Phase or dark
phase or Zernicke phase microscopy is a microscopic method
which allows the viewing of unstained specimens by using
the light phase amplitude differences within microscopic
objects. When an unstained biological specimen is observed
in the normal brightfield microscope, it is quite difficult
to see because most biological material is uncolored and
transparent. To overcome this problem without staining
or otherwise treating the specimen, microscopists have
used a trick to change the amplitude or brightness of
the light passing through the specimen. The amplitude
of a light wave was shown earlier (see Basics). Amplitude
can be changed in two ways. First, it can be reduced by
placing a neutral density filter in the light path. This
is not very useful because all the light, that passing
through the object as well as the background light, is
reduced the same amount. If one looks at an unstained
specimen, it is difficult to see without altering the
amplitude of the light passing through it making darker
or lighter relative to the background. However, the second
way that the amplitude can be altered adoes allow to create
differences in brightness between the object and the background.
This is by making use of phase differences of the light.
When a light wave passes through an object, it is deviated,
that is, it becomes phase retarded or phase shifted. The
diagram below shows the difference between the deviated
wave passing through an object and undeviated wave
from the background.
(Redrawn
from Gray)*
Notice that
the deviated wave is no longer parallel to the undeviated
one and it has also become slightly diminished in amplitude.
it is also retarded by about one quarter of a wavelength
What the eye actually sees, however, is the resultant
wave which comes about from the two waves being superimposed
upon each other. Therefore a particle seen in brightfield
would be slightly brighter than the background.
If the resultant
wave is what the eye actually sees, then it should be
obvious that if one were able to superimpose the deviated
and undeviated waves, then the resultant wave would be
almost doubled in amplitude. This is shown in the following
diagram.
(Redrawn
from Gray)*
Since brightness
is proportional to the square of the amplitude, the object
in this case would be almost four times as bright as the
background. Also, if the one of the waves could be shifted
one half wavelength relative to the other, the crest of
one would correspond to the peak of the other, the object
would be four times darker than the background. The latter
situation is what we normally have in dark phase
optics.
How is this
accomplished in the microscope. The image below shows
the setup of the dark phase optical path.
(Redrawn
from Gray)*
By the use of
a annular stop in the condenser and a phase plate within
an objective lens aligned with the annular stop, a light
beam can be split and each of the separated beams will
pass through the same transparent medium. The light passes
through the annular stop and forms a cone of light which
comes to its vertex at the focal point of the specimen.
Any background light which is not deviated by the specimen
goes through the phase ring in the phase plate and is
advanced about a quarter of a wavelength. Deviated light
passing through the specimen is retarded by about a quarter
of a wavelength and passes through the phase plate without
going through the ring. When the beams are recombined
further along the light path, the differences in the phase
of the deviated and undeviated light beams become additive
and subtractive. The resultant wave is sum of the two
waves which have their crests and troughs opposite each
other and is four times darker than the background. Therefore,
the specimen appears four times darker than the background.
The net result is that features of the object are either
lighter or darker than the surrounding field.
Dark phase is
the most common optical method used for viewing living
cells. All of the inverted tissue culture microscopes
employ it for quick examination of cells in tissue culture.
Tissue culture microscopes should be cleaned periodically
and their phase rings adjusted in order to obtain the
best possible images of cells.
*Diagrams
redrawn from Gray, P. 1964. Handbook of Basic Microtechnique.
McGraw-Hill: New York.
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