Scanning Electron Microscopy 



G. W. BOEHLERT 



SCANNING electron microscopy is an ideal tool for descrip- 

 tion of microstructure in taxonomic studies. The scanning 

 electron microscope (SEM) provides a surface image character- 

 ized by high resolution and depth of field and a three-dimen- 

 sional quality unavailable with other techniques. In many cases 

 this allows one to objectively describe microstructure where only 

 subjective descriptions were available in the past. It is the pur- 

 pose of this contribution to describe the techniques and use of 

 scanning electron microscopy and its application to systematic 

 investigations of fish eggs and larvae. 



The SEM has been used in a wide variety of systematic and 

 evolutionary investigations. With available magnifications from 

 10 to greater than 100,000 times, the SEM covers the range 

 from dissecting and compound light microscopy to transmission 

 electron microscopes. It has thus been immensely important to 

 progress in classification in the study of micropaleontology, bot- 

 any, insects and mites, and a wide variety of microorganisms, 

 among other taxa (Heywood, 1971; Kormandy, 1975). Taxo- 

 nomic applications of the SEM to fishes have been more limited. 

 Several studies have used the SEM for studies of morphology, 

 including epidermis, gill tissue, optic capsules, eggs, sperm, and 

 embryosof fishes (Dobbs, 1974, 1975). 



Microstructural analysis of otoliths of fishes with the SEM is 

 now common (Pannella, 1 980). For early life history stages, the 

 most frequent use in identification and classification has been 

 with the egg stage. The chorion, or external membrane, of many 

 species is variously ornamented with filaments, spines, patterns 

 of ridges, loops, blebs, and pustules ( Ahlstrom and Moser, 1 980; 

 Robertson, 1981; Matarese and Sandknop, this volume). These 

 ornamentations and the ultrastructure of the chorion are species- 

 specific (I vankov and Kurdyayeva, l973;Lonning, 1972). While 

 many of these structures may be easily visualized with light 

 microscopy (Hubbs and Kampa, 1946; Kovalevskaya, 1982), 

 the SEM often provides the best means of adequately describing 

 structures which are very small or transparent under the light 

 microscope. The egg chorion of Maurolicus muelleri, for ex- 

 ample, was described as "drawn up into hexagonally arranged 

 points," by Robertson (1976) based upon light microscopy but 

 as "drawn up into hexagonal ridges . . . and slightly raised at 

 the point of intersection" under the SEM (Robertson, 1981). 

 Similarly, Boyd and Simmonds ( 1 974), among others, suggested 

 that the chorion of southern populations of Fundulus fietero- 

 clitus lacked fibrils using light microscopy, whereas the SEM 

 showed the presence of numerous short and thin fibrils (Brum- 

 mett and Dumont, 1981). Thus for purposes of classification, 

 the SEM allows visualization of surface structures that are dif- 

 ficult to describe with light microscopy. 



Methodology 



Preparation of biological material for examination under the 

 SEM is concerned with preservation, dehydration, and coating 

 with a conductive material. Fixation of labile biological speci- 

 mens is necessary because removal of water during the stages 



of dehydration may result in collapse of cells and other artifacts. 

 Depending upon the method of fixation and dehydration, the 

 artifacts can range from shrinkage to collapse or fracture of the 

 structures to be observed. It is preferable to begin with fresh, 

 live material. For eggs this requires either laboratory spawning 

 or abundant eggs from the field which can be reliably collected. 

 For larvae at different stages, it is diflicult without laboratory 

 rearing facilities. Results with formalin-fixed material from 

 plankton collections will generally be satisfactory for lower mag- 

 nification analysis of surface morphology, but may not reflect 

 the quality of freshly prepared material. 



Fresh material should be fixed for electron microscopy. Larval 

 stages may first be relaxed in anesthetant solution (such as MS- 

 222). Initial fixatives for both eggs and larvae are generally based 

 upon glutaraldehyde, with concentrations ranging from 0.5 to 

 4.0%; lower concentrations are typically followed by post-fix- 

 ation. A fixative which I have found acceptable is that from 

 Dobbs (1974) as follows: 70% glutaraldehyde-2.0 ml, flounder 

 saline— 34 ml, and distilled water— 34 ml. The flounder saline 

 follows Forster and Hong (1958) and contains the following (in 

 grams per liter): NaCl, 7.890; KCl, 0.186; CaCK, 0.167; MgCK- 

 6H,0, 0.203; NaH,FO,H_,0, 0.069; NaHCO,, 0.84. The fix- 

 ative has a final osmolarity of 380 mOsm/l. Fixation should be 

 for 24 hours. Other authors provide several other fixatives. One 

 suggested by Stehr and Hawkes (1979), while more difficult to 

 prepare, is also useful should transmission electron microscopy 

 be desired for the same material. Post-fixation in osmium te- 

 troxide is recommended by several authors as a means of hard- 

 ening particularly soft tissues. Generally, 1-2% osmium tetrox- 

 ide in buffered saline is used. I have found this unnecessary with 

 fish eggs and larvae, as suggested by Dobbs (1974) and Stehr 

 and Hawkes (1979). It may be considered, however, if collapse 

 is a problem. Lonning and Hagstrom (1975) suggested that egg 

 chorions not post-fixed would rupture under the electron beam; 

 I have not noticed this. 



It is the process of dehydration where the greatest artifacts 

 are likely to occur. With larvae, shrinkage of tissue may occur, 

 while eggs may suffer complete collapse. On larger eggs, punc- 

 turing the chorion with a sharpened dissecting needle may fa- 

 cilitate transfer of fluids and prevent this collapse (Stehr and 

 Hawkes, 1979). 



Removal of water from the tissues is prerequisite to coating 

 and observation, which are both conducted under high vacuum. 

 Two methods are available, freeze drying and critical point 

 drying. For freeze drying, unfixed fresh material may be used. 

 Fixed material should first be rinsed with distilled water to 

 remove salts, and then plunged with little adhering water into 

 liquid nitrogen. Damage here may result from formation of ice 

 crystals if freezing rate is too slow, but this is typically not a 

 problem with small eggs and larvae in liquid nitrogen. Boyde 

 and Wood (1969) recommend using 20 ml chloroform per liter 

 of distilled water to increase nucleation rates and decrease ice 

 crystal formation. After freezing, the material is immediately 



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