A
PHOTOMICROGRAPHY
PRIMER
It’s worth a closer look
Light microscopy has classically been viewed
as an experimental tool for the biological and
medical sciences. In this respect, the
microscope has proven useful in countless
investigations into the mysteries of life.
However, as lens and coatings
technologies have improved over
the years, visible light microscopy
has slowly found applications in
such diverse disciplines as
chemistry, geology, physics,
materials sciences, and even the
semiconductor and computer
industries.
Most biological microscopes
are equipped only with brightfield
and darkfield illumination for
examining pre-stained or live
specimens. To enhance contrast
of unstained samples, many of the
more modern microscopes are also
equipped with phase contrast
optics. These optical techniques
are generally of little use for
imaging specimens in the physical
sciences where alternative
methods such as light plane crosspolarization, differential interference contrast, and Rheinberg
illumination are commonly
employed. Unfortunately, these
alternative optical illumination
techniques add considerably to
the initial expense when a microscope is purchased. In this article,
I discuss how low-cost methods
can be implemented to convert the
biological microscopes found in
most high schools into polarized
light microscopes useful for
instructional purposes in the
physical sciences. Also, the basic
principles of photomicrography
are discussed.
MICROSCOPE SETUP
Several science supply distributors are an excellent source
of high quality optical components at reasonable prices (see the
list in Figure 1). Polarizers
suitable for performing crossedpolarized light microscopy can be
purchased from these dealers for
less than $25.00 and adapters
which couple most cameras to the
microscope are available for
$15.00 to $100.00. Expensive
high-powered microscope objectives (lenses) have a very narrow
depth-of-field and are not really
useful for a majority of work in
the physical sciences, therefore
1
Figure 1. Microscope and accessory manufactures
and distributors
OLYMPUS CORPORATION
4 Nevada Drive
Lake Success, N.Y. 11042
Telephone: 516-488-3880
FISHER SCIENTIFIC
50 Fadem Road
Springfield, N. J. 07081
Telephone: 201-379-1400
CAROLINA BIOLOGICAL
SUPPLY COMPANY
2700 York Road
Burlington, N.C.
Telephone:800-334-5551
EXCEL TECHNOLOGIES
90 Phoenix Avenue
Enfield, Ct. 06082
E. LEITZ, INC.
24 Link Drive
Rockleigh, N. J. 07647
Telephone: 201-767-1100
CARL ZEISS, INC.
One Ziess Drive
Thornwood, N.Y. 10594
Telephone: 914-747-1800
NIKON INC. INSTRUMENT
GROUP
623 Stewart Ave.
Garden City, N. Y. 11530
Telephone 516-222-0200
EDMUND SCIENTIFIC CO.
101 E. Gloucester Pike
Barrington, N. J. 08007
Telephone: 609-573-6250
McCRONE ACCESSORIES
AND COMPONENTS
850 Pasquinelli Dr.
Westmont, IL 60559
Telephone 312-887-7100
VVVR SCIENTIFIC
P.O. Box 330348
Houston, TX 77233
Telephone 800-392-3338
Telephone800-527-1576
AO SCIENTIFIC
INSTRUMENTS
P.O. Box 123
Buffalo, N. Y. 14240
lower cost 5x and 10x objectives,
which usually come as standard
equipment on most microscopes,
are sufficient.
Two polarizers will be needed
to convert the microscope. The
first polarizer is inserted into the
lightpath at the base of the
substage condenser (see Figure 2).
This polarizer can be held in place
with tape, or if the microscope has
a built-in light source, the polarizer can be placed directly over
the field lens. The second polarizer, commonly termed the
analyzer, is place inside the body
of the microscope between the
main body tube and the eyepiece
tube. There is usually a lens
mount at the top of the body tube
and the analyzer can be placed
directly on this mount. Because of
the restricted space within the
main body tube, the analyzer must
be limited in size to 1-2 centimeters in diameter. An analyzer of
the proper size can be acquired by
cutting a piece of polarized sheet
plastic or buying a small polarizer
from a dealer (Figure 1). After
installation of the polarizer and
analyzer, the microscope illumination is turned on and the polarizer
at the microscope base is rotated
until the viewfield becomes very
dark (total extinction).
At this point, the polarization
direction is perpendicular between
the polarizer and the analyzer and
you then have what is termed
crossed polarizers. When purchasing polarizers, it is important
to select polarizing materials that
are very close to a neutral gray in
color such as the threaded
polarizers made for the front of a
camera lens. Avoid polarizing
materials that are green or amber
in color as these will not produce
total extinction. Some microscope
manufacturers offer a low-budget
polarization kit ($150.00 to
$300.00) which is easily installed.
It is advisable to contact your
microscope’s distributor on the
availability of these items if your
budget allows.
The adjustments described
above apply only to transmitted
light microscopy where polarized
visible light passes through the
sample. An alternative method of
microscopy utilizes reflected light
where a beam of light is reflected
off the surface of the sample to be
examined. To avoid investing in
expensive reflected light attachments, oblique illumination from
an external light source can be
substituted to achieve a reflected
light effect. A high-intensity light
source such as a fiber optics lamp
provides an excellent substitute.
Attaching a camera to the
microscope is the last step.
Microscope viewing heads come
in three varieties: monocular (one
eyepiece), binocular (two eyepieces), and trinocular (two
eyepieces and a photography
tube). A camera can be adapted to
each of these viewing heads.
Commercial aftermarket camera
adapters usually are attached to
one of the viewing tubes with a
thumbscrew and adjusted to be
parfocal with the eyepieces by
sliding the adapter up or down on
the viewing tube. A simple
camera back will be sufficient for
photomicrography because the
camera is only required to store,
expose, and advance the film. The
microscope itself acts as the
camera lens.
2
Figure 2. Schematic illustration of a visible light microscope equipped
for photomicrography.
For photomicrography, it is very
important to ensure that your
microscope is aligned to produce
even illumination across the
viewfield. Information on microscope alignment is available in the
owners manuals or in textbooks
dealing with microscopy.
SAMPLE PREPARATION
The Laboratory chemicals found
in high school chemistry stockrooms provide an excellent source
for samples. The chemicals listed
in Figure 3 are easily obtained and
are known to produce good
crystals for viewing with a polarized light microscope. Most
crystals are anisotropic and
birefringent, which means that will
refract plane polarized light
emitted from the polarizer and will
“bend” it until it is visible through
the analyzer under crosspolarized
illumination.
the slide to cool slowly before
examination, or place the melted
chemical on the microscope stage
and examine the crystallization
process as it occurs.
Certain chemicals recrystallize
very rapidly (within a few minutes) while others may recrystallize slowly over a period of days,
weeks, or even months. Urea and
benzoic acid are excellent examples of common laboratory
chemicals that will recrystallize
rapidly enough to be examined
directly after melting. The
instructor and students should
experiment with safe, familiar
chemicals that are available to
identify those that are optimal for
microscopic analysis.
Another very effective method
of preparing crystals is to dissolve
the chemical in a suitable solvent
such as water, ethanol, or mineral
spirits. A drop of the solution is
sandwiched between the microscope slide and coverslip and the
solvent slowly allowed to evaporate, resulting in formation of
crystalline patterns. This method
is especially useful for chemicals
in the salt family that usually
decompose upon heating and
leave a tar-like mess. Chemicals
can display a wide spectrum of
polymorphic crystalline patterns
To prepare crystals for examination in the microscope, deposit a
few milligrams of the appropriate
chemical on a glass microscope
slide and carefully
place a glass coverCLASSICAL PHOTOGRAPHY
slip over the powder.
Next, heat the bottom
ASSIGNMENTS CAN BE COUPLED
side of the microWITH SCIENCE MICROSCOPY
scope slide carefully
with a bunsen burner
STUDIES TO PROVIDE A
or hot plate until the
MULTIDISCIPLINARY PROGRAM
powder has completely melted.
IN PHOTOMICROGRAPHY
(NOTE: Some chemicals decompose upon
heating and will
provide poor subjects for microdepending on whether they are
scopic examination. Others
melt-recrystallized or recrystalproduce harmful vapors and
lized by solution evaporation.
should be avoided.) When molten,
Samples for reflected light
the chemical will flow underneath
microscopy usually require very
the coverslip and fill the entire
little preparation. Reflected light
volume between the coverslip and
microscopy can be likened to
the microscope slide. Either allow
topographical surface examina3
tion with a high-power magnifying glass, and almost anything can
be examined in microscopic detail
with this technique. For example,
the fine details of surface structure can be revealed on leaves,
coins, printed paper, insects, and a
variety of other specimens.
Perhaps the most interesting
subjects for reflected light
examination are integrated
circuits. These electronic “chips”
generally are packaged either by
being molded into plastic cases or
cemented into ceramic cases. It is
virtually impossible to recover an
integrated circuit from a plastic
case because the epoxy resin
flows into the microstructure on
the circuit surface and cannot be
easily removed. However, the
cement that secures the two halves
of an integrated circuit ceramic
case can be scored with a hacksaw
and split with a fine chisel to
reveal the internal chip. The chip
is cemented into a depression on
the bottom section of the ceramic
case and can be viewed directly
without removal from the case.
Leaving this portion of the casing
intact also serves to protect the
delicate silicon surface of the
integrated circuit. Most programmable read-only memory (EPROM), random access memory
(RAM), microprocessors, and
many other digital and analog
integrated circuits are protected
with ceramic cases. Defective
integrated circuits are quite
satisfactory for examination
because the defect is usually not
apparent on the surface of the
circuit.
An excellent source for nonworking integrated circuits is
computer or electronics repair
shops. These shops normally
stockpile a large quantity of
defective integrated circuits and
will usually give them away at no
cost. Alternatively, many new
integrated circuits are available
from dealers for less than $2. Be
sure to specify that you require
ceramic cases when ordering new
integrated circuits.
Examination of integrated
circuits with reflected light can
serve two purposes. Details of a
particular circuit structure are
readily apparent and, by observing differences in the architecture
of various integrated circuits,
students can begin to get a handle
on the complex electronics
involved in modern devices such
as radio, television, and computers. Reflected light microscopy
has become an indispensable tool
for the semiconductor industry
due to its usefulness in characterizing manufacturing defects and
monitoring the successive stages
of integrated fabrication.
PHOTOMICROGAPHY
A necessary responsibility of
microscopy is to capture the
images seen in the microscope
onto photographic film to obtain
“hard copy” for research records.
In a high school environment,
classical photography assignments can be coupled with science
microscopy studies to provide a
multidisciplinary program in
photomicrography.
Photomicrography encompasses
the techniques of both black-andwhite and color photography.
Black-and-white film processing
is substantially lower in cost than
color film, if processing is done
in-house. Many commercial film
processors no longer offer blackand-white processing services or
charge exorbitant amounts for this
service. If budget restrictions
force the exclusive use of blackand-white photomicrography, it is
advisable to invent in darkroom
equipment so students can
develop and print their own
photomicrographs.
A green filter should be inserted
into the microscope lightpath
between the light source and the
first polarizer (see Figure 1) for
black-and-white photomicrography. I recommend the use of
Kodak™ Technical pan film with
HC-110 developer for crisp
images with excellent resolution.
Printing can be done on Kodak
Polycontrast™ paper with
Dektol™ developer. After pro4
cessing a roll of film, carefully cut
the negatives into sections of 5
frames each and store in specially
made polyethylene storage sheets.
You can make contact prints by
placing a sheet of negatives
directly onto an 8" x 10" piece of
polycontrast paper and exposing
for a few seconds with the enlarger lens aperture wide open.
Contact sheets are an ideal way of
cataloging data and they provide a
compact method for storing or
sorting through many images.
When an enlargement is needed,
simply remove the appropriate
negative strip.
Color photomicrography is
considerably more complicated
than black-and-white photomicrography because color film emulsions are color balanced for a
particular spectrum of light. The
term “color temperature” refers to
the wavelength spectrum emitted
by a particular light source. For
instance, films intended to be used
outside in ordinary daylight or
under fluorescent lighting are
balanced during manufacture for a
color temperature of 5500º K
while films made for indoor
tungsten light bulb use are
balanced for a color temperature
of 3200ºK.
The majority of microscopes use
a tungsten-halide lightbulb as a
films are not available, a Kodak
light source that emits a wave80A or equivalent filter can be
length spectrum centered in the
3200º K color temperature region. inserted into the lightpath between
the light source and the first
Therefore, films color balanced
polarizer to allow the use of
the best results. All major film
manufacturers have
one or several 3200º
K films available in
COLOR PHOTOMICROGRAPHY
35-mm transparency
IS CONSIDERABLY MORE
format. Transparency
film is preferable to
COMPLICATED THAN
color negative film
BLACK-AND-WHITE
for several reasons:
PHOTOMICROGRAPHY
Color negative films
are balanced for
5500º K and must be
manipulated during
daylight balanced films with
printing to avoid a decidedly
minimal color shift. But, if this
yellowish cast. Most
filter is used, exposure times must
photoprocessors cannot or will
be increased one to three f-steps to
not produce satisfactory results
allow for a reduction in light
with photomicrographs on color
intensity.
negative film. The contrast and
When photographing new
color saturation in transparency
samples or after making changes to
film cannot be equaled by color
the microscope (such as installanegative film. Color transparention of polarizers), the new expocies are easier to label, store, and
catalog, and they can be projected sure characteristics should be
determined on a test roll of film.
at seminars.
Bracket several exposures of the
With a 20-to-50 watt tungstensame viewfield at least one and
halide bulb in your microscope,
preferably two f-steps over and
exposure times are usually very
under previous exposure times.
short and allow the use of slow
This will assure at least one or
films such as Ektachrome 50 or
Fujichrome 64T. Using slow films several good exposure times. This
will assure at least one or several
reduces the grain in photomicrogood exposures and will yield
graphs. If tungsten-balanced
exposure time information useful
for photomicrography of future
samples.
The best film, in my opinion, is
Fujichrome 64T, a highly colorsaturated E-6 transparency film
with excellent contrast. Recently, a
new emulsion of this film was
introduced that is designed to
allow push processing with very
little reduction in image quality.
Push processing is a method
developed to increase contrast
(inherently low in photomicrographs) and color saturation. This
is accomplished by underexposing
the film one to two f-steps and
increasing the process time in the
first developer during the E-6
process.
5
Figure 3. Common chemicals suitable for recrystallization.
CHEMICAL
COMMENTS
Alka-Seltzer
Best crystals from aqueous solution.
Ascorbic acid (vitamin C)
Can either be melt-recrystallized or
recrystallized from alcohol.
Aspartame (Nutra-sweet)
Good crystals from meltrecrystallization or aqueous solution.
Aspirin (acetylsalicylic acid)
Equally good crystals from meltrecrystallization or aqueous or ethanol
solutions.
Acetaminophen (Tylenol)
Equally good crystals from meltrecrystallization or aqueous solution.
Benzoic acid
Best crystals from melt-crystallization.
Biotin (vitamin H)
Best crystals from melt-cystallization.
Citric acid
Good crystals by evaporation from
aqueous ethanol solutions.
Epsom salts
Recrystallize from aqueous solution
only.
Glucose and sucrose
(common sugars)
Crystals quickly obtained by evapora
tion of solution in water. Very beauti
ful crystals after melt-crystallization.
Ibuprofen (Advil)
Best crystals after evaporation of
rubbing alcohol from ethanol.
Kodak Dektol paper developer
Best crystals after evaporation of
working stock solution.
Kodak D-76 film developer
Best crystals after evaporation of
working stock solution.
Kodak rapid fixer
Best crystals after evaporation of
working stock solution.
Niacin (a B vitamin)
Beautiful crystals after meltcrystallization.
Nicotinic acid
Good crystals from melt-or
evaporation from alcohol solutions.
Tartaric acid
Very good crystals from ethanol
solutions.
Urea
Crystals form rapidly after meltrecrystallization.
INDEPENDENT STUDENT
RESEARCH PROJECTS
A variety of specific applications are available to teachers and
students in the form of individualized student research projects.
The following paragraphs should
serve as an introductory guide to
the myriad of projects which are
possible.
1. By examining a number of
different integrated circuit types,
the student can become familiar
with details of various electronic
design motifs. For instance,
microprocessor integrated circuits
generally contain a ROM and
RAM section for internal calculations and storage of information.
These memory sections differ
from one circuit to another and
also from the same type of circuits
provided by different manufacturers. Also, transistor size has
steadily decreased as more
transistors are packed onto a
single integrated circuit. Many
older, non-working integrated
circuits are available from electronics repair facilities, and these
circuits can be examined for
transistor size versus manufacturer date. In addition, memory
circuits can be studied to relate
microscopic features to the actual
storage capacity of the integrated
circuits. These types of investigations should be undertaken by
students who have an interest in
engineering and electronics
technology.
2. The crystallization patterns of a
single set of biochemicalsvitamins, for instance-can be
observed by assembling a collection of recrystallized
biochemicals. In a large biochemical family, such as the
vitamins, many different types of
organic chemical groupings are
presented. This leads to a large
spectrum of different crystalline
morphologies. By combining
specific information about the
individual vitamins with photomicrographs of the vitamins, the
student should succeed in producing a interesting and informative
photographic display. Students
interested in biology, chemistry,
and biochemistry would benefit
from this experiment.
3. For students who are primarily
interested in photography, the
beautiful colors provided by
polarized light microscopy can
serve to sharpen color photography skills. Because the microscope has a relatively fixed setting
when compared to standard
photography, students can compare the results produced by
different films under indentical
conditions. Also, students can
easily see the effect, on photomi6
crographs, of small differences in
color processing variables.
4. The topic of art in science is
becoming increasingly more
popular. Students interested in
this area can benefit by producing
a selection of color photomicrographs and mounting them for
display in local art galleries and
libraries. This project will provide
students with experience in
photomicrography as well as the
details pertaining to preparing
photographs for display.
By introducing polarized light
microscopy and photomicrography to high school students, you
give them experience with a
technique that is becoming a
mainstay of modern science and
industry. Students will find that
their creativity in photomicrography is limited only by the boundaries of their own imagination.
NOTE
The author would like to thank
the Nikon Instrument Group for
providing photomicrography
equipment and the FSU Center for
Materials Research and Technology for continued support.
REFERENCES
Delly, J.G. 1988. Photography
Through the Microscope. New
York: Eastman Kodak Company
publication.
Davidson, M.W., and R.L. Rill.
1989. Photomicrography: Common ground for science and art.
MICROSCOPY and Analysis 4:712.
Davidson, M.W. 1991. Fascinating photography with a simple
light microscope. PHOTOgraphic
Magazine (April): 92.
M ichae l W. Davidson is a resea r ch a s s o ci a t e
at the National High Magne tic Fi el d
Laboratory and the S u pe rc omp u t er
Com putations R e se arch Instit u t e i n t h e
D epartment of Phy sics at the F l o r i d a S t a t e
U nive rsity, Tallah asse e, Florida 3 2 3 0 6 .
7