Describe the principles and limitations of transmission and scanning electron microscopes. Specific reference should be made to magnification and resolution

Principles and Limitations of light microscopy

    Light microscopes function by focussing a beam of light on the object to be examined.The beam passes through a series of lenses after passing through the specimen.The condenser lens focuses the light onto the specimen for maximum illumination.The lower objective lens magnifies the object ,and the image from this lens is further enlarged when viewed through the upper eyepiece lens.The beam from the light source directly enters the observer’s eye.It has a few limitations.Material to be examined by light microscopy must be sufficiently transparent and thin enough for light to pass through .They are cheap,portable and easy to handle.However they cannot resolve anything that is less than 0.2 micro metre apart This limit is due to the wavelength of light.For this reason they can’t examine minute organisms like viruses nor can they readily allow scientists to examine individual tiny parts of cells in detail.It has low resolution(200nm) and low magnification.(X1500).Howver light microscopes only allow us to determine the shape of whole cells or large organelles.It does not let us see smaller organelles.Those which are visible lack clarity (appear fuzzy).Hence the magnification is less important than the resolution.The low resolution of light microscopes is due to the low wavelength of light (500nm)

Principles and Limitations of electron microscopy

The other type of microscope is the electron microscope (which consists of transmission electron microscope and scanning electron microscope).It works by focussing a beam of electrons at the specimen.The electrons have a higher wavelength than photons of light hence the resolving power is greater.The beam of electrons are produced by a heated filament ,focussed on the specimen by the condenser lens and the image then magnified by the objective and projector lenses.The beam can be bent and focussed by electromagnetic lenses,in the same way the glass lenses are used in a light microscope.Unlike light microscopes ,the electron microscopes,have a high resolution(0.1 nm) and high magnification(1000000X) .The image formed by the electron microscope is projected onto a cathode ray tube so that it is visible to the operator ,looking rather like a black and white TV screen.

The second type is the scanning electron microscope.They examine the surface of specimens.The surface is coated with a film of gold and palladium.A fine beam of electrons is passed backwards and forwards over the specimen.and the electrons that are reflected from it are collected and amplified to form an image on a cathode ray tube.Although the scanning electron microscope gives a much lower resolution(5-20nm) than the transmission electron microscope they can investigate a greater depth of field which is not possible in light microscopes.However they are much more expensive to set up.

The most serious limitation of electron microscopy is the necessity of vacuum and metallic surfaces ,meaning only dead specimens can be inspected.Consequently the specimen must be dehydrated .Another big problem is that the specimen must be subjected to severe treatment involving corrosive chemicals and physical shock.This may alter the actual appearance of the cells.Any deviation from the real appearance of cells is known as an artefact.There could be a deviation in the shape or structure.Sometimes new structures could be formed.Hence it is very difficult to tell whether  the resulting micrographs are true representatives of the original cell structure.

Electron microscopes are very expensive pieces of equipment .They are large,complex and have to be maintained under controlled conditions ,such as constant temperature,as well as needing expensive pumps to maintain their internal vacuum.Their use is normally limited to universities and other research establishments.

Conclusion

Electron microscopes have been far more successful in determining cell ultrastructure compared to light microscopes.The high magnification and high resolution enables us to see cells and organelles much bigger and in much more detail.It has brought about the complete reappraisal of the microanatomy of biological tissues,organisms and cells.In early days of it’s applications to biological materials,it was a tool of anatomists and histologists.The detailed knowledge of internal organisation of organelles has helped in our understanding of how they work.The fact that electron micrographs appear in many research papers and books in cell biology and anatomy emphasize the importance of electron microscopy to the biologists not only in the present times but also in future.Today electron microscopy is widely used in metallurgy,biology,material science,physics,chemistry and many other biological fields.It has been an integral part in the understanding of the complexities of cellular structure,the fine structure of metals and crystalline materials as well as numerous other areas of the microscopic world.Although new inventions like the laser microscope may seem to put the use of electron microscopes under threat they will always play an integral part in determining the ultrastructure of cells.