Which reason best explains why dead specimens must be used with transmission electron microscopes?

A.Electrons pass over the specimen.

B.The lights that are used are harmful to the specimens.

C.Specimens are placed in a vacuum.

D.The image that is produced is two dimensional.

Diagram of a transmission electron microscope
Diagram of a transmission electron microscope

The correct answer is C. Specimens are placed in a vacuum.

Electron microscopy works by using electrons rather than light which is also the reason a vacuum is needed. Without a vacuum, the electrons that are aimed at a specimen would most likely be deflected as they hit air particles. This would be a problem since the electrons need to make contact with the specimen in order for an image to be formed.

Living tissue would not survive the process of electron microscopy. The preparation of the tissue uses harsh chemicals that would kill any living cells and they would likely also not survive in a vacuum environment.

In addition, the preparation time is much longer and often more complicated than is the case with light microscopy. There are also concerns about how much distortion may be occurring during the process of fixing the cells and tissues. Scientists also worry about distortions when staining tissues for light microscopy.

A transmission electron microscope (TEM) is used to look inside of structures and cells. In comparison, a scanning electron microscope (SEM) is used to view the surface of things. Both these methods of microscopy require a careful preparation of specimens.

Electron microscopy

The electron microscope was invented many years after the light microscope was invented. It was really created to overcome some of the limitations that were evident with microscopy using light. For one thing, the light microscope was very limited when it came to resolution and magnification.

This was due to the wavelength of light particles which imposed a limit on resolving power. Electrons have a considerably shorter wavelength compared with light and thus an instrument using electrons would give better resolution.

The electron microscope uses a vacuum because electrons would not be able to move very far in the presence of air. The electrons would likely change direction and not reach the specimen if there were air particles present.

To achieve a vacuum a series of pumps are used which function to remove air from inside the machine. A system of airlocks is used to maintain the integrity of the vacuum when a specimen is placed inside the machine.

History

Max Knoll and Ernst Ruska developed the first electron microscope in 1931, and the first commercial microscope became available in 1938. The EM was first used to look at particles in the material sciences. It wasn’t until the 1940s that improved techniques in sectioning allowed the use of such microscopes for viewing biology specimens.

The most important advance made here was in the ability to make thin sections using a knife-edge microtome. Since TEM was aimed at looking through a section it meant that tissue had to be extremely thin.

It was only in the 1960s that the first scanning electron microscope was made. Starting at about this time, there were also improvements in the source of electrons and the invention of stage tilting.

This meant that brighter images could be created and specimens could be tilted at different angles to provide different views.

Transmission electron microscopy

The TEM is a machine that is comprised of an electron gun and a way to produce and record an image. The electron beam is produced by the gun and it passes through a condenser that functions to focus the beam onto the specimen.

Various lenses help to focus the electrons and create an image. This image is formed on a fluorescent screen and then a system is in place to capture a photograph, an electron micrograph. In transmission electron microscopy the electrons pass through the specimen. This is different from scanning electron microscopes.

The image that is generated in all types of electron microscopy is monochromatic and today this can be further enhanced using computer software, and false colors can be used to emphasize particular features.

The magnification of TEM is usually between 1000 to 250,000 times.  Further magnification may be achieved using computer software.

Specimen preparation

In transmission electron microscopy specimen preparation can be time-consuming. Specimens are fixed and then usually dehydrated using chemicals such as acetone.

The objects are then embedded in blocks of resin in preparation for sectioning. They are cut using an ultramicrotome which usually uses a diamond knife.

The sections that are cut are very thin and in the order of 50 to 70nm thick. The process is done using a microscope and sectioned pieces are caught on a metal grid.

This is then stained using an electron-dense material. Heavy metals may be used to stain the grids with the specimen. Uranium and lead can be used as stains to allow contrast between structures when viewed by the electron microscope.

Scanning electron microscopy

The SEM works by scanning electrons over a specimen and generating an image. The electrons used in this instrument are relatively low energy and the beam hits the surface of the specimen. Backscattered electrons are then used to produce an image of the surface of the object of interest.

Larger specimens and thick specimens can be used in SEM which is not the case in TEM. Objects do however need to be coated with an electrically conductive coating.

This is usually done by sputter coating with a thin metal layer of gold particles. The layer is only from 50 to 100 angstroms in thickness in order to prevent a decrease in resolution.

It is much easier and quicker to prepare a specimen for scanning electron microscopy than for transmission electron microscopy. This is especially the case because specimens do not have to be sectioned. It is also much easier if you are working with larger specimens that you can see and easily handle.

References

  1. DC Joy, S Bradbury, BJ Ford (2018). Transmission Electron Microscopy. Retrieved from Encyclopedia Britannica.
  2. DC Joy, S Bradbury, BJ Ford (2018). Electron Microscope. Retrieved from Encyclopedia Britannica.
  3. DC Joy, S Bradbury, BJ Ford (2018). Scanning Electron Microscopy. Retrieved from Encyclopedia Britannica.
  4. AM Glauert, PR Lewis (1998). Biological specimen preparation for transmission electron microscopy. Princeton: USA, Princeton University Press.
  5. RH Raven, RF Evert, SE Eichhorn (1987). Biology of plants, 4th edition. New York: USA, Worth Publishers.

LEAVE A REPLY

Please enter your comment!
Please enter your name here