TRANSMISSION ELECTRONMICROSCOPE The first TEM was built by Max Knoll and ErnstRuska in 1931, with this group developing thefirst TEM with resolution greater than that oflight in 1933 and the first commercial TEM in1939. It is capable of imaging at a significantlyhigher resolution than light microscopes, owingto the small wavelength of electrons. TEM is far more useful for medical investigationsthan SEM TEM forms a major analysis method in a range ofscientific fields, in both physical and biologicalsciences. Cancer research, virology, materials science aswell as pollution , nanotechnology,and semiconductor research.
Electrons transmitted through the specimen arefocused and the image magnified by usingelectromagnetic 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
PARTS OF TEMVacuum system To increase the mean free path of the electrongas interaction allowance for the voltage difference between thecathode and the ground without generating anarc, and secondly to reduce the collisionfrequency of electrons with gas atoms tonegligible levels
Specimen stage to allow for insertion of the specimen holder into thevacuum with minimal increase in pressure in otherareas of the microscopeElectron lens are designed to act in a manner emulating that of anoptical lens, by focusing parallel rays at some constantfocal length
Apertures annular metallic plates, through which electronsthat are further than a fixed distance fromthe optic axis may be excluded
HOW TO VIEW SLIDES UNDERTRANSMISSION ELECTRONMICROSCOPE The imaging systems of TEM consist of a phosphorscreen, which may be made of fine (10–100 μm)particulate zinc sulphide, for direct observation by theoperator. Optionally, an image recording system suchas film based or doped YAG screen coupledCCDs.Typically these devices can be removed orinserted into the beam path by the operator asrequired.
OPTICScondensor lenses responsible for primary beam formationobjective lense focus the beam that comes through the sampleitselfprojector lenses used to expand the beam onto the phosphorscreen or other imaging device, such as film.
PRINCIPLES OF TEM Illumination - Source is a beam of high velocityelectrons accelerated under vacuum, focused bycondenser lens (electromagnetic bending ofelectron beam) onto specimen. Image formation - Loss and scattering ofelectrons by individual parts of the specimen.Emergent electron beam is focused by objectivelens. Final image forms on a fluorescent screenfor 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, stainedwith toluidine blue Selection of sample blocks Ultrathin sections at 0.1 micron thick, stainedwith lead citrate and uranium acetate
HOW ARE ELECTRONS EXCITED When an electron beam passes through a thin-section specimen of a material, electrons arescattered.
ADVANTAGES OF TEM high magnification at high resolution technique largely standardized some ultrastructural features are highly specificfor certain cell types or diseases
DISADVANTAGES OF TEM equipment is expensive procedures time consuming (staff costly) small samples lead to possible sampling errorand misinterpretation optimum tissue preservation requires specialfixative and processing much experience is needed for interpreting theresults Time consuming Works in the dark Photography required