A
main obstacle in the microscopy of biological objects is their poor
contrast. Only where a contrast exists or where it can be achieved by
contrast-enhancing dyes, structures can be made visible.
Light-absorbing parts of a preparation weaken the amplitude of the
light waves that pass through them. It is thus also spoken of
amplitude preparations. The
change of stronger and feebler light is perceived by the eye as a
difference in brightness. The invisible parts of the preparation are
went through by the light without a change of amplitude, but the
phase of the light may be changed
depending on the consistency of the material. This change is due to
the altered speed of the lightwaves. Differences in phase can be
perceived neither by the eye nor by a photographical film.
The Dutch physicist F. ZERNIKE succeeded in 1935 to convert phase to amplitude differences. He was awarded the Nobel price for this achievement in 1953. Today his method is known as phase-contrast microscopy and it is by now an integral part of nearly all research and many teaching microscopes. Its eminent advantage is that it allows the examination of living objects and thus to follow the processes within cells. It was the phase-contrast microscope that made it possible to make mitosis visible and even to film it (K. MICHEL, Company CARL ZEISS, 1943).
For the procedure itself a special condenser with a ring-shaped mask and an additional "phase-ring" that is fixed within the back focal plane of the objective is needed. The "phase-ring" has two important tasks:
- It has to achieve an alignment of the brightness of refracted and unrefracted light as the rays that pass through the preparation are weakened in their intensity. In contrast to a conventional image gained with the help of a light microscope, the background of a phase-contrast image is thus dark.
- The phase difference of most biological preparations is one-quarter of a wavelength or less (lambda / 4). The phase-ring is constructed so as to achieve an additional difference of lambda / 4. The complete phase difference is thus lambda / 2 and crest and trough of refracted and unrefracted rays extinguish each other. A disadvantage of this method is the appearance of light halos around some objects ("halo-effect").
To the left: Arrangement of the ring-shaped mask below the objective and of the phase-ring within the objective. Only the direct light beams are influenced by the phase-ring (according to a works photography of CARL ZEISS).To the right: The path of light rays within a phase-contrast microscope. 1. ring-shaped mask, 2. condenser, 3. specimen, 4. objective, 5. phase plate, 6. focal plane of the objective. The wave character of the light is indicated by the change of light and dark areas (according to a works photography of CARL ZEISS)
Dark Field Microscopy: This method uses a special condenser with an aperture that big that the light beams that go through it pass by the objective. Only if the object is brought into the center of the light, the light is diffracted, collected by the objective, and used for image formation. Shiny structures are seen in front of a dark background. This method has no very important role in biology, but is impressive with crystals.
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