Optika Italy B-380 Manuel D'utilisation page 4

Amplitude objects show excellent contrast when the diffracted and direct light are out of step (display a phase
difference) by 1/2 of a wavelength. Zernike's method was to speed up the direct light by 1/4 wavelength so that
the difference in wavelength between the direct and deviated light for a phase specimen would now be 1/2
wavelength. As a result, the direct and diffracted light arriving at the image level of the eyepiece would be able to
produce destructive interference. Such a procedure results in the details of the image appearing darker against
a lighter background. This is called positive phase contrast (see Figure 2).
Another possible course is to arrange to slow down the direct light by 1/4 wavelength so that the diffracted light
and the direct light arrive at the eyepiece in step and can interfere constructively. This arrangement results in a
bright image of the details of the specimen on a darker background, and is called negative contrast (see Figure
3).
The accessories needed for phase contrast work are a substage phase contrast condenser equipped with an-
nuli and a set of phase contrast objectives, each of which has a phase plate installed. The phase outfit, usually
includes a green filter (to increase the resolution) and a phase telescope (to center the annuli).
Phase microscopy techniques are particularly useful with specimens that are thin and scattered in the field of
view. There are some limitations of phase contrast microscopy:
• Phase images are usually surrounded by halos around the outlines of details. Such halos are optical arti-
facts, which sometimes obscure the boundaries of details.
• The phase annuli do limit the working numerical aperture of the optical system to a certain degree,
thus reducing resolution.
• Phase contrast does not work well with thick specimens because of shifts in phase occur from areas slightly
below or slightly above the plane that is in focus.
• Phase images appear gray if white light is used and green if a green filter is used.
Figure 2
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Figure 3