Histological Techniques - Electron Microscopy

So what is electron microscopy?. Electron microscopy involves a highly specialised machine called the transmission electron microscope (TEM). This is similar to a light microscope but there are several differences. For example instead of light, it uses electrons in a high voltage beam to focus the image. Also instead of glass it uses electromagnetic lenses to focuse the image. The transmission electron microscope settings complexity means it can only be altered by trained engineers. It operates using the same basic principles as a light microscope except using electrons in the form of a high voltage beam instead of light for visualisation.

To produce the image, electrons are emitted from the electron gun when a high voltage is passed through. The electron gun is normally fitted with a tungsten v-shaped wire filament which when heated in a vacuum, releases the free electrons required for the process. Surrounding the filament is the wehnet shield, the aperture used to focus and control the electrons emitted through the anode aperture and accelerated at between 30 -100 kV speed into the column.

Here the electromagnetic coils are used as lenses to focus, by altering the strength of the magnetic field. This involves the condenser lens being used to collect the electrons and determine the beam diameter (Spot size) on the specimen. After that the electrons that pass through the specimen enter the objective, intermediate and projector lenses to produce the image by focusing, magnifying and directing the beam onto the imaging screen. The subsequent image is produced by electrons either being absorbed by the specimen or being deflected at small angles and so helping to produce the final image, as several million electrons pass through the specimen to produce the final image.

Diagram 1 - How the transmission electron microscope works to produce images

Comparison of Light with Electron microscopes
Features		LIGHT MICROSCOPE		TRANSMISSION EM		SCANNING EM
Main function		Surface morphology and sections 1-10 microns		Sections 40 – 150 microns or small particles		Surface morphology can scan bigger specimens than TEM
Source of illumination		Light		High speed elctrons in vacuum		High speed elctrons in vacuum
Normal Resolution		200nm		0.2nm		3-6nm
Magnification range		10 – 1000X		500x-500kx		20x-250kx
Lens type		Glass		Electromagnetic		Electromagnetic
Image formation		On eye by lenses		On phosphorescent plate by lenses		On cathode ray tube by scanning device

In the laboratory, the most common diagnostic application of the transmission electron microscope is to assist in the diagnosis of renal biopsy diseases. This is used in cases where further analysis that can only be provided by electron microscopy use, to provide a definitive diagnosis eg Alport’s disease for example or thin membrane nephropathy.

Photo 1 – Renal Biopsy with electron Microscopy used

Electron microscopy can also be used for muscle biopsies. This is useful where the results will help to confirm a patient diagnosis for muscle disorders. This is able to differentiate between normal and abnormal muscle, as well as certain structures like nemaline rods and identify inclusions.

Photo 1 – Muscle biopsy with Electron microscopy used

Electron microscopy also has a role for the laboratory with nerve biopsies. This is in the investigation of peripheral neuropathies, although these are rarely seen in the laboratory.

Photo 2 – Electron Microscopy of Myelinated axon of nerve

 

The EM Epon processing Schedule, as shown below involves the use of Osmium Tetroxide. This is to fix the lipids as a secondary fixative. It also uses Uranyl Acetate to allow highlighting of the proteins and membranes for later on. It also requires complete dehydration of the tissue before Epon or epoxy resin can penetrate the tissue.

EPON OVERNIGHT processing schedule
Reagents					Time
Em Buffer					10 min
Em Buffer					10 min
Em Buffer					10 min
2% Osmium Tetroxide					1 hour
Distilled water					10 min
Distilled water					10 min
Distilled water					10 min
2% Aqeuous Uranyl Acetate					45 min
Distilled water					10 min
35% Alcohol					10 min
70% Alcohol					15 min
Spirit					15 min
Acetone (dried)					30 min
Acetone (dried)					30 min
Acetone (dried)					30 min
Acetone/Epon 1:1					1 hour
Acetone/Epon 1:3					2 hour
Epon					2 hour
Epon					6 hour
Total Time					16 hours 5 min