Page 123 - Fundamentals of Light Microscopy and Electronic Imaging

106 PHASE CONTRAST MICROSCOPY AND DARK-FIELD MICROSCOPY

It is also possible to produce optics giving negative phase contrast, where the S
wave is retarded relative to the D wave, causing high-refractive-index objects to appear
bright against a gray background. In this case, the phase plate contains an elevated ring
that retards the phase of the 0th-order S wave relative to the phase of the D wave. The
effect of this action in generating negative phase contrast is shown on the right-hand side
of Figure 7-8.

Alignment

To form a phase contrast image, the rings of the annulus and phase plate must have
matching diameters and be perfectly aligned. A multiple-position condenser with a
rotating turret may contain two or three annuli intended for use with different phase con-
trast objectives. Small annuli are used for low-power dry objectives, whereas large
annuli are employed with high-power, oil immersion lenses. The nomenclature used by
different microscope companies varies, but the proper selection can always be made by
matching the designation on the edge of the turret with the corresponding designation
on the barrel of the objective lens. Whenever a lens is changed and a new annulus is
brought into position, it is important to inspect the objective back aperture to make sure
the annulus and the phase plate match and that they are aligned. Since the objective lens
is usually fixed, alignment is performed by moving the condenser annulus with special
annulus positioning screws on the condenser. The annulus adjustment screws, not to be
confused with the condenser centration screws, are either permanently mounted on the
condenser turret or come as separate tools that must be inserted into the condenser for
this purpose. After bringing the rings into sharp focus with the telescope focus, move the
bright image of the annulus to exactly coincide with the dark ring on the phase plate
(Fig. 7-9). Improper alignment gives a bright, low-contrast image, because the bright
background rays are not properly attenuated or advanced in phase as required by phase
contrast theory.

Interpreting the Phase Contrast Image

Phase contrast images are easiest to interpret when the cells are thin and spread out on
the substrate. When such specimens are examined in positive contrast mode, the con-
ventional mode of viewing, objects with a higher refractive index than the surrounding
medium appear dark. Most notably, phase contrast optics differentially enhance the con-
trast of the edges of extended objects such as cell margins. Generally, positive phase
contrast optics give high-contrast images that we interpret as density maps. As an
approximation, this interpretation is usually correct, because the amplitude and intensity
in an object image are related to refractive index, and optical path length. Thus, a series
of objects of increasing density (such as cytoplasm, nucleus, and nucleolus) are typi-
cally seen as progressively darker objects. However, the size and orientation of asym-
metric objects also affect intensity and contrast. Further, there are optical artifacts we
need to recognize that are present in every phase contrast image.
Interpreting phase contrast images requires care. In positive phase contrast optics,
cell organelles having a lower refractive index than the surrounding cytoplasm generally
appear bright against a gray background. Examples include pinocytotic vesicles, lipid
droplets, and vacuoles in plant cells and protozoa. For objects that introduce relatively