Page 129 - Fundamentals of Light Microscopy and Electronic Imaging

112 PHASE CONTRAST MICROSCOPY AND DARK-FIELD MICROSCOPY

more concentrated is hemoglobin in an erythrocyte than tubulin (2 M) or
aldolase (20 nM) in a fibroblast?
4. To what do you attribute the range of phase densities observed in the same
erythrocyte sample? What are the estimated extremes of molarity of
hemoglobin in your cells? How would you test if this is an artifact or rep-
resents real variation?

DARK-FIELD MICROSCOPY

In most forms of transmitted light microscopy, both the diffracted rays (rays that inter-
act with the specimen) and nondiffracted rays (rays that pass undeviated through the
specimen) are collected by the objective lens and contribute to image formation. For
unstained transparent specimens, we have seen that the component of nondiffracted
background light is very large, resulting in bright, low-contrast images in which details
are poorly visible. Another solution for viewing such objects is dark-field microscopy,
in which the nondiffracted rays are removed altogether so that the image is composed
solely of diffracted wave components. This technique is very sensitive because images
based on small amounts of diffracted light from minute phase objects are seen clearly
against a black or very dark background. Dark-field microscopy is most commonly used
for minute light-diffracting specimens such as diatoms, bacteria and bacterial flagella,
isolated organelles and polymers such as cilia, flagella, microtubules, and actin fila-
ments, and silver grains and gold particles in histochemically labeled cells and tissues.
An example of a dark-field image of labeled neurons is shown in Figure 7-12. The num-
ber of scattering objects in the specimen is an important factor, because the scattering of
light from too many objects may brighten the background and obscure fine details.

Theory and Optics

Dark-field conditions are obtained by illuminating the specimen at an oblique angle
such that direct, nondiffracted rays are not collected by the objective lens. The effect of
dark-field optics can be obtained quickly with bright-field optics by rotating the con-
denser turret so that rays illuminate the specimen obliquely. Only diffracted light from
the specimen is captured by the objective, and the direct waves pass uncollected off to
one side of the lens. The disadvantage of this technique is that unidirectional illumina-
tion of highly refractile objects can introduce large amounts of flare. Much better
images are obtained with a special dark-field condenser annulus, which is mounted in
the condenser turret. Special oil immersion dark-field condensers must be used for oil
immersion objectives. Dark-field microscopy resembles phase contrast microscopy in
that the specimen is illuminated by rays originating at a transparent annulus in the con-
denser. However, in dark-field optics only diffracted rays are collected by the objective
and contribute to the image; nondiffracted rays are pitched too steeply and do not enter
the lens (Fig. 7-13). Since nondiffracted background light is absent from the image,
light-diffracting objects look bright against a dark field.