LEAF SURFACES 



Rhodix, J., "Electron microscop}' of the kidney," 

 Am. J. Med., 24, 661 (1958). 



Johannes A. G. Rhodin 



LEAF SURFACES 



The electron microscope and its associated 

 techniques have now reached a stage in de- 

 velopment where they can be used by biolo- 

 gists as tools of research, not simply as 

 instruments for making interesting new mor- 

 phological discoveries or for confirming in- 

 formation gained from other sources. Our 

 investigations, using the new techniques, 

 have demonstrated the existence of a fine 

 structure on the surfaces of many plants. Al- 

 though some such structure beyond the reso- 

 lution of the light microscope had been in- 

 ferred, because plant surfaces vary widely 

 in their ability to repel water droplets, its 

 morphology, diversity, and the problems of 

 its development had never been suspected. 



Prior to this work several attempts had 

 been made to examine the surfaces of animal 

 and plant cuticle with the electron micro- 

 scope. Holdgate, Menter and Seal (1954) 

 used reflection electron microscopy to study 

 insect cuticle. This technique, although suc- 

 cessful in demonstrating changes in the insect 

 cuticle, suffered from the disadvantages as- 

 sociated with reflection electron microscopy; 

 the difficulty of interpretation is due to dis- 

 tortion and a restricted magnification only a 

 little above that of the light microscope. 



Most of the work on plant cuticle has 

 used some form of replica technique. The 

 pioneer work by Mueller, Carr and Loomis 

 (1954) and Schieferstein and Loomis (1956) 

 used liquid polyvinyl alcohol as the first stage 

 of a two-stage replica method. However, it 

 was necessary in their technique to wet the 

 surface of the leaf with wetting agents to 

 make the liquid plastic adhere. The liquids 

 used to wet the surface ought to affect both 

 the behavior and fine structure of those sur- 

 faces, and their results appear to confirm 

 this. 



A technique which does not involve wet- 

 ting the leaf's surface has therefore been de- 

 vised. It is basically the single-stage carbon 

 method of Bradley (1954) in which the speci- 

 men is coated with a film of carbon, and 

 everything but the carbon is subsequently 

 dissolved away. The technique is as follows 

 with leaf specimens (Juniper and Bradley, 

 1958). The leaf to be examined is fixed to a 

 glass slide with cellulose adhesive tape, and 

 the slide is then placed in the evaporating 

 chamber along with a porcelain/oil marker 

 to gauge the thickness of carbon deposited. 

 The chamber, which in the work described 

 was an Edwards Coating Unit Model 12 EA, 

 is then evacuated. While pumping is in prog- 

 ress very little gas would appear to be given 

 off by the leaf and a level of vacuum (10~' 

 mm Hg) sufficient to allow the evaporation 

 of carbon is easily obtained. Carbon is evapo- 

 rated by passing a heavy alternating current 

 through the points of two carbon rods lightly 

 pressed together. The points of the rods are 

 15 cm above the specimen to be coated. A 

 film of carbon 15-20 m/x thick is deposited on 

 the leaf surface and the leaf is then removed 

 from the vacuum. 



In spite of the level of \-acuum reached in 

 the chamber, the leaf does not suffer any 

 superficial distortion due to the escape of 

 gas provided that the pumping time is kept 

 short. The pumping time for most leaves is 

 about 9 minutes, but succulent leaves may 

 take up to 11 minutes. The subsequent stages 

 of the technique are described with reference 

 to Fig. 1. The carbon film is backed succes- 

 sively with thin layers of "Formvar" and 

 "Bedacryl" 122x, allowing each in turn to 

 dry completely (Fig. lb). "Formvar" and 

 "Bedacryl" 122x are quick-setting liquid 

 plastics, flexible when dry and extremely 

 soluble in certain solvents. Then the com- 

 bined film of carbon and plastic (Fig. lb) is 

 backed with cellulose adhesive tape and the 

 leaf is peeled away from this composite film 

 (Fig. Ic). The film is then immersed in ace- 

 tone (Fig. Id). This removes the "Bedacryl" 



177 



