How the T.E.M. works?
The T.E.M. works on the same basic principles as a light microscope does, but instead of the T.E.M. using light, it uses beams of electrons that act as a “light source”, making the wavelength lower to get a thousand time better resolution. This makes the study of the object easier to see, as you can observe the object to the nearest angstrom (1 angstrom = 10-10 m). This gives the object in great detail; all these possibilities for high magnification have made the T.E.M. a valuable tool in medical, biological and material research.
What do we use the T.E.M. for?
- The T.E.M. is used for looking at the structure within the specimen in fine detail, which is also known as ultrastructure.
- The ability to see the ultrastructure enables us to see the cellular organelles in detail.
- We can quantify changes in the ultrastructure within the cells (for example after a certain treatment). This method is called morphometric analysis.
- Morphometric analysis is carried out by taking images with a digital camera. These images are then analysed on a computer to measure the changes of the organelles within a cell.
- The T.E.M. can also be used to see specific proteins in a cell. This is done by a method called immunogold labelling.
The scanning electron microscopes (S.E.M.)
The preparation for the S.E.M.
Firstly the specimen has to be fixed; it’s then dehydrated with an alcohol (e.g. ethanol). This is done because the electron microscope operates under a high vacuum, and it is therefore that water is not allowed to be present in the microscope. The specimen is then dried in a specialised pressure vessel; this involves the replacing of ethanol with liquid carbon dioxide, which is ‘sublimed’ instantaneously by heating. The drying part produces a turgid specimen, which is then stuck on a special S.E.M. sample holder and coated with a thin layer of gold using a coating device. This gives the sample a conductive layer and prevents the S.E.M. from charging and it is then the specimen is ready for viewing.
How the S.E.M. works?
The S.E.M. also uses electron beams rather than light, however these electron beams are finally focused and scan to and fro across the specimen. The electrons that reflected from the surface are collected to form the basis of a television like image on a cathode ray tube. The advantage of using this technique is so that the surface features are shown in great depth giving a 3D effect. Although the resolution is poorer, larger specimen can be seen. The S.E.M. also has a vacuum, which acts as a water seal, as electrons have limited energy, anything like gas or water would disrupt the passage of the electrons.
What do we use the S.E.M. for?
- The S.E.M. is used for looking at different specimens surface features in detail.
- The samples that are commonly looked in labs are Drosophila’s (fruit flies), feather mites, the cochlea (inner part of the ear) and floral parts.
- The S.E.M. can also be used to find out which elements are present within the specimen. This is done by using an elementary analysis detector, which can collect the x-rays given off by a specimen when the electron beam hits it.
- The useful feature to this is that the x-rays that are given off can not only be collected, but also be used to identify which elements have been emitted.
The Freeze Fracture Technique.
This technique is used to obtain a detail structure of the cell; this technique is done by firstly flooding the sample with glycerol and water, and then rapidly freeze the sample. This is done to preserve the cell features and also at the same time to prevent any damages or enzyme action to the sample.
Next the sample is fractured and a carbon template is made out of it using a microtome knife. Cells will then split along the path of least resistance, forming what is known as the fracture plane. The things that can cause the changes to the fracture plane are, elevation, depression, and ridges, which are cellular organelles. A heavy metal is then placed on the fracture surface at an angle, so that a shadow is produced on the surface, which enhances the contours. Finally a solid uniform layer of carbon covers the sample over the top, producing a carbon replica template of the cellular constituents, which is viewed under the microscope. The remains of the sample are no longer needed and are thrown away. Below are some pictures of different cells or organelles.
