Olympus BH2 Series User manual

Phase Contrast on the Olympus BH-2 Microscopes Revision 1 Page 1 of 6
Phase Contrast on the
Olympus BH-2 Microscopes
Revision 1
Copyright © 2021 Carl Hunsinger
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Revision History
Revision
Description of Changes
Date
1
Initial release
May 5, 2021

Phase Contrast on the Olympus BH-2 Microscopes Revision 1 Page 2 of 6
Table of Contents
Introduction ............................................................................................................................................................................3
Scope of this Document......................................................................................................................................................3
What is Phase Contrast?.........................................................................................................................................................3
How Does Phase Contrast Work? ...........................................................................................................................................3
Phase Contrast Types..............................................................................................................................................................3
Setting Up for Phase Contrast Microscopy.............................................................................................................................3
Match Selector Dial with the Low-Power Objective...........................................................................................................4
Replace Eyepiece with Phase-Centering Telescope............................................................................................................ 4
Focus the Phase-Centering Telescope ................................................................................................................................4
Center the Selected Phase Annulus....................................................................................................................................5
Center the Remaining Phase Annuli ...................................................................................................................................5
Replace Centering Telescope with the Eyepiece ................................................................................................................5
Place Green/Yellow Filter into Filter Receptacle ................................................................................................................5
Using Phase Contrast ..............................................................................................................................................................6
Periodically Verify Centering of the Phase Annuli ..............................................................................................................6
About the Phase Contrast Filter.......................................................................................................................................... 6
Phase Contrast Artifacts..........................................................................................................................................................6
How To Contact the Author....................................................................................................................................................6
Table of Figures
Figure 1 –Set condenser dial to match the objective ............................................................................................................4
Figure 2 –Replace right eyepiece with phase telescope........................................................................................................ 4
Figure 3 –Phase telescope improperly focused .....................................................................................................................4
Figure 4 –Adjust knurled focus ring to focus the image ........................................................................................................4
Figure 5 –The phase telescope is properly focused...............................................................................................................4
Figure 6 –Centering screws of BH2-PC/PCD condenser.........................................................................................................5
Figure 7 –The phase ring and phase annulus images ............................................................................................................5
Figure 8 –The phase annulus properly centered ...................................................................................................................5
Figure 9 –Place green/yellow filter into filter receptacle ......................................................................................................6

Phase Contrast on the Olympus BH-2 Microscopes Revision 1 Page 3 of 6
Introduction
This document describes the proper procedures to set
up an Olympus BH-2 microscope for phase contrast
viewing.
Scope of this Document
The procedure described here was performed using an
Olympus BHT microscope, but this procedure also
applies to the BHS, BHSP, BHSU, BHTP, and BHTU
models as well.
What is Phase Contrast?
The technique of phase contrast microscopy was
developed in the 1930s by Dutch physicist Frits Zernike
and began to be broadly used by 1942. Zernike was
awarded the Nobel Prize in Physics for his achievement
in 1953. Phase contrast techniques are most useful for
studying living, non-stained specimens, since live
specimens cannot typically be stained without affecting
their behavior or killing them outright. For these types
of unstained specimens, phase contrast provides
significantly increased contrast as compared to
conventional brightfield microscopy. In a nutshell,
phase contrast optics exaggerate the differences in the
phase relationships between the light waves in the
background illumination and the light waves passing
through the specimen, so that they can constructively
or de-constructively interfere with each other at the
intermediate image plane, thereby converting invisible
phase differences into visible image contrast.
How Does Phase Contrast Work?
In conventional brightfield microscopy, a visible image is
formed by wave interference at the intermediate image
plane of the background illumination (the background
illumination is the light that does not pass through the
specimen, which is also known as the “S”, or surround
wave-front) and of the diffracted light (the diffracted
light is the light that passes through the specimen under
observation, which is also known as the “D”, or
diffracted wave-front). The image of the specimen is
visible to the observer due to the diffraction,
absorption, and phase-shifting that occurs as the D
wave-front passes through the specimen under
observation. Diffraction in the D wave-front occurs as a
result of detail in the specimen. Absorption occurs as a
result of the specimen being not completely
transparent. Phase shift occurs as a result of
differences in the refractive index of the specimen, as
compared to the surrounding medium.
When viewing live, unstained specimens in brightfield,
the specimen is often difficult to see since light
absorption can be minimal and since the
constructive/destructive interference that occurs as a
result of the phase-shifted D wave-front is minimal as
well. Phase contrast microscopy utilizes special optics
(both in the condenser and in the objectives) to
accomplish two things: 1) The S wave-front (i.e., the
background illumination) is decreased in amplitude by
the phase ring in the objective so that the intensity of
the D wave-front will not be swamped by the otherwise
bright background lighting. 2) The S wave-front is phase
shifted by a quarter wavelength by the phase ring in the
objective, thereby exaggerating the constructive or
destructive interference that occurs between the S and
D wave-fronts at the intermediate image plane. The
result of these two things is that images of live,
unstained specimens have significantly higher contrast
than could otherwise be obtained using convention
brightfield microscopy.
Phase Contrast Types
Olympus phase contrast optics for the BH-2 are
available in two basic types: Positive and Negative,as
indicated by the markings on the objective barrel.
Positive phase contrast objectives are marked with a
“P” (such as “PL” or “PLL”) and negative objectives are
marked with an “N” (such as “NH” or “NM”).
In positive phase contrast, the phase ring in the
objective advances the S wave-front by a quarter
wavelength, relative to the D wave-front, whereas in
negative phase contrast, the phase ring retards the S
wave-front by a quarter wavelength, relative to the D
wave-front. In both cases, the phase of the D wave-
front is retarded by areas of the specimen which have a
higher refractive index than the surrounding medium,
and advanced by the areas of the specimen which have
a lower refractive index than the surrounding medium.
In positive phase contrast, the advanced S wave and the
retarded D wave destructively interfere at the
intermediate image plane, resulting in the areas of the
specimen with a higher refractive index than the
surrounding medium appearing darker than the neutral-
gray background. In negative phase contrast, the exact
opposite occurs. The retarded S wave and the retarded
D wave constructively interfere at the intermediate
image plane, resulting in the areas of the specimen with
a higher refractive index than the surrounding medium
appearing lighter than the neutral-gray background.
Setting Up for Phase Contrast Microscopy
The following equipment will be needed to utilize phase
contrast on Olympus BH-2 microscopes.
•Olympus BH-2 microscope
•BH2-PC or BH2-PCD Zernike-style phase contrast condenser
•Olympus CT-5 or CT-30 phase-centering telescope (as
appropriate)

Phase Contrast on the Olympus BH-2 Microscopes Revision 1 Page 4 of 6
•Olympus IF-550 (interference) or 45G533 (absorptive) filter
•One or more Olympus LB phase contrast objectives
Match Selector Dial with the Low-Power Objective
Select the lowest-powered phase contrast objective
available on your microscope. Rotate the selector dial
on the front of the phase contrast condenser until the
number visible on the front of the dial matches the
selected phase contrast objective (see Figure 1). Make
sure the selected number on the dial clicks in place.
Figure 1 –Set condenser dial to match the objective
Replace Eyepiece with Phase-Centering Telescope
Carefully remove the eyepiece from the right-hand
ocular tube and insert an Olympus phase-centering
telescope
1
in its place (see Figure 2).
Figure 2 –Replace right eyepiece with phase telescope
Focus the Phase-Centering Telescope
Look into the phase-centering telescope. With the
lighting intensity set to a comfortable viewing level, you
1
Use the “CT-5” for 23mm tubes and use the “CT-30” for 30mm
tubes. Note that other manufacturer’s centering telescopes may be
substituted for the Olympus CT, so long as the barrel diameter of the
phase telescope is compatible with your ocular tubes.
should see a bright ring of light and also a darker ring,
similar to that shown in Figure 3.Note that the image
may be so blurry that these features cannot be readily
discerned at this point. Also note that the relative
positioning of the two rings may vary from that shown
in Figure 3.
Figure 3 –Phase telescope improperly focused
Rotate the knurled focusing ring on the phase-centering
telescope (see Figure 4) until the image seen through
the phase-centering telescope is sharply focused (see
Figure 5).
Figure 4 –Adjust knurled focus ring to focus the image
Figure 5 –The phase telescope is properly focused

Phase Contrast on the Olympus BH-2 Microscopes Revision 1 Page 5 of 6
Center the Selected Phase Annulus
Per the following procedure, carefully adjust the two
orthogonal, spring-loaded centering thumbscrews on
the rear of the phase contrast condenser (see Figure 6)
in order to center the bright image of the phase annulus
within the dark image of the phase ring in the objective
(see Figure 7 and Figure 8).
Figure 6 –Centering screws of BH2-PC/PCD condenser
Figure 7 –The phase ring and phase annulus images
To adjust the position of the phase annulus, carefully
depress both of the spring-loaded centering
thumbscrews on the rear of the phase contrast
condenser (see Figure 6) until the thumbscrews engage
with the internal centering screws for the selected
phase annulus. Then, while watching through the
phase-centering telescope, adjust the thumbscrews to
get a feel for how the centering adjustments work and
how the two thumbscrews interact with each other.
Once you have gained a feel for it, use the thumbscrews
to adjust the position of the selected phase annulus so
that its image (the bright ring) falls within the image of
the phase ring in the objective (the dark ring) as shown
in Figure 8.
Figure 8 –The phase annulus properly centered
After the selected phase annulus has been properly
centered, release the two centering thumbscrews and
allow them to disengage from the internal centering
screws for the phase annulus.
Caution: Never turn the thumbscrews counterclockwise
beyond the point where the phase annulus ceases to
move in the field of view, or clockwise beyond the point
where their rotation begins to feel firm, and do not
rotate the selector dial on the phase contrast condenser
while one or both centering thumbscrews are engaged
with the internal centering screws, otherwise damage
to the phase contrast condenser may result.
Center the Remaining Phase Annuli
Repeat the centering procedure described above to
center the phase annuli in the condenser for the
remaining phase contrast objectives on the microscope.
Replace Centering Telescope with the Eyepiece
Once the various phase annuli have been properly
centered, remove the phase-centering telescope from
the right-hand ocular tube and replace it with the
eyepiece.
Place Green/Yellow Filter into Filter Receptacle
Carefully place the green/yellow phase contrast filter
(the IF-550 interference filter provides the best results,
but lacking this, the 45G533 absorptive filter may be
used instead) into the 45mm filter receptacle beneath
the stage (see Figure 9).

Phase Contrast on the Olympus BH-2 Microscopes Revision 1 Page 6 of 6
Figure 9 –Place green/yellow filter into filter receptacle
Congratulations, you have now properly setup your
BH-2 microscope for phase contrast viewing.
Using Phase Contrast
The hard part is now done. From here on out, to use
phase contrast, just be sure to always match the
number on the selector dial on the front of the phase
contrast condenser with the magnification of the
selected phase contrast objective. That is basically all
there is to it. Don’t forget to return the selector dial on
the phase contrast condenser to the “0” setting
whenever you wish to switch back to regular brightfield
viewing.
Periodically Verify Centering of the Phase Annuli
It is good practice to re-check the centering of the
phase annuli before starting a phase contrast
observation session. With practice, you will come to
see that once the phase annuli have been centered,
they rarely need to be re-adjusted.
About the Phase Contrast Filter
Note that it is necessary to use the green/yellow filter in
the illumination system, in order to provide light of the
proper wavelengths with which the phase contrast
objectives were designed to operate. This is critical if
you wish to obtain optimal phase contrast results. This
filter is particularly important when you wish to
perform photomicrography, since that’s when image
quality is most critical. The inevitable green/yellow cast
that the filter imparts may be removed from the images
by converting them to grayscale.
For routine viewing, however, you may find that you
prefer to omit the green filter from the illumination
system, knowing that the phase contrast effects will be
less than ideal, but the resulting color rendition will be
much closer to normal.
Phase Contrast Artifacts
Certain visual artifacts will inevitably appear when
viewing specimens under phase contrast, which can
sometimes make it difficult to accurately interpret the
resulting images. The bad news is you really cannot get
rid of these artifacts. You will find that certain
specimens are ideally suited for phase contrast,
whereas others are better suited to regular brightfield
viewing, because of these artifacts.
Halos: The most obvious phase contrast artifacts are the
halos which you will see surrounding your specimens.
These halos are caused by some of the diffracted light
from the specimen passing through the phase ring in
the objective. Ideally, only the background illumination
should pass through the phase ring, and only diffracted
light should pass through the areas inside and outside
the phase ring. In positive phase contrast imaging (i.e.,
when using PL or PLL phase contrast objectives), this
effect causes larger objects have a brighter edge,
whereas in negative phase contrast (i.e., when using
NM or NH phase contrast objectives), this effect causes
them to have a darker edge.
Shade-Off Effect: Another phase contrast artifact is
known as the Shade-Off Effect. In this case, the
homogeneous parts of the image show up at the same
brightness level as the background (i.e., the same as the
surrounding medium). This occurs because although
the light experiences a phase shift as it passes through
these regions of the specimen, only minimal diffraction
occurs and the angle it scatters is therefore limited,
causing it to pass through the phase ring and therefore
not experience the desired interference.
Contrast Inversion: A third phase contrast artifact is
known as Contrast Inversion. In the case of positive
phase contrast, objects with a high index of refraction
situated next to objects with a low index of refraction
will appear brighter than the background, instead of
darker. This happens because in these cases the phase
shift is not the usual quarter wave that should occur,
and instead of the expected destructive interference
occurring, constructive interference occurs instead. The
opposite of this is true for negative phase contrast.
How To Contact the Author
Please feel free to direct any questions or comments
regarding this document (or BH-2 microscopes in
general) to the author as listed on the cover page of this
document.