Page 109 - Fundamentals of Light Microscopy and Electronic Imaging

92 DIFFRACTION AND SPATIAL RESOLUTION

black, and that the overall contrast is reduced. Similarly, fine details in the histological
specimen such as collagen bundles, cell organelles, and the edges of membranes and
nuclei have low contrast and are difficult to distinguish. The overall impression is that
the image looks milky and washed out—in short, unacceptable. The poor definition at
this setting of the condenser diaphragm is due to polychromatic illumination, scattered
light, and reduction in the degree of coherence of the contributing waves. However, con-
siderable improvements in the quality of the image can be made by using a bandpass fil-
ter to restrict illumination to a limited range of wavelengths and by partially closing the
condenser diaphragm.
Monochromatic light assures that chromatic aberration is eliminated and that unit
diffraction spots in the image are all of uniform size. With white light, diffraction spot
size varies by nearly a factor of 2, due to the presence of different color wavelengths,
with the result that diffraction features in the image are somewhat blurred. Monochro-
matic illumination sharpens the image and increases contrast, particularly for objects
with inherently low contrast. For color objects such as histological specimens that are to
be examined visually or recorded with a gray-scale camera or black-and-white film,
contrast can be improved dramatically by selecting filters with complementary colors: a
green filter for pink eosin dye or a yellow filter for blue hematoxylin stain. A green fil-
ter, for example, removes all of the pink eosin signal, reducing the amplitude of eosin-
stained structures and making them look dark against a bright background in a
gray-scale image.
To our surprise, closing down the condenser diaphragm also has a pronounced effect:
It increases the contrast and greatly improves the visibility of the scene (Fig. 6-6). The
grid bars now look black and certain features are more readily apparent. There are sev-
eral reasons why this happens: (1) Part of the improvement comes from reducing the
amount of stray light that becomes reflected and scattered at the periphery of the lens.
(2) Reducing the aperture increases the coherence of light; by selecting a smaller por-
tion of the light source used for illumination, the phase relationships among diffracted
rays are more defined, and interference in the image plane results in higher-amplitude
differences, thus increasing contrast. (3) With reduced angular aperture, the unit dif-
fraction spots comprising the image become larger, causing lines and edges to become
thicker and cover a greater number of photoreceptor cells on the retina, thus making the
demarcations appear darker and easier to see. Thus, the benefits of improved contrast
and visibility might lead us to select a slightly stopped-down condenser aperture, even
though spatial resolution has been compromised slightly in the process. If the condenser
aperture is closed down too far, however, the image loses significant spatial resolution
and the dark diffraction edges around objects become objectionable.
Thus, the principles of image formation must be understood if the microscope is to
be used properly. A wide aperture allows maximal spatial resolution, but decreases con-
trast, while a smaller, constricted aperture improves visibility and contrast, but
decreases spatial resolution. For all specimens, the ideal aperture location defines a bal-
ance between resolution and contrast. A useful guideline for beginners is to stop down
the condenser aperture to about 70% of the maximum aperture diameter, but this is not
a rigid rule. If we view specimens such as a diffraction grating, a diatom, or a section of
striated muscle, stopping down the diaphragm to improve contrast might suddenly oblit-
erate periodic specimen details, because the angular aperture is too small to allow dif-
fracted light to be collected and focused in the image plane. In such a situation, the
aperture should be reopened to whatever position gives adequate resolution of specimen
detail and acceptable overall image visibility and contrast.