MICROSCOPES 



204 



MICROSCOPES 



the elevations on an object by differen- 

 tial focussing. 



Three lens systems, condenser, ob- 

 jective, and ocular cooperate to form 

 the final image. The purpose of the 

 condenser is two-fold: to gather light 

 for illumination, and to focus this light 

 on the object in a cone of the proper 

 dimensions so that the full resolving 

 power of the objective lens can be 

 gained. By means of the objective 

 lens a real, magnified, inverted, re- 

 versed image of the object is formed at 

 the upper end of the tube. The ocular 

 further magnifies this primary image to 

 yield a virtual, inverted, reversed sec- 

 ondary image. Total magnification is 

 calculated by multiplying the magnify- 

 ing powers of the objective and the 

 ocular. 



Inspection of the markings on a set 

 of objectives will show their magnifying 

 powers and another optical property 

 called the numerical aperture (N. A.). 

 Usually the 10 X (also called 16 mm) 

 objective is marked with the number 

 "0.25;" the 44X (4 mm, "high dry"), 

 "0.66;" and the oil immersion, 95X 

 (1.8 or 2.0 mm), "1.25." Numerical 

 aperture, calculated from a geometrical 

 property of the front lens element, is 

 proportional to the theoretical resolving 

 power of the objective and is related 

 to the maximum power of the ocular 

 that can be profitably used with that 

 objective. In order to understand 

 this relation between magnification, 

 resolution, and N. A. it is necessary to 

 consider certain fundamentals. 



As successively higher powers of mag- 

 nification are brought to bear on a tis- 

 sue section more and more fine detail 

 becomes visible, which is, after all, the 

 only purpose of microscopy. But to 

 fineness of detail a limit is set, not by 

 magnification, but by the nature of 

 light and by the optical properties of 

 lenses. As long as the object to be 

 seen is large compared to the wave 

 length of the light which illuminates it 

 the microscopic image will be sharp. 

 If structural details are so small that 

 their size nearly approaches the wave 

 length of light, the image becomes 

 fuzzy. Although magnification may 

 be secondarily raised by employing 

 stronger eyepieces no further resolu- 

 tion of detail takes place; the image 

 remains fuzzy, and we have what is 

 called "empty magnification." 



The formula R = X/2 N. A. gives a 

 general relationship, where X = wave 

 length of light used, N. A. is a given 

 constant of the objective lens, and R is 

 the size of minimum resolvable detail 

 (given in /i if X is given in fx). Taking 



an average value for X in white light to 

 be 0.5 fi and substituting the N. A. 

 values mentioned above, we have for 

 the lOX objective, R = 1.0 m; for the 

 44X, R = 0.4 m; and for oil immersion, 

 R = 0.2 /x. Thus, with the best lenses, 

 details lying less than 0.2 m apart are 

 not discerned. 



Another general rule in microscopy 

 states that the maximum total magnifi- 

 cation should not be pushed higher than 

 about one thousand times the N. A.; 

 otherwise empty magnification results. 

 With the lOX objective, 1000 X 0.25 = 

 250. Therefore a 25X ocular is the 

 maximum. For the oil immersion, a 

 13X ocular gives the limiting useful 

 magnification. 



It is thus evident that the objective 

 lens is the heart of the microscope. 

 The ocular brings out only the details 

 which already have been resolved by 

 the objective. 



The role of the condenser may be 

 reviewed in the light of these interpre- 

 tations. Historically the condenser 

 received its name from its original pur- 

 pose: it was simply a lens used to con- 

 centrate light on the subject. E. 

 Abbe (1840-1905), who contributed a 

 great deal to theoretical and practical 

 microscopy, including the concept of 

 numerical aperture, came to the con- 

 clusion that an objective lens could not 

 work at its maximum N. A. unless it 

 were matched with a condenser lens 

 system of equal N. A. Hence the 

 modern condenser is also rated in terms 

 of N. A. and is provided with a variable 

 iris diaphragm to alter its N. A. in order 

 to match with that of the objective. 



Since the standard microscope pro- 

 vides all the convenient magnifications 

 and even more resolution than is usually 

 required in routine histology, it is not 

 necessary to take extraordinary pains 

 to coUimate (parallel) the microscope 

 with the light source or to worry over 

 the fine points of diaphragm control. 

 However, good photomicrography de- 

 mands such attention to details, be- 

 cause the plate is much more sensitive 

 to inequalities of lighting than the ob- 

 server's eye. On the other hand the 

 eye is subject to fatigue unless the 

 proper illumination is employed. In 

 student classes one commonly finds that 

 the lighting with monocular micro- 

 scopes is too bright and with binoculars 

 too dim. To minimize visual fatigue 

 the brightness of the microscopic field 

 of view should about equal the bright- 

 ness of the table-top and be in keeping 

 with the general level of illumination 

 in the room. 



The following suggestions for setting 



