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Electron Microscopy Core

Electron Microscopy Core
The Electron Microscopy Core is available to investigators wishing to define ultrastructural details in their experimental systems. The EM Core assists with all aspects of studies brought to the lab including training, experimental design and interpretation of results.
 
A variety of preparation methods are available for transmission and scanning electron microscopy including ultra-thin sections of conventionally fixed samples, immunogold labeling, plunge-freezing cryo-EM and tomography.
 
Services:
Thin sectioning of resin-embedded specimens
High pressure freezing/freeze substitution
Cryo-EM of specimens plunge-frozen with FEIVitrobot
Negative Staining
Immunogold labeling of Lowicryl-embedded sections
Scanning electron microscopy
Atomic force microscopy
Tomography
Single particle analysis
 

Abstract for Grants

The Electron Microscopy Core Facility is available to investigators wishing to define ultrastructural details in their experimental systems.

The electron microscopes: 1) the transmission electron microscope is a FEI Tecnai 12 Twin is equipped with a Gatan 894 Ultrascan 1000 CCD camera, cryo-stage/box, and tomography holder. In addition to traditional images, single-tilt tomography series and low-dose exposure images of specimens frozen in vitreous ice can be collected; 2) the scanning electron microscope is a FEI Quanta 200 with environmental SEM capabilities. Both microscopes are user-friendly and fully digital microscopes. They can be operated by investigators following brief training.

Sample preparation: In addition to providing conventional ultra-thin resin sections, the facility provides ultra-thin sections of samples prepared by high pressure freezing in a LEICA EM PACT2 high pressure freezer then fixed and resin infiltrated at low temperature in a LEICA EM Automatic Freeze Substitution System. Specimens prepared in this manner are suitable for immunogold labeling. Immunogold labeling is available. A fully automated vitrification device, the FEI Vitrobot Mark IV, for plunge-freezing of aqueous (colloidal) suspensions is available for preparing samples for particle analysis. Other forms of sample preparation include negative staining and shadow replicas. Work stations and software for particle analysis and tomographic reconstruction and visualization are available.
 
Financial Support
Please acknowledge these funding sources when publishing work derived from the EM Core:
 
  • TEM -ONR N00014-02-1 0958
  • SEM-DOD 1435-04-03GT-73134
  • Cryo-Ultra Microtome -NSF DBI-9970143
  • Vitrobot & Cryo-Stage -San Diego Construction Industries
  • High Pressure Freezer and Freeze Substitution System - NIH 1S10RR025569-01

Equipment

1. FEI Tecnai Twin 120kV Transmission Electron Microscope (TEM) equipped with:
 
  • Gatan Ultrascan 1000 2K CCD camera with ultra sensitive phosphor scintillator
  • 70° Tilt Cryo-transfer Holder Tomography enables frozen suspensions or sections to be loaded into the TEM holder and transferred frost-free at temperatures below -170 °C
  • Software and hardware needed for acquiring and aligning tomography data for subsequent tomographic reconstruction of tilt series images.
 
One of three specialized microscopes in the core, this instrument is used to examine specimens prepared by negative staining, conventional embedding, and cryo-specimens.  The tilting stage provides a means for collecting tomographic images.
 
 
FEI Tecnai 12 Twin Transmission electron microscope (TEM) allows direct visualization of the molecular complexes within cells and the structure of individual macromolecules.
 
 
Gatan sample holder and pumping station are used to introduce frozen specimens into the TEM.
 
2. FEI Quanta 200 Scanning Electron Microscope (SEM) is a versatile, high-performance scanning electron microscope with three operation modes (high vacuum, low vacuum and ESEM) equipped with a motorized stage and providing secondary and backscatter electron detection.
 
 
Core user is operating this easy to use SEM to define the surface structure of cells differentiated in culture.  This is only one application for which the SEM is well suited.
 
3. Bruker Multimode 8 Scanning Probe Microscope (SPM).  This microscope, often described as an atomic force microscope, provides a means for imaging the surfaces of specimens using a physical probe.  The Multimode 8 performs all major SPM imaging techniques.  These include contact and non-contact atomic force, lateral force, Tapping Mode (air), magnetic force, electric force microscopy and ScanAsyst (Peak Force Tapping) techniques.  A cantilever holder is also available for scanning in fluid in Tapping and force modulation modes.
 
 
4. FEI Vitrobot Mark IV is a fully automated vitrification device for plunge-freezing of aqueous (colloidal) suspensions.
 
 
This instrument provides a powerful means for determining the 3D structure of macromolecules and macromolecular complexes at sub-nanometer resolution.  Single-particle analysis or EM reconstruction often partners nicely with X-ray crystallography and NMR spectrometry to provide a more complete understanding of molecular structures.
 
5.  Leica EM PACT2 High Pressure Freezer with Rapid Transfer System (RTS) provides the preparation of samples for subsequent techniques.  Also available is a Leica Microbiopsy Transfer System (not shown) which provides the means for rapidly collecting microbiopsy specimens and rapidly e microbiopsies (0.3x0.6x1.2mm in size) from donor to the high pressure freezer for freezing within 30-60 seconds.
 
Leica EM PACT2 high pressure freezer equipped with RTS allows ultra-rapid freezing of specimens.   High pressure freezing (hpf) is ultra-rapid and the best way to preserve the native structure of cells and molecules.  Antigenic sites in specimens are better preserved with hpf opening greater possibilities for localization of molecules of interest within cells.
 
6. Leica EM Automatic Freeze Substitution System is the follow-on procedure for high pressure freezing.
 
In the Leica EM automatic freeze substitution system specimens are fixed and resin-infiltrated at low temperature providing specimens with high quality structural preservation and specimens suitable for immunolabeling for electron microscopy.
 
7. Leica Ultracut UCT Microtome for ultrathin sectioning.FCS attachment allows low temperature ultrathin sectioning of biological samples at temperatures as low as -185°C.
 
This versatile ultramicrotome is the workhorse instrument in the EM Core that provides sections for TEM from specimens prepared by conventional and cryo methods.  Specimens can also be frozen and embedded is sucrose for sectioning and immunogold labeling following the method of Tokuyasu.  Specimens are typically cut using a diamond knife.
 
8. Leica VT1000S Vibrating-blade Microtome for sectioning of specimens in medicine, biology and industry, especially for sectioning fixed or fresh tissue immersed in buffer solution.
 
Like a microtome, a vibratome provides tissue sections, but in a vibratome a vibrating razor blade is used for cutting.  Tissues sections from the vibratome are sometimes used in the EM Core in immunolabeling procedures.
 
9. CRESSINGTON 308 COATING SYSTEM
 
Cressington coating system provides thermal evaporation of various metals, sputtering (often of gold/palladium SEM samples, carbon evaporation and glow discharge.  Samples for SEM and grids for TEM applications are often modified in the Cressington in preparation for microscopy.

Links to Resources

Pricing

Prices and availability vary. Please Contact Us  for current information.
 
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Services

Ultra-thin Section from Resin Embedded Samples for TEM
 
The most commonly used approach to obtaining ultrastructural details of cells and tissues.
 
Cryo-EM of Plunge-Frozen Samples
 
Frozen-hydrated liposomes.
 
Toluidine Blue Stained 1-um-thick Section
 
One-micron sections provide an overview as illustrated here with a colony of pancreatic stem cells.
 
Negative Staining
 
Negative stained exosomes.
 
High Pressure Freezing and Freeze Substitution
 
Compared to harsh chemical fixation, HPF-FS provides superior preservation of cellular structure.
 
Immuno-gold labeling on specimen prepared by high pressure freezing/freeze substitution
 
Indirect immunolabeling with antibody conjugated 10-nm gold particles was used to monitor uptake of FITC-CpG-siRNA.
 
Immunogold Labeling SEM
 
In the SEM the immunogold particles are bright. Here immunogold labeling reveals CEACAM-1 associated with microvilli on the cell surface.
 
Visualization of Nucleic Acid
 
Rotary shadowing of spread DNA
 
Scanning Electron Microscopy
 
Surface structure of a monocyte
 
Atomic Force Microscopy
 
Structure of mRNA molecules (~1000 nt)

Transmission Electron Microscopy
 
Ultra-thin section of avian LMH cells prepared with membrane-enhancing reagents.
 
Transmission Electron Microscopy
 
Ultra-thin section of an avian LMH cell. Sample preparation was done on an adhesive slide using only ~2000 cells. This method can be used to image small numbers of cells (for example, directly from flow cytometry).
 
Transmission Electron Microscopy
 
Single wall carbon nanotubes with a diameter of about 1.4 nm resolved in the Tecnai 12 transmission electron microscope.

Electron Microscopy Team

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