What
is phase contrast?
Phase
contrast is a method used in microscopy and developed in the early 20th century by Frits
Zernike. Zernike discovered that if you speed up the
direct light path, you can cause destructive interference
patterns in the viewed image. These patterns make details
in the image appear darker against a light background. To
cause these interference patterns, Zernike developed a system of
rings located both in the objective lens and in the condenser
system. When aligned properly, light waves emitted from the
illuminator arrive at your eye 1/2 wavelength out of phase.
The image of the specimen then becomes greatly enhanced. Phase is only
useful on specimens that are colorless and transparent and usually difficult to distinguish from their surroundings.
We call these specimens "phase objects". Examples of phase objects include cell parts in
protozoans, bacteria, sperm tails and other
types of unstained cells. This phase contrast technique proved to be such
an advancement in microscopy that Zernike was awarded the Nobel prize (physics)
in 1953. (see more: Frits
Zernike biography)
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(click on image for larger picture)
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(click on image for larger picture)
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Image
above with regular brightfield objectives.
Notice the air bubbles at three locations, some cells are visible
at the left side
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Same image
with phase contrast objectives.
White dots inside each cell are the nuclei.
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| About
the images above: A common activity in a
high school biology class is to observe cheek cells. To do this, you
take a flat toothpick and gently scrape the inside of your cheek. You
then smear the saliva specimen on a flat microscope slide and cover it with a cover
slip. The cheek cells are epithelial cells and will be seen in large
numbers. The students then add a drop of iodine to the sample and the
nuclei of the cells become visible as small round dots inside the cell.
In the two pictures above, we see the cells as they would appear without
iodine with a regular microscope. At the right, you see the same
specimen using a phase contrast microscope (actually, we used the same
microscope with different lenses). This clearly shows that, for some
specimens (called Phase Objects), a phase contrast microscope will greatly improve you image quality. |
Setting
up your microscope for phase contrast observations
(you
must have special phase objective lenses and a phase substage
condenser)
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Shown
at left is the phase contrast kit used on the National Optical 160
series microscopes. It consists of 4 objective lenses, a centering
telescope and Zernike phase condenser lens.
The long adjustment screws on
the phase condenser push
in to engage set screws for proper alignment of the phase ring. There
is a thumb wheel on the opposite side used to dial in the proper setting
to match the power of the objective lens. There is also a
"BF" setting on the thumb wheel for brightfield. This
allows you to use the phase objectives as standard brightfield lenses.
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Not all
phase contrast microscopes are the same but generally they rely on
similar techniques to set up the system for optimum
results. In the system shown at the left, the phase
condenser has five settings that you spin with your thumb ( 10X, 20X, 40X,
100X and BF) BF is "brightfield", no phase.
To
set up your microscope for phase optics, you first set it at BF
and focus on the specimen. Adjust the height of the condenser for
optimum image quality. Next, set the condenser turret to
the phase setting for that particular lens and remove the
specimen.
The controls that stick out
from both sides on the back are for centering the condenser.
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Next, you
remove one of the eyepiece lenses and insert the centering
telescope in its place. The set screw is used to focus the
centering telescope. When looking through the telescope,
you will see two rings. They may or may not be
concentric. By turning centering adjustment screws on the
condenser, you align the rings so that they become concentric.
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Above, what you might see before
alignment of the phase condenser |
Once
aligned and optimized for phase contrast microscopy |
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then remove the centering telescope and replace the eyepiece lens.
Put your specimen back on the stage and you are ready for phase observations.
When you change objectives, you should go through this procedure
again (although you may discover that the alignment remains consistent with all
objectives). |
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One final
note. Your phase
contrast microscope might also be a darkfield microscope. You will
likely discover that when you set the condenser to a different phase
setting than the chosen lens you will get a nice darkfield effect.
So you are getting a pseudo darkfield microscope as well as a phase
contrast instrument! This will show your specimen, not as a phase
image, but illuminated on a black background. |
