated into liquid epoxy resin (Epon/Araldite or Spurr), embedded into molds and polymerized at 70oC.  The epoxy blocks are cut by a technician using an ultramicrotome to cut semi-thin (1.0-2.0 um) sections with a glass knife which are stained with Toluidine Blue. Examination of sections by light microscopy are necessary to determine the appropriate area to be trimmed and thin (70nm) sectioned for electron microscopy.  A diamond knife with attached water filled boat is substituted for the glass one and thin sections are cut and placed onto grids for subsequent staining with heavy metals such as uranyl acetate and lead citrate.  Researchers/graduate students are present when the grids are examined under the TEM with the presence of the EM Core Director who helps guide and interpret the ultrastructural morphology or immunolabeling.  Recently, Ms. Bentley has acquired funds from the Department of Defense/Air Force to purchase a new Hitachi 7650 TEM with a Cantega 11 megapixel digital camera and Gatan software for morphometric analysis.  Additional features of this microscope include the ability to perform “darkfield” STEM and Electron Dispersive X-ray Spectroscopy (EDS) for elemental analysis.  Both of these features will provide “high tech” support to established and future funded research projects involving nanoparticles and nanotechnology.  Scanning electron microscopy service is also available and the Core technical staff utilizes the River Campus/Optics Department’s LEO 982 SEM for these types of requests.

Specialized services include:

  • Detection of antigen/antibody reactions with Immunoelectron Microscopy embedding of cells or tissue for either pre-embedding or post-embedding labeling of the antigen/antibody complex using biotin-HRP, or gold conjugated markers to precisely localize antigens in a subcellular compartment or organelles.
  • Cell Culture “Pop-off” technique to remove and examine ultrastructurally cells grown on glass slides or coverslips.  An additional “Pop-off” technique involves the removal of  a rare cell(s) from a 2 micron epoxy section adhered to a glass slide previously made only for light microscopy
  • Viral Negative staining using phosphotungstic acid or uranyl acetate to identify viral particles or viral-like particles (VLPs) placed onto formvar coated grids.
  • Organelle Subcellular fraction identification of RER, SER, Golgi, mitochondria, peroxisomes, melanosomes and/or immunolabeling of organelles using a pelleting/embedding  technique.
  • Morphometric analysis of various subcellular structures such as, muscle fibers, viral particles, bacteria, collagen and amyloid fibrils.  Nanoparticles are measured for size verification and quality control of those projects involving Nanotoxicology/Nanotechnology.
  • Nanoparticle Identification Using Darkfield STEM is a contrast mode allowing positioning of a fine probe of high energy electrons over a thin section on a grid.  This causes an “edge effect” and nanoparticles appear as bright objects against the “darkfield” making it extremely useful for localization of very small (<20nm) “suspect” nanoparticles translocated into cells or tissue.
  • Elemental Analysis using EDS , an attached detector cooled with liquid nitrogen and used in biological studies to provide exact elemental identification or confirmation of above mentioned “suspect” nanoparticles identified visually by Brightfield TEM or detected using Darkfield STEM.

Specific Instrumentation/Equipment:

  • Transmission Electron Microscope Hitachi 7650 with EDS (IF Instruments) and STEM (scanning/transmission unit for darkfield EM), (will be 100% dedicated EM Core microscope, funded for purchase Spring 2007).
  • Ultramicrotomes (2) LKB III (1971) & Reichert UltraCut III (1986) for sectioning epoxy embedded tissue blocks. 
  • Critical Point Dryer (1991), BALZERS, CPD 030 used for specimen drying process prior to SEM examination.
  • Light Microscopes (2), Olympus BX-40(1997 & BH-2(1984), Double Headed.
  • Dissecting Microscope, Baush & Lomb(1980).
  • Diamond knives (4), Diatome, for thin-sectioning epoxy blocks.

Faculty User Group

This core does not have a  formal faculty user/advisory group.  Major faculty users include: Brad Berk, Nick Crispe, Jack Finkelstein, Joe Miano and Gunter Oberdoerster.