38 



ONTOGENY AND SYSTEM ATICS OF FISHES -AHLSTROM SYMPOSIUM 



of larvae for skeletal deformities. The consensus among ichthy- 

 ologists who have used both techniques is that, although clearing 

 and staining methods provide the detail necessary for describing 

 developmental osteology, radiography is a simple and quick way 

 of obtaining counts from large numbers of specimens. 



Hard (shortwave) x-rays have been used to form shadow pic- 

 tures, or radiographs, of large, well-ossified fish for almost four 

 decades (Goshne, 1948; Bartlett and Haedrich, 1966), but the 

 use of soft (longwave) x-rays for small specimens is relatively 

 new. Although first suggested by Bonham and Baylifr( 1953) and 

 used by Watson and Mather (1961 unpubl. manusc), useful 

 techniques for larval radiography have only recently been de- 

 scribed (Miller and Tucker, 1979). Potential larval fish radiog- 

 raphers should consult Miller and Tucker's paper for method- 

 ological details and Quinn and Sigl ( 1 980) for basic radiographic 

 principles. Although specimen fragility determines the mini- 

 mum size of larvae that can be x-rayed, sensitivity of the tech- 

 nique, which depends to a large degree on spectral characteristics 

 of the radiation, determines the amount of detail present in the 

 finished radiograph. This section, therefore, reviews the prin- 

 ciples and current methods useful for maximizing detail in ra- 

 diographs of fish larvae. 



Radiographic sensitivity refers to the clarity of details in the 

 radiographic image and depends on a combination of two fac- 

 tors, definition and radiographic contrast. Definition is sharp- 

 ness of the image. Radiographic contrast refers to the density 

 (darkness) range of the image and depends on two factors, sub- 

 ject contrast and film contrast. Subject contrast refers to the 

 ratio of radiation intensities that pass through different parts of 

 the specimen. Film contrast refers to the ratio of densities in 

 parts of the film that have received different degrees of exposure. 



In larval fish work, radiographic sensitivity can be improved 

 by several means. Definition can be improved by using the 

 longest possible radiation wavelengths, by using the finest grained 

 film available, and by minimizing geometric production of over- 

 lapping shadows at tissue discontinuities in the specimen. Ab- 

 sorption by x-rays of a given wavelength depends mostly on the 

 atomic numbers of components in the x-rayed material, and to 

 a lesser degree on thickness and density of the material. Larval 

 skeletons, which are thin, poorly calcified, and of relatively uni- 

 form composition and thickness, do not contrast radiographi- 

 cally with the rest of the body as much as in older fish. High 

 contrast techniques should, therefore, be employed. Subject con- 

 trast can be increased by increasing wavelengths and by de- 

 creasing the thickness of non-skeletal tissue by dehydrating the 

 specimen. Film contrast can be increased by using a high con- 

 trast film and by increasing development time; however, over- 

 development will also increase graininess and reduce definition, 

 and probably should be avoided. 



The longwave (soft) end of the x-ray spectrum is the portion 

 most useful for x-raying small fish, because this low energy 

 radiation does not pass through materials as easily as that at 

 the shortwave (hard) end. Decreasing the tube voltage (kv) caus- 

 es a shift of the emitted spectrum toward longer wavelengths. 

 Resultant elimination of some of the hard radiation contributes 

 to better subject contrast and improves definition by reducing 

 clumping of silver grains in the film emulsion (graininess). The 

 x-ray unit should be equipped with a thin beryllium window, 

 which allows passage of soft rays. A 25 mil (0.63 mm) window 

 allows work at a kv of 20; a 10 mil (0.25 mm) window extends 

 capabilities to about 8 kv (Joseph Fowler, Hewlett Packard, pers. 

 comm.). However, the lower practical limit for fish larvae may 



be governed by restrictions on exposure time, rather than kv 

 limitations. 



Another relevant factor is the source-to-specimen distance, 

 to which image definition is directly related. Increasing the source- 

 to-specimen distance improves definition by minimizing en- 

 largement and distortion. Practical limits are set by air atten- 

 uation, loss of radiation intensity (roughly as the square of the 

 ratio of the distances), and dimensions of the x-ray unit. Geo- 

 metric unsharpness is the maximum width of the zone of over- 

 lapping shadows that are caused by a non-point source. This 

 factor can be calculated to determine the minimum source to 

 specimen distance that can be tolerated. Use of the minimum 

 distance will permit the shortest possible exposure time and 

 reduce relative attenuation of soft rays, thus contributing to 

 subject contrast. The formula for geometric unsharpness, Ug 

 (Quinn and Sigl, 1980) is: 



U„ 



D, 



in which F is the radiation source size. Do is the source-to- 

 specimen distance, and t is the specimen to film distance (max- 

 imum specimen thickness). For F = 0.5 mm, D,, = 460 mm, 

 and t = 1 mm, U^ is 0.00 1 mm. This level of unsharpness would 

 not be visible without magnification and could be tolerated at 

 moderate magnification depending on the requirements of the 

 investigator. To ensure that geometric unsharpness is not large 

 enough to affect quality of radiographs, it should be calculated 

 for the set of factors relevant to each operation, keeping in mind 

 the level of magnification to be used. With most modem x-ray 

 units, a distance of 46 cm or less can be used. 



Because air attenuates soft rays more than hard, elimination 

 of air between the x-ray source and specimen allows a greater 

 proportion of soft radiation to reach the specimen. Decreasing 

 the source to specimen distance helps some, but also increases 

 geometric unsharpness, unless the source is very small. A vac- 

 uum would be ideal but is impractical. Replacement of the air 

 in a cabinet unit with helium allows the use of lower kv with 

 reasonably short exposure times and provides an increase in 

 subject contrast. Helium can be conserved and reused if it is 

 placed in a small volume plastic cylinder that has its ends sealed 

 with dry-cleaning plastic. 



Before a specimen is x-rayed it should be dehydrated as much 

 as can be tolerated to increase the signal (skeleton) to noise 

 (non-skeleton) ratio. For best results, the specimen should be 

 placed in 50-75% ethyl alcohol for a short period, maybe 30- 

 60 min, depending on size. Then the specimen should be placed 

 on the film holder, blotted to remove surface liquid and bubbles, 

 and quickly x-rayed and returned to a container of liquid before 

 desiccation damage occurs. 



The specimen should be placed as close as possible to the film 

 emulsion. This can be accomplished without wetting the film 

 by sandwiching it between two thin sheets of black polyethylene. 

 Details for construction of a convenient film holder (cassette) 

 are presented in Miller and Tucker (1 979). Polyethylene is trans- 

 parent to soft x-rays and is good cassette material. Vinyl, as well 

 as wood, paper, and any metal are relatively opaque to soft 

 x-rays, and vinyl or metal make good labels. 



Single coated Type R (now Type XAR) film has provided the 

 best quality radiographs of larvae. High resolution plates give 

 better resolution but are too slow. Type R film is slow relative 

 to other films but within practical limits. It has ultra-fine grain 



