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Electron microscopy

Electron microscopy is the process of greatly magnifying tiny particles in order to see and analyse them. The use of electrons allows 10,000x plus magnification, which is not possible using light microscopy, and atomic resolution. Electron microscopy can yield the following information:

  • Topography: the surface features of an object or "how it looks"
  • Morphology: the shape and size of the particles making up the object
  • Composition: the elements and compounds that the object is composed of and the relative amounts of them 

In particular, the use of colloidal gold provides analytical structural data of the specimen.

Equipment

LBIC has two electron microscopes both placed at Biomedicinskt Centrum (BMC), floor C11

Transmission electron microscope

Transmission Electron Microscope: FEI Tecnai Biotwin 120kv

Standard embedding

The aim of the embedding procedure is to produce blocks that are suitable for ultra thin sectioning and still preserve the fine structure with as little alteration as possible. The procedure starts with fixation of the specimen, usually a double fixation with an aldehyde and osmium tetroxide in buffer. After washing, with buffer, the specimens are dehydrated in a graded series of ethanol or acetone. The solvent is then replaced by a monomeric resin (usually an epoxy resin) which is polymerized into a hard block that can be sectioned with an ultra microtome into 50- 80 nm slices. This procedure is suitable for tissue and large particles (usually bacteria and organelles.


Embedding for immunolabeling

Antigens in an embedded specimen can often be localized with a complex of colloidal gold and antibody if the antigenesity is preserved, since the structures are protruding out of the section surface. To preserve the antigenesity of the specimen the above procedure is altered somewhat. The fixation is milder, only aldehyde, and the epoxy resin is replaced with a methacrylate. The whole procedure is usually performed at low temperature, -50 – 0 degrees.

 
 

Negative staining

Suspensions of small particles, molecules ore viruses, are prepared with negative staining. The principle is that the specimen is adsorbed on to a support film and a heavy metal salt solution is applied, which dries down around the specimen. As a result, the specimen remains electron-translucent while its immediate surrounding does not.

Fibrills

Click here for larger image

 

Scanning electron microscope

Scanning electron microscopy (SEM) depends on a focused electron beam scanned over the surface of a sample. When the beam hits the sample surface, electrons are emitted from a very shallow area around the beam impact point. SEM provides detailed topographic images of the surfaces of cells, tissue and whole organisms that is not possible with TEM.

  • SEM (Jeol JSM-7800F)

Our system is equipped with secondary and backscatter detectors for both high and low vacuum work. This makes it possible to study both the topography (secondary electrons) and detect contrast between areas with different atomic compositions (backscatter electrons), for example, the surface distribution of electron dense immuno-labels (nanogold or Quantum dots). Working under low vacuum allows non-conductive samples (e.g. biological samples) to be examined with minimal preparation work. The microscope is also equipped with a retractable STEM detector (Scanning Transmission Electron Microscopy) that allows us to image thin, electron transparent samples with sub nanometer resolution.

Eye of a Drosophila
Eye of a Drosophila
Hanging yest
Hanging yest