TRANSMISSION ELECTRON MICROSCOPE

TRANSMISSION ELECTRON MICROSCOPE The first TEM was built by Max Knoll and Ernst

Ruska in 1931, with this group developing the first TEM with resolution greater than that of light in 1933 and the first commercial TEM in 1939.

It is capable of imaging at a significantly higher resolution than light microscopes, owing to the small wavelength of electrons.

TEM is far more useful for medical investigations than SEM

TEM forms a major analysis method in a range of scientific fields, in both physical and biological sciences. 

Cancer research, virology, materials science as well as pollution , nanotechnology, and semiconductor research.

Electrons transmitted through the specimen are focused and the image magnified by using electromagnetic lenses (rather than glass lenses) to bend the trajectories of the charged electrons.

Image is focused onto a viewing screen or film.

Used to study internal cellular ultrastructure

STRUCTURE

STRUCTURE

PARTS OF TEM

Vacuum system To increase the mean free path of the

electron gas interaction allowance for the voltage difference between

the cathode and the ground without generating an arc, and secondly to reduce the collision frequency of electrons with gas atoms to negligible levels

Specimen stage to allow for insertion of the specimen holder into

the vacuum with minimal increase in pressure in other areas of the microscope

Electron lens are designed to act in a manner emulating that of

an optical lens, by focusing parallel rays at some constant focal length

Apertures annular metallic plates, through which

electrons that are further than a fixed distance from the optic axis may be excluded

HOW TO VIEW SLIDES UNDER TRANSMISSION ELECTRON MICROSCOPE The imaging systems of TEM consist of a phosphor

screen, which may be made of fine (10–100 μm) particulate zinc sulphide, for direct observation by the operator. Optionally, an image recording system such as film based or doped YAG screen coupled CCDs.Typically these devices can be removed or inserted into the beam path by the operator as required.

OPTICS

condensor lenses responsible for primary beam formationobjective lense focus the beam that comes through the

sample itselfprojector lenses used to expand the beam onto the phosphor

screen or other imaging device, such as film.

PRINCIPLES OF TEM

Illumination - Source is a beam of high velocity electrons accelerated under vacuum, focused by condenser lens (electromagnetic bending of electron beam) onto specimen.

Image formation - Loss and scattering of electrons by individual parts of the specimen. Emergent electron beam is focused by objective lens. Final image forms on a fluorescent screen for viewing

Image capture – on negative or by digital camera

FIXATION OF TISSUES FOR EM

Must be prompt Cut to 1-2 mm cubes Use sharp razor blade, avoid crushing 2.5% glutaraldehyde for 4 to 12 hours Postfixation in 1% osmium tetroxide

TISSUE PREPARATION FOR TEM Dehydration in alcohol Embedding in resin Semithin sections cut at 0.5 micron thick,

stained with toluidine blue Selection of sample blocks Ultrathin sections at 0.1 micron thick, stained

with lead citrate and uranium acetate

SEMITHIN SECTIONSSTAINED WITH TOLUIDINE BLUE

ULTRATHIN SECTIONS ON GRID

HOW ARE ELECTRONS EXCITED

When an electron beam passes through a thin-section specimen of a material, electrons are scattered.

ADVANTAGES OF TEM

high magnification at high resolution

technique largely standardized some ultrastructural features are highly

specific for certain cell types or diseases

DISADVANTAGES OF TEM

equipment is expensive procedures time consuming (staff costly) small samples lead to possible sampling

error and misinterpretation optimum tissue preservation requires special

fixative and processing much experience is needed for interpreting

the results Time consuming Works in the dark Photography required