Scanning Electron Microscopy

Notes

of Drosophila H. HARTMAN AND T. L. HAYES

T

HIS paper deals with a simple method for visualizing the morphology of the Drosophila eye using the scanning electron microscope (SEM) in the secondary electron mode of operation. The techniques of preparation of the specimen are very simple and the results can be useful in augmenting information obtained by the standard techniques of light microscopy or electron microscopy. In the scanning electron microscope the image is formed by a timed sequence of points rather than by a spacially focused beam of radiation. The principle of the instrument and several of 3its modes of operation have been recently reviewed , and its ability to probe the specimen for a variety of kinds of physical, chemical, and electrical information has proven to be a useful technique in a variety of biological studies. The scanning electron microscope has been particularly useful in identifying the micro architecture of insects2' 8, and might help to correlate the surface structure of the eye with the extensive information already available concerning eye formation in Drosophila}-5'7'9i 10. It was hoped that by providing a simple technique for the study of morphological detail that lies at the limit of resolution of the binocular stereoscopic light microscope, a large number of mutants could be examined with an ease that had not previously been obtainable. The mutants selected for study of the eye were bar (1-57.0), eyeless-Russian (4-2.0), bubble-eye (4-locus unknown) and lozenge (1-27.7) in addition to the wild type.

FIGURE 1—Wild-type eye. Hexagonal packing of facets and hexagonal lattice of bristles between the ommatidia is shown. There is a low proportion of missing bristles in the array but the bristle lattice does not extend to the edges of the eye. Note that the bristles do not occur at every corner of the hexagonal packing and therefore the bristle lattice is face centered hexagonal (rather than degenerating into a face centered triangular lattice). The number of bristles and ontmatidia are equal except for the edge effect ( 3 0 0 X ) .

From the Group in Logic and Methodology of Science and Donner Laboratory, Lawrence Radiation Laboratory, University of California, Berkeley 94720. This work has been supported in part by the United States Atomic Energy Commission. The authors wish to thank Professor Curt Stern and Dr. Collin Murphy of the Department of Zoology for their very helpful comments.

41

Methods Flies were obtained from the stocks of Professor Curt Stern of the University of California, Berkeley, and, just prior to study in the scanning electron microscope, were anesthetized with ether. The flies were then attached to a copper mount using silver paint as a conducting adhesive. They were then put directly into the viewing chamber of the scanning electron microscope without prior coating with metal for conduction, as is required for nonliving, nonconducting material. It has been reported previously4 that living insects do not require this conductive coating presumably because of the escape of ionized water from the specimen. This ionizing stream constitutes an electrical current that balances the charging effect of the electron beam. The SEM, (a modified Japan Electron Optics Laboratory Model JSM-1) was operated at 25 kV with a specimen current of 2 X 10~n amps. The specimen-beam angle (viewing aspect) was 45°. All pictures were photographed using type 42 Polaroid roll film. Results The eye morphology of several Drosophila mutants were studied in order to test the applicability of this simple electron microscope technique to the inspection of living Drosophila. Figure 1 shows the wild-type eye with the classical hexagonal facet packing and bristle array. Bristles occur at 3 of the.

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Scanning Electron Microscopy

The Journal of Heredity

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E H—TBar-eye mutant. Irregular packing and much lowered bristle to facet ratio is shown (325X)-

6 corners of each hexagonal facet. If we adopt the convention of describing a 2-dimensional lattice according to the pattern of nearest neighbors to any point, the bristles of the wild type form a hexagonal lattice. Figure 2 is a bar-eye mutant showing the irregular packing found in this mutation. Figure 3 shows the mutant, eyeless-Russian, in which tetragonal packing and tetragonal bristle lattice is most WILD TYPE predominant. Figure 4 is a schematic representation Bristle of the typical facet packing and bristle lattice found in the eyeless-Russian as comjaared to the wild type. The bubble-eye mutant in Figure 5 shows irregular facets and an anomalous part of the hypodermis extending into the center of the eye. A part of the large range of magnification available with the scanning electron microscope is demonstrated in Figures 3 and 4, and Figure oA and B. The irregular facets and a doubled bristle can be seen in the 3,000 X magnification picture (5B). Figure 6 shows the lozenge eye with no facets at all and only a very few bristles. Many of these images strengthen the information obtained by light optical methods in terms of the increased resolution and very large depth of field available. On the other hand, because of the 45° viewing angle and the 3 dimensional surface picture EYELESS RUSSIAN that the SEM provides, direct measurement of FIGURE 4—Schematic representation of facet packdistances in various directions is quite complex. ing and bristle array in wild type (hexagonal) and eye> The ease of preparation of the sample allowed very less-Russian (tetragonal) mutant.


FIGURE 3—Eyeless-Russian mutant. Tetragonal packing of facets and face centered tetragonal lattice of bristles is shown. Very few missing bristles in the array. As in the wild type, the number of bristles ami the number of ommatidia are equal except at the edges of the eye ( 3 0 0 X ) .

Notes

43

FIGURE 5—A shows the irregular packing and bristle array in bubble-eye mutant. Some doubling of bristles at single facet intersections can be seen (300X). B— higher magnification of bubble-eye mutant shows base of bristles with double bristle in center (3,000.X)-

defects, particularly of eye structure, at a magnification range of approximately 100 to 10,000 X. The method of preparation used in this study is extremely simple and involves only the anesthetizing of these insects and attaching them to the copper holder for scanning electron microscopy. No coating with metal film is necessary in the production of these photographs. The entire preparative procedure can be completed in a matter of a few minutes, and this convenience, coupled with the reduction of artifacts, would indicate that scanning electron microscopy will be a useful adjunct for the identification of mutants of interest in genetic investigations. Literature Cited

rapid visualization of eye morphology and elimi1. HANLY, E. W. and W. H. HEMMERT. Morphology and nated preparative steps that could contribute development of the Drosophila eye: II. In vitro development artifacts to the final image. The increased resolution of ommatidial bristles. J. Embryol. Exp. Morph. 17:501-511. of the scanning electron microscope and the ability 1967. 2. HAYES, T. L., R. F. W. PEASE, and A. S. CAMP. Stereoto experience the complete specimen visually are scopic scanning electron microscopy of living Tribolium factors in considering the possible usefulness of confusum.J. Insect Physiol. 13:1143-1145. 1967. scanning electron microscopy in the identification 3. and R. F. W. PEASE. The scanning electron of Drosophila morphology. It is hoped that informa- microscope: Principles and applications in biology and tion transferred by this experiential mode can medicine". In Advances in Biological and Medical Physics. H. Lawrence and J. W. Gofman, Eds. Academic Press help our understanding of the architecture of the J. New York. Vol. 12, pp. 85-137. 196S. Drosophila eye and its interrelated parts. Such Inc.,4. PEASE, R. F. W., T. L. HAYES., A. S. CAMP, and N. M. subjective experience, when added to the results of AMER. Electron microscopy of living insects. Science 154:1185— objective investigation, may evoke new ideas in the 1186. 1966. 5. PERRY, M. M. Further studies on the development of mind of the observer. Erich Fromm has said: "Creativity is the ability to see (or be aware) and to the eye of Drosophila melanogasler: I. The ommatidia. / . 124:227-24S. 1968. respond". Perhaps this technique can help us to Morph. 6. . Further studies on the development of the see a little better. eye of Drosophila melanogasler; II. The interommatidial Summary Scanning electron microscopy can rapidly survey mutants of Drosophila and can detect structural

bristles. ./. Morph. 124:249-262. 196S. 7. PILKINGTON, R. W. Facet mutants of Drosophila. Proc. Zool. Soc. Lond. Her. A. 111:199-222. 1941. 8. SOKOLOFF, A., T. L. HAYES, R. F. W. PEASE, and

M.

ACKERMA.NX.

Tribolium caslaneiim: Morphology of


FIGURE 6—Lozenge-eye mutant with no facets at all and very few bristles (300X)-

The Journal of Heredity

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"aureate" revealed by the scanning electron microscope". Science 157:443-445. 1967. 9. WADDINGTON, C.

H.

and

M.

M.

PERRY. The

ultra-

structure of the developing eye of Drosophila. Proc. Roy. Soc. Lond. Ser. B, Biol. Sd. 153:155-178. 1960. 1.0.

and R. W. PILKINGTON. The structure and

development of four mutant eyes in Drosophila. J. Genet. 45: 44-50. 1943.

Cellular Esterases of the Ringneck Dove LARRY G. BOEHM AND M. R. IRWIN

in the genetic aspects of enzymes in IbyNTEREST both erythrocytes and serum has been stimulated Smithies' - starch gel electrophresis technique, 9 10

Materials and Methods Blood was collected via brachial venipuncture in 2 percent sodium citrate. The red cells were washed three times with physiological saline, then lysed with two volumes of cold distilled water and centrifuged at 4° C for 30 minutes at 20,000 g. Starch gels were made with 12g of starch/100 ml of buffer diluted 1 in 20 from a stock solution containing 0.9M Tris (hydroxymethyl aminomethane), 0.5M boric acid, and 0.1M EDTA. The electrolyte was a 9 in SO dilution of the same solution. Electrophoretic runs of the samples were done for 17 hours at 4° C with 5 v/cm, the gels were then sliced and stained for esterase activity. The stain solution was 69.6 ml of 0.2M tris maleate pH 6.4, 6 ml« naphthyl acetate, and 200 mg of Fast Blue RR made up to 100 ml with distilled water. Staining took place on a shaking water bath at 37° C until bands appeared (usually about 15-20 minutes). The gels were then washed with distilled water, photographed, and discarded. Results Three esterase band patterns represented diaThe authors are, respectively, formerly research specialist, Laboratory of Genetics; and emeritus professor of genetics, University of Wisconsin, Madison 53706. Contribution from the Laboratory of Genetics (No. 1449), University of Wisconsin. This project was supported in part by PHS Grant No. E-1643, National Institute of Allergy and Infectious Diseases. Please address requests for reprints to Dr. Irwin.

1

2

3


and Market and Moller's5 characterization of multiple forms of enzymes as "isozymes". Genetically controlled variations of esterases of the red cells have11been reported in cattle8, mice6' 7, rabbits3, and man . Studies of various esterases in the sera of birds 4 include those by Kaminski , Beckman and Nilson1, 2 and Boehm and Irwin . This report deals with the inheritance of the cellular esterases of Streptopelia risoria (the ringneck dove). Three esterase phenotypes have been demonstrated, and those presumably are controlled by two codominant autosomal alleles.

FIGURE 1—Diagrammatic representation of ringneck dove cellular esterases. 1—A/A; 2—A/B, and 3— B/B.

grammatically in Figure 1 were found in the red cell hemolysates. Pattern no. 1 consists of a single fast moving band, phenotype A/A. Pattern no. 2, phenotype A/B, has a fast band corresponding to the single band of pattern no. 1 and a second slower band. Pattern no. 3, phenotype B/B, has a single slow moving band located so that the slower band of pattern no. 2 is intermediate between the single fast band of pattern no. 1 and the single band of pattern no. 3. The bands of these patterns always appeared without change from test to test when fresh samples were used. Hemolysates can be kept frozen at — 20°C for 6 to S weeks without noticeable change in the band patterns. Pattern no. 3 was subject to alteration after storage of S weeks with the appearance of two additional bands migrating faster than the single band found in fresh samples. Red cells frozen in 40 percent ethylene glycol, then freshly washed and lysed, showed no alteration of their esterase patterns after long term storage (greater than 6 months). Mixtures of the three phenotypes were tested. Each of the possible mixtures separated into the band patterns of the phenotypes constituting the mixture. A mixture of phenotype A/A and A/B separated into two bands corresponding to the bands found in the two phenotypes, with no new bands formed. Mixtures of A/A and A/B and of A/B and B/B also separated into their respective band patterns with no new bands formed. Where bands were identical the patterns of the mixtures showed a greater intensity of staining in that region. In the initial survey of more than 125 ringneck doves in our colony only phenotypes A/B and B/B (Figure 1) in a ratio of 2:1 were observed. As shown