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PHOTOMICROGRAPHY

Photomicrography;

The purpose of this section is to collect together information about the types of film used in the Rubin lab., with brief comments on their processing and uses. Information on the use of compound microscopes in general, for photography or otherwise, is not included and can be found in publications such as the handbooks published by the Royal Microscopical Society. Useful titles include:

Handbook #1 "An introduction to the optical microscope", by S. Bradbury, Oxford University Press, 1989 ISBN 0-19-856419-8.

Handbook #13 "An introduction to photomicrography", by D. J. Thomson and S. Bradbury, Oxford University Press, 1987 ISBN 0-19-856414-7.

Black-and-white negative film

Kodak Technical Pan is the film of choice for most purposes. Technical Pan can be processed to give a wide range of contrasts according to need. When used at lower contrasts a large range of tones are retained in the image; at higher contrasts small differences are accentuated that pictorial films have more difficulty recording. Table 1 contains a set of developing conditions for Technical Pan at different contrast levels, and typical examples of their uses. When in doubt, lower contrast will preserve more information and can be followed by printing on high contrast paper.

TABLE 1

A cautionary note: processing schedule 4. results in negatives of extremely high contrast. Small gradations in lighting not visible to the eye will be recorded. Typical problems that can arise include diffraction patterns caused by dirt outside the plan of focus (eg: on the top of the coverslip or undersurface of the slide), by air-bubbles in immersion oil, or imperfect centering of the microscope light source. With short exposures the time taken for the shutter to traverse the field may be enough to produce an exposure gradient across the negative. It will be appreciated that only an exceptionally well-maintained microscope can be used for such work.

Technical Pan is less useful when very fast exposure ratings are required, eg for photographing fluorescence images. Instead use Kodak Tmax P3200. Processing conditions for this film with HC110 are given in Table 2.

TABLE 2

HC110 dilution B

Use of monochromatic illumination with B&W film

The signal-to-noise ratio of black-and-white films can sometimes be increased by using a monochromatic light source. This is because parts of the spectrum not absorbed by the specimen (and which therefore contribute only "noise") are not used. Also a narrow bandwidth should increase lens resolution. Examples are the use of a green bandpass filter (550-600nm) to photograph orcein-stained chromosome preparations, or a blue bandpass filter (450-500nm) to highlight pigment granules in Drosophila eye sections.

Colour negative film

Colour negative films have some advantages over black-and -white. They contain more information, and are developed and printed commercially both rapidly and with little effort to yourself. We have preferred Fujicolour 100, but are trying the new high-definition Kodak Ektar films. The main disadvantage is loss of control over the printing process. Only the standard (low) contrast level is available, cropping and enlargement are expensive, and variation in colour balance and exposure are hard to avoid. These last two points result from the strong red colour of the negatives, which must be filtered out by the printer. Inexpensive commercial labs invariably expose and balance prints automatically with respect to some standard. Consequently there is little point bracketing the exposure of your negatives or using colour filtered illumination, since the printer will likely correct all of this. Printing your own requires equipment and skill.

Colour slide film

Colour reversal (slide) films do not suffer these disadvantages, since what you get back is the film you originally exposed, and the processing is invariant. Accurate prints can readily be made from slides if required. The most used films are Kodachrome and Kodak Ektachrome. These two have a different construction that affects their properties. Kodachrome comprises three separate light-sensitive layers, each of which is processed separately to generate one of the primary colours. Also a non-reflective backing reduces halation. The resolution and colour-rendition of Kodachrome are unparalleled. Also the thorough nature of the processing effectively removes uncoupled dyes, increasing the stability of the image. In Ektachrome films all the colour reactions occur within the same layer, limiting the accuracy of the colour reproduction, but making processing easier. Ektachrome films are available in a variety of speeds. Also, Ektachrome slides are supposedly more stable in intense illumination, such as found in a slide-projector. We tend to use Ektchrome 160ASA routinely, because of its speed and ease of processing, Ektachrome P800/1600 for fluorescent images, and Kodachrome only when the very highest standards are required.

Daylight/Tungsten films

Ektachrome is availible balanced for daylight or tungsten lamp (3200K) illumination. The colour balance of a lamp changes with its output. With an Axiophot it is therefore best to use the tungsten light source always, and use the neutral-density filters to adjust the light level. An 87A filter (which approximately corresponds to the blue microscope filter) converts tungsten illumination for use with daylight film.

"Professional" films

Both Kodachrome and Ektachrome are availible in a "Professional" grade that is slightly more expensive. These offer better quality control, more natural colour in the case of Ektachrome, and more accurate speed rating. These films are sold and stored refrigerated, and there is little point paying the extra unless you intend to do likewise.

Reciprocity

At low light levels the response of photographic films (like X-ray films) ceases to be linear. The exposure time necessary increases out of proportion to the decrease in illumination, an effect called "reciprocity failure". Typically this becomes significant for exposures >1sec. The axiophot control panel will correct for this effect if a reciprocity factor, specific for each film, is entered. Some reciprocity factors recommended by Zeiss are:

KODAK Technical Pan 3

EKTACHROME 160 5

EKTACHROME 400 (=P800/1600) 6

For colour films, however, reciprocity failure will be variable with different wavelengths, a problem not completely overcome by these exposure compensations.

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