The Electron and Confocal Core Facility provides a broad spectrum of techniques for examination of specimens by Transmission Electron Microscopy (TEM) or Laser Scanning Confocal Microscopy.
Transmission Electron Microscopy
Several techniques can be utilized to examine the ultrastructure of cells, tissues and macromolecules using TEM:
- Standard TEM – This method involves preserving a specimen in a fixative (usually glutaraldehyde, formaldehyde or a combination of both). The specimen is then subjected to several buffer washes, stained with heavy metal solutions, dehydrated in ethanol and embedded in epoxy resin. This process typically requires two days. Specimens are then sectioned on an ultramicrotome, placed on plastic coated EM grids and stained with additional heavy metal solutions. The specimen is then imaged by placing the grid in a specimen holder that is inserted into the main column of the TEM. A beam of electrons is passed through the specimen and imaged on a fluorescent screen, which is then captured by a high resolution digital camera.
- Immunoelectron Microscopy – Preparation of specimens for immunoEM differs somewhat from standard TEM. In order to preserve immunogenicity of the sample, fixation is done with formaldehyde and a very small percentage of glutaraldehyde. The specimen is then dehydrated in ethanol and embedded in LR white, which is a hydrophilic resin. Once the resin is polymerized, the specimen is sectioned, placed on nickel grids and labeled with the primary antibody of interest followed by conjugation to a secondary antibody that is attached to a gold particle. The specimen is then stained with a heavy metal solution and imaged in the TEM. The gold particles are visible as small black dots in the TEM.
- Negative staining – This method is used to image whole mount isolated macromolecules such as cytoskeleton proteins, vesicles and virus particles. The solution containing the specimen is placed on a plastic coated grid and the macromolecules are allowed to settle on the surface of the grid. The grid is then rinsed in water and incubated with uranyl acetate. The uranyl acetate is then removed from the grid, which is allowed to dry before being imaged in the TEM.
Laser Scanning Confocal Microscopy
Laser Scanning Confocal Microscopy is used to provide high resolution fluorescent images of whole mount or sectioned specimens. Confocal microscopes have several advantages over widefield epi-fluorescence microscopy. In traditional widefield epi-fluorescence microscopy, the specimen is subjected to intense illumination from a white light source, which is then viewed directly through the eyepieces or projected to a camera. The resulting secondary fluorescence emitted from the specimen frequently occurs through an excited volume, which obscures resolution of fine detail in the objective plane. Confocal microscopy significantly improves resolution of a specimen by utilizing lasers of a single wavelength to excite the fluorescent molecules thereby reducing background fluorescence and improving signal-to-noise. In addition, the excited fluorescent light is directed through a pinhole aperture that excludes out of focus light. Finally, the confocal microscope can produce three dimensional renderings of a specimen through optical sectioning using a stepper motor to acquire sequential images through the Z axis of a specimen. The resulting images are then stacked together to form a 3D image.