GENERAL MICROSCOPY 



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Fig. 1. Schematic illustration of sectioning at 

 an angle of 90° between the cutting direction and 

 the edge line, c = clearance angle, h = bevel angle, 

 r = rake angle, h = the feed of the microtome, 

 <2 = section thickness. 



(c). The sum of these three angles is 90°. The 

 larger the rake angle employed, the slighter 

 the distortion of the section, i.e., the more 

 the ratio hi a approach 1. It is therefore de- 

 sirable to have the clearance and the bevel 

 angles as small as possible. However, the 

 clearance angle cannot be smaller than 6°, if 

 the risk of compression of the tissue block 

 is to be avoided. The possibilities of decreas- 

 ing the bevel angle are also limited, because 

 of the difficulties involved in producing a 

 sufficiently even edge line. If the facet sur- 

 faces meet in a small angle, even minimal 

 abrasions may cause rather serious irregular- 

 ities in the edge line. The solution must 

 therefore be a compromise. To produce a 

 bevel angle of 20°, e.g., which meets precise 

 demands on the evenness of the edge line, 

 a disproportionately large amount of work 

 is required, while it is easy to make a good 

 edge if the bevel angle is allowed to exceed 

 50°. 



The sharpening of a microtome knife in- 

 volves imparting to the two facet surfaces 

 such a planarity and finish that their line 

 of sectioning is exactly even. Fig. 2 shows an 

 interference microscopical picture of facet 

 surfaces produced by different methods. The 

 shape of the interference fringes reveal the 

 irregularities of the surface. The interfringe 

 distance represents a profile depth of 0.27 ^l. 

 The rate of removal of the various methods 



is, of course, inxcrsely proportional to the 

 resulting surface finish. The treatment of the 

 facet surface, therefore, has to be made 

 stepwise; the last step generally consists of 

 lapping against glass in an immersion me- 

 dium. To prevent this step from becoming 

 too time-consuming, it is necessary that the 

 facet surfaces be pretreated with coarser 

 methods. Unfortunately, stropping is still 

 widely used as the last step in the sharpen- 

 ing of microtome knives. As indicated in 

 Fig. 2b, the facet surface exhibits a rather 

 good finish after stropping and the edge is 

 therefore sharp. However, the decreasing 

 distance between the interference fringes as 

 the edge line is approached shows that the 

 facet surface is curved, so that the bevel 

 angle is considerably larger than the original 

 one, and is not well defined. The other angles 

 also are unknown, and accurate cutting be- 

 comes undependable. Stropped knives there- 

 fore are to be avoided. 



The control of the knife edge is a fre- 

 quently neglected detail in microtomy. It is 

 still customary to judge the sharpness by 

 hair cutting or by passing the thumb over 

 the edge. These procedures are unworthy of 

 the instrumental equipment which we pos- 

 sess today. A microtome knife used for quan- 

 titative biological work ought to be Avell 

 defined with respect to the bevel angle and 

 to the width of the facet surfaces, and the 

 evenness of the edge line should be con- 

 trolled in incident dark-field microscopy at 

 700 times magnification. 



Measurement of Section Thickness. 

 In all investigations intending to supply 

 quantitative morphological and cytochemi- 

 cal information, in which sectioned material 

 is used, it is necessary to know the thickness 

 of individual sections. This determination is 

 often difficult. The lack of hardness in the 

 sectioned tissue means that not even a mini- 

 mal measuring pressure may be applied to 

 the surface of the section without causing 

 plastic deformation. Secondly, to control 

 what is being measured, the method em- 



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