oil immersion lens into focus is described in most 

 of the instruction booklets provided by manu- 

 facturers of microscopes. 



After the oil immersion lens has been focused 

 on the cells or tissues on the slide, the lamp dia- 

 phragm is again closed and the mirror adjusted 

 slightly so that the circle of light is centered in 

 the field. Then the lamp diaphragm is opened 

 slowly until its margin barely passes out of the 

 microscope field. If one goes much further a 

 flare is produced across the objects on the slides, 

 which is disturbing. 



Next the eyepiece lens is removed and the 

 microscope condenser diaphragm is opened 

 slowly; this action enlarges the circle of light 

 seen through the back lens of the objective. The 

 movement should be stopped as soon as a point is 

 reached where further opening of the diaphragm 

 no longer enlarges the circle. An unlighted 

 rim remains on the margin of the back surface 

 of the lens. This is due to the fact that air sepa- 

 rates the top of the condenser from the bottom 

 of the slide, setting a theoretical limit of a nu- 

 merical aperture ( N. A. ) of 1.0 for the entire lens 

 system. If oil is placed on the condenser and 

 the condenser is again brought to a focus at the 

 level of the objects on the slide, it will be found, 

 after the microscope diaphragm has been opened 

 still further, that the back lens of the objective 

 is fully illuminated. This will occur if the nu- 

 merical aperture of the condenser is as high as, 

 or higher than, the numerical aperture of the oil 

 immersion objective. If less — for example, an 

 N. A. 1.25 condenser and an N. A. 1.3 ob- 

 jective — there will still remain a narrow dark 

 ring. 



Oil on the condenser and on the bottom of the 

 slide often makes a mess if the slide is moved 

 around much. The use of oil between condenser 

 and slide is necessary if a single cell or part of a 

 cell is to be photographed or if objects within 

 the cell are much less than a micron in diameter. 

 The procedure outlined gives maximum resolv- 

 mg power. The resolving power can be esti- 

 mated by means of a monogram (Richards, 

 1938). The objects usually studied in blood 

 cells are large enough to preclude the need for 

 oil between the condenser and slide. 



It has been said that use of an air-space in- 

 stead of oil gave a theoretical limit of N. A. 1.0 

 but Spitta (1920) suggests that in actual prac- 

 tice it is an average of the theoretical limit of 1.0 



224 



and the N. A. of the objective; thus for an N. A. 

 1.3 the effective N. A. would be 1.15. 



Ill selecting the most desirable filter, each 

 worker sliould consider tlie sensitivity of his eyes 

 to light; he should try a number of filters and se- 

 lect the one that enables him to study cellular 

 details without suffering eye fatigue. 



Do not close the microscope diaphragm or drop 

 the condenser out of focus to reduce the intensity 

 of light. Closing the diaphragm reduces the ef- 

 fective N. A. and hence the resolving power, but 

 it increases the apparent refractivity of cells and 

 their parts. Sometimes individuals mistake this 

 effect for what is described as "seeing the ob- 

 jects better." A worker must have considerable 

 experience in the correct use of the microscope 

 before he realizes that he can see more with the 

 condenser focused and the diaphragm open. If 

 preparations of unstained cells, either living or 

 dead, are to be studied satisfactorily, the dia- 

 phragm must be closed nearly all the way. The 

 cells are distinguished by differences in refrac- 

 tibility, and closing the diaphragm emphasizes 

 these differences. 



On page 223 it was said that the problem of fill- 

 ing the field at low magnification with a lamp 

 giving collimated light would be discussed later. 

 The procedure for the high dry (4 mm. focal 

 length objective) lenses is the same as for the oil 

 immersion lenses. In general, there are lenses 

 manufactured at three numerical apertures — ■ 

 0.66. 0.85, and 0.95. Each has its advantages 

 and disadvantages; only the N. A. 0.66 has suf- 

 ficient working distance to be used with the usual 

 thick cover glass of the blood-counting chamber. 



When the 8 or 16 mm. objectives are used the 

 light does not fill the field. If the work is not 

 critical, a quick examination of a section is suffi- 

 cient to see whether it is flat and whether the 

 stain and counterstains are balanced. For count- 

 ing objects in the sections, a frosted blue light 

 bulb in a gooseneck lamp (or similar adjustable 

 lamp) brought close to the flat mirror of the 

 microscope is adequate. If the lamp is close 

 enough it will fill the field with light. Wlien 

 the condenser is focused, an image of the light 

 bulb appears in the field. To avoid the print- 

 ing, take the light from the side of the bull) rather 

 than from the end. The mirror should not be 

 moved when the frosted bulb is brought into posi- 

 tion. If movement is limited to the lamp bulb 



