Sensitometry, Resolution and Contrast

1) Density Charachteristics of Emulsions

In film or paper, photons or electrons striking the emulsion will result in an increase on the optical density of the exposed portion of the emulsion. Under ideal conditions, an increase in exposure (intensity * time) will cause an increase in density.

Unfortunately, no ideal film or paper exists. In most cases, the curve looks similar to the one below.
At low exposures, no increase in density is seen. At high exposures, once maximum density is reached, no further response is seen with increased exposure. Between these two extremes is a straight line portion in which an increase in exposure results in an increase in density. This, obviously is the useful range of the emulsion.

2) Resoloution

If you increase the number of electrons hitting the negative you will increase the resolution of the negative. If the exposure is too short then resolution is lost in the negative. This is why the proper exposure is necessary at the TEM level.

3) Contrast

The contrast of an emulsion is its ability to render different amounts of exposure as different densities of silver. The contrast of an image can be affected by:

a.choice of emulsion
b.choice of developer
c.temperature of development
d.time of development

Some emulsions have inherent low, medium, or high contrast. An example of this is enlargement paper, which can be purchased in different grades (1,2,3,4,5,6) of increasing contrast (meaning a given negative printed on #5 paper will have more contrast thatn the same negative printed on #3 paper). This allows one to compensate for a negative with less than ideal contrast level; if a negative has little contrast, use a high contrast paper. An alternative to using different grades of paper is to use "polycontrast" paper in concert with polycontrast fibers. The paper consists of two different kinds of silver halide grains; high and low contrast. The filters are numberes 0 to 5 in one half steps increments. Using polycontrast paper with a #4 filter will give similar results to using a #4 graded paper. This is the most common system of contrast control used today.

A given emulsion can have contrast increased by increasing the developer action (stronger developer or higher temperatures) or the time of development. This result is due to the following important characteristics:

a.The areas of low exposure can be developed to completion quickly. The latent image is converted or fully developed in a short time.
b.Areas of high exposure may be partially developed, more developed, or fully developed with increasing time.
c.Thus in an image of 2 points, high and low exposure, the resultant difference in density (contrast) will be influenced by development time.

In this way, contrast of film or paper can be controlled to suit the exposure renge of the image. Low contrast images will be enhanced by high contrast film, paper, or developent while high contrast images can be "compressed" onto low contrast film or paper.

1) Exposure Process in TEM

Photographic imulsions react to photons and electrons in different ways. Generally photons have low energy and several need to strike a grain before a latent image will form. Electrons generated in the electron microscope have high energy and one electron can cause a latent image to form. Additionally, photons will interact with the upper emulsion surface while electrons must slow down as they travel through the emulsion. If a thin emulsion is used for TEM film, then the electrons will reach the base material before slowing completely. When this happens the electron can backscatter and expose extra silver halide grains. For this reason a thicker emulsion (12um) is used for TEM film.

The sensitometry of electron exposed emulsion is also different.

As a result, increased exposure results in increased density with high contrast at higher exposure levels. The film doesn't show the shoulder seen in photon exposure. In practical terms, this means that the contrast of an EM negative can be increased with increasing exposure. This can be accomplished through either increased image brightness at a fixed time or by increased time at a fixed image brightness. Additionally, the microscopist has the ability to extend developer time, increase developer action, or print on a higher contrast enlarging paper. It follows that print contrast can be lowered by the opposite of these steps.

2) TEM Image Recording

1. Critical Focus
2. Spread electron beam.
3. Lower fluorescent screen.
4. Cover viewing area.
5. Depress auto brightness button (ABS on H7000) or manually adjust brightness (H600).
6. Check intensity setting (10.5 for 75kV).
7. Check exposure time (4 seconds in ideal conditions).
8. Pull exposure lever forward and hold until exposure is complete.
9. Replace exposure lever.
3) SEM Imaging Recording

Images are recorded in a different fashion on the scanning electron microscopes. Instead of being directly exposed to the electrons, the film is exposed to a special TV screen. The CRT is a high resolution, low persistence screen whose light is focused on the film. In addition, a different type of film is used. We use Polaroid Type 52 and Type 55 P/N. These are instant developing films that produce positives (Type 52) or positive/negatives (Type 55). The resolution of these films is not as good as with EM film, but can be positive, so if high quality prints are desired, a photographic enlargement of the negative should be done.

SEM photos are obtained by the following steps:

1. Compose desired image on the main viewing screen.
2. Depress the ABC (auto brightness and contrast) button.
3. Make sure the lever on the front of the film holder is set to "L" (load) and insert film as indicated on the cover of the film.
4. Make sure film is fully inserted, then pull the paper cover back out until it stops. This will leave just the negative to be exposed.
5. Press the photo button and re-insert cover after exposure is completed.
6. Switch lever to "P" (process). and briskly pull film out of the holder.
7. Wait 20-25 seconds and separate the paper cover by pulling the two tabs apart.
8. Place the negative in the 18% sodium sulfite solution (at least 5 minutes) and coat the positive with the supplied applicator and allow to dry.
9. After 5 minutes the negative should be placed in the water bath and washed in running lukewarm water for at least 10 minutes.
10. Dip the negative in Photo-Flo solution and hang to dry.

Photographic Processing

1. Development

All developers are complex solutions containing water, developing agents, preservatives, activators, and restrainers. Developer is used to provide electrons to silver halide crystals containing latent images, crystallizing the conversion of the remainder of the crystal to metallic silver. Agents such as metol and hydroquinone are the donors of electrons to the exposed grains. During development, the speck of free metallic silver serves as a nucleation site to which more free silver is added. The additional free silver serves as a development center for even more silver to be laid down. In this way the minute latent image silver speck is "amplified" into a large, visible silver mass, the developed grain.

2. Stop Bath

The stop bath is used to halt the developer's action and neutralize the high pH of the developer. A good water wash accomplishes the same thing, though not as quickly.

3. Fixer

After development, unexposed and undeveloped silver halide crystals are present in the emulsion and are still light sensitive. The fixer is made from a solution of sodium thiosulfate (also known as sodium hyposulfate hence the common name "Hypo") which acts to dissolve and remove these crystals. Therefore, exposed portions of the emulsion are black (due to free metallic silver) and unexposed areas are clear (if on glass or plastic) or white (if on paper). The emulsion may now be exposed to light without damage to the image.

4. Washing

Washing is necessary to remove soluble silver salts and other residual chemicals. Failure to remove these materials can result in the subsequent formation of silver sulfate, a yellow stain. Washing should be followed with a dip Photo-Flo solution which will prevent water spots. When wet, the emulsion is easily damaged and must be "set" by drying. This should be carried out in a dust free environment.