Photomicrography-Instant Photography Through

POLAROID
Instant Photography
Photomicrography
Through the Microscope
Polaroid
Instant Photography
Photomicrography
Introduction
Through the Microscope
Polaroid and the Art of
Photomicrography
Introduction
On the front cover
L. ascorbic acid (vitamin C)
crystallized from hot aqueous
solution. Rheinberg
illumination and crossed
polarizing filters show the
crystal growth from scratched
supersaturates.
Photographed on Polaroid
Polacolor ER Type 59 film.
Photomicrograph by M.I.
“Spike” Walker.
On the back cover
Instant photomicrography, the
technique of making photographic
images through the microscope using
instant film, gives microscopists an
invaluable tool for capturing and
conveying the images revealed
through the microscope. In many
ways, camera and film tend to be more
demanding than the human eye.
Similarly, good photomicrography
consists of more than good visual
microscopy. Photomicrography:
Instant Photography Through the
Microscope is written for readers with
a basic working knowledge of the
microscope. It is designed to help the
photomicrographer use the best
techniques to achieve the highest
quality instant imaging results.
The techniques in Photomicrography:
Instant Photography Through the
Microscope are useful with the
microscopes of every major
manufacturer. Polaroid is a leader in
instant photomicrography, delivering
imaging solutions for microscopists in
every discipline. Students and
researchers, biologists and
metallurgists, amateurs and
professionals alike can make use of
the photomicrography skills in this
guidebook to capture microscopic
images.
L. ascorbic acid (vitamin C).
Photographed on Polaroid
Polachrome instant 35mm
slide film. Photomicrograph
by M.I. “Spike” Walker.
Polaroid offers a variety of instant film
types for different micrographic needs.
These films give excellent
photographic results right away,
without the need for complex
darkroom procedures and with the
immediacy that’’ often required for
scientific research.
The convenience of Polaroid film is
unmatched: if your first result is not
quite satisfactory because of incorrect
microscope adjustment, filtration, or
exposure, you can see right away what
changes need to be made. With
Polaroid film, perfect
photomicrographs are always within
reach. Polaroid also offers
photomicrographic hardware to
complement its selection of films.
What’s in this Guide Book
This book is intended as an overview
of microscopy, basic
photomicrographic cameras and films.
We have included information on
appropriate illumination techniques as
well as discussions on the use of filters
for black and white and for color
photomicrography. In addition, we
discuss special contrast enhancement
techniques and troubleshooting with a
view toward improving the overall
quality of photomicrographs.
This document is meant to serve as a
helpful guide for a range of microscope
and photomicrographic procedures,
not to represent comprehensive
operating instructions.
For additional help, please call the
Polaroid Technical Assistance Hotline
at 1-800-1618.
2
Polaroid
Instant Photography
Photomicrography
Table of Contents
Through the Microscope
Microscopes and Cameras for Photomicrography
Introduction ................................................................................................................................................................
Basic Components of the Compound Microscope .....................................................................................................
Understanding Aberrations ........................................................................................................................................
What is Numerical Aperture? .....................................................................................................................................
Cameras for Photomicrography .................................................................................................................................
Setting up the Microscope and Camera to Create Instant 35mm Slides ....................................................................
Understanding Parfocalization ...................................................................................................................................
The Camera Magnification Factor and Photographic Magnification ...........................................................................
Controlling Exposure in Photomicrography ................................................................................................................
Setting Up Your Microscope for Photography ............................................................................................................
5
7
8
9
11
12
12
13
14
14
Kohler Illumination
Introduction ................................................................................................................................................................
Understanding Kohler Illumination as Two Paths of Light ..........................................................................................
Microscope Components that Provide Kohler Illumination .........................................................................................
Adjustments to the Microscope for Kohler Illumination ..............................................................................................
Aligning and Focusing the Substage Condenser and the Field Diaphragm ...............................................................
Controlling the Size of the Aperture Diaphragm .........................................................................................................
Centering the Aperture Diaphragm ............................................................................................................................
Manually Aligning the Light Source ............................................................................................................................
The Condenser’s Illuminating Cone ...........................................................................................................................
Typical Field Views for Kohler Illumination .................................................................................................................
The Reflected Light Microscope ................................................................................................................................
Setting Up Kohler Illumination for a Reflected Light Microscope ................................................................................
15
16
17
18
18
19
19
20
21
21
22
22
Further Understanding Kohler Illumination
Introduction ................................................................................................................................................................
How the Specimen Affects Light ................................................................................................................................
Controlling Light and Image Quality ...........................................................................................................................
The Mix of Deflected and Direct Light ........................................................................................................................
The Effect of Aperture Setting on Image Quality ........................................................................................................
Determining the Best Aperture Diaphragm Setting ....................................................................................................
The Aperture Diaphragm and Its Effect on Depth of Field .........................................................................................
23
24
25
25
26
28
28
Instant Film Characteristics for Photomicrography
Introduction ................................................................................................................................................................
Film Speed Choices ..................................................................................................................................................
Photographic Image Resolution .................................................................................................................................
Contrast .....................................................................................................................................................................
Color Temperature .....................................................................................................................................................
Spectral Sensitivity of Instant Films ...........................................................................................................................
Reciprocity Failure in Black and White Films .............................................................................................................
Reciprocity Failure in Color Films ..............................................................................................................................
Using a Graduated Exposure Test Strip .....................................................................................................................
3
29
30
30
30
31
32
32
32
34
Instant Photography
Photomicrography
Through the Microscope
Using Filters for Black and White Photomicrography
Introduction ................................................................................................................................................................
Where to Obtain Filters..............................................................................................................................................
Placing Filters in Your Microscope .............................................................................................................................
Understanding Filter Factors .....................................................................................................................................
Filtration for Optimum Image Resolution ...................................................................................................................
Using Filters for Contrast Control ..............................................................................................................................
Selecting Filters for Contrast Control .........................................................................................................................
Filter and Stain Techniques .......................................................................................................................................
Using Interference Filters ..........................................................................................................................................
Using Neutral Density Filters .....................................................................................................................................
Using Heat-Absorbing Filters .....................................................................................................................................
Using Ultraviolet-Absorbing Filters .............................................................................................................................
35
36
36
36
37
38
39
41
42
42
42
42
Using Filters for Color Photomicrography
Introduction ................................................................................................................................................................
The Relationship of Color Film, Illumination, Exposure Time and Filters ...................................................................
Establishing a Standard Exposure Time for Standard Filtration .................................................................................
The Effect of Lamp Voltage and Exposure Duration on Color Balance ......................................................................
Placing Filters in Your Microscope .............................................................................................................................
Filters for Color Photomicrography ............................................................................................................................
Color-Conversion and Light-Balancing Filters ............................................................................................................
Color Imbalances ......................................................................................................................................................
Ultraviolet-Absorbing Filters .......................................................................................................................................
Didymium Filters ........................................................................................................................................................
Heat-Absorbing Filters ...............................................................................................................................................
Neutral Density Filters ...............................................................................................................................................
Sample Applications of Color Filters ..........................................................................................................................
43
44
44
45
45
46
46
49
50
50
50
51
52
Special Contrast-Enhancement Techniques
Introduction ................................................................................................................................................................
Polarized Light ...........................................................................................................................................................
Darkfield Illumination .................................................................................................................................................
Reflected Light Darkfield ...........................................................................................................................................
Phase-Contrast Illumination .......................................................................................................................................
Hoffman Modulation Contrast ....................................................................................................................................
Differential Interference Contrast ...............................................................................................................................
Reflected Light differential Interference Contrast .......................................................................................................
53
55
56
56
58
60
62
64
Troubleshooting Common Problems ..................................................................................................................... 65
Instant Films for Photomicrography ...................................................................................................................... 69
4
Polaroid
Instant Photography
Microscopes and Cameras
for Photomicrography
Photomicrography
Through the Microscope
Introduction
Throughout the world, in laboratories,
factories, classrooms and hospitals,
microscopes are used to provide
insight into materials, processes and
dynamic events. Since 1958, Polaroid
has been an important partner to the
microscopist, providing instant
photomicrographs. Polaroid films, film
holders and cameras allow the
microscopist to instantly document
what is seen in the microscope and to
share that view with others.
Menthol, 10x
PolanPan CT
Yoko Miyake
5
Instant Photography
Photomicrography
Through the Microscope
A compound microscope
6
Instant Photography
Photomicrography
Through the Microscope
Basic Components of the
Compound Microscope
Modern light microscopes are
compound microscopes. That is, they
have more than one stage of
magnification.
• The objective lens is the first
magnification stage. It forms an
enlarged image of the specimen at
the intermediate image plane,
located about 10mm below the end
of the eyepiece tube.
• The eyepiece is the second stage of
magnification. It magnifies the
intermediate image. The viewer
sees this further enlarged image as
if it were at about 250mm, the
normal close-viewing distance.
The objective forms an enlarged image of the
specimen, which is further enlarged by the
eyepiece, and is seen by the viewer as if it were
250mm away.
The Objective Lens Barrel
Offers Valuable Information:
•
Color band gives a visual
clue of the lens magnifying
power
•
Type of lens indicates the
amount of correction for
optical aberrations
•
•
•
•
Magnifying power
•
Immersion medium (oil, water,
glycerine).
•
An immersion lens has a
black ring engraved near the
front of the lens
The total magnification of the
microscope is the product of the
magnifying powers of the objective and
the eyepiece. For example, a 10X
objective with a 10X eyepiece will
produce a visual magnification of
100X.
The compound microscope has a
variety of choices of its principal optical
components: the objective, the
condenser and the eyepiece. A brief
discussion in this chapter will help you
choose the most appropriate
components for your
photomicrography needs.
The objective
The objective has the greatest influence
on the resolution of detail in the
specimen and on the clarity of the
image. The degree of correction for
optical aberrations affects the
usefulness of the objective for
photomicrography. The type of lens
denotes the degree of correction and
is shown on the barrel of the lens.
Numerical aperture
Mechanical tube length
Coverslip thickness assumed
in designing the lens
7
Instant Photography
Photomicrography
Through the Microscope
Understanding Aberrations
Objectives
Objective Lenses for Photomicrography
Plan
Corrected for curvature of field, plan objectives give an in-focus
image of a flat specimen across the entire field of view. Both
achromats and apochromats are available as plan objectives.
Achromat
The most common objectives, achromats are corrected for axial
chromatic aberrations in the red and blue ranges of the spectrum, and for
spherical aberrations in the green. For sharpest black and white
photomicrographs, use a green filter.
Apochromat
Apochromats are corrected for axial chromatic aberrations in red, blue,
and violet, and for spherical aberrations in two colors. The are better
corrected than the achromats-and consequently are more expensive.
Apochromats usually have higher numerical apertures, and therefore
better resolving power, than achromats of the same magnifying power.
These lenses are best for color photomicrography.
Fluorite
Fluorites are intermediate in their corrections between achromats and
apochromats, suitable for color photomicrography, and priced between
achromats and apochromats.
Polarizing
Polarizing objectives are made of strain-free optics that do not distort the
polarization of the light entering the objective. Consequently, they are
most suitable for use in polarized light and DIC.
DIC (Differential Interference Contrast)
Objective lenses marked DIC are also strain-free lenses that do not distort
the polarization of the light entering the objective. They are for use in
Differential Interference Contrast.
Reflected Light Brightfield Darkfield
Reflected light objectives can be configured to direct light onto a specimen
at an oblique angle to provide darkfield illumination. Reflected light
objectives are normally designed for use without coverslips. They are
usually marked BD or HD.
Axial chromatic aberration
Because the refractive index of glass
varies across the spectrum, light of
different wavelengths is not focused at
the same point on the optic axis. Lens
designers are able to bring the focal
points for different wavelengths closer
together by using different glasses in
combination.
Lateral chromatic aberration
The red, green, and blue images
produced by highly corrected
objectives differ slightly in
magnification. This is called lateral
chromatic aberration or Chromatic
Difference in Magnification (CDM).
The eyepiece is designed to
compensate for the CDM in the
intermediate image. Since the CDM of
manufacturers’ designs may differ, it’s
best not to mix optics from different
manufacturers.
Spherical aberration
A lens exhibits spherical aberration
when the outer and inner portions of
the lens focus light at different points
on the optic axis. The objective lens
design is calculated with assumptions
of a specific thickness of the glass
coverslip and the specific optical tube
length of the microscope. However, if
the coverslip is missing, or it differs
from the assumed thickness, spherical
aberration may occur. The result is a
low contrast image with poor definition.
This is troublesome, particularly with
high numerical aperture dry (nonimmersion) objectives.
Note that low power, low aperture
lenses may not require a specific
coverslip thickness, and reflected-light
objectives are usually designed for use
without a coverslip.
Axial chromatic aberration and its partial
compensation.
Spherical aberration
8
Instant Photography
Photomicrography
Through the Microscope
What Is Numerical Aperture?
Both the objective and the condenser
are characterized by their numerical
aperture (NA) of a lens is:
NA=n sine u
Where u is ½ the angular aperture of
the lens, and n is the refractive index of
the medium between the lens and the
object. Since the refractive index of air
is 1.0, the highest theoretical numerical
aperture of a dry objective or condenser
is 1, but in practice it is 0.95. The
refractive index of immersion oil is
1.515, and immersion lenses may have
numerical apertures up to 1.4.
Condensers
Condenser Characteristics
Condensers vary in their degree of optical correction. There are four types of
condensers commonly available.
Abbe condenser
The simplest and least corrected type is the Abbe condenser. For
photomicrography, it can be used with a low power objective (10X or less).
The top lens can be swung out from the optical path to allow it to illuminate the
large visual field seen with low power objectives. An Abbe condenser is not
adequate for use with objectives having numerical apertures of 0.60 or higher.
Achromatic condenser
The achromatic condenser is corrected for chromatic aberrations and is
suitable for black and white photography with a green filter.
Aplanatic-achromatic condensers
For critical work, an aplanatic-achromatic condenser is the best choice. It is
corrected for chromatic aberration to give a fringe-free image of the field
diaphragm, and is corrected for spherical aberration so that it has the same
effective focal length at small and large aperture settings.
Angular apertures and numerical apertures of
a range of objectives.
The resolving power of the objective is
limited by its numerical aperture.
Resolving power is the ability of the
objective to clearly separate two
points, which are close together in a
sample. There are a number of
expressions for resolving power, but
one often used is the equation
according to Lord Rayleigh:
R=0.61/NA
R is the minimum between two points
that can still be resolved. The factor
0.61 results from the theoretical
calculation, and is the wavelength of
the light being used. This equation
shows that the larger the numerical
aperture of the objective, the smaller
details it can resolve. In addition,
using shorter wavelength light will
decrease the size of features, which
can be resolved.
The numerical aperture of the
microscope system depends on the
numerical apertures of both the
objective and the condenser.
NA system=
½ (NA objective + NA condenser)
Therefore, choose the numerical
aperture of the condenser to be at least
as large as the numerical aperture of
your highest power objective.
A condenser having a numerical aperture of
1.00 or larger must have its top lens “oiled” to
the bottom of the specimen slide. (This is in
addition to the drop of oil placed between the
objective lens and the specimen slide.) Failure
to do this would reveal spherical aberration
(top right) in an otherwise well-corrected
condenser. With the condenser properly oiled
to the slide, accurate focusing of the field
diaphragm is made easier (bottom right). In
addition, the objective lens is able to use a
greater proportion of the illuminating beam, to
form an image of good resolution and contrast.
9
Instant Photography
Photomicrography
Through the Microscope
Eyepiece
Eyepiece Characteristics
Magnifying Power
The magnifying power of an eyepiece is marked by a number followed by an X.
Eyepieces range in power from 2X to 20X.
Field of View
The field of view number follows the magnifying power. It shows the diameter in
mm of the primary image that is magnified by the eyepiece.
Eyepoint of Exit Pupil
The eyepoint, or exit pupil, of the microscope is the point above the eyepiece
where the image comes to its smallest diameter. The pupil of your eye is
positioned at the eyepoint during observation. Note that high eyepoint
eyepieces provide a more comfortable view if you wear eyeglasses. Look for a
symbol of spectacles on the equipment.
Compensating Eyepiece
Compensating eyepieces correct for the Chromatic Difference in Magnification
in the intermediate image produced by highly corrected objectives. They
should be used with all plan objectives and with fluorites and achromats.
Compensating eyepieces are designated by K, C, or Comp. They can be
identified by a yellowish fringe at the periphery of the image.
Adjustable Eyepiece
Adjustable eyepieces have an eyelens, which can be used to focus on a reticle
positioned at the intermediate image plane. When used in the viewing
eyepiece, adjust the eyelens so that the reticle is in sharp focus before
focusing the specimen. It is convenient to use an adjustable eyepiece in the
phototube when shooting photomicrographs with a bellows camera.
Projection Eyepiece
A projection eyepiece cannot be used for normal viewing. It is used for
photomicrography with cameras that use no other lenses between the
projection lens and the film plane. Some projection eyepieces are adjustable
for different distances to the film plane while others may be fixed for a specific
distance.
Diagram showing eyepoint, the eyelens, the
field lens, and the diaphragm defining the
intermediate image plane.
10
Instant Photography
Photomicrography
Through the Microscope
Cameras for Photomicrography
The Polaroid MicroCam
A microscope camera serves many
functions, including:
Polaroid offers the MicroCam, a
lightweight SLR microscope camera
that attaches to virtually any light
microscope instantly and easily. It can
be used on both compound and stereo
microscopes that have standard
diameter eyepiece tubes, even those
that have no provisions for
photography.
·
·
·
·
holding the film in a fixed position
in relation to the microscope
Producing a real image of the
specimen in the film plane
Facilitating focusing and framing of
the specimen
Providing a means to control
exposure of the film
A variety of camera options are
available for photomicrography:
Cameras from microscope
manufacturers
Microscope manufacturers supply
cameras that accept the most
commonly used instant
photomicrography film formats, 4 x 5”
and 3 ¼ x 4 ¼”.
These cameras are designed to
function as an integral part of the
microscope. In some systems, the
cameras are part of the microscope
body. However, most camera systems
are attached to the phototube of a
trinocular microscope, which has a
binocular arrangement for viewing and
a vertical third tube for
photomicrography.
In the most common design, the
camera body contains a shutter
mechanism, a photo sensor for
determining light levels, and a lens to
focus the image of the specimen on
the film plane, which is parfocal with
the binocular image. It may also have
a viewing telescope for focusing and
framing.
To install the MicroCam, remove the
microscope eyepiece and insert the
camera into the microscope tube. The
MicroCam’s 10X eyepiece replaces the
microscope eyepiece. The MicroCam
has a unique rotary shutter that allows
through the lens viewing for focusing
and framing the sample. Other
positions of the shutter measure
brightness and exposure. You can use
either black and white or color integral
film. The picture is automatically
ejected after exposure.
Bellows cameras
A bellows camera can also be used
with microscopes with a vertical
phototube. Polaroid offers the MP 4+
bellows camera. Leica and Nikon also
have made bellows cameras for
photomicrography. Note that the
magnification factor of a bellows
camera is dependent on the bellows
length, so adjust the bellows to the
desired length before installing it over
the microscope.
The MicroCam can be used on the
microscopes that have no provision for
photomicrography.
8 x 10 large-format cameras
Some of the more sophisticated
microscopes can accommodate 8 x
10” film holders for large format
photomicrography. Alternatively, a
large bellows camera head for an
8x10” camera back is available for the
MP 4+ camera.
Use planapochromat lenses, which are
the best corrected, for large format
photomicrography because of the
magnification may exceed the
maximum useful magnification of
microscope objectives. Wide-field
eyepieces are also helpful to minimize
the bellows length. For illumination,
use a 100-watt tungsten/halogen light
(or a more powerful light source) to
keep exposure times short.
Many current designs have reduced
the optics within the camera and use
projection eyepieces that do not need
a lens in the camera to focus the
image on the film plane.
Photomicrographic camera adaptable for instant print or 35mm film.
11
Instant Photography
Photomicrography
Through the Microscope
Setting Up the Microscope and
Camera to Create Instant 35mm
Slides
You can make instant
photomicrographic slides with Polaroid
instant 35mm transparency films,
Polachrome, Polachrome HC,
Polapan, and Polagraph. (See the film
chart on pages 70-71). Most camera
systems supplied by microscope
manufacturers accept a 35mm back.
An SLR 35mm camera can be
attached to the microscope with a Tmount adapter and a microscope tube
adapter. The T-mount adapter is
specific for the lens mount mechanism
of your 35mm camera. It should be
joined to the tube adapter which holds
the microscope eyepiece and locks on
the microscope eyepiece tube. The Tmount adapter is available from
camera manufacturers, microscope
manufacturers, or scientific supply
houses. For framing, use the SLR
viewing screen of the camera and the
microscope focus control. Expose the
film with the camera focal plane
shutter. Since exposure is controlled
only by time, se t the exposure control
to the “aperture preferred” mode.
Focusing and framing
Understanding Parfocalization
Microscope cameras provide one of
the three methods for ensuring proper
focus of the specimen in the film
plane:
Parfocalization is more than a
convenience; it ensures proper use of
the objective. It is achieved when the
image of the specimen is
simultaneously in focus in the
microscope and on the camera film
plane. The microscope objective then
allows optimal direct viewing. If the
focus of the objective must be
readjusted for photography, the
microscope objective will no longer be
in its proper position. Spherical
aberrations may result in a degraded
image, especially with high numerical
aperture dry objectives.
·
A viewing telescope in the camera
body, which is parfocal with the
film plane of the camera. The
telescope contains a reticle with a
double cross hair. It is helpful,
especially at low magnifications, to
use a focusing magnifier in
conjunction with the viewing and
telescope to critically focus the
reticle and then the specimen.
·
Some cameras use an adjustable
eyepiece with a focusing eyelens
and a reticle delineating the area
of the photomicrograph as one of
the binocular eyepieces. It is
important that the reticle be in
sharp focus before focusing the
specimen.
·
A ground glass screen at the film
plane may also be used for
focusing, but it has the
disadvantage of low light levels at
the film plane. This may
necessitate dimming room lights
for focusing.
Basics
Depth of field: the total distance
within the sample between the
nearest and farthest points of
acceptable focus in the image.
Depth of focus: the distance of
acceptable focus in image space
where the film plane can be
placed.
Increasing the magnifying power
of the objective decreases the
depth of the field and increases
the depth of focus.
12
Cameras supplied by the microscope
manufacturer should be parfocal when
the viewing reticle is in focus with the
specimen. To make a camera parfocal
with the viewing eyepiece, use an
adjustable eyepiece in the phototube.
This may be a projection eyepiece or a
viewing eyepiece with a focusing
eyelens. With a low magnification
(10X) objective, focus carefully through
the viewing eyepiece on a thin, highcontrast specimen or a stage
micrometer. Then, without touching
the microscope focusing knob, adjust
the eyepiece in the phototube to the
point where the image of the specimen
is sharpest on the camera viewing
screen. Use the clear central areal of
the viewing screen to check focus in
the aerial image. (It will be necessary
to remove the light baffle temporarily for
this step.)
Setting parfocality is most critical with
a low power objective because the
depth of focus is smallest.
Instant Photography
Photomicrography
Through the Microscope
The Camera Magnification Factor
and Photographic Magnification
The visual magnification of a
microscope is the product of the
magnifying powers of the objective and
the eyepiece. Photographic
magnification is determined by the
microscope magnification and the
magnification factor of the camera.
With a bellows camera, the camera
magnification factor is dependent on
the bellows length, which is the
distance from the eyepoint of the
ocular to the film plane (also called
projection distance). The camera
magnification factor is equal to the
bellows length (in millimeters) divided
by 250mm, the reference distance for
visual magnification. Thus, when the
projection distance is 250mm, the
camera magnification factor is 1X, and
the photographic magnification is
equal to the visual magnification.
Most cameras for 4 x 5” or 3 1/4 x 4 ¼”
formats have a magnification factor
between 0.8X and 1.25X, and that
factor is marked on the camera itself.
Some currently available cameras
designed for use with projection
eyepieces (of low magnifying power)
have a larger camera factor 3X or 4X.
As an illustration, the following two
systems will each give photographic
magnification of 200X:
·
·
A 20X objective used with a 10X
eyepiece and a camera factor of
1X.
A 20X objective used with a 2.5X
projection eyepiece and a camera
factor of 4X.
A 35mm camera usually has a smaller
magnification factor (0.25X to 0.5X)
because of the shorter projection
distance. However, the image on the
slide is subsequently enlarged in
printing or projection.
Determining exact magnification
Successful Photomicrography Tips
Microscope objectives may differ from
their nominal magnifying power by a
few percent. If you are making critical
measurements on your micrographs
and want to know the exact
magnification, photograph a stage
micrometer and measure the
photomicrograph directly.
Controlling Exposure in
Photomicrography
Photographic exposure is the length of
time that the shutter remains open.
For photomicrography, camera
systems with automatic exposure
control offer greatest flexibility and
efficiency. In general, older or less
expensive automatic cameras
measure the amount of light in the
whole image and determine exposure
based on the average brightness. The
design is ideal for specimens of
uniform brightness. However, nonuniform specimens may require some
adjustment of exposure. More
sophisticated cameras offer a choice
of averaging mode or spot-metering
mode that measures the brightness in
a selected spot and automatically
registers the best exposure for that
area.
Simple microscope cameras pride only
a shutter for manual exposure control;
however, consistent exposures are
possible with such simple cameras if
you standardize your conditions for
photomicrography. Use the graduated
test strip described on page 32 to find
optimum exposure. Keep a record of
the type of specimen, lamp voltage,
magnification, filtration, contrast
techniques, and speed of the film.
13
· When working with a specimen
that is not uniformly bright, you can
modify the camera’s automatic
exposure setting to correct the
exposure. For example, if your
specimen has small, bright particles
on a dark background, the average
brightness will be low and the
camera will give too much exposure.
You can either reduce the exposure
factor using a feature available on
some microscope cameras, or set
the camera for a higher film speed
value than what you’re actually
using.
Instant Photography
Photomicrography
Through the Microscope
lamp manufacturer. Set your lamp
consistently. Bear in mind that the
color temperature of the
microscope lamp increases with
increasing lamp voltage.
Setting Up Your Microscope for
Photography
1. Install the camera
·
·
·
For a MicroCam: Remove the
eyepiece from the phototube or
from the binocular tube and insert
the MicroCam.
For a camera supplied by
microscope manufacturer: Secure
the camera on the phototube after
installing the recommended
eyepiece or photo eyepiece.
For a bellows camera, set the
desired bellows length and position
the shutter at the height of the
ocular eyepoint. (There should be
no contact between the eyepiece
and the shutter.)
2. Remove the viewing filters and
diffusers
4. Add photographic filters
done through a parfocalized
microscope eyepiece, a viewfinder
eyepiece in the microscope
camera system, or a camera
viewing screen.
6. Expose the film
Use the appropriate color
conversion filters for color
photography or the desired contrast
filters for black and white work.
·
Note: The Polaroid MicroCam
incorporates a blue filter for
exposure of 339 Color Autofilm. No
other color conversion filter is
necessary.
·
Successful Photomicrograph Tip
5. Focus the specimen carefully in
the film plane of the camera
and ensure that the microscope
is set for Kohler illumination
Some trinocular microscopes have
an adjustable prism, which is used
to change the light path from the
viewing eyepieces to the photo
tube. Ensure the prism is set for
photography before you make an
exposure.
Follow directions for Kohler
Illumination described on pages 15
through 22. The focusing method
depends on the photomicrographic
system. Generally, focusing will be
Remove any viewing filters and
diffusers from the light path.
3. Adjust the lamp voltage
Follow the recommendations of the
1
4
For manual exposure cameras:
Determine the exposure and
expose the film accordingly. Start
by making a graduated test strip,
as described on page 34.
For automatic or semi-automatic
exposure systems, refer to the
speeds of Polaroid films shown on
pages 70 and 71.
2
3
5
6
14
Polaroid
Instant Photography
Photomicrography
Kohler Illumination
Through the Microscope
Introduction
Camera and film are in many ways
more demanding than the human eye,
so that good photography through the
microscope consists of more than
good visual microscopy.
Arctium
Lappa stern,
50x
PolaPan CT
Cristina Zeni
Illumination is the most critical element
in high-quality microscopy and
photomicrography. With careful
attention to illumination, you can reveal
the full color and detail of a specimen
and produce the best
photomicrographs. To produce a
satisfactory image, you must meet
special illumination criteria, adjust the
microscope carefully, and align
components properly.
In 1893, August Kohler of the Carl
Zeiss organization developed a
method for producing optimum
illumination conditions in the light
microscope.
Kohler Illumination is essential for
quality photomicrography at high
magnifications. Without it, the sample
is not uniformly illuminated, there is
insufficient light intensity at the film
plane, and the objective lens is
severely limited in its ability to resolve
fine detail.
15
Instant Photography
Photomicrography
Through the Microscope
Understanding Kohler Illumination
as Two Paths of Light
Kohler Illumination is designed to
satisfy two needs. First, it enables the
light source to fully and evenly
illuminate the required specimen area.
Second, it enables the light source to
completely fill the back focal plane of
the objective lens with image-forming
light, a theoretical requirement for
optimum image resolution.
To understand the optical
characteristics of Kohler Illumination, it
is useful to look at the light in two
ways: as image-forming rays and as
illuminating rays. In practice, of
course, the two co-exist.
Conjugate planes
In each ray path there are four planes,
called conjugate planes, which are in
common focus. When we arrange the
microscope for Kohler Illumination, we
are ensuring the common focus of the
conjugate planes.
Whatever occurs in one conjugate
plane will be seen in other conjugate
planes. The special contrast
techniques discussed in this book
depend on manipulations in the
conjugate planes to create contrast in
the image of the specimen.
To make the following diagrams easily
comprehensible, each optical
component is shown as a single unit.
Generally, this is not an exact
representation of the components in
an actual instrument, but a simple
representation of more complex
systems.
Image-forming ray path
The field diaphragm, the specimen, the primary
image plane at the eyepiece field stop, and the
retina of the eye for the film plane are the
conjugate planes of the image-forming ray path,
and must be in common focus.
16
Illuminating ray path
The light source, the substage condenser
aperture diaphragm, the back focal plane of the
objective lens, and the eyepoint of the eyepiece
are the conjugate planes of the illuminating ray
path, and must be in common focus.
Instant Photography
Photomicrography
Through the Microscope
Microscope Components That
Provide Kohler Illumination
Collector and field lenses
A collector lens in the illuminating
system brings the image of the light
source into focus at the plane of the
substage condenser aperture
diaphragm.
If the illuminating lens system contains
a pair of lenses, the lens closer to the
light source is called the collector lens
(a), as it collects the light rays from the
lamp. It directs the rays to the field
lens (b), which is closer to the field
diaphragm. The field lens is
responsible for bringing the rays into
focus at the aperture diaphragm of the
substage condenser.
Field diaphragm
The field diaphragm © limits the area
of illumination to that part of the image
field that is actually in view or being
photographed. This restriction
prevents unneeded light outside the
field of view from entering the
microscope and causing imagedegrading flare. The elimination of
extraneous light is particularly
important with highly refractive
specimens of low inherent contrast.
While setting Kohler illumination, the
circular image of the field diaphragm
(b) must be brought into focus at the
specimen plane by raising or lowering
the substage condenser.
Substage condenser
A properly adjusted substage
condenser © will focus the light rays to
uniformly illuminate the field of view of
the specimen and to fill the back focal
plane of the objective lens with imageforming light.
The substage condenser (d) has an
aperture diaphragm (e) that controls
the angle of illumination – and has an
aperture diaphragm (e) that controls
the angle of illumination – and thus the
amount of light to the objective lens.
The adjustment of the aperture
diaphragm has a decisive effect on the
contrast and resolution of the image of
the specimen.
Learn more about its effect in the
chapter on Further Understanding
Kohler Illumination.
By selecting the appropriate optics, the
microscope designer can place the
field diaphragm anywhere between the
collector lens and the substage
condenser. In most modern
microscopes, it is in the base of the
apparatus.
17
These diagrams show the position of the
collector lens (a), the field lens (b), the field
diaphragm (c), the substage condenser (d),
and the aperture diaphragm (e).
Instant Photography
Photomicrography
Through the Microscope
Adjustments to the Microscope for
Kohler Illumination
The adjustments for Kohler illumination
are carried out in these general steps:
• Align and focus the substage
condenser and field diaphragm
- by moving the optical components
along the optical axis of the
microscope system to achieve focus
-by laterally aligning the optical
components to center the entire
optical system along a common
optical axis.
• Adjust the size of the aperture
diaphragm.
• Center the aperture diaphragm.
• Align the light source.
Aligning and Focusing the Substage
Condenser and the Field
Diaphragm
1. Place the specimen slide on the
stage and focus using a low power
objective (10X).
To focus the specimen, raise the stage
while viewing from the side until the
slide is close to the objective. Then,
while viewing through the eyepiece,
slowly lower the stage until the image
is in sharp focus. If focus of the
specimen is difficult to find, a small
movement of the slide helps locate the
plane of focus. Slowly make slight
movements of the slide, either with the
position control or a rotation of the
stage while it is lowered. This
technique will prevent contact of the
specimen and objective.
Slowly close the aperture diaphragm of
the substage condenser to a point
where you see a distinct reduction in
brightness through the eyepiece. Then
open very slightly.
2.Adjust the separation of the
binoculars to your interpupillary
distance; You should be able to see
the microscope image with both eyes
without moving your head. (Your
interpupillary distance is constant
and you can make that adjustment
when you first approach the
microscope.) Then adjust the
eyelens for sharp focus of the reticle.
A
3.Close the field diaphragm (generally
situated in the base of the
microscope) to its smallest setting.
(a)
Viewing through the eyepiece, raise or
lower the substage condenser until the
edge of the field diaphragm appears
sharp with the specimen image. A
condenser with good chromatic
correction will yield a sharp outline,
neutral in color. A condenser of low
chromatic correction will allow only an
approximate focus, in which case it is
generally best to adjust for a red-blue
edge in the diaphragm.
B
4.Open the field diaphragm to about ¾
of the visual field and refocus the
edge of the diaphragm. (b)
Align the substage condenser by
centering the image of the field
diaphragm, using the condenser’s
radial centering screws. If necessary,
refocus the condenser to keep the field
diaphragm in sharp focus with the
specimen image ©
5.Open the field diaphragm until it is
just outside the field of view. For
photography, open the field
diaphragm just beyond the area of
the film format. Do not open it any
farther, since this could cause flare
and a loss in contrast. (d)
Repeat steps 3 through 5 every time
you change the objective.
18
C
D
Instant Photography
Photomicrography
Through the Microscope
Controlling the Size of the Aperture
Diaphragm
Set the size if the substage condenser
aperture diaphragm to ensure the best
possible image of the specimen. As a
rule of thumb, the diaphragm should
be closed down sufficiently to provide
the desired image contrast, but not so
far as to cause a loss of resolution of
detail.
The best setting will vary with the
nature of the specimen, as well as with
the information or effect that is to be
derived from the image. Most
commonly, the setting will be such that
the circle of light within the diaphragm
blades has a diameter of 2/3 to ¾ the
size of the entire light disc, as seen
down the eyepiece tube with the
eyepiece removed.
Centering the Aperture Diaphragm
Remove the microscope eyepiece and
look down the tube at the back focal
plane of the objective lens. The
aperture diaphragm is visible in the
back focal plane of the objective lens
when Kohler illumination is properly
set.
Viewing is easier if a phase telescope,
available from your microscope
manufacturer, is inserted in place of
the eyepiece. If the microscope
features a Bertrand Lens system, you
can view the back focal plane without
removing the eyepiece. Focus the
phase telescope or the Bertrand lens
on the back focal plane of the objective
lens.
If the edge of the 3/4 open diaphragm nearly
touches the edge of the illuminated back focal
plane of the objective lens, centering is
necessary.
Close the aperture diaphragm to about
¾ of the diameter of the field of view.
If the edge of the diaphragm nearly
touches the edge of the objective back
focal plane, the misalignment should be
corrected.
Follow the manufacturer’s directions for
centering. In some microscopes, the
aperture diaphragms fixed in a centered
position. When the condenser
contains phase contrast or darkfield
elements, a centering mechanism is
provided.
Misalignment is corrected and the image of the
aperture diaphragm is centered.
Successful Photomicrography Tip
Illustration of aperture diaphragm adjusted to
diameter of 2/3 to 3/4 of the entire light disc.
Use a high-dry 40X objective lens
when centering the aperture
diaphragm to avoid a frequent need
to re-center. When you have
centered the aperture diaphragm,
the lower-powered lenses will almost
always fall within acceptable
tolerances.
Improvising When you Don’t have
a Phase Telescope or a Bertrand
Lens
For accurate viewing of the aperture
diaphragm, your eye must be
centered on the eyepiece tube. In
the absence of a phase telescope or
Bertrand lens, the following method
helps ensure that your eye is
centered.
Press a piece of household aluminum
foil over the empty eyepiece tube and
punch a small hole in the center, no
larger than 1/8 inch in diameter, as
shown.
19
Instant Photography
Photomicrography
Through the Microscope
Manually Aligning the Light Source
Summary of Kohler steps
Although most microscopes have precentered and pre-focused lamps, some
require that the light source be aligned
and focused by the user. Most of the
older systems still in use do allow this
control. Refer to the instruction
manual, or follow this procedure:
1.Place a piece of white paper
immediately below the aperture
diaphragm of the substage
condenser. If the filter holder is next
to the aperture diaphragm, the paper
can be conveniently placed in it.
2.Using a suitably small mirror, view
the underside of the paper, which is
facing the light source.
3.Center and focus the projected image
of the lamp filament by whatever
means are provided by the
manufacturer.
4.Carefully remove the paper and check
that the filament’s image is also
centered on the closed aperture
diaphragm. If you have a Bertrand
lens, you can look directly at the
back of the focal plane of a high
power objective (40X or 100X) and
check filament centering there. Once
aligned, the light source should not
require realignment until the lamp is
replaced.
Sometimes a diffusing surface on the
collector lens, or a diffusion screen in
the illuminating system, prevents the
projection of a recognizable image of
the filament. If the design of the
microscope permits, center and focus
the diffused illumination so the
projected circle of light is uniform in
brightness.
1. Position the slide. Using a 10X
objective lens, focus the image of
the specimen.
2. Adjust the binoculars and
eyepiece for your eyes.
3. Close the field diaphragm and
raise or lower the substage
condenser to obtain a sharp
image of the field diaphragm.
The manufacturer of this instrument has
provided a lamp target for convenient
centering and focusing of the light source.
The filament of the light source is focused
and centered on the target by means of
the lamps’s focusing knob and radial
centering screws. The method of
focusing and centering the light source
may differ in other microscope models.
4. Open the field diaphragm to ¾.
Center and refocus, as necessary.
5. Open the field diaphragm to just
outside the field of view.
6. Adjust the diameter of the
aperture diaphragm for optimum
contrast and resolution.
7. Check the centering of the
aperture diaphragm by viewing the
back focal plane of the objective.
An additional adjustment is necessary
when a mirror is incorporated in the lamp
housing. The primary image and reflected
image of the lamp filament must both be in
sharp focus, positioned next each other,
and centered.
Summary of Kohler Steps
20
Instant Photography
Photomicrography
Through the Microscope
The Condenser’s Illuminating Cone
The illustrations below show how the
angle of the illuminating cone, and thus
the numerical aperture of the
condenser, are controlled by the
aperture diaphragm of the condenser.
As the aperture of the diaphragm is
closed down, the cone of light becomes
NA 1.20
narrower and more sharply delineated
(while the lower part of the cone, which
represents the specimen plane,
remains constant in size).
If the field diaphragm were reduced in
size, the lower part of the cone would
become smaller (but the angle of the
cone, and thus the numerical aperture,
would remain essentially unaltered).
NA 0.60
NA 0.30
NA 0.15
To show the path of light rays emitted from the condenser,
a small block of uranium glass, which fluoresces when it
absorbs visible light of short wavelengths, was oiled to the
top of a highly corrected substage condenser.
Typical Field Views for Kohler Illumination
A typical field of view when the substage
condenser and the field and aperture
diaphragms have been set properly. The
aperture diaphragm setting will affect the ability
of the objective lens to resolve fine detail. It
will also control image contrast and depth of
field.
When the conditions of Kohler illumination
have been met, the partially closed field
diaphragm will be in focus together with the
specimen. (This requirement is indicated in
the diagram of the image-forming ray path,
page 16.)
21
When the substage condenser is not focused
properly, a reduction in either field or aperture
diaphragm setting produces uneven
illumination over the visual field. This effect
may be recorded noticeably on film, even when
it is barely perceptible when viewing through
the microscope.
Instant Photography
Photomicrography
Through the Microscope
The Reflected Light Microscope
In reflected light microscopes, the
objective also acts as the condenser.
The objective both focuses the
illumination onto the specimen, and
then images the light reflected from
specimen. Consequently, setting up for
Kohler illumination in reflected light
microscopes is simpler, and does not
require positioning of the condenser.
The field diaphragm is the aperture
closest to the objective. The aperture
diaphragm is further away, but is
imaged by a relay lens in the back
focal plane of the objective.
The setting of the field diaphragm has a
large effect on flare in the image and
should be closed down to illuminate
only the area being photographed. As
in transmitted light, the aperture
diaphragm controls the cone of
illumination and affects the resolution of
fine detail.
Successful Photomicrography
Tips
• If you are focusing on a sample
that is nearly featureless, the plane
of focus is hard to find. Close the
fielddiaphragm to its smallest
diameter and look for the sharp
image of the field diaphragm.
Optimum focus of the specimen
will be close to that setting.
• If you need to examine the surface
of a highly scattering sample, an
opaque evaporated metal or carbon
coating diminishes the amount of
light scattered from subsurface
features, and increases the
contrast of the image.
• If you need to examine the surface
of a clear specimen, oil the
specimen toa blackened glass
slide. This eliminates reflection
from the back surface, increasing
contrast of the image of the front
surface.
Setting Up Kohler Illumination for a
Reflected Light Microscope
1.Adjust the binoculars to your
interocular distance and focus the
eyepiece reticle.
2.Place the specimen on the stage,
checking to ensure that there is no
coverslip.
3.Center the specimen under a low
power objective lens (approximately
10X).
22
4.Close the field diaphragm
5.Focus the specimen. It will be in
focus when the image of the field
diaphragm is near focus. The
objective acts as its own condenser
and proper positioning of the field
diaphragm occurs automatically.
6.Center the field diaphragm and open
to the edge of the field of view.
7.Adjust the aperture diaphragm for
optimal contrast and resolution.
Polaroid
Instant Photography
Further Understanding
Kohler Illumination
Photomicrography
Through the Microscope
Introduction
Beech, myrtle,
grapvine and
maize stems, 2x
Polachrome
Roland H. Gebert
Proper illumination is the most
important feature of photomicrography.
The substage condenser’s aperture
diaphragm controls the angular cone of
illumination, and thus the amount of
light that reaches the objective lens.
The adjustment of the aperture
diaphragm is one of the most important
steps. It has a decisive effect on the
contrast and resolution of the image.
Understanding proper techniques for
illumination and the function of the
aperture diagram is essential for highquality photomicrography.
23
Instant Photography
Photomicrography
Through the Microscope
How the Specimen Affects Light
Absorption
Diffraction
As it encounters the specimen, light is
affected in several ways, dictated by
the characteristics of the specimen. In
transmitted light brightfield microscopy,
the three dominant effects are
absorption, refraction, and diffraction.
Absorption is the reduction in the
intensity of light as it is transmitted
through a medium. When the
absorption of light is spectrally
selective, the light will change color as
it passes through the medium. Certain
microscope specimens are stained, so
that the selective absorption of light can
be utilized to reveal the greatest
possible detail and information.
Diffraction is a deflection of light rays at
an “edge” or interface between small
details of the specimen having different
absorptive or refractive properties.
Diffracted light plays an important part
in the creation of a microscope image.
The more diffracted light rays an
objective lens can accept, the better it
can resolve the specimen.
Smaller features in a specimen diffract
light to a greater angle than large
features. Red light is diffracted to a
greater angle than blue light.
A red filter absorbs blue and green light
and transmits its own color, red.
Refraction
Refraction is the deflection, or
changing of course, of light rays as
they pass obliquely from one medium
to another of different refractive index
(that is, between media in which the
velocity of light is different). The two
media could be two different specimen
parts, or they could be the specimen
and the mounting medium.
The path of a single oblique ray.
24
Diffraction at the edge of a specimen.
Instant Photography
Photomicrography
Through the Microscope
Controlling Light and Image Quality
Image quality depends on a subtle
interplay between the objective lens
and the substage condenser’s aperture
diaphragm.
The Objective lens
The more diffracted light rays an
objective lens can accept, the better its
resolving power. The larger the
numerical aperture of the objective, the
greater its light-gathering power, and
the smaller the features it can resolve.
The substage condenser
The substage condenser supplies the
specimen, as well as the objective
lens, with a concentrated beam of light.
The substage condenser also has a
numerical aperture. It indicates the
condenser’s light-concentrating ability,
or its maximum cone of illumination.
When the effective numerical aperture
of the substage condenser, as
controlled by the aperture diaphragm,
closely matches the numerical aperture
of the objective lens, there is the
greatest potential for resolving detail.
As the aperture diaphragm is
progressively closed down, it changes
the proportion of direct image-forming
light to deflected or refracted) light
reaching the objective. The ability to
alter this proportion allows some
control of image quality.
The Mix of Deflected and Direct
Light
deflected (diffracted and refracted)
light is hardly reduced at all.
The light at the back focal plane of the
objective is a mix of direct rays and
rays diffracted or refracted by the
specimen. You can observe the mix of
direct rays and deflected rays by
removing the microscope eyepiece and
viewing the back focal plane of the
objective. An enlarged and more
detailed view will be obtained if the
eyepiece is replaced by a phase
telescope, described on page 19.
The central circle of direct illuminating
rays represents light that was affected
by absorption in the specimen. The
less intense light that fills the entire
back focal plane of the objective lens is
diffracted and refracted light. The
intensity of this light depends on the
amount of diffraction and refraction
present and thus on the nature of the
specimen. Deflected rays actually
occupy the full aperture of the objective
lens. They are not discernible in the
central illuminating beam simply
because they are overwhelmed by the
intensity of that beam. Deflected light
does not represent unwanted flare. It is
very important and useful for forming the
image.
The angle of the direct light rays
reaching the objective lens from the
specimen depends on the setting of
the aperture diaphragm. As the
aperture size is reduced, the central
circle of direct light decreases
proportionally, while the surrounding
Aperture diaphragm settings and angle of direct light rays reaching objective lens.
Left: The aperture diaphragm is almost fully open and the sample is illuminated by a
wide cone of direct rays. Right: The aperture diaphragm is closed down. The cone of
illumination is smaller and the deflected rays have a greater effect on the image.
25
Instant Photography
Photomicrography
Through the Microscope
The Effect of Aperture Setting on Image Quality
The photomicrographs below illustrate how different aperture diaphragm settings affect three different specimens.
Photomicrographic Data
90%
Aperture diaphragm settings
These photomicrographs show a step-by-step
reduction in the effective numerical aperture of
the condenser’s illuminating beam-from 90, 50,
25, and 12 percent of the diameter of the back
focal plane of the objective lens-illustrating
how aperture diaphragm settings affect
photomicrographs of each specimen.
If the objective lens numerical aperture were
1.35, the substage condenser numerical
aperture in the above settings would be 1.20,
0.68, 0.34, and 0.16, respectively.
1
2
3
Diatom, stauroceis phoenicenteronUnstained
This diatom is visible primarily because of
diffraction and refraction. It has negligible
absorption.
Objective lens: 100X Planapochromat, NA 1.35
Condenser: Achr/Apl, NA 1.40, oiled to slide
Film: Black and white panchromatic
(Polaroid Type 55 film)
Filters: Wratten #58 + #8
Magnification: 800X
Human kidney tissue, glumerulusFrazer/Landrum stain
This stained kidney tissue section features
strong absorption characteristics and moderate
amounts of diffraction and refraction.
Objective lens: 40X Planaphrochromat, NA 0.95
Condenser; Achr/Apl, NA 1.40, oiled to slide
Film: Black and white panchromatic
(Polaroid Type 55 film)
Filters: Wratten #32+#22
Magnificaton: 400X
Human blood smear, myelocytesWright stain
This stained blood smear has distinct
absorption properties, very limited diffraction
effects, and virtually no refraction.
Objective lens: 100X Planapochromat, NA 1.35
Condenser: Achr/Apl, NA 140, oiled to slide
Film: Black and white panchromatic
(Polaroid Type 55 film)
Filters: Wratten #44A + #15
Magnification: 1500X
26
50%
Instant Photography
Photomicrography
Through the Microscope
25%
Visual and Photographic Effects
12%
1. When the condenser aperture
diaphragm is at 90 percent of the
numerical aperture of the objective lens,
the direct illuminating beam is large and
powerful. A diatom has no appreciation
absorption qualities, so the direct illuminating
beam has relatively little useful effect. The
important contribution of diffraction and
reflection, provided by the many small
punctae and other delicate glass-like
structures of the diatom, is overwhelmed by
this much stronger illuminating beam. Thus,
the specimen is only faintly visible.
As the aperture diaphragm is reduced, the
illuminating beam is diminished so that the
diffracted and refracted rays can play a
decisive role in making the diatom clearly
visible.
1
Extreme reduction of the aperture leads to a
loss of image resolution due to disturbing
diffraction patterns that interfere with the
clear reproduction of the significant small
punctae in the diatom.
2. This biological kidney tissue is thinly
sectioned and selectively color-stained to
reveal fine detail and indicate chemical
characteristics. As the colors of such a
stained specimen are revealed by light
absorption, they are equally apparent at all
condenser apertures.
2
Important details within the kidney specimen
are visible when the condenser is in the 90
and 50 percent settings. Upon further
reduction of the aperture diaphragm, fine
image detail becomes obscured by
unwanted diffraction and refraction
phenomena.
At the smallest aperture setting (12
percent), all important structures in the
specimen are overwhelmed by the
broadening diffraction pattern, as well as
by refraction to the extent that only the
color and general irregular shape of the
kidney specimen remain to be seen.
3
27
3. Blood, which is commonly stained for
best visibility, has very limited diffractive
and refractive qualities. As the aperture
diaphragm is closed down, the specimen
gains only slightly in contrast due to the
almost total absence edge effects from
diffraction and refraction. Only after
extreme reduction of the aperture (12
percent or less) will the resolution of the
image deteriorate to a noticeable extent.
Instant Photography
Photomicrography
Through the Microscope
Determining the Best Aperture
Diaphragm Setting
In General, the aperture diaphragm
setting controls image quality in the
following ways:
• When the full numerical aperture of
the objective lens is used, the
potential for optimum image
resolutions is at its highest, but
contrast is relatively low.
• As the aperture diaphragm is closed
down, image resolution tends to
deteriorate, but contrast increases.
Setting the aperture diaphragm, which
provides the most satisfactory mix of
direct and deflected light, depends on
the proportions of absorption,
diffraction, and refraction in the
specimen. It also depends on the
information that is sought from the
specimen, and whether resolution of
detail or image contrast is of primary
importance. Familiarize yourself with
the specimens, and understand the
optical characteristics each displays.
If the fully illuminated numerical
aperture of the condenser is higher
than that of the objective lens,
unwanted flare or stray light is present.
Under such conditions the image loses
contrast, and the image detail is
obscured. Literature on photomicrography often suggests an
“average” aperture setting of about ¾
the diameter of the entire disk visible
when you look at the back focal plane
of the objective. However, this should
serve only as an approximate setting.
The examples in this chapter will help
you determine the aperture setting
most appropriate for your specimen.
Critical Factors that Affect Image
Quality and Resolution for
Photomicrography
The Aperture Diaphragm and Its
Effect on Depth of Field
• The staining technique.
• The objective lens numerical
aperture.
• The condenser numerical aperture
and the setting of its aperture
diaphragm.
• The wavelength (color) of the light.
• The nature of the mounting
medium.
• The thickness of the sample.
Closing the aperture diaphragm
increases the zone of sharpness, or
depth of field, through the thickness of
the specimen. This is significant with
lower power instruments such as
stereo zoom microscopes.
When the aperture diaphragm is closed
down too far, the deflected light overpowers the direct illuminating rays to
that the diffraction causes visible and
disturbing fringes, bands, or patterns in
the image. Unwanted refraction
phenomena can produce apparent
structures in the image that do not
represent the actual specimen. This
can lead to erroneous deductions about
the specimen.
28
Polaroid
Instant Photography
Photomicrography
Instant Film Characteristics
for Photomicrography
Through the Microscope
Introduction
A variety of film types and formats are
available for instant photography. To
select the best film to record the
information you need, it’s important to
understand the special characteristics
of each.
See-crab
carapace, 25x
PolaChrome
Ignaz Kaelin
Base the choice between black and
white or color film on the
characteristics of the specimen. For
example, a monochromatic specimen,
which inherently has only shades of
gray or a single color, is best recorded
in black and white. For stained
specimens, whose colors are the result
of staining to differentiate structure, use
black and white film and filters to
dramatically enhance the rendering of
stains and communicate the
specimens’ features most clearly. For
specimens that are multicolored in their
natural state, use color film to
communicate the appearance
accurately.
Instant films are available in black and
white print, print with usable negative,
positive black and white transparency
and color print or positive transparency
formats. Sizes range from 35mm to
8 x 10”.
29
Instant Photography
Photomicrography
Through the Microscope
Film Speed Choices
Photographic Image Resolution
Film speed, or sensitivity, varies by film
type. Make your selection of film
speed according to the specimen’s
characteristics and the available l light
level in the instrument. Generally,
Resolution is expressed as the number
of line pairs per millimeter which the
film can resolve. (The resolution of
each film is listed on the chart at the
back of this book.) For general
recording, any instant film has sufficient
resolution to capture the detail present
in a microscope image. High-quality
publication illustrations or presentation
transparencies of detailed specimens
require the higher resolution of peelapart films.
• Higher speed films are used in lower
light levels
• Higher speed films render higher
contrast images
Film Instruction Sheets and
Technical Data Sheets
For the most up to date information
on the characteristics of the film you
are using, please read the instruction
sheet packed with the film.
Technical data sheets are available
for most films. These contain more
complete information. They are
available through the Hotline.
Why There Are No Exposure
Tables for Photomicrography
It isn’t practicable to provide specific
exposure tables for photomicrography
because there are simply too many
variables to consider, including:
• Image magnification
• Numerical aperture of the objective
lens
• Nature of the illumination
• Film speed
• Higher speed films tend to have more
structure or grain
If a specimen is moving or if the light
level is low (such as with a fluorescent
stain), choose a 3000 speed film, for
shortest exposure time.
Among the highest resolution films
available for either instant or
conventional photography are the
negatives of the Polaroid positive/
negative films. Polaroid offers Types
665, 55, and 51HC film for applications
that require further enlargement or
multiple copies.
Successful Photomicrography Tip
The microscope objective is the
component that limits the resolution
achieved by the microscope. In
general, the resolution of the film and
the microscope optics are well
matched with a 10X to 15X eyepiece
and a camera with a 1X magnification
factor. When choosing the
photographic magnification,
remember the general rule for
Maximum Useful Magnification
(MUM): 1000 x NA of the objective.
Magnification higher than that is
empty magnification and will not
produce further detail. The maximum
useful magnification for a 10X
objective with a 0.3 NA is 300X. The
maximum for a 100X with 1.3 NA
objective is 1,300X.
• Reciprocity failure
• Effects of filtration
• Microscope optics light absorption
characteristics
• Nature of the specimen
30
Polapan and Polagraph HC instant
35mm films have a resolution of 90 line
pairs per millimeter and may also be
used for enlargements.
Contrast
Polaroid black and white print films are
all medium contrast, with the exception
of high contrast Type 51HC.
The contrast of the print films can be
slightly increased by extending
processing time to up to double its
suggested time.
You will have greatest control of
contrast by using Kohler illumination
and by the filtration techniques and
contrast enhancement techniques
discussed in later chapters.
Instant Photography
Photomicrography
Through the Microscope
Polacolor 64 Tungsten,
balanced for tungsten
light, requires little or
filtration for
photomicrography.
Color Temperature
Most microscopes use tungsten or
tungsten/halogen light sources. Films
balanced for tungsten/halogen lighting,
having color temperature of 3200
degrees Kelvin (3200K), usually provide
the most accurate renderings for
photomicrographs. Polaroid offers
Polacolor 64 Tungsten instant print film
in 3 ¼ x 4 ¼” pack and 4 x 5” sheet
formats.
Polacolor ER, balanced for
daylight, gives a red-yellow
image if no filtration is used.
Daylight films are designed to be used
with daylight or electronic flash
(5500oK); filtration is needed for
photomicrography. When you use
tungsten or tungsten/halogen lighting
with daylight film, you may get a more
red/yellow photomicrograph image than
the specimen appears through the
microscope.
When a Wratten 80 Series
light balancing filter is used
to correct color rendition
with Polacolor ER film, the
color balance is more
neutral.
Xenon arc lamps and cesium iodide
lamps have outputs similar to
photographic daylight. Polaroid
recommends Polacolor ER film,
available in all formats except 35mm,
for these light sources.
Successful Photomicrography Tips
Polachrome and Polachrome HC 35mm
films are also balanced for daylight.
They require light-balancing filters when
you use tungsten or tungsten/halogen
lamps.
• Polacolor 64 Tungsten film requires
no filtration at exposures between
1/8 second and eight seconds. If
the light is bright and exposure
time is less than 1/8 second, add
neutral density filters to increase
the exposure time.
• Similarly, Polacolor ER film
requires no filtration at
approximately four seconds.
If you use Polacolor ER
film with tungsten lighting, use
neutral density filters to increase
the exposure time to four seconds.
• Automatic exposure systems
cannot correct for color shifts due
toreciprocity failure, even with built
in exposure corrections. However,
the MicroCam contains a blue light
balancing filter to balance the color
temperature over a range of
exposures.
31
Instant Photography
Photomicrography
Through the Microscope
Spectral Sensitivity of Instant Film
Silver halides are inherently sensitive in
the blue and ultraviolet regions of the
spectrum. Sensitizing dyes are added
to extend their sensitivity to longer
wavelengths. The absorption peaks of
the sensitizing dyes cause the peaks
in the sensitivity curve.
Reciprocity Failure in Black and
White Films
For general photography using instant
and conventional film, the reciprocity
law states that the total exposure of
film relates directly to the total light
energy it absorbs. Exposure is the
product of the exposure time and light
intensity.
If the light intensity is halved, and the
exposure time is doubled, the film
response should remain the same.
This response law does not hold true at
low light levels. Reciprocity failure, or
the failure of the reciprocity law, is a
common phenomenon in
photomicrography, where low light
levels are frequently encountered.
A graph showing the spectral sensitivity of
Polaroid Type 52 film, typical of the sensitivities
of most Polaroid black and white films. The
emulsion has maximum sensitivity in the 400 to
450 nanometer range with further sensitivity
peaks near 550 and 630 nanometers. The
spectral sensitivity diminishes rapidly at
wavelengths above 650 nanometers.
The sensitivity of instant black and
white film decreases at exposure times
longer than one or two seconds. The
expected exposure time for a high
quality image as measured by a light
metering system or calculated
mathematically yields increasingly
underexposed images as light
intensities decrease. For black and
white film, the exposure time should be
increased as shown on the following
page.
This graph shows the sensitivity curve of
Polacolor 64 Tungsten film. The curves
illustrate the spectral sensitivities of each of
the three emulsion layers in the negative. The
overlaps of sensitivities can affect the
rendition of very narrow band illumination,
such as light from sodium arc lamps.
32
Reciprocity Failure in Color Films
Most color films form images by
combining three separate emulsion
layers that are sensitive to red, green
and blue light. Color films have
reciprocity failure at low light levels with
one additional complication-the three
layers lose sensitivity at different rates,
creating a change in color rendition.
Color shifts progress in intensity with
decreasing light levels. Thus, with long
exposure times, the decrease in film
speed due to a reciprocity failure is
accompanied by a shift in apparent
color balance. When using Polacolor
films balanced for daylight, the shift can
be corrected by diminishing the amount
of blue filtration.
Successful Photomicrography Tip
Many camera systems have built-in
corrections for reciprocity failure.
They automatically increase the
exposure time sensed by the meter
to compensate for loss of film
sensitivity with long exposures.
Instant Photography
Photomicrography
Through the Microscope
Typical Reciprocity Failure Compensation for Instant Black and White Films
1 sec.
2 sec.
4 sec.
8 sec.
15 sec.
30 sec.
1 sec.
2-12 sec.
6 sec.
14 sec.
40 sec.
100 sec.
Expected Time
Actual Time
Required
The micrographs above show the effect of
reciprocity failure in black and white films.
Each step on the upper row shows the
micrograph obtained when the light level is
diminished by 50% and the exposure time was
doubled. The lower row shows the corrected
exposures for the same light levels.
Exposure Range and Color Shift of Polaroid Color Films at Low Light Levels
Exposure Range and Color Shift of Polaroid Color Films at Low Light Levels
FILM EMULSION
EXPOSURE RANGE
COLOR SHIFT
Polaroid 64 Tungsten
1/2 second to 8 seconds
None
8 seconds to 30 seconds
Negligible
Polacolor ER Types 669,
691, 59, 559, 809, 891,
and Autofilm Type 339
1/2 second and longer
Cyan/Blue/Green
Polachrome Instant
1/60 second to 30 seconds
longer
No color shift
Polachrome is uniquely
constructed with a single,
panchromatic emulsion exposed
through microscopic red,
green, and blue stripes embedded
in the film base-rather than
three separate layers. At low light
levels, it will show the speed losses
typical of black and white films, but
no color shift.
(virtually any exposure time)
Note: Polacolor PRO 100 and Type 779, 600+ are not appropriate for photomicrography because of their color shift with reciprocity failure.
33
Instant Photography
Photomicrography
Through the Microscope
Using a Graduated Exposure Test
Strip
A graduated test strip is a series of
progressively longer exposure times on
different sections of one piece of film. If
you don’t have an automatic exposure
system, it is the most economical and
effective way of determining correct
exposure.
Proper film for a graduated test
strip
To make a graduated test strip, use
Polaroid instant films in a film holder
with a dark slide, or Polaroid 4 x 5”
sheet films, which have an envelope
that functions as a dark slide.
Making a graduated test strip
Follow these procedures for making a
graduated test strip. Each successive
exposure will be twice as long as the
preceding one.
• Focus the specimen for photography.
• Exposure step 3: Push in the dark
slide or envelope by another 1/5 of
the total length of film. Double the
exposure time of step 2:
• Exposure step 4: Push in the dark
slide or envelope by another 1/5 of
the total length of film. Double the
exposure time of step 3.
• Add the appropriate filter.
• Exposure step 5: Push I the dark
slide or envelope by another 1/5 of
the total length of film. Double the
exposure time of step 4.
• Insert film into the camera.
• Withdraw the dark slide or film
envelope from the film holder.
• Estimate a satisfactory exposure
time (one second in the example
below). Aim to place this exposure
time in the center of the strip,
exposure step 3.
• Exposure step 1: With the dark slide
or envelope fully withdrawn, give one
quarter of the exposure time selected
(1/4 second in the example below.)
• Completely push in the dark slide or
envelope. Process the film.
You may notice a small amount of
image movement in the steps with
multiple exposures, even when the dark
slide or envelope is pushed in carefully.
However, this is still a valid exposure
test.
• Exposure step 2: Push in the dark
slide or envelope about 1/5 of the
total length of film (approximately ¾
inch) and give the same exposure as
step 1.
Successful Photomicrography Tip
To make your photomicrograph, use
the exposure time given to the step
that looks closest to the desired
result. If all the steps are too dark,
make another exposure test strip,
increasing each of the original
exposure times 16X. If all the
steps are too light, make another
test strip by decreasing exposure to
1/16 of the original time or by using
neutral density filters – or both.
Exposure Steps
1
2
3
4
5
Exposure Time
1/4 sec.
1/4 sec.
1/2 sec.
1 sec.
2 sec.
Cumulative exposure 1/4 sec.
1/2 sec.
1 sec.
2 sec.
4 sec.
34
Polaroid
Instant Photography
Photomicrography
Using Filters for Black and
White Photomicrography
Through the Microscope
Introduction
Color filters are essential for producing
high-quality black and white
photomicrographs. There are two major
reasons to use color filters in black and
white photomicrography – to control
contrast in a colored specimen and to
confine the illumination to the part of
the color spectrum for which the
microscope lens has been optimized.
Cupric oxide thin
film, 50x
Polaroid type 52
Darell Schlom,
James Harris,
Jim Eckstein, &
Ivan Bozovic
Filtration techniques used with black
and white film are distinctly different
from those designed for color
photography,. This chapter outlines
filtration techniques that allow you to
produce top-qualilty black and white
instant photomicrographs.
35
Instant Photography
Photomicrography
Through the Microscope
Where to Obtain Filters
Understanding Filter Factors
Filters are available from professional
photographic dealers or microscope
manufacturers or suppliers. They
come in many sizes, suitable for
different microscopes, and in many
formats, such as glass mounts for
frequent use or gelatin squares of
various densities. The Kodak Wratten
filter series, described in this
publication, is indexed with a standard
numbering system. You can reference
equivalent filters from other
manufacturers using this established
system.
Filter Factors indicate the approximate
amount by which an exposure time
must be multiplied in order to
compensate for light absorption by the
filter. Filter factors provide only a rough
guide for photomicrography. Many
other factors can influence the filter’s
effect on exposure: the spectral
sensitivity of the film, color temperature
of the light source, the nature of the
specimen and stain, and the precise
effect desired in the photomicrograph.
Placing Filters in Your Microscope
Place Filters in Your Microscope
Place your filter in the filter holder, if a
filter holder is available. (a)
Or, place the filter where light exits from
the microscope base. (b)
On an automatic exposure system, the
meter reading is made through the filter
and the exposure time is automatically
lengthened. In theory, you do not need
to apply a filter factor. In practice,
however, the automatic exposure
increase may not be accurate because
of differences between the spectral
sensitivities of the photocell and the
film. In such a case, you will need to
adjust the exposure manually.
Experiment
Filter Factor Experiment
Determine the length of exposure in
white light. Then add your chosen
filter to the light path and determine
the new indicated exposure time. If
the increase in time is not similar to
the filter factor (listed on the next
page), adjust the camera manually
to obtain that time.
Successful Photomicrography Tip
If you place your filters where light
exists from the microscope base,
ensure that they are clean and
undamaged. Proximity to the field
diaphragm increases the likelihood
that a filter’s blemishes will be
focused in the plane of the specimen
with the field diaphragm blades.
36
Instant Photography
Photomicrography
Through the Microscope
Empirical Filter Factors for Selected Wratten Filters
Filter Number
These empirical filter
8
factors were determined 12
from tests made with
15
Wratten filters, black and 22
white film (Polaroid
25
Type 52), and tungsten
29
lighting. They apply to
34A
all Polaroid black and
44A
white films with tungsten 47
or halogen lighting.
47A
47B
58
Filter Color
Filter Factor
Yellow
Deep yellow
Deep yellow
Deep Orange
Red
Deep red
Magenta
Cyan
Blue
Light Blue10X
Deep Blue
Green
1.5X
2X
2X
2.5X
3X
7.5X
6X
15X
15X
20X
10X
Filtration for Optimum Image
Resolution
For black and white photomicrographs,
filtration can also help you attain the
best possible resolution of fine detail.
Objective lenses are not equally well
corrected for optical aberrations over
the entire spectrum. Using only the
part of the spectrum where the lenses
are best corrected, you can obtain the
finest possible image quality.
Achromatic lenses have their best
correction in the green region of the
spectrum. Illumination which is
exclusively in this region can be
attained by using green filters of a fairly
narrow band, such as a Wratten No.
58. Using a No. 58 in conjunction with
a No. 15 (deep yellow), yields an even
narrower spectral band of transmission.
The examples to the right demonstrate
the superior resolution achieved by
using this combination of filters to
photograph carbon black (animal bone).
A No. 99 (green) filter would limit the
spectral transmission to an even
narrower band.
Apochromatic lenses correct
aberrations over a wider range of the
spectrum. Green filtration is still
suitable, however, narrow band
transmission is not necessary.
Filtration can contribute to image
resolution in another way, too. The
potential for high resolution of detail
increases as the wavelength of light
decreases. (Blue light will yield higher
resolution than red light.) Thus, a green
No. 58 filter can contribute to image
resolution simply by eliminating the
long-wavelength red component from
the illumination. The No. 47B (blue)
filter will do so to an even greater extent
because it transmits shorter wavelength
light.
This technique can be used only with
black and white film, since illumination
for color photomicrography must have a
full and fairly uniform color spectrum.
Carbon black (animal bone), 1250x.
Photomicrographs made with a
100x planachromatic objective lens
and an achromatic-aplanatic
condenser, using 3000K tungsten
illumination.
Top: Without filtration
Bottom: No. 58 and No. 12 filters.
37
Instant Photography
Photomicrography
Through the Microscope
Using Filters for Contrast Control
In a black and white photograph, the
colors of a specimen are translated into
shades of gray. Color filters selectively
transmit or absorb the colors
transmitted by the specimen. They
affect the shades of gray in which the
specimen colors are reproduced. In
general, each filter or filter combination
transmits light of its own color and
absorbs light of all other colors.
You can enhance the contrast of a
light-colored feature in a specimen
against the right background by
darkening that feature. Also, you can
control contrast by rendering the gray
of one specimen part distinctly lighter
or darker than that of another specimen
part.
In order to decisively suppress some
colors and enhance others, contrast
control filters must transmit light within
a relatively narrow band of the
spectrum. For this reason, these filters
differ significantly from those used in
color photomicrography.
38
Basic Filter Colors for Darkening
Colored Specimens
SPECIMEN COLOR TO
BE DARKENED
CONTRAST FILTER
COLOR
Blue
Blue/Green
Green
Yellow
Brown
Red
Magenta (Blue/Red)
Violet
Red
Red
Magenta (Blue/Red)
Blue
Blue
Cyan (Blue/Green)
Green
Yellow
Instant Photography
Photomicrography
Through the Microscope
Selecting Filters for Contrast
Control
• To make a specimen stand out more
clearly from a bright background,
render the specimen darker by using
a filter of the opposite, or
complementary, color.
Tissue and cells
cross section
• To enhance contrast and detail in a
specimen made up of two colors, use
filtration to darken the part of the
specimen that contains the most
important information. The exact
amount the colors should be
darkened and lightened respectively
must be determined by
experimentation.
Tissue cross
section
Lightly stained
• To enhance detail in a stained
specimen which is very pale, choose
a filter of the opposite, or
complementary color, which will
darken the specimen and enhance
detail.
Puccinia
• To enhance detail in specimen with a
very dense stain, choose a filter of
the same color.
Techniques for contrast control
Good contrast control depends on more
than just selecting an appropriate filter
color. You must determine which part
of the specimen you want to darken,
and by how much. Too much contrast
can defeat your purpose, which will
generally be to record the greatest
39
possible amount of specimen
information or image detail. You can
determine the filtration visually, either
by viewing through the microscope with
the appropriate filter, or filters, in the
light path or by comparing the filter
transmission and stain absorption
characteristics.
Instant Photography
Photomicrography
Through the Microscope
Color Characteristics of filters aand Filter Cobinations for Black and White Photomicrography
Filter or Filter Combination
Light Transmittance Over 10%
47B (deep blue)
400-470
47B +2E (pale yellow
420-470
47B + 3
440-470
47 (blue)
410-500
47 +8 (yellow)
440-500
47A (light blue)
380-520
47A+ 8 (yellow)
480-520
44 (blue green)
440-540
44 + 8 (yellow)
480-540
44 + 12 (deep yellow)
500-540
58 (green)
500-580
58 + 15 (deep yellow)
520-580
29 (deep red)
610 into infrared*
25 (red)
590 into infrared*
22 (deep orange)
560 into infrared*
15 (deep yellow)
520 into infrared*
12 (deep yellow)
510 into infrared*
8 (yellow)
480 into infrared*
400 Nanometers
500
Blue
Green
600
Yellow
700
Red
* The spectral sensitivity of panchromatic Polaroid instant films extend to about 660-690 nanometers and does not include sensitivity to infrared.
The chart above shows the
characteristics of filters and filter
combinations commonly used in black
and white photomicrography.
The chart on the opposite page lists
some of the more widely used
biological stains with their color
characteristics.
By using the two charts together, you
can select filtration for contrast control
with considerable accuracy.
The light areas in each chart show
where light is transmitted and the dark
areas show where light is absorbed.
Basics
• Nanometer (nm) is the unit of
measure for light wavelengths.
• Spectral range of light
transmittance from filters is
presented in nm.
• The visible color spectrum
extends approximately from 400
nanometers (blue) to 700
nanometers (red).
40
Instant Photography
Photomicrography
Through the Microscope
Color Characteristics of Commonly Used Biological Stains
Stain
Spectral Absorption
Acid Fuchism
530-560
Aniline Blue
550-620
Azure C
580-640
Basic Fuchism
520-570
Brilliant Cresyl Blue
550-650
Carmine
500-570
Congo Red
400-560
Crystal Violet
550-610
Darrow Red
450-550
Eosin Y
490-530
Erythrosin B
510-540
Ethyl Eosin
510-550
Light Green SF
590-650
Methyl Green
560-640
Methylene Blue
590-680
Neutral Red
480-570
Phloxine B
520-560
Orange G
450-510
Safranin O
470-550
Sudan lV
470-580
Tartrazine
400-460
Toluidine
560-660
400 Nanometers
500
Blue
Filter and Stain Techniques
For maximum darkening of a stain,
use a filter or filter combination that
transmits light along the nanometer
scale only where the chosen stain
blocks, or absorbs, light.
Example: With the stain basic fuchsin
(absorption about 520 to 570 nm), the
filtration should be No. 58 + No. 15
(transmission 520 to 580 nm).
Green
For less darkening, use filtration with
a transmittance range wider than the
absorption range of the stain.
Example: With the stain basic fuchsin
(absorption about 520 to 570 nm), use
a No. 58 filter (transmittance 500 to 580
nm) or a No. 12 filter (transmittance
510 nm to the red end of the
panchromatic film’s spectral
sensitivity).
41
600
Yellow
700
Red
To lighten a stain, use filtration that
transmits light primarily along a section
of the nanometer scale where the
chosen stain also transmits light.
Example: For a specimen stained with
basic fuchsin (absorption about 520 to
570 nm), use a No. 47 filter
(transmittance 410 to 580 nm) or a No.
22 filter (transmittance 560 nm to the
red end of the panchromatic film’’
spectral sensitivity).
Instant Photography
Photomicrography
Through the Microscope
Using Interference Filters
Interference filters, made up of a
number of thin evaporated layers, can
also be used to control contrast.
They are designated by:
• The center of the wavelength range
they transmit, in nanometers or
Angstroms.
• The width of the wavelength range
they transmit, designated as fwhm,
full width of the transmission band
measured at the level of one-half the
maximum intensity.
Interference filters are available from
microscope dealers and from optical
component suppliers.
Successful Photomicrography Tip
• A basic set of filters includes 0.10
(2) 0.30, 0.60 and 0.90 densities.
You can use two filters together, if
necessary.
For example, 0.30 and 0.90 give a
total density of 1.20 (with a
transmittance of about 6 percent
of the original illumination). It is
advisable to use no more than two
neutral density filters together.
• Deeply colored filters may slightly
displace the effective focus of the
microscope, due to optical
characteristics of the objective
lens. Check the focus after you
put the filter in place – before
making the photomicrograph.
Using Neutral Density Filters
The intensity of the illumination at the
eyepiece or film plane can vary greatly
with image magnification or other
optical factors. Neutral density filters,
which are gay or colorless, reduce the
intensity of the illumination in a precise
manner. They help to keep exposure
times as constant as possible or to
provide for more comfortable viewing.
Neutral density filters provide an
excellent alternative to adjusting the
light intensity through the lamp’s
voltage control. Variations in lamp
voltage will change the color
temperature of the light and make
standardization of filtration difficult.
With tungsten or halogen lamps, an
excessive reduction in voltage also
prevents the lamp from functioning
properly.
At high magnification and with slower
black and white film, you may need the
total output of the light source in order
to make a reasonably short exposure.
In such a case, you may still want to
reduce the intensity of the light for
viewing purposes. If you use neutral
density filters, the visual assessment of
the effect of a color filter will be more
accurate than it would be if the lamp
voltage were reduced.
Keep a Record of Filter Use
For each filter, keep a record of
photomicrographic data, including
details about the specimen, the
objective and eyepiece, and the
total photographic magnification.
Also list the nature of the illumination,
the film type, and the exposure time.
42
Neutral Density filters and their
Light transmission Characteristics
Neutral Density
0.10
0.20
0.30
0.40
0.50
0.60
0.90
1.00
Percentage of
Light transmitted
80
63
50
40
32
25
13
10
Using Heat-Absorbing Filters
A microscope lamp generates infrared
radiation, which produces heat that can
damage specimens and filters. A heat
absorbing filter is often included as
standard equipment on a microscope.
If it is not, insert such a filter into the
light path.
Using Ultraviolet-Absorbing Filters
Black and white films are inherently
sensitive to ultraviolet (UV) radiation
which is not visible to the human eye.
However, in some microscopes it can
be a problem for photography and may
appear as an even haze in a
photomicrograph. Use a Wratten 2A,
2B or 2E (or equivalent) filter to absorb
the unwanted UV.
Polaroid
Instant Photography
Photomicrography
Using Filters for Color
Photomicrography
Through the Microscope
Introduction
In color photomicrography, filters are
used to control the color quality of the
light reaching the film and to achieve
the best possible color balance or
effect in your photomicrograph. This
chapter introduces many filters and
special filtration techniques that can
correct color imbalances to achieve a
full range of desired effects.
Perovskite
Ianthanum
aluminate, 2.5x
Polarized light
Polachrome HC
Michael Davidson
43
Instant Photography
Photomicrography
Through the Microscope
The Relationship of Color Film,
illumination, Exposure Time and
Filters
For color photomicrography, select film
in anticipation of the quality of light you
will use. The spectral quality of light,
measured as color temperature in
degrees Kelvin, follows these general
rules:
• The higher the color temperature, the
higher the proportion of blue light in
the illumination.
• The lower the color temperature, the
higher the proportion of red light.
Most microscopes use tungsten or
tungsten/halogen lamps, rich in red
light. If you select tungsten-balanced
film such as Polacolor 64 Tungsten
film, intended for use with 3200K light,
Effects of Neutral Density Filters
Neutral
Density
0.10
0.20
0.30
0.40
0.50
0.60
0.90
1.00
Percentage of
Light Transmitted
80
63
50
40
32
25
13
10
Photographic
Stop Equiv.
1/3
2/3
1
1-1/3
1-2/3
2
3
3-1/3
Successful Photomicrography Tip
A xenon arc lamp gives illumination
that closely resembles daylight.
Whenyou use xenon illumination
with any daylight-balanced
film-such as Polaroid Types 59,
559, 669, 108, T339, TimeZero,
or Polachrome HC-you won’t need
a filter.
you’ll need little or no filtration for
exposure times up to 30 seconds.
On the other hand, daylight-balanced
film is designed for use in light that has
a high proportion of blue light, with a
color temperature of 5500K. When
daylight films are used with tungsten
light, color conversion or light-balancing
filters are necessary.
Polaroid color films (except for
Polacolor 64 Tungsten) are balanced for
use in daylight at short exposure times.
With the daylight-balanced films, you
will need to use filters to make the
microscope illumination more similar to
daylight. Film tip sheets recommend
80 series filters or a combination of
color-correcting filters, depending on
exposure times.
Establishing a Standard Exposure
Time for Standard Filtration
In photomicrography, the level of
illumination at the film plane can vary
over a wide range, depending upon
magnification and other optical
variables. Color filtration is easiest if
the film’s color response is held
constant. This can be done by limiting
exposure times to a range of one to
four seconds. Neutral density filters
placed in the light path can be used to
regulate exposure.
Base your standard exposure on the
exposure time needed at the lowest
level of illumination that you are likely
to encounter in your everyday workprobably between one and four
seconds. Don’t base it on unusual
conditions that call for extremely long
exposure times, such as 30 seconds or
longer.
44
Successful Photomicrography Tip
• Daylight-balanced films will need
less blue filtration as exposures
increase.
• Tungsten-balanced films will need
red-yellow filtration at exposure
times longer than 30 seconds.
Long exposure times affect the color
balance of the both daylight and
tungsten films. As the exposure times
increase to several seconds, reciprocity
failure causes a color shift toward more
cyan or blue prints.
The lowest light level at the film plane
generally occurs at high magnifications,
or when a relatively high degree of color
filtration is in the light path. Make a
test print under such conditions, and
establish what exposure time gives the
best result.
For example, if your longest exposure
time is normally about two seconds,
and you change to an objective lens of
much lower power, the exposure time
may change to ¼ second. To keep the
reciprocity failure of the film constant,
you need to return to a two-second
exposure time. You can accomplish
this by adding a 0.9 neutral density
filter, which transmits 13 percent (or
about 1/8) of the original illumination,
bringing your required exposure time
back to two seconds.
Instant Photography
Photomicrography
Through the Microscope
The Effect of Lamp Voltage and
Exposure Duration on Color
Balance
Before you use filters, ensure the most
consistent color balance in both the
illumination and the response of the film
by:
• Controlling the lamp voltage properly
Successful Photomicrography Tip
If you place your filters where light
exits from the microscope base,
ensure that they are clean and
undamaged. Proximity to the filed
diaphragm increases the likelihood
that a filter’s blemishes will be
focused in the plane of the specimen
with the field diaphragm blades.
• Establishing one standard exposure
time for as much of your color
photomicrography as possible.
Used at the voltage setting specified by
the lamp manufacturer, most
microscope tungsten filament lamps
have a color temperature of
approximately 3200K. When you
decrease the voltage, the color
temperature drops and the light
becomes richer in red rays.
Always use the specified voltage
setting for color photomicrography to
minimize the need for filtration and to
keep the color quality of the illumination
consistent.
Placing Filters in Your Microscope
Place your filter in the filter holder, if a
filter holder is available, (a) Or, place
the filter where light exits from the
microscope base. (b)
45
Instant Photography
Photomicrography
Through the Microscope
Filters for Color Photomicrography
Color filters are available in various
types with characteristics designed to
serve a specific purpose. Major filters
for color photomicrography include
color-conversion, light-balancing, colorcompensating, ultraviolet-absorbing,
didymium, neutral density (gray), and
heat-absorbing filters.
A filter’s apparent color does not signify
its characteristics. Color-conversion,
light-balancing, color-compensating and
under certain conditions, didymium
filters may all appear blue to the eye.
However, as the information that follows
shows, their spectral features and
purposes for color photography are
different.
Keep a Record of Filter Use
For each filter, keep a record of
photomicrographic data, including
details about the specimen, the
objective lens and eyepiece, and the
total photographic magnification.
Also list the nature of the illumination,
the film type, and the exposure time.
Color-Conversion and LightBalancing Filters
To fine-tune color balance, you can
use two filters together-such as 80C
plus 82.
Color-conversion and light-balancing
filters are most often used to modify
tungsten illumination for color film
whose balance is between daylight and
tungsten light, depending on the
reciprocity effect at any specific
exposure time. Thus, they’re generally
blue.
The weaker red-yellowish filters in the
right half of the following chart are
needed for extremely long exposures,
when the film shows a strong color shift
toward the blue end of the spectrum.
Filters increasingly bluish
(to raise color temperature of light)
Color-Conversion Filters
80A
4.0
80B
3.4
80C
2.0
80D
1.3
82B
1.7
82A
1.3
Please note that with automatic
exposure systems, the required
exposure is increased automatically
when an exposure reading is made with
the chosen filter or files in place.
Filters increasingly yellowish
(to lower color temperature of light)
Light-Balancing Filters
82C
1.7
The filter factors indicate the
approximate amount by which the
exposure time without that filtration
must be multiplied when using a filter.
Light-Balancing Filters
81
1.3
82
1.3
Approximate filter factors
One of these filters may be necessary for daylight
film and tungsten light with short exposures.
81A
1.3
81B
1.3
81C
1.3
81D
1.7
Color-Conversion Filters
81EF
1.7
85C
1.3
Approximate filter factors
One of these filters may be necessary for very
long exposures.
46
85
1.7
85B
1.7
Instant Photography
Photomicrography
Through the Microscope
Color-conversion filters
Color-conversion filters are designed to
convert the color balance of the
illumination to match the color balance
of the film. These filters shift the entire
spectral balance either to the cool
(blue-cyan) or to the warm (red-yellow)
end of the spectrum. They are not
intended to control individual colors.
This chart indicates the characteristics
of filters in the 80 (blue) series, which
are used to balance 3200K tungsten
lighting for use with daylight-balanced
color film.
Light-balancing filters
Light-balancing filters are similar to
color-conversion filters. However, lightbalancing filters are weaker. When
illumination is of the required color
temperature, these filters make
possible additional minor adjustments
in the color balance of the light.
This chart indicates the characteristics
of filters in the 82 (bluish) series. The
curves are similar to those of the 80
series filters, but less steep.
Successful Photomicrography Tips
Color – compensating (CC) filters
• Polacolor 64 Tungsten film is a
good choice for simple color
photomicrography under tungsten
or tungsten/halogen illumination.
This film does not require a colorconversion filter, and exposures
will be two-to-four times shorter.
Color-compensating filters are
designed to give you control of
individual colors in the spectrum, once
the color temperature of the light has
been balanced to match the film’s
requirements.
• Always use the recommended
The solid line on this chart is
characteristic of a CC Green filter,
which transmits relatively more green
light and less blue and red. The broken
line represents a CC Magenta (bluered) filter, which transmits relatively
more blue and red light and less green.
lamp voltage setting and keep to a
standard exposure time whenever
possible.
47
Instant Photography
Photomicrography
Through the Microscope
More on Color compensating filters
Color Wheel Basics
Each of the primary colors-red,
green, and blue-in the color wheel is
equivalent to the two adjacent colors.
For example, red is made up of
yellow and magenta, and a CC20
yellow filter plus a CC20 magenta
filter is equivalent to a CC20 Red
filter.
Filters of the same color can also be
added together: a CC 20 yellow filter
plus a CC 10 yellow filter are
effectively the same as a CC 30
Yellow filter.
Normally, any specific color can be
suppressed in a photograph by using a
filter of its color complement located
directly opposite on the color wheel.
For example, a CC Magenta filter will
suppress green and enhance blue and
red, while a CC Green filter will subdue
blue and red and enhance green.
Each filter factor indicates the
approximate amount by which the
original exposure time must be
multiplied when the filter is added.
However, with automatic exposure
systems, you don’t have to make an
allowance for the filter factor when you
make an exposure reading with the
chosen filter or filters in place. The
exposure will be increased
automatically.
Color-Compensating Filters
Filter Colors
Colors Absorbed
Filter Strength (increasing from left to right) and Approximate Filter Factors
Cyan
(blue/green)
Red
CC05C
1.0
CC10C
1.3
CC20C
1.3
CC30C
1.5
CC40C
1.7
CC50C
1.7
Magenta
Green
CC05M
1.3
CC10M
1.3
CC20M
1.5
CC30M
1.7
CC40M
2.0
CC50M
2.0
Yellow
(red/green)
Blue
CC05Y
1.0
CC10Y
1.3
CC20Y
1.3
CC30Y
1.3
CC40Y
1.5
CC50Y
1.5
Red
Blue and Green
CC05R
1.3
CC10R
1.3
CC20R
1.7
CC30R
2.0
CC40R
2.5
CC50R
3.0
Green
Red and blue
CC05G
1.0
CC10G
1.3
CC20G
1/5
CC30G
1/7
CC40G
2.0
CC50G
2.5
Blue
Green and red
CC05B
1.3
CC10B
1.3
CC20B
1.5
CC30B
1.7
CC40B
2.0
CC50B
2.5
48
Instant Photography
Photomicrography
Through the Microscope
Color Imbalances
Successful Photomicrography Tip
If you have balanced the lighting for the
film and its reciprocity failure, the
imbalance is due to another cause.
This chart gives an approximate idea of
the CC filtration that might be needed
under a variety of conditions.
Choose the color and strength of
CC filtration you might need to
correct an imbalance in a
photomicrograph you’ve made.
Then view that photomicrograph
through the selected filter. When
the colors in the photo look well
balanced-as shown in the right-hand
column of the chart below-add that
filter to make the next
photomicrograph.
Color Imbalances
Color Imbalance
Amount
CC Filtration
(Approximate Range)
Satisfactory
Color Balance
Too
Moderate
As illustrated at left
15 to 30 Cyan
Red
Slight
Imbalance just noticeable
05 to 10 Cyan
Too
Moderate
As illustrated at left
15 to 30 Magenta
Green
Slight
Imbalance just noticeable
05 to 10 Magenta
Too
Moderate
As illustrated at left
15 to 30 Yellow
Blue
Slight
Imbalance just noticeable
05 to 10 Yellow
Too
Moderate
As illustrated at left
15 to 30 Red
Cyan
Slight
Imbalance just noticeable
05 to 10 Red
Moderate
As illustrated at left
15 to 30 Green
Slight
Imbalance just noticeable
05 to 10 Green
Moderate
As illustrated at left
15 to 30 Blue
Slight
Imbalance just noticeable
05 to 10 Blue
Too
Magenta
Too
Yellow
49
Instant Photography
Photomicrography
Through the Microscope
Ultraviolet-Absorbing Filters
Microscope illuminators, and
particularly some tungsten/halogen
lamps, generally emit unwanted
ultraviolet radiation. It’s not visible to
the eye-however, color film is sensitive
to it. The result may be an unexpected
bluish haze over the image in a color
photomicrograph. A Wratten 2A or 2B
filter, or equivalent, generally eliminates
unwanted UV. If the excess blue in the
photomicrograph persists, try a
Wratten 2E or equivalent, which is
stronger than the 2A.
Didymium Filters
The color rendition of certain stained
specimens can be improved markedly
through the use of a didymium filter,
which has the ability to enhance both
blues and reds. It is most effective with
certain common histological stains,
such as eosin, fuchsin and methylene
blue.
The didymium filter has a spectrum
with distinct and narrow color
absorption bands . The strongest is in
the yellow range with total absorption
occurring at about 580 to 590
nanometers. Didymium filters are
made in thicknesses of one and two
millimeters. The amount of light
absorbed varies with the thickness of
the filter.
Top: No Didymium filter was used.
Bottom: A significant difference
can be seen with the use of a
didymium filter.
Because of unpredictable differences in
specimens and staining practices,
there are no specific directions for
using a didymium filter. Make a test
exposure to determine whether the filter
achieves the desired effect with a
specific stain. This filter is rarely used
for general purpose photography.
50
Top: No ultraviolet-absorbing filter
was used. Bottom: A Wratten 2E
filter removed the undesirable effect
of the ultraviolet radiation.
Heat-Absorbing Filters
A microscope lamp generates infrared
radiation, which produces heat that can
damage specimens and filters. Often,
a heat-absorbing filter is included as
standard equipment on a microscope.
If not, insert one in the light path.
Successful Photomicrography Tip
For best image quality, always try
to keep to a minimum the number
of filters used at one time.
Instant Photography
Photomicrography
Through the Microscope
Neutral Density Filters
Sources of Color Imbalance and Filter Solutions
Cause of Color Imbalance
Filter Type Needed
Polar color ER color films ar daylightbalanced. As exposure time increases
to several seconds, reciprocity failure
in the film causes a blue-cyan color
shift. Up to a specific point, this shift
makes the film increasingly compatible
with tungten illumination. The exact
filtration needed to balance the
illumination with the film characteristics
decreases with longer exposure times.
For good color balance with little or no
filtration, use Polacolor 64 Tungsten
film at standard exposure times of 1/2
to ten seconds.
Use color-conversion filters or lightbalancing filters, depending on the
amount of imbalance.
The optical components of the
microscope absorb light selectively, so
that the light reaching the film has
different spectral characterics than the
light emitted by the source.
Generally use color-compensating
filters. Occasionally use ligt-balancing
filters.
The spectral transmission properties of
some biological specimen stains
require modification to suit the
characteristics of the color film being
used to enhance the separation
between certain specimen colors.
Use color-compensating filters.
Occasionally us a didymium filter.
A heat-absorbing filter in the optical
system can cause a color imbalance.
Use color-compensating filters.
A Abbe or aplanatic substage
condenser may introduce a sligh color
imbalance to the image area when an
objective lens with a high numerical
aperture (aboce 0.65) is used. Teh
color of this imbalance will change as
you adjust the focus of the condenser.
First, keep the color consistent by
always focusing the condenser to
achieves the same effect - the color
fringe at the edge of the image of the
field diaphragm blades should always
look the same. To correct an
imbalance, use appropriate lightbalancing or color-compensating
filters.
Two or more of the above.
Use a combination of filter types.
Neutral density filters are gray, or
colorless. They reduce light intensity
without changing the color
characteristics of the light, and by
controlling light intensity they help
control exposure time and color
balance in a predictable manner.
Neutral density filters are available in a
wide range of densities. For example,
Kodak Wratten neutral density filter No.
96 is available in 3 inch (75 mm) or
larger gelatin filter squares. Inconel
coated filters on a glass base exhibit
best color neutrality. They are available
from microscope dealers or optical
supply houses.
A basic set of neutral density filters
includes two 0.10 and one each of
0.30, 0.60, and 0.90 densities. You
can use two filters together, if
necessary. For example, 0.30 and
0.90 give a total density of 1.20 (with a
transmittance of about 6 percent of the
original illumination). For best quality,
it is advisable to use no more than two
neutral density filters together.
Successful Photomicrography Tip
51
It’s very important to evaluate a print
for color quality only when that print
has been exposed accurately.
Incorrect exposure can cause a
color imbalance of its own.
Instant Photography
Photomicrography
Through the Microscope
Sample Applications of Color Filters
1. This distinctly “warm” reddish color cast is
the result of Polacolor ER film exposed to
unfiltered tungsten illumination at a short
exposure time.
2. An appropriate blue color-conversion filter
helped record the specimen’s colors more
accurately.
1
2
3
4
5
6
7
8
3. The dark spots in this pair of
photomicrographs of lung-coccidiodes
mycosis are fungi. Given the density of the
stain and the incompatibility of the stain and
the incompatibility of the stain with the film
characteristics, standard filtration rendered
the fungi dark gray.
4. The true color of the fungi, dark brown, was
captured using a CC20R and a CC10Y filter.
To record an important part of a specimen in
its true color, sometimes you must sacrifice
the neutrality of the background or the color
accuracy of other parts of the specimen.
5. The spectral characteristics of the optical
glasses, the anti-reflection coatings, and the
heat-absorbing filters used in a microscope
system can change the color of the
illumination. This photomicrograph was
made with filtration considered standard for
the color temperature of the light source and
the known reciprocity failure of the film.
6. This photomicrograph was made with the
same filtration, plus CC40M and CC20C filters
to compensate for the color bias introduced
by the microscope optics.
7. The standard filtration for a two-second
exposure time was used in this
photomicrograph, but the actual exposure
time was 36 seconds. Reciprocity failure
caused the bluish color imbalance.
8. This photo was made with filtration adjusted
to compensate for reciprocity failure.
52
Polaroid
Instant Photography
Special ContrastEnhancement Techniques
Photomicrography
Through the Microscope
Introduction
Some specimens are of such low
contrast that their microscopic image is
barely discernible to either the eye or
film. A specimen that absorbs almost
no light and is virtually colorless calls
for a contrast-enhancement technique
beyond the use of color filters.
Pine stem,
65x
Polarized light
Red l Plate
Polacolor Type
778
Mary McCann
Keep this rule of thumb in mind; aim for
a level of contrast that best reveals the
information you need without sacrificing
other important image qualities such as
resolution.
You will find the following items
convenient for the special contrastenhancement techniques in this
chapter.
High-intensity light source
Contrast-enhancement techniques
make effective use of only a small
portion of the total illumination.
Therefore, you will need a high-intensity
light source and high-speed films to
keep exposure times to a minimum.
Read the Kohler Illumination section
first. Properly aligned Kohler
illumination is essential for all special
contrast-enhancement techniques.
Rotating stage
With some techniques the orientation
of the specimen on the stage
influences the appearance of its image.
A rotating stage allows you to control
the orientation of the sample to suit the
requirements of the specific technique.
High-speed film
You need to keep exposure times to a
minimum for special contrastenhancement techniques; therefore,
choose high-speed film.
53
Instant Photography
Photomicrography
Through the Microscope
Polarized Light
Purpose
Suitable Specimens
Equipment
• To use the birefringence of an
anisotropic specimen for pictorial,
analytical, or identification purposes.
•
•
•
•
•
•
•
•
•
•
• One polarizer blow the specimen
• One polarizer (called the analyzer)
above the specimen that can be
rotated at least 90 degrees.
• Strain-free objective lenses and
condensers
• Specimen stage that rotates about
the optical axis
• Compensators or retarders (full-wave
and quarter-wave plates) for
quantitative work and for adding
color.
• To differentiate isotropic specimen
areas (those having only one index
of refraction) and anisotropic
specimen areas (those having more
than one index of refraction)
Chemical crystals
Mineral preparations
Plant and animal tissue
Pharmaceutical preparations
Fats and waxes
Natural and synthetic fibers
Starch grains
Bone and horn sections
Animal and plant hair
Wood sections
The Physics of Polarized Light
The polarizer below the specimen
transmits light vibrating in one direction
and absorbs the remaining light.
When the analyzer is crossed with this
polarizer, their transmission directions
are perpendicular to each other. All
light is absorbed and the image field
will appear dark. The isotropic parts of
the specimen will appear dark, too, as
they will have no effect on the polarized
illumination.
The polarized light that passes through
a birefringent area of the specimen is
split into two beams that are polarized
perpendicularly to each other. The
specimen introduces a phase difference
between the two beams, depending on
its birefringence and thickness. Some
of each of these beams will pass
through the analyzer, where they
recombine and interfere.
The image may appear gray, white, or
brightly colored. Colors and tones are
dependent on the interference between
the two out-of-phase beams when they
reunite on passing through the
analyzer. The interference colors in the
image indicate the approximate
thickness or birefringence in this
specimen.
Successful Photomicrography Tip
If the microscope does not have a
built-in location for the analyzer,
place the analyzer in the microscope
tube or over the eyepiece. If the
analyzer is located where it cannot
easily be rotated, rotate the lower
polarizer, which can be placed over
the light exit of the microscope.
Polarized light reveals starch grains
in a cross section of a pelargonium
stem. 160X (left) brightfield
illumination without polarizers;
(right) specimen between crossed
polarizers.
54
Instant Photography
Photomicrography
Through the Microscope
Techniques
• To enhance the visibility of significant
specimen contours, set the prism to
create an apparent shadow effect by
darkening one side of a specimen
interface and lightening the other.
When you rotate an anisotropic
specimen between crossed
polarizers, the various image areas
will alternate between bright and dark
with each 90 degrees of rotation.
The colors of each area will remain
the same, but their brightness will
change. This change in brightness
with change in orientation can be
used for contrast control-a slight
rotation of the specimen can reduce
an excessive rightness difference
between specimen parts.
Thin, transparent petrographic
specimen, 60X magnification.
Specimen between crossed
polarizers. (Left) the low
birefringence of the specimen
yielded an image lacking in color;
(right) the use of a full-wave,
phase-changing plate helped to
produce a colorful image.
• Image analysis may require that the
brightness of the specimen remain
constant, regardless of its
orientation. Circular polarizers, used
in place of the linear polarizers
normally used, will eliminate
orientation effects. Circular
polarizers can be obtained from your
microscope manufacturer, or from
Polaroid Corporation, Polarizer
Division, Upland Road, Norwood, MA
0202-1598.
Resorcinol crystals, 20X magnification.
Specimen between crossed polarizers,
(left) brightness range is high, and
some detail is lost; (right) specimen
was rotated by a few degrees to reduce
brightness range. Note increased
detail on right side.
• Use a full-wave compensator plate
between the polarizer and the
analyzer to enhance color contrast
considerably. It can improve the
image of a weakly birefringent
specimen by adding its own phase
change to that imparted by the
specimen. The black background
produced by the fully crossed
polarizer and analyzer changes to
magenta. Gray or white articles
change to blue, red, and yellow. The
full-wave plate can also be helpful in
indicating the optical properties of the
specimen.
• If contrast is too great, reduce the
tonal range by rotating the specimen
between the crossed polarizers.
Rotating the analyzer by 10 to 20
55
degrees will lighten the background
without destroying the color
information. A slightly lighter
background will allow the detection of
isotropic particles.
• Use the normally recommended color
filtration for best color rendition. A
didymium filter will often help to yield
an optimum result.
Photographic Consideration
• To provide information on the optical
properties of the specimen, use a fullwave compensator and color film.
Specimens of low birefringence,
which yield an image of mainly gray
tones, may be best recorded on
black and which film.
• When the polarizer and analyzer are
fully crossed and the background is
very dark, the exposure time
indicated by an automatic exposure
time indicated by an automatic
exposure system will generally need
to be reduced by about 25 to 50
percent to avoid overexposing the
bright birefringent features.
Instant Photography
Photomicrography
Through the Microscope
Darkfield Illumination
• Diatoms, both living and dead
Purpose
• Dust-count specimens
• To show the specimen as a bright
image against a dark background
• To greatly enhance the visibility of a
transparent or semi-transparent
specimen which scatters or absorbs
light only slightly.
• To render visible tiny particles, too
small for the limits of resolution of the
microscope’s optics.
Suitable Specimens
• Unstained bacteria
• Yeast
Equipment
• A special darkfield condenser
• Or, a brightfield condenser (with a
numerical aperture higher than that of
the objective lens) used with a central
drop.
• Colloidal Specimens
• Colloidal particles
Some objective lenses are
equipped with an iris diaphragm that
The Physics of Darkfield Illumination
Reflected light darkfield illumination
Darkfield is used in reflected light and
transmitted light. In transmitted-light
darkfield, only the light deflected by the
specimen enters the microscope
objective lens to form the image. The
central stop keeps direct light from
entering the objective lens to form the
image. The central stop keeps direct
light from entering the objective lens
and provides the dark image
background. The refractive indices of
subject matter and mounting medium
must be sufficiently different to make
possible an adequate deflection of light.
Reflected Light Darkfield
Light scattering particles or scratches
on a flat or rough surface are detected
best in darkfield. Voids in thin films
scatter light and are more visible in
darkfield illumination. Semi-opaque
specimens, such as polished mineral
specimens or paint, ink, or dye images
on paper also are conveniently
examined with this technique.
Transmitted light darkfield illumination
In reflected light, the illuminating beam
strikes the sample at such an angle
that the directly reflected rays fall
56
enables the effective numerical
aperture to be reduced to less than
that of the condenser. High
numerical aperture condensers are
available for high-magnification
objective lenses (that have an
accordingly high numerical aperture).
Special illuminators are available for
work at very low magnifications, such
as with stereo microscopes.
Equipment for Reflected Light
• Annul reflector –usually on a slider
• Brightfield/darkfield objectives that
allow the light an outer pathway in
the objective.
outside the acceptance angle of the
objective. Only the light scattered by
the specimen is imaged by the
objective.
For reflected light darkfield, you’ll need
an annular reflector, usually on a slider
that directs the light down an outer path
in the objective. You’ll also need
brightfield/darkfield objectives that
provide the outer pathway for the
illuminating beam and include a conical
mirror that directs light onto the
specimen at the proper angle.
Techniques
• Features in the specimen above and
below the plane of focus can
contribute to the image if they scatter
light.
• On a black background, dust and dirt
scatter light. For this reason, clean
the slide and coverslip thoroughly.
• The thickness of the specimen will
have an effect on scatter. In general
a thin specimen is preferable.
• In reflected light microscopy, the field
diaphragm must be fully open to
allow the light down the outer portion
of the objective.
Instant Photography
Photomicrography
Through the Microscope
Photographic Considerations
Fiber rayon, 120X magnification,
Green filter, (left) brightfield; (right)
darkfield. Photomicrographs by
John P. Vetter, R.B.P.
• Color film is generally not useful for
darkfield work. For short exposure
times, use high-speed black and
white film.
• Because of the dark background,
automatic exposure systems tend to
give gross overexposure. In general,
to expose the specimen correctly,
reduce the indicated exposure time.
The amount of reduction depends on
the proportion of the sample that is
scattering light. If there are only a
few scattering entities, very little light
is detected. The indicated exposure
is very long and those few points of
light will be overexposed. The
indicated exposure should be
reduced considerably. If there are
many scattering entities, more light
is detected. Reduce the indicated
exposure by a smaller factor.
Benard cells in polymer coating,
(Left) Brightfield; (right) Darkfield.
57
Instant Photography
Photomicrography
Through the Microscope
Phase-Contrast Illumination
Purpose
Suitable Specimens
• To increase contrast and reveal
structural detail in a cell or other
specimen where very slight
differences in thickness and refractive
index are normally invisible
Usually very thin and colorless:
• Blood cells
• Latex dispersions
• Glass fragments
• Protozoa
• Replicas
• Tissue cultures
• To record a bright image on a darker
background
Equipment
• Bacteria
• Annular ring in the condenser
• To record a relatively dark image on a
brighter background, depending on
the phase optics
• Yeasts
• Phase-changing ring in the back
focal plane of the objective lens
• Molds
• Diatoms
The Physics of Phase-Contrast
Techniques
Phase-contrast relies on the
interference between the direct
illuminating beam, as defined by the
annular ring, and the light deflected
(refracted or diffracted) by the different
parts of the specimen.
• In aligning the system, the circular
image of the direct illuminating beam
must be superimposed accurately on
the phase-changing ring to achieve
enhanced contrast.
The deflected image-forming light
experiences phase differences caused
by the various parts of the specimen.
The phase-changing ring introduces a
uniform phase change to the
undeflected beam.
The phase-changing ring also reduces
the intensity of the direct illuminating
beam, so that the direct and the
scattered light beams are of relatively
equal intensity. This enables them to
interfere effectively.
The direct illuminating beam and the
deflected light beam interfere when
they recombine at the eyepiece. This
interference changes the effective light
intensity from the various specimen
parts to enhance image contrast.
This technique is not for making
dimensional measurements of an
image, because phase-contrast tends
to cause disturbing diffraction halos
within the image.
58
• For easy alignment, use a phase
telescope in the place of one of the
eyepieces to view a magnified image
of the back focal plane of the
objective.
• Clean the slide and coverslip
thoroughly, as dirt could contribute
unwanted and misleading phase
information to the image.
• Halo effects, characteristic of phasecontrast, may conceal useful
information. Use a mounting medium
of appropriate refractive index to
lessen halo effects. Changing the
mounting medium can also affect
image contrast.
Instant Photography
Photomicrography
Through the Microscope
• The phase-changing ring is designed
to provide a phase change with
green light of a specific wave-lengthnormally 546 nanometers. For best
results, limit the illumination to this
region of the spectrum. For
example, use an appropriate
interference filter, centered on a 546
nanometers or a Wratten No. 58
green filter.
The phase-contrast optics are usually
achromatic and have optimum
correction for optical aberrations in
the green region of the spectrum.
The green filter limits the illumination
to this part of the spectrum, to further
ensure optimum contrast and image
resolution.
Trichuris trichiura, 600X
magnification. Green filter, (left)
brightfield; (right) phasecontrast. Photomicrographs by
John P. Vetter.
Photographic Considerations
• The amount of light reaching the film
may be 1/10 to 1/20 of that in normal
brightfield work, so exposure times
will be longer. The exact exposure
correction depends on the nature of
the specimen and on the phase
system.
• For excellent resolution and gray
scale rendition, use black and white
print film such as Polaroid type 52 or
552. For black and white negatives
suitable for enlargement, Polaroid
Type 55 or 665 is recommended.
Color film is not appropriate for
phase-contrast.
Sepedonium, 300X magnification.
Green filter. Both photomicrographs
made in phase-contrast illumination.
Photomicrographs by John P. Vetter.
Successful Photomicrography Tip
Phase-contrast is more sensitive to
refractive index differences than
brightfield illumination. Use phasecontrast for precise refractive index
determination.
59
Instant Photography
Photomicrography
Through the Microscope
Hoffman Modulation Contrast
Purpose
Suitable Specimens
• To enhance contrast by generating an
image with apparent shadow effects,
similar to those seen in Differential
Interference Contrast
• To render images without the halo
effects associated with phasecontrast
• To allow reliable measurements, as
edges are well defined
• To enhance the contrast of
birefringent samples, or samples in
birefringent containers
•
•
•
•
•
•
•
•
Tissue cultures
Unstained tissue sections
Blood cells
Protozoa
Polymer fibers
Crystalline samples
Etched glass
Birefringent samples and birefringent
culture dishes
• Specimens for phase-contrast
illumination
The Physics of Hoffman Modulation
Contrast
The Hoffman Modulation Contrast
system detects optical gradients, or
slopes, in a specimen and converts
them into intensity variations.
At an edge or interface where there are
differences in refractive index, or at a
slope in a specimen, light will be
deflected to either side. The amount of
light deflected in each direction at any
one point depends on the
characteristics of the specimen at that
point.
The modulation plate in the back focal
plane of the objective lens absorbs the
light deflected in each direction to
Equipment
Basic parts:
• A slit aperture (A), just below the
condenser
• A modulation plate, mounted in the
back focal plane of the objective lens
Variable contrast system parts:
• A polarizer in the slit aperture (B)
• A rotatable polarizer
Each objective lens requires its own
Hoffman modulation plate and alit
aperture of appropriate size. You can
fit your present equipment with these
parts.
different extents. In the resultant
image, one side of a feature in the
specimen is darkened and the other is
lightened. This creates the shadow
effect that enhances contrast and
subject visibility.
The shadow effect created in the image
is directional. Thus, by orienting the
specimen appropriately, you can make
the apparent shadows fall in the most
advantageous way for optimum
enhancement of contrast and detail.
Since the slit direction is constant, the
light and dark sides of slopes are
predictable. (In differential interference
contrast, a specimen can be made to
look like a protrusion or depression.)
Etched surface of transparent birefringent
calcite crystal, 200X magnification. (Left)
brightfield; (right) Hoffman Modulation
Contrast. Photomicrographs by Dr.
Robert Hoffman.
60
Instant Photography
Photomicrography
Through the Microscope
Techniques
• Align your equipment so that the
desired illuminating beam from the
slit (A) is imaged in the gray section
of the modulation plate. Use a phase
telescope for easiest alignment.
• The variable contrast system (B)
incorporates a polarizer in the slit
aperture and a rotatable polarizer
below. Rotate the polarizer to vary
the size and intensity of the direct
illuminating beam to attain optimum
contrast for specimens having widely
differing optical characteristics.
Transparent replica of a
microelectronic circuit. 200X
magnification. This pair of
Hoffman Modulation
photomicrographs shows the
effect of specimen orientation.
(Left) both horizontal and vertical
channels are clearly visible;
(right) specimen having been
rotated by 45 degrees, the
horizontal channels are lost.
Photographic Considerations
• The amount of light reaching the film
may be about 1/10 to 1/20 of that
available in normal brightfield work.
The slit aperture reduces the
illumination considerable and a
further reduction is caused by
absorption by the modulation plate.
Live algae, 400X magnification.
This pair of photomicrographs
shows the effect of the polarizer
in the slit aperture and the
rotatable polarizer just below.
(Left) by fully crossing the
polarizers, very high contrast
was achieved: (right) as the
polarizers were uncrossed,
contrast was reduced.
Photomicrographs by Dr. Robert
Hoffman.
• For excellent resolution and tonal
rendition, use Polaroid Type 52, 552,
57, or 667 film. Use an appropriate
interference filter or a green filter
(such as the Wratten No. 58) for
optimum contrast and resolution.
• When using color film, omit the
green filter. Use appropriate filtration
for best color rendition.
• Depth of field is limited because of
the relatively high numerical aperture.
Therefore, selective focusing or
optical sectioning is possible through
the thickness of a specimen.
61
Instant Photography
Photomicrography
Through the Microscope
Differential Interference Contrast (DIC)
Purpose
Suitable Specimens
Equipment
• To enhance contrast in a specimen of
very low contrast by making use of
the differences in thickness and
refractive index at the various
interfaces, edges, and slopes within
the specimen.
•
•
•
•
•
•
•
•
•
•
•
• A polarizer
• A condenser that incorporates a
beam-splitting Wollaston prism
• Strain-free objective lens
• A second prism that recombines the
beam near the back focal plane of the
objective lens
• An analyzer (the second polarizer)
This equipment is easily
interchangeable between Differential
interference Contrast and normal
brightfield illumination.
• To bring out detail that is otherwise
hidden in an unstained and
transparent specimen.
• To utilize the full numerical aperture
of the objective, ensuring optimum
resolution.
Smears
Cell cultures
Blood cells
Organelles in protoplasm
Unstained tissue sections
Chromosomes
Constrained protozoa
Diatoms
Polymers and polymer coatings
Replicas
Relatively thick specimens, due to
limited depth of field
The Physics of Differential
Interference Contrast
Like Hoffman Modulation Contrast,
Differential Interference Contrast
detects optical gradients, or slopes, in
a specimen and converts them into
intensity differences.
Transmitted light differential Interference Contrast
The enhancement of contrast in
Differential Interference Contrast is due
to interference between two beams of
light that travel through the specimen
adjacent to each other. When there is
interference between the two beams of
light, the intensity from various parts of
the specimen is different and contrast
is enhanced.
The polarizer defines the plane of
polarization. The first Wollaston prism
splits the polarized light into two beams
that are parallel and separated by an
extremely small distance. The planes
of polarization of the two beams are
perpendicular, at 45 degrees, to the first
polarizer. These totally independent
light beams cannot interfere with each
other.
62
The two adjacent beams travel through
parts of the specimen which are
separated by the same small distance.
At an edge, interface, or slope in the
specimen where there are differences in
refractive index and thickness, the
beams experience a relative change in
phase. Opposite sides of an edge or
interface, or different parts of a slope,
produce opposite phase differences.
After traversing the specimen, the
beams are recombined by the second
Wollaston prism. Only when the light
has passed through the analyzer do the
beams interfere. This interference
translates the phase differences into
light intensity differences and thus
generates contrast enhancement.
Techniques
• To ensure optimum contrast, the
polarizer and analyzer must be fully
crossed, at 90 degrees to each other.
• You can vary the colors and tonal
values in the image, as well as the
density of the background, by
rotating the analyzer. A few degrees
rotation slightly lightens the darker
areas. Ninety-degree rotation turns
the dark areas bright and transforms
the colors in the specimen to their
complements.
Instant Photography
Photomicrography
Through the Microscope
• Vary the phase difference to achieve
different contrast and color effects by
making a simple adjustment in one of
the Wollaston prisms.
• The shadow effect created in the
image is unidirectional. Orient the
specimen so the shadows reveal
features of interest.
• The sensitivity of DIC, and the
placement of the shadows on an
image, can create the impression of
either a protrusion or a depression.
To prevent confusion, compare the
shadows of unknown features with
those of known features.
Motor neuron, 160X
magnification. Green filter.
(Left) brightfield; (right)
Differential Interference
Contrast.
• Birefringent specimens are generally
not suitable for Differential
Interference Contrast because they
interfere with the decisive polarization
of the light and produce a confusing
image.
Photographic Considerations
• For optimum resolution and contrast
in black and white photomicrography,
use a green filter, such as the
Wratten No. 58 or an appropriate
interference filter.
Glass shards, in oil of 1,500
refractive index. 200X
magnification. Green (546 nm)
interference filter. This pair of
photomicrographs shows clearly
that, by making an appropriate
adjustment in the setting of the
Wollaston prism, the apparent
“shadow” created by the
Differential Interference Contrast
method can be placed on either
side of the specimen.
• With color film, which yields
attractive and informative color
images, use normal color filtration
and a didymium filter, where
appropriate. Do not use a green filter.
• The amount of light reaching the film
may be from ½ to about 1/30 of that
available in normal brightfield work.
The exact light reduction depends on
the amount of phase difference
introduced and on the setting of the
beam-splitting Wollaston prism.
• Selective focusing, or optical
sectioning, is possible because the
Differential Interference Contrast
method makes use of the full
numerical aperture of the objective
lens, and the depth of the field is very
limited. Only the specimen plane
that has been focused will be sharp.
The remainder of the depth through
the specimen will not be sharp and
will not intrude into the desired
image.
63
Instant Photography
Photomicrography
Through the Microscope
Reflected Light Differential
Interference Contrast
Differential Interference Contrast in
reflected light requires a polarizer and
analyzer, a brightfield reflected light
objective, a reflector, and a Wollaston
prism which acts as both the beam
splitter and beam recombiner.
Reflected light differential Interference Contrast
Slopes on the surface of the specimen
create the phase differences between
adjacent beams, and lead to brightness
differences which delineate the
topography of the specimen.
To obtain the greatest contrast on the
samples with subtle slopes, adjust the
prism so that the background is gray,
with one slope dark and opposite slope
bight.
Uncoated surface of a diamond-turned
lens: The directional sensitivity of DIC
shows the grooves most clearly when
they are oriented in the NW-SE
direction. The chatter marks which
are perpendicular to the grooves are
shown most clearly when they are in
the same presentation.
The shadow effect in DIC is
variable, and the same feature can
appear to be a depression or a
protrusion. (Left) features appear
to be depressions, and (right) with
a different setting of the beam
splitter, the same features appear
to be protrusions.
64
Polaroid
Instant Photography
Troubleshooting
Common Problems
Photomicrography
Through the Microscope
Introduction
Many factors can affect image quality –
improperly adjusted illumination or
optics; dirt, dust, or grease; or the use
of the wrong filter. The following trouble
shooting guide can help you correct
common mistakes and better
understand the reasons behind them.
Basswood stern
Polarized light
Red l Plate
Polacolor ER
Type 59
Mary McCann
65
Instant Photography
Photomicrography
Through the Microscope
Blurry Photomicrograph Image When Viewed Image is in Focus
• If the photomicrograph is out of focus
when the viewed image is sharp, the
film plane and the viewing optics may
not be parfocal. This is more likely at
low magnifications where depth of
focus is shallow.
• In a camera with a focusing
telescope, check to see if the reticle
in the telescope is in sharp focus.
• In microscopes with a reticle in the
viewing eyepiece, ensure that the
reticule is in focus before focusing
the specimen. In older
microscopes, make sure that the
tubes of the binocular are set for the
proper interpupillary distance.
• if there is still a problem, check to
see that the eyepiece in the
phototube is properly set. First
check the focus of the eyepiece
reticule and focus the specimen
carefully using a 10X objective. See
page 12 “Understanding
Parfocalization.”
• Vibrations may also cause blurriness
of the photomicrograph. Use a cable
release with a mechanical shutter,
and if shutter vibration persists, use
neutral density filters to lengthen
exposure times. Cameras supplied
by microscope manufacturers.
Slightly Out-of-Focus Spots
• Dark spots in your photomicrograph
may be the result of shadows from
dust particles in the following places:
- the field lens of the microscope
camera
- the top surface of the eyepiece
- the glass adjacent to the field
diaphragm
- any filters adjacent to the field
diaphragm
• Remove the camera back and the
cone from the shutter assembly.
Clean the field lens with compressed
air or a camel hair brush.
Bright Rectangles
• Bright rectangles on a
photomicrograph, particularly when
long exposures are required, may be
the images of overhead lighting. The
problem occurs with microscopes
that have a beam splitter between the
viewing binocular and the phototube.
66
You can remedy the problem by
adjusting the beam splitter to the
position where all the light is directed
to the phototube. If this isn’t
possible, cover the viewing eyepiece
that is directed toward the overhead
light.
Instant Photography
Photomicrography
Through the Microscope
Blurring of Viewed Image
• If your image is blurry, there may be
fingerprints or grease on the font of
the objective lens. On the top lens of
the eyepiece, or on the slide. Use a
very small amount of solvent to clean
these surfaces. (Avoid using too
much solvent, as this may affect the
objective cement or the slidemounting medium). Refer to your
microscope instruction book for
directions for cleaning lens surfaces.
• Improper adjustment of the optics
may be the cause of blurring. Check
if either the field diaphragm or the
aperture diaphragm is open too far or
not properly centered.
• Blurring may also result when the
specimen is too thick.
Gradual Darkening at the Edge of the Image
• If the photomicrograph shows gradual
darkening at the edge of the image
area, check that the substage
condenser is properly aligned and the
microscope is properly set for Kohler
illumination. If the condenser isn’t set
to its proper height, closing the
aperture diaphragm can cause
darkening at the edge of the field.
The objective is usually set in a fixed
position, so center the substage
condenser with the field diaphragm.
• If you have centered the objective
and the condenser, and the
illumination is still uneven, check
that the lamp filament is centered.
Sharply Focused, Dark Corners
• Check that the field diaphragm is
opened sufficiently.
• If the corners of your
photomicrograph are in focus but
dark, check that the eyepiece
magnifications is sufficient to fill the
full diagonal. For example, the
diagonal of a 4x5” photomicrograph
is 145 mm. Using a camera with a
1X magnification factor, an
67
8X/21 mm field of view eyepiece will
give an image 168 mm in diameter,
sufficient to fill the whole
photomicrograph. However, using
this eyepiece with a camera
magnification factor of 0.8 will
give an image 134 mm in diameter.
This eyepiece would be more
appropriate for the 3 1/4X 4 1/4” film.
Instant Photography
Photomicrography
Through the Microscope
Concentric Areas In and Out of Focus
• If the image is sharp in the center but
out of focus around the edges,
ensure that you have a plan objective.
A non-plan objective is not corrected
for curvature of field of the image and
will not give a sharp image across the
total field of view.
• In reflected light samples, a focused
center but out-of-focus edges may
result when the specimen is not
mounted flat. Mount the specimen
on a small amount of modeling clay
to allow leveling.
Lack of Contrast and Poor Sharpness
• A high numerical aperture objective
with an improper coverslip over the
specimen may lack contrast in a
photomicrograph due to spherical
aberration. Check the side of the
barrel of the objective to see what
coverslip thickness is required
(usually 0.17 mm, the thickness of a
#1 1/2 coverslip). Ensure that you
have the correct thickness coverslip
when you work with objectives of
high numerical aperture.
• Remember, reflected light objectives
usually need no coverslip. If a
coverslip is used, the micrograph will
lack contrast.
• Photomicrographs lack contrast
when the substage diaphragm is
opened too far. Close the substage
diaphragm to reduce flare. In
reflected light, the settings of both
the field diaphragm and the aperture
diaphragm affect contrast.
Image Too Light or Too Dark, Using an Automatic Camera
• The average brightness of the
specimen may not be representative
of the brightness in the areas of
interest. For example, if the
specimen has small dark features on
a bright background, the image will
be underexposed. Use the spot
meter setting if available. If the meter
averages the whole field of view, set
the film speed indicator to
compensate. If the specimen is
bright in a large dark surround, the
image will be overexposed. Use the
spot meter setting if available, or
increase the film speed indicator to a
lower value to increase the exposure.
68
• When you use an automatic camera
with deeply colored filters and the
resulting photomicrograph image is
either too light or too dark, the
sensitivity of the camera photocell
may not be uniform across the
spectrum. Keep a record of the
photo results with different color
filters, and adjust the film speed
indicator to compensate.
• If automatic exposure with polarized
light varies with the presence of the
polarizer or the analyzer, the
photocell may be sensitive to
polarization effects. Keep a record of
the exposure adjustment necessary
or inquire about remedy from
microscope manufacturer.
Polaroid
Instant Photography
Instant Films
for Photomicrography
Photomicrography
Through the Microscope
Copper screen plated with
copper, 100x Polaroid
Type 53 JoAnn H.
Montoya
69
Instant Photography
Photomicrography
Through the Microscope
Polaroid Films for Instant Photomicrography
FILM
TYPE
SIZE AND HOLDER
SPEED (ISO/DIN)
Print-Tungsten Balance
Polacolor 64 Tungsten
3 1/4 x 4 1/4” pack film, 405 film holder
4 x 5” sheet film, 545i film holder
64/19 0
64/190
Print-Daylight Balance
669
559
59
809
3 1/4 x 4 1/4” pack film, 405 film holder
4 x 5” pack film, 550 film holder
4 x 5” sheet film, 545i film holder
8 x 10” sheet film, 8106 8 x10” film hoder
80/200
80/200
80/200
80/200
Transparency
691
891
Polachrome HC
Polachrome CS
3 1/4 x 4 1/4” pack film, 405 film holder
8 x 10” sheet film, 8106 8 x 10” film holder
35mm instant film, any 35mm back
35mm instant film, any 35mm back
80/200
80/200
40/170
40/170
Self-developing Print
339 Autofilm
778
3 x 4” pack film, MicroCam and CB 33 back
3 1/8 x 3 1/8” pack film, SX-70 camera
640/290
150/230
3 1/4 x 4 1/4” pack film, 405 film holder
4 x 5” sheet film, 545i film holder
4 x 5” sheet film, 545i film holder
80/200
50/18, 32/160 neg
200/24, 32/160 neg
Color
Black and White
Print with Printable Negative 665
55
51HC
Print with Print Coating
552
52
57
107D
4 x 5” pack film, 550 film holder
4 x 5 sheet film, 545i film holder
4 x 5 sheet film, 545i film holder
3 1/4 x 4 1/4” pack film, 405 film holder
400/270
400/270
3000/360
3000/360
Coaterless Print
553
53
803
Polapan 400
667
4 x 5” pack film, 550 film holder
4 x 5” sheet film, 545i film holder
8 x 10” sheet film, 8106 8 x 10” film holder
4 x 5” sheet film, 545i film holder
4 x 5” pack film, 550 film holder
3 1/4 x 4 1/4” pack film, 405 film holder
3 1/4” x 4 1/4” pack film, 405 film holder
800/300
800/300
800/300
400/270
400/270
400/270
3000/360
Self-developing
331 Autofilm
337
3 1/4” pack film, MicroCam and CB 33 back
3 1/4” pack film, MicroCam and CB 33 back
400/270
3000/360
Transparency
Polapan CT
Polagraph HC
35mm instant film, any 35mm back
35mm instant film, any 35mm back
125/220
400/270
For the environment: Types 59, 55, 52, and 57 and Polacolor 64 Tungsten, in 4 x 5” format, are available with reduced packaging in cartons
of 200 exposures. Types 559 and 552 are available in cartons of 30 packs. Types 809, 891, and 803 are available in bulk packages of 45
exposures.
70
Instant Photography
Photomicrography
Through the Microscope
CONTRAST
RESOLUTION (LINE PAIRS/MM)
SPEED CHARACTERISTICS
Medium
10-12 1p/mm
No filtration necessary with tungsten illumination, exposures between
1/2 and 8 seconds
Medium
10-12 lp/mm
Medium
Medium
Medium
Medium
10-12 lp/mm
10-12 lp/mm
10-12 lp/mm
10-12 lp/mm
Filtration necessary (see your current film data sheet).
Medium
Medium
High
Medium
10-12 lp/mm
10-12 lp/mm
90 lp/mm
90 lp/mm
Same sensitivity as T669, daylight balanced
Requires Polaroid 8 x 10 film processor
Daylight balanced, requires Polaroid 35mm processor
Daylight balanced, requires Polaroid 35mm processor
Medium
Medium
90 lp/mm
8-10 lp/mm
Daylight balanced, filtration necessary betwen 1/60 and 3 seconds
Medium
Medium
High print, med. neg
22-25 print, 160-180 neg
20-25 print, 160-180 neg
20-23 print, 100-120 neg
Fine grain print, high-resolution negative
Medium
Medium
Medium
Medium
20-25 lp/mm
22-25 lp/mm
16-22 lp/mm
16-22 lp/mm
Fine grain, good gray-scale rendition
Medium
Medium
Medium
Medium
Medium
Medium
Medium
16-22 lp/mm
16-22 lp/mm
16-22 lp/mm
20-22 lp/mm
20-22 lp/mm
20-22 lp/mm
16-22 lp/mm
Print does not require coating
Medium
Medium
20 lp/mm
20 lp/mm
Self developing, self timing
Medium
High
90 lp/mm
90 lp/mm
Requires Polaroid 35mm film processor
High contrast, requires Polaroid 35mm film processor
Requires Polaroid 8 x 10 film processor
High-contrast print, high resolution negative
Exceptionally high-speed film
Requires Polaroid 8 x 10 film processor
Same speed as Type 52, but does not require coating
Exceptionallly high-speed film
71
Instant Photography
Photomicrography
Through the Microscope
Acknowledgement
Mary McCann, a Research Associate
in the Microstructures Characterization
Laboratory of Polaroid’s Research
Division, provided principle technical
assistance in preparing
Photomicrography: Instant Photography
through the Microscope. Her
contributions have helped to update and
supplement information that was
previously available in a series of
photomicrography booklets written by
John P. Vetter and Vernon Gorter.
Thin cast polymer film,
observed with Zeiss
Jamin-Lebedeff
interference optics. 50 x
magnification.
Photomicrograph made
with Polaroid SX-70
camera and SX-70
microscope adapter.
Mary has been making instant
photomicrographs since she joined
Polaroid as a chemical engineer in
1960. She is a teacher, lecturer, and
researcher whose particular interests
are polarized light microscopy,
interference microscopy, and
microscopy of imaging materials.
72
Photomicrography: Instant Photography Through the Microscope
brings together principles and techniques of photography and
microscopy to help photomicrographers in any industry capture
superior photomicrographs. This book covers:
•
•
•
•
•
•
•
Polaroid Technical Assistance
Hotline 1-800-225-1618
Microscopes and cameras used for photomicrography
Kohler illumination practices
Film characteristics for instant photomicrography
How to use filters for black and white photomicrography
How to use filters for color photomicrography
Special contrast-enhancement techniques
Troubleshooting assistance