Page 27 - Fundamentals of Light Microscopy and Electronic Imaging

10 FUNDAMENTALS OF LIGHT MICROSCOPY

• Focus and center the condenser. With the specimen in focus, close down (stop
down) the field diaphragm, and then, while examining the specimen through the
eyepieces, focus the angular outline of the diaphragm using the condenser’s focus-
ing knob. If there is no light, turn up the power supply and bring the condenser
closer to the microscope slide. If light is seen but seems to be far off axis, switch to
a low-power lens and move the condenser positioning knobs slowly to bring the
center of the illumination into the center of the field of view. Focus the image of the
field diaphragm and center it using the condenser’s two centration adjustment
screws. The field diaphragm is then opened just enough to accommodate the object
or the field of a given detector. This helps reduce scattered or stray light and
improves image contrast. The condenser is now properly adjusted. We are nearly
there! The conjugate focal planes that define Koehler illumination are now at their
proper locations in the microscope.
• Adjust the condenser diaphragm while viewing the objective back aperture with an
eyepiece telescope or Bertrand lens. Finally, the condenser diaphragm (and the
built-in objective diaphragm, if the objective has one) is adjusted to obtain the best
resolution and contrast, but is not closed so far as to degrade the resolution. In view-
ing the condenser front aperture using a telescope, the small bright disk of light
seen in the telescope represents the objective’s back aperture plus the superimposed
image of the condenser’s front aperture diaphragm. As you close down the con-
denser diaphragm, you will see its edges enter the aperture opening and limit the
objective aperture’s diameter. Focus the telescope so that the edges of the
diaphragm are seen clearly. Stop when 3/4 of the maximum diameter of the aper-
ture remains illuminated, and use this setting as a starting position for subsequent
examination of the specimen. As pointed out in the next chapter, the setting of this
aperture is crucial, because it determines the resolution of the microscope, affects
the contrast of the image, and establishes the depth of field. It is usually impossible
to optimize for resolution and contrast at the same time, so the 3/4 open position
indicated here is a good starting position. The final setting depends on the inherent
contrast of the specimen.
• Adjust the lamp brightness. Image brightness is controlled by regulating the lamp
voltage, or if the voltage is nonadjustable, by placing neutral density filters in the
light path near the illuminator in specially designed filter holders. The aperture
diaphragm should never be closed down as a way to reduce light intensity, because
this action reduces the resolving power and may blur fine details in the image. We
will return to this point in Chapter 6.

The procedure for adjusting the microscope for Koehler illumination seems invari-
ably to stymie most newcomers. With so many different focusing dials, diaphragm
adjustments, viewing modes, eyepiece changes, image planes, filter placements, and
lamp settings to worry about, this is perhaps to be expected. To get you on your way, try
to remember this simple two-step guide: Focus on a specimen and then focus and cen-
ter the condenser. Post this reminder near your microscope. If you do nothing else, you
will have properly adjusted the image and aperture planes of the microscope, and the
rest will come quickly enough after practicing the procedure a few times. Although the
adjustments sound complex, they are simple to perform, and their significance for opti-
cal performance cannot be overstated. The advantages of Koehler illumination for a
number of optical contrasting techniques will be revealed in the next several chapters.