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Kohler Illumination

Kohler illumination needs a high density illumination source, field diaphragm, condenser
diaphragm, and collector and condenser lenses.

Although all major manufacturers of high quality microscopes provide Kohler illumination, if
a microscope doesn’t have Kohler illumination it can be retrofitted as long as certain criteria
can be met.

A true Kohler lamp has a very large filament but a standard lamp can be used. If your
microscope doesn’t have a field diaphragm your unit will need to allow for the addition of
this and the necessary lenses mounted above the lamp. Also, you will need to be able to
raise and lower the condenser.

Working Principle
In Kohler illumination, four separate planes combine to form conjugate planes in both the
illumination and image-forming light pathways.

The lamp filament, aperture diaphragm, back focal plane of the objective lens, and the eye
point which is approximately one centimeter above the top lens of the ocular, form the
illumination conjugate plane.

The conjugate planes of the imaging light path are the field diaphragm, specimen, the fixed
diaphragm of the ocular, and the retinal plane of the viewer.

In Kohler illumination the collector lens or field diaphragm collects light from the
illumination source and focuses it at the front focal plane of the sub-stage condenser’s
aperture diaphragm which, in essence, projects an image of the lamp filament onto the

The condenser transmits the light to illuminate the specimen. Often, the condenser must be
adjusted to guarantee that the filament image appears in the focal plane and fills the
aperture. The image of the filament must fill the aperture diaphragm and the field
diaphragm and they must share the same conjugate image planes as the specimen.

This results in extremely even illumination because the filament in the specimen image
planes is completely defocused and forms a clear image of the specimen.

Closing the field diaphragm does not reduce the brightness of the image but merely controls
the width of the light beam being transmitted to the condenser and restricts the light to the
part of the specimen which is actually being observed.

Adjusting the condenser aperture affects the angle of the light being transmitted to the
specimen. Setting this diaphragm and the aperture of the objective determines the actual
working numerical aperture of the microscope. Resolution and contrast increase as the
condenser diaphragm is opened.
Using Kohler Illumination
The condenser and field diaphragms must be adjusted each time the microscope is used and
each time the objective is changed.

When changing to a higher objective the field diaphragm should be closed down because
the viewing field is reduced and the aperture diaphragm should be opened to match the
higher numerical aperture number of the more powerful objective.

To properly set up Kohler illumination on a microscope:

 Turn on the light source and ensure that light is coming through the field diaphragm.
Also, ascertain that your specimen is being illuminated.
 Using the coarse focus and then the fine focus, bring the specimen into focus.
 Begin closing the field diaphragm and begin adjusting the condenser height to bring
the edges of the diaphragm into focus. At this point the field diaphragm and the specimen
should be in focus.
 Next, center the image of the field diaphragm using the condenser centering knobs.
Open the diaphragm until its edges are barely outside the view field of the microscope.
 Adjust the contrast by adjusting the condenser diaphragm and adjust the
illumination intensity.

To get the best contrast and resolution close the diaphragm until the image begins to
darken. Closing the condenser diaphragm any further will compromise resolution.

Advantages and Disadvantages

The main advantages are high contrast and evenly distributed illumination.

Also, less specimen heating occurs and helps prevent thermally induced changes in the
specimen. Reflection and glare are eliminated by using the field diaphragm controlling the
width of the light beam.

While this method of illumination works with darkfield viewing the results are poor unless
the diaphragm is completely open. But, other than that, this is the preferred method of

Quick Summary
With sophisticated microscopes, all segments of the optical train significantly contribute to
the quality of the image but, often neglected, proper configuration of the light path is crucial
for all imaging methods.

Kohler illumination works with all methods of microscopy including DIC,phase

contrast, polarization, brightfield, darkfield and all epi-illuminations.
Sometimes referred to as double diaphragm illumination, it uses both a field and an
aperture diaphragm to focus the illumination and provides an evenly illuminated viewing
field, a bright specimen image and eliminates glare.

Dark Field
What is Dark Field Illumination?

Dark Field illumination is a technique used to observe unstained

samples causing them to appear brightly lit against a dark,
almost purely black, background.

Pictured right: Highly magnified image of sugar crystals using

darkfield microscopy technique

When light hits an object, rays are scattered in all azimuths or

directions. The design of the dark field microscope is such that it
removes the dispersed light, or zeroth order, so that only the
scattered beams hit the sample.

The introduction of a condenser and/or stop below the stage ensures that these light rays
will hit the specimen at different angles, rather than as a direct light source above/below
the object.

The result is a “cone of light” where rays are diffracted, reflected and/or refracted off the
object, ultimately, allowing you to view a specimen in dark field.

Dark Field Transformation

Most stereo and standard compound microscopes have the potential for dark field


If a microscope has built-in elements to easily modify for dark field illumination, the
manufacturer usually lists this amongst the observation specifications.

You can achieve dark field by using condensers, mirrors and/or a “stop.” Some microscopes
come with these accessories or researchers can purchase dark field kits, or even use some
common items to adapt a microscope for dark field illumination.
In bright field illumination, the object is lit from below the stage, resulting in a larger,
contrasted image that can be studied.

A dark field microscope blocks this central light with a condenser so that only oblique rays
hit the object.

An Abbe condenser, for example, contains a concave orb that collects light rays in all
azimuths that bounce off a sample to form a cone of illumination.

If there is nothing on the stage, the aperture of the condenser is greater than the objective
and the view will be completely black.

A stop is an opaque object that blocks the central light when placed underneath the stage

This also causes light to scatter in all azimuths, resulting in a cone of light that allows for
dark field observation.

Too expensive? What you can do...

If you do not have access to these accessories and cannot afford a dark field kit, there are
alternative ways to adapt your microscope for dark field illumination.

The expensive stops are all made of opaque material.

Any possible substitutions cannot have any transparent properties.

One option is to use a circular object, such as a coin; adhere the coin to a larger disk and
place below the stage.

You can also cut out a round piece of thick paper, such as construction paper, cardboard or
poster-board, and attach to the condenser.

Whatever you use, the trick is to find the right diameter so that the makeshift stop will
block the light and only allow the oblique rays to illuminate the specimen.

Advantages of Dark Field Microscopy

A dark field microscope is ideal for viewing objects that are unstained, transparent and
absorb little or no light.

These specimens often have similar refractive indices as their surroundings, making them
hard to distinguish with other illumination techniques.
You can use dark field to study marine organisms such as algae and plankton, diatoms,
insects, fibers, hairs, yeast and protozoa as well as some minerals and crystals, thin
polymers and some ceramics.

You can also use dark field in the research of live bacterium, as well as mounted cells and

It is more useful in examining external details, such as outlines, edges, grain boundaries
and surface defects than internal structure.

Dark field microscopy is often dismissed for more modern observation techniques such
as phase contrast and DIC, which provide more accurate, higher contrasted images and can
be used to observe a greater number of specimens.

Recently, dark field has regained some of its popularity when combined with other
illumination techniques, such as fluorescence, which widens its possible employment in
certain fields.

Disadvantages of Dark Field Microscopy

A dark field microscope can result in beautiful and amazing images; this technique also
comes with a number of disadvantages.

 First, dark field images are prone to degradation, distortion and inaccuracies.

A specimen that is not thin enough or its density differs across the slide, may appear to
have artifacts throughout the image.

 The preparation and quality of the slides can grossly affect the contrast and accuracy
of a dark field image.

You need to take special care that the slide, stage, nose and light source are free from
small particles such as dust, as these will appear as part of the image.

 Similarly, if you need to use oil or water on the condenser and/or slide, it is almost
impossible to avoid all air bubbles.

These liquid bubbles will cause images degradation, flare and distortion and even
decrease the contrast and details of the specimen.

 Dark field needs an intense amount of light to work. This, coupled with the fact that
it relies exclusively on scattered light rays, can cause glare and distortion.

It is not a reliable tool to obtain accurate measurements of specimens.

 Finally, numerous problems can arise when adapting and using a dark field
microscope. The amount and intensity of light, the position, size and placement of the
condenser and stop need to be correct to avoid any aberrations.
Dark field has many applications and is a wonderful observation tool, especially when used
in conjunction with other techniques.

However, when employing this technique as part of a research study, you need to take into
consideration the limitations and knowledge of possible unwanted artifacts.

Darkfield Illumination

All of us are quite familiar with the appearance and visibility of stars on a dark night, this despite their
enormous distances from the Earth. Stars can be readily observed at night primarily because of the
stark contrast between their faint light and the black sky.

Yet stars are shining both night and day, but they are invisible during the day because the
overwhelming brightness of the sun "blots out" the faint light from the stars, rendering them invisible.
During a total solar eclipse, the moon moves between the Earth and the sun blocking out the light of
the sun and the stars can now be seen even though it is daytime. In short, the visibility of the faint
star light is enormously enhanced against a dark background.

This principle is applied in darkfield (also called darkground) microscopy, a simple and popular
method for making unstained transparent specimens clearly visible. Such objects often have
refractive indices very close in value to that of their surroundings and are difficult to image in
conventional brightfield microscopy. For instance, many small aquatic organisms have a refractive
index ranging from 1.2 to 1.4, resulting in a negligible optical difference from the surrounding
aqueous medium. These are ideal candidates for darkfield illumination.
Darkfield illumination requires blocking out of the central light which ordinarily passes through and
around (surrounding) the specimen, allowing only oblique rays from every azimuth to "strike" the
specimen mounted on the microscope slide. The top lens of a simple Abbe darkfield condenser is
spherically concave, allowing light rays emerging from the surface in all azimuths to form an inverted
hollow cone of light with an apex centered in the specimen plane. If no specimen is present and the
numerical aperture of the condenser is greater than that of the objective, the oblique rays cross and
all such rays will miss entering the objective because of their obliquity. The field of view will appear

Interactive Java Tutorial

Specimen Imaging in Darkfield Condensers

Explore how a specimen reflects, refracts, and diffracts light emitted

from the hollow cone of light produced by a darkfield
The darkfield condenser/objective pair illustrated in Figure 1 is a high-numerical aperture
arrangement that represents darkfield microscopy in its most sophisticated configuration, which will
be discussed in detail below. The objective contains an internal iris diaphragm that serves to reduce
the numerical aperture of the objective to a value below that of the inverted hollow light cone emitted
by the condenser. The cardioid condenser is a reflecting darkfield design that relies on internal
mirrors to project an aberration-free cone of light onto the specimen plane.

When a specimen is placed on the slide, especially an unstained, non-light absorbing specimen, the
oblique rays cross the specimen and are diffracted, reflected, and/or refracted by optical
discontinuities (such as the cell membrane, nucleus, and internal organelles) allowing these faint
rays to enter the objective. The specimen can then be seen bright on an otherwise black
background. In terms of Fourier optics, darkfield illumination removes the zeroth order (unscattered
light) from the diffraction pattern formed at the rear focal plane of the objective. This results in an
image formed exclusively from higher order diffraction intensities scattered by the specimen.

The photomicrographs in Figure 2 illustrate the effects of darkfield and brightfield illumination on
silica skeletons from a small marine protozoan (radiolarian) in a whole mount specimen. In ordinary
brightfield, skeletal features of the radiolarian are not well defined and tend to be washed out in
photomicrographs recorded either with traditional film or digitally captured. The photomicrograph in
Figure 2(a) was taken in brightfield illumination with the condenser aperture diaphragm closed to a
point where diffraction artifacts obscure some of the sample detail. This enhances specimen contrast
at the expense of image distortion. Under darkfield illumination (Figure 2(b)), more detail is present,
especially in the upper portion of the organism, and the image acquires an apparent three-
dimensional appearance. When a red filter is used in conjunction with a darkfield stop (Figure 2(c)),
the radiolarian takes on a colorful appearance that is more pleasing, although no additional detail is
produced and there is even some reduction in image quality.

Specimens that have smooth reflective surfaces produce images due, in part, toreflection of light
into the objective. In situations where the refractive index is different from the surrounding medium or
where refractive index gradients occur (as in the edge of a membrane), light is refracted by the
specimen. Both instances of reflection and refraction produce relatively small angular changes in the
direction of light, allowing some to enter the objective. In contrast, some light striking the specimen is
also diffracted, producing a 180-degree arc of light that passes through the entire numerical
aperture range of the objective. The resolving power of the objective is the same in darkfield
illumination as observed under brightfield conditions, but the optical character of the image is not as
faithfully reproduced (except when a specially-designed iris diaphragm is utilized to lower the
effective numerical aperture with high-magnification oil immersion objectives designed specifically
for darkfield microscopy).

As in the example of starlight described above, the visibility is greatly enhanced by the contrast
between the brightly shining specimen and the dark surround. As discussed above, what has
happened in darkfield illumination is that all the ordinarily undeviated rays of the zeroth order have
been blocked by the opaque stop. Oblique rays, now diffracted by the specimen and yielding first,
second, and higher diffracted orders at the rear focal plane of the objective, proceed onto the image
plane where they interfere with one another to produce an image of the specimen.

Ideal candidates for darkfield illumination include minute living aquatic organisms, diatoms, small
insects, bone, fibers, hair, unstained bacteria, yeast, cells in tissue culture, and protozoa. Non-
biological specimens include mineral and chemical crystals, colloidal particles, dust-count
specimens, and thin sections of polymers and ceramics containing small inclusions, porosity
differences, or refractive index gradients. Care should be taken when preparing specimens for
darkfield microscopy because features that lie above and below the plane of focus can also scatter
light and contribute to image degradation. Specimen thickness and microscope slide thickness are
also very important and, in general, a thin specimen is desirable to eliminate the possibility of
diffraction artifacts that can interfere with image formation.

The substage condensers illustrated in Figure 3 demonstrate the effect of an opaque stop on light
pathways through a simple refracting condenser. On the left (Figure 3(a)), is a typical Abbe
brightfield condenser positioned with the aperture diaphragm opened to maximize the numerical
aperture of the light cone. Light from the source passes through the aperture diaphragm and is then
refracted through several lens elements to form an inverted cone of light with a numerical aperture of
approximately 1.20. When an opaque spider-style light stop (Figure 3(b)) is inserted below the
completely opened aperture diaphragm, the central light rays are blocked, allowing only peripheral
light rays to pass through the lenses to form an inverted oblique hollow cone of light with no change
in numerical aperture (1.20). The illuminating hollow light cone is formed by refraction of light at
perimeter of the lens elements, where optical correction is often poorest. Even so, this condenser
will perform adequately using low magnification objectives, and produces very nice results for
qualitative darkfield work. For more exacting quantitative microscopy, it is necessary to use aplanatic
condensers (corrected for both chromatic and spherical aberrations), which perform much better by
producing images with clearer detail and more reliable features.
In a darkfield microscope, if you were to look at the back of the objective through a Bertrand lens or
phase telescope, it would appear filled with light. This faint diffracted light is reconstituted into the
visible image at the plane of the eyepiece diaphragm with its contrast reversed, bright image on
black background. Since darkfield microscopy eliminates the bright undeviated light, this form of
illumination is very wasteful of light and thus demands a high intensity illumination source.
Microscope slides must be of the appropriate thickness, approximately one millimeter +/- 0.1 mm.
Specimen slides and all optical surfaces in the light path must be scrupulously clean because every
dirt speck will be mercilessly bright.

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Abbe Darkfield Condensers

Discover how changing the opaque stop size affects light cone
formation and numerical aperture in a simple Abbe
There are several pieces of equipment that are utilized to produce darkfield illumination. The
simplest is a "spider stop" placed just under the bottom lens (in the front focal plane) of the substage
condenser (Figures 3(b) and 4(a)). Both the aperture and field diaphragms are opened wide to pass
oblique rays. The central opaque stop (you can make one by mounting a coin on a clear glass disk)
blocks out the central rays. This device works fairly well, even with the Abbe condenser (Figure 3),
with the 10x objective up to 40x or higher objectives having a numerical aperture no higher than
0.65. The diameter of the opaque stop should be approximately 16-18 millimeters for a 10x objective
of numerical aperture 0.25 to approximately 20-24 millimeters for 20x and 40x objectives of
numerical apertures approaching 0.65.

The set of stops illustrated in Figure 4(a) vary in size from 8 millimeters to 30 millimeters and provide
excellent darkfield opaque stops for virtually any numerical aperture objective (below 0.65).
Individual stops can be interchanged simply by removing the screw in the bottom of the support
spider and replacing the stop with a new size. The outer size of the spider holder will vary depending
upon the housing opening diameter at the bottom of the condenser.

The light stop illustrated in Figure 4(b) is an ingenious device that expands and contracts a "reverse
iris" diaphragm to increase or decrease the size of the stop using a lever control arm. As this lever is
turned, the size of the central leaves changes from about 10 millimeters to 25 millimeters in
diameter, creating a larger stop for higher magnification objectives. This type of variable light stop
diaphragm eliminates the need for changing light stops each time a higher power objective is
inserted into the optical path. It also has the additional advantage of being "tunable" for slight
differences in the stop diameter necessary to achieve the best performance while observing the
specimen. Although these types of diaphragm light stops are now very rare, they do provide a
unique method of achieving highly desirable effects with darkfield illumination.

Almost any brightfield laboratory microscope can be easily converted for use with darkfield
illumination. As discussed above, central opaque stops can be fashioned from a small coin,
cardboard, plastic, or black paper that can be placed in a filter carrier beneath the condenser (or
taped to the condenser bottom with adhesive tape) to block light from entering the front lens of the
objective. The diameter of the opaque stop will vary from objective to objective and should be
carefully measured by placing a transparent ruler in the substage filter carrier and holding it steady
against the bottom of the condenser. Next, determine the opening size by removing an eyepiece and
observing the image of the ruler at the back focal plane of the objective using a phase telescope (or
by inserting a Bertrand lens). Make certain that both the substage condenser aperture and field
diaphragms are opened to their widest position before performing this maneuver. The number of
ruler divisions visible in the back focal plane will be equal to the size of the stop necessary to block
zeroth order light from entering the objective. Change to the next largest size objective and take
another measurement, repeating until stop sizes for all objectives are known.

A guide to approximate opaque stop size versus magnification is given in Table 1. The actual size
will vary, depending upon several factors including the proximity of the stop with respect to the
condenser aperture diaphragm, the numerical aperture of both the objective and condenser, the
degree of aberration correction for the condenser, and the field number of the eyepiece. Also
important in determining the stop size is the diameter of the condenser back lens, the magnification
power of the eyepiece (smaller magnifications require slightly larger stops), and the type of mounting
medium. Stop size varies proportionally to the refractive index of the mounting medium: higher
refractive index requires a larger stop. A dry mount will also need a smaller stop than an aqueous

Approximate Field Stop Diameter Size

Magnification Stop Size (mm)

1X 0.03 25-30

2x 0.05 8-11

4X 0.10 8-14

10x 0.25 16-18

20X 0.40 18-20

20x 0.65 20-22

40X 0.65 22-24

Table 1 

Use scissors or (preferably) a brass cork borer to cut a set of stops matched to all of the objectives,
and glue them to a sturdy sheet of clear acetate or glass. The acetate or glass substrate should be
easily mountable onto the underside of the substage condenser, either through a filter holder or by
other means, such as adhesive tape. Alignment of the stop can be done by observing it through a
Bertrand lens or removing the eyepiece and viewing through a phase telescope while adjusting the
condenser centering screws.

Darkfield Microscopy at High Magnifications

For more precise work and blacker backgrounds, you may choose a condenser designed especially
for darkfield, i.e. to transmit only oblique rays. There are several varieties: "dry" darkfield condensers
with air between the top of the condenser and the underside of the slide--and immersion darkfield
condensers which require the use of a drop of immersion oil (some are designed to use water
instead) establishing contact between the top of the condenser and the underside of the specimen
slide. The immersion darkfield condenser has internal mirrored surfaces and passes rays of great
obliquity and free of chromatic aberration, producing the best results and blackest background.

Perhaps the most widely used darkfield condenser is the paraboloid, consisting of a solid piece of
glass ground very accurately into the shape of a paraboloid, as illustrated in Figure 5(b). Light
incident upon the reflecting surface (between the glass and condenser housing in Figure 5(b)) of a
paraboloid condenser will be focused at the focal point of the reflector. Most paraboloid condensers
are cut to ensure that the focal point is slightly beyond the top of the condenser so that parallel light
rays will be focused at a position that maximizes illumination of the specimen. The light stop at the
bottom of the glass condenser serves to block central rays from reaching the specimen. Light rays
that are reflected by the condenser are angled higher than the critical angle of reflection and
converge at the principal focus of the condenser. The combination of a glass slide, mounting
medium, and immersion oil (between the condenser and the microscope slide) complete the optical
homogeneity of the paraboloid shape.

As discussed above, the dry darkfield condenser is useful for objectives with numerical apertures
below 0.75 (Figure 5(a)), while the paraboloid and cardioid immersion condensers (Figures 1 and
5(b)) can be used with objectives of very high numerical aperture (up to 1.4). Objectives with a
numerical aperture above 1.2 will require some reduction of their working aperture since their
maximum numerical aperture may exceed the numerical aperture of the condenser, thus allowing
direct light to enter the objective. For this reason, many high numerical aperture objectives designed
for use with darkfield as well as brightfield illumination are made with a built-in adjustable iris
diaphragm that acts as an aperture stop. This reduction in numerical aperture also limits the
resolving power of the objective as well as the intensity of light in the image. Specialized objectives
designed exclusively for darkfield work are produced with a maximum numerical aperture close to
the lower limit of the numerical aperture of the darkfield condenser. They do not have internal iris
diaphragms, however the lens mount diameters are adjusted so at least one internal lens has the
optimum diameter to perform as an aperture stop.

Table 2 lists several properties of the most common reflecting high numerical aperture darkfield
condensers. This table should be used as a guide when selecting condenser/objective combinations
for use with high numerical aperture darkfield applications.
High Numerical Aperture Darkfield Condenser Specifications
Hollow Cone Number of
Condenser Maximum
Numerical Reflecting Optical Corrections
Type Numerical
Aperture Surfaces

Paraboloid 1.00-1.40 0.85 1 Parabolic Achromatic

1 Spherical
Cardioid 1.20-1.30 1.05 Achromatic/Aplanatic
1 Cardioidal

1 Cardioidal
Bicentric 1.20-1.30 1.05 Aplanatic
1 Spherical

Bispheric 1.20-1.30 1.05 2 Spherical Aplanatic

1 Aspheric
Cassegrain 1.40-1.50 1.30 Aplanatic
1 Spherical

Spot Ring
1.40-1.50 1.30 2 Spherical Aplanatic

Nelson 1 Aspheric
1.30-1.45 1.20 Aplanatic
Cassegrain 1 Spherical

Table 2

The condensers illustrated in Figure 5 are designed specifically to produce oblique hollow light
cones of high numerical aperture for darkfield illumination. In both instances, the upper surface of
the condenser is planar and perpendicular to the optical axis of the microscope. The condenser on
the left (Figure 5(a)) is designed to be used "dry" with no oil between the condenser and the
underside of the microscope slide. In contrast, the paraboloid condenser in Figure 5(b) is intended to
be "oiled" to the bottom of the microscope slide, directly underneath the specimen. Omission of
immersion oil when using this condenser (or any of the other condensers listed in Table 2) will
preclude any light from reaching the specimen. The oblique hollow cone of light rays emitted by
these condensers cannot emerge from the top lens without oil and will be totally reflected back into
the condenser. Light emitted from the illumination source is reflected at the mirrored glass surfaces
within the interior of the condensers and exits the top of the condensers at much higher angles of
inclination than the critical angle (approximately 41 degrees) at which total reflection occurs for
passage of light from glass to air. In the situation of the oiled paraboloid condenser (Figure 5(b) and
the condensers in Table 2) where the refractive index of the condenser glass, immersion oil, and
glass slide are equal, light emitted from the condenser passes through the specimen unrefracted by
glass-air interfaces.

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Hollow Light Cone Numerical Aperture

Use this tutorial to visualize how the hollow cone of light changes
with numerical aperture in reflecting darkfield

Reflecting high numerical aperture condensers listed in Table 2 cover a wide range of designs used
to produce the oblique hollow cone of light necessary for high-magnification darkfield microscopy.
The paraboloid darkfield condenser has been discussed in detail above. Another very useful design
is the cardioid condenser that is illustrated in Figure 1. This condenser design utilizes a mirrored
hemisphere in the center of the condenser that serves as both a light stop and a reflector to direct
light onto a second reflecting surface shaped to resemble a cardioid of revolution, from which the
condenser derives its name. The combination of spherical and cardioid reflecting surfaces produces
a condenser that is free from coma and both spherical and chromatic aberration. There are several
technical drawbacks to using a condenser of such high numerical aperture. The cardioid condenser
is very sensitive to alignment and must be carefully positioned to take advantage of the very sharp
cone of illumination, making it the most difficult darkfield condenser to use. In addition, the
condenser produces a significant amount of glare, even from the most minute dust particles, and the
short focal length may result in poor illumination on objects that exceed a few microns in size or
thickness. When choosing microscope slides for quantitative high-magnification darkfield
microscopy, make certain to select slides made from a glass mixture that is free of fluorescent

High numerical aperture reflecting condensers (Figures 1, 5, 6 and Table 2) with darkfield
illumination provide the method of choice for observing and photographing collections of very small
particles or colloidal suspensions, even when the particle diameter is significantly lower than the limit
of resolution for the objective. This is due to light diffracted by the particles, which passes through
the objective and becomes visible as bright diffraction disks. Each particle is visible as a minute
diffraction disk, provided the lateral distance between adjacent particles is greater than the limit of
resolving power of the objective. As illumination intensity is increased, the optical difference between
minute diffracting particles and their background increases. Simultaneously, even smaller particles
(detectable solely by their ability to scatter light) now diffract enough light to become visible and
suspended particles can be seen even when their diameters are smaller than 40 nanometers, which
is about one-fifth the 200 nanometer resolution limit with oil immersion objectives of the highest
numerical aperture. In biological applications, the movements of living bacterial flagella that average
about 20 nanometers in diameter (too small to be seen in brightfield or DIC illumination) can be
observed and photographed using high numerical aperture darkfield condensers.

Careful attention should be paid to the details of oiling a high numerical aperture condenser to the
bottom of the specimen slide. It is very difficult to avoid introduction of tiny air bubbles into the area
between the condenser top lens and the bottom of the microscope slide, and this technique should
be practiced to perfection. Air bubbles will cause image flare and distortion, leading to a loss of
contrast and overall image degradation. Problems are also encountered when using microscope
slides that are either too thick or too thin. Many darkfield condensers contain the range of usable
slide thickness inscribed directly on the condenser mount. If the slide is too thick, it is often difficult to
focus the condenser without resorting to a higher viscosity immersion oil. On the other hand, slides
that are too thin have a tendency to break the oil bond between the condenser and the slide. It is a
good idea to purchase precision microscope slides of the correct thickness to avoid any of the
problems mentioned above.

Interactive Java Tutorial

Darkfield Condenser Adjustment

Explore how alignment and configuration of a darkfield condenser

affects image quality.  

A unique situation arises when specimens immersed in aqueous medium are being imaged using a
high numerical aperture darkfield condenser. Under these conditions the refractive index of the
aqueous solution limits the angle of inclination under which light can pass from the glass microscope
slide (n = 1.515) into the water (n = 1.336) surrounding the specimen. The maximum numerical
aperture of light passing from glass to water is given by the following equation:

NA (illumination) = 1.555 × sin(i) = 1.336 × sin(90°)

and because sin(90°) = 1
NA (illumination) = 1.336

Even though reflecting darkfield condensers designed for oil immersion are listed with upper limits of
numerical aperture as high as 1.50 (see Table 2), light contributing to the illumination of specimens
in aqueous media must have a numerical aperture no greater than 1.336, reducing the effective
upper limit of darkfield illumination. In the case of specimens immersed in liquids of higher refractive
index, the effective upper limit of the numerical aperture of darkfield illumination can approach a
maximum of 1.50, although this is difficult to achieve in practice.

High numerical aperture condensers, whether intended for use dry or with oil, must be accurately
centered in the optical path of the microscope to realize optimum performance. To achieve this,
many darkfield condensers are built with a small circle engraved onto the upper surface to aid in
centering the condenser. Centering is performed with a low power (10x-20x) objective by imaging
the engraved circle and using the condenser centering screws to ensure the circle (and condenser)
are correctly centered in the optical path. For more detailed information about microscope alignment
for darkfield illumination, consult our section on darkfield microscope configuration elsewhere in
the microscopy primer.

In general, objects imaged under proper conditions of darkfield illumination are quite spectacular to
see (e.g. try a drop of fresh blood in darkfield). Often specimens containing very low inherent
contrast in brightfield microscopy shine brilliantly in darkfield. Such illumination is best for revealing
outlines, edges, boundaries, and refractive index gradients. Unfortunately, darkfield illumination is
less useful in revealing internal details.

Other types of specimens, including many that are stained, also respond well to illumination under
darkfield conditions. Figure 7 illustrates darkfield photomicrographs of three types of specimen, all of
which produce good contrast in both brightfield and darkfield illumination. Details in the body of the
deer tick (Ixodes demmini) shown in Figure 7(a) can be washed out in brightfield, unless the
condenser aperture is stopped down to maximize contrast. However, in darkfield, most of the
specimen detail in the tick becomes visible and can be easily captured on film. The heavily stained
helminth trematode (Echinostoma revolutum, Figure 7(b)) also reveals considerably more detail
when illuminated under darkfield conditions, as does the silkworm trachea and spiracle illustrated in
Figure 7(c). In addition to the examples presented above, a number of other specimens can also be
viewed and photographed under both brightfield and darkfield illumination to achieve the desired

During the first half of the twentieth century, darkfield microscopy had a very strong following and
much effort was expended in optimizing darkfield condensers and illuminators. This intense interest
slowly began to fade with the emergence of more advanced contrasting-enhancing techniques such
as phase contrast, differential interference contrast, and Hoffman modulation contrast. Recently, a
renewed interest in transmitted darkfield microscopy has arisen due to its advantages when used in
combination with fluorescence microscopy.

Darkfield microscopy is still an excellent tool for both biological and medical investigations. It can be
effectively used at high magnifications to photograph living bacteria, or at low magnifications to view
and photograph cells, tissues, and whole mounts. Marine biologists continue to use darkfield
illumination at low powers to observe and record data about fresh and salt water organisms such as
algae and plankton.

Rheinberg Illumination

A variation of Darkfield Illumination was invented by Julius Rheinberg in 1896, and

is called Rheinberg Illumination. It uses colored filters in place of the DF stop.

Rheinberg illumination uses a Color Darkfield Stop to illuminate the sample with two different colors of light. The
central area, where the DF stop would be, is one color (e.g., green) and the outer ring (annulus) a contrasting color
(e.g., red). Unmodified light (light that does not impinge on the sample) fills the background with uniform light the
color of the central circle. Modified light (light that impinges on the sample and is refracted) is the color of the outer
annulus. In this example, the sample would be red on a green background.

Rheinberg stops can be almost any color combination.

You can make your own Rheinberg color stop using a color laser printer and
transparency film. The stop will be the diameter of the condenser filter holder. The
diameter of the central circle will be the diameter of the field of view for any given
objective. Adjust the condenser for Köhler illumination, remove an eyepiece, open the
Condenser Iris until it just fills the objective (seen through the eyepiece tube),
measure the diameter of the condenser aperture.
Rheinberg Illumination

Rheinberg illumination, a form of optical staining, was initially demonstrated by the British
microscopist Julius Rheinberg to the Royal Microscopical Society and the Quekett Club (England)
over a hundred years ago. This technique is a striking variation of low to medium power darkfield
illumination using colored gelatin or glass filters to provide rich color to both the specimen and

The Rheinberg technique can be compared to the more familiar darkfield illumination. In darkfield
microscopy, the substage condenser is arranged so that the rays of light from the lamp, coming
through the condenser, will pass through the specimen only at very oblique angles. The central area
of the cone of light traversing the condenser is occluded by means of an opaque stop, large enough
in diameter so as to prevent light from directly entering the microscope objective. In more
sophisticated darkfield condensers--paraboloid, cardioid, Cassegrain, or Leuchtbild--the occlusion of
the direct light and the utilization of oblique rays only are achieved by use of specially designed
mirror surfaces.

Interactive Java Tutorial

Rheinberg Illumination

Experiment with color variations in central and annular Rheinberg

illumination filters.  

In darkfield illumination, the objective's numerical aperture is chosen (or reduced via an objective iris
or funnel stop) so that light from the condenser cannot enter the objective directly. The only light that
does enter the objective is light reflected, refracted, or diffracted by the specimen when the oblique
rays from the condenser "strike" the specimen. The specimen then appears bright on an otherwise
black field; hence the name darkfield illumination. The stark contrast of a bright object on a black
field increases the visibility of already-resolved detail. A darkfield condenser can be approximated
with the illustration in Figure 1 when the green central filter is substituted with an opaque stop, and
the red oblique illumination filter is removed, allowing unfiltered white light to pass through.
In Rheinberg illumination (illustrated in Figure 1), which is related to darkfield, several kinds and
shapes of filters are used. The oblique or outer light rays coming through a wide-open bright-field
condenser pass through an annular (doughnut-shaped) filter of one or more colors (shown as the
red filter in Figure 1 and the annular filters in Figure 2); the central rays of light pass through another
spot-shaped filter fitting into the circular opening of the annular-shaped filter as shown for several
combinations in Figure 2. The objective is used at full aperture. In this particular case, the specimen
would appear as either red on a green background (Figure 2(a)), yellow on a blue background
(Figure 2(b)), or green on a red background (Figure 2(c)).

The Rheinberg filters are prepared, or manufactured, in the form of transparent, annular colored
rings with a circular opening in the center as depicted in Figure 3. In commercial versions of these
filters, an accompanying set of transparent colored spots or "central filters" is made to fit snugly into
the openings of the annular rings (Figure 4). The diameter of the central filter can be varied (to
modulate background illumination intensity) by increasing the size of the narrow black rings that
separate the filter from the outer ring (Figure 4(a-c)). Effective combinations include: red ring, violet
central filter; yellow-orange ring, blue central filter, and so forth. These combinations can be explored
using ourRheinberg Illumination Java Tutorial to adjust both the annular ring and central filter
colors. Other visually effective images are produced by using a clear uncolored ring with a colored
central filter. For example, a clear ring with a red central filter will produce white or colorless images
on a red background.
In effect, the outer ring "becomes" the color of the specimen and the central filter "becomes" the
color of the background. The outer ring can also be divided into alternating sectors of color as
illustrated in Figure 5. The sector filters are especially effective in the study of warp-proof fabrics,
crystal faces, diatoms, and wood sections where the length-width dimensions are displayed in
contrasting colors.

For years, some microscope manufacturers sold Rheinberg filters in colored gels (similar to Kodak
Wratten filters) that were precut to fit the substage filter ring of their microscopes. The annular rings
had outside diameters of 31-35 millimeters and central filters usually had a diameter of 15-18
millimeters for use with a 10x - 0.25 NA objective. These filters can also be readily fabricated in the
laboratory using either Kodak Wratten filters or colored filters that are widely available at scientific
supply houses and optical component distributors. Gelatin filters can be prepared by cutting the
central filter with a cork-borer of the proper diameter, and the annular rings can be prepared by
carefully drawing a circle on the filter paper and cutting it out with a pair of scissors. In our
laboratory, the support machinists have constructed a brass die that cuts annular filter circles that
are the same diameter as our Rheinberg filter holder. This die can quickly stamp very smooth circles
from sheets of acetate or gelatin colored filters. The center of the annular filter can be cut with the
same cork-borer used to prepare the central filter.

Interactive Java Tutorial

Darkfield Cardioid Condensers

Explore how a darkfield condenser works when the specimen

scatters light into the objective.  

Sometimes, it is difficult to provide a solid housing for the central filter within the annular filter when
these are both made of very thin acetate or gelatin filters. In this case, you can just tape the central
filter over the center of the annular filter with double-sided tape, but remember that the central filter
and annular filter colors will add, so use a very dark filter for the central filter. The central filter, in
general, should be much darker than the annular filter to allow the specimen highlights to be in sharp
contrast to the background. We often place two or three pieces of the same color central filters in a
stack to adjust the transmittance of light through the central filter. Rheinberg filters can also be made
using glass filters and this is very convenient when the substage condenser is fitted with a housing
for these filters. In this case, it is best to simply tape the central filter onto the center of the glass
filter. For those interested in making their own filters, a more complete discussion is presented in
Needham's Practical Use of the Microscope, listed in our bibliography.
We have compared brightfield, darkfield, and Rheinberg illumination techniques using
photomicrographs of a Deer tick (Ixodes dammini) in Figure 6. The first photomicrograph (a)
illustrates the tick under brightfield illumination. The image is lacking in contrast and many details are
hard to resolve. Figure 6(b) shows the same tick under darkfield illumination, where more contrast
and detail are present and many of the features on the tick are apparent. Rheinberg illumination
(Figure 6(c)) of the tick using a blue central filter and a yellow annular ring (see Figure 2(b)) cause
an increased contrast effect, similar to darkfield, but with a pleasant blue background. In this
example, the darkfield and Rheinberg illumination techniques yield remarkably better
photomicrographs than does brightfield.

During the late 1930s, Carl Zeiss manufactured a special condenser, the Mikropolychromar,
designed to produce beautiful Rheinberg images. This condenser is long out of production and is
now virtually unobtainable, but it consisted of an aplanatic condenser under which there were three
separately controlled diaphragms. The outermost diaphragm controlled the diameter of the field, and
two smaller diaphragms controlled the light passing through the central disk. Annular transparent
rings of various colors were part of the set. Accompanying these was a set of transparent glass
central disks which fitted neatly into the central opening of the annular ring. This ingenious
condenser has been used by one of the authors (M. Abramowitz) to produce striking
photomicrographs at various magnifications, some of which have appeared in the
publications Omni, Time-Life, Scientific American, and National Wildlife.

Rheinberg illumination is suitable for objectives ranging from 2x to 100x. However, in order to clearly
separate the inner and outer colors, opaque metal or paper rings should be placed around the
central filter. For example, the opaque rings might have an outside diameter of 15-18 millimeters for
a 10x objective, and an outside diameter of 22 millimeters might be best for a 60x oil immersion
objective. It is a good idea to experiment using central filters like the ones illustrated in Figure 4 to
achieve the best results with Rheinberg illumination.

For studying fibers, protozoa, textiles, insects, wood sections, crystal, or other unstained low-
contrast subjects, microscopists should consider adding Rheinberg illumination to their library of
contrast techniques. It will be possible to view and photograph specimens yielding images that are
visually enhanced, as well as aesthetically beautiful.

One final note from the authors: It is strongly suggested to the microscope manufacturers and/or
independent accessory manufacturers that they produce Rheinberg condensers and filters for use
by microscopists. The necessary components would be very inexpensive to produce and would
greatly aid microscopists in setting up their microscopes for Rheinberg Illumination.

Rheinberg Illumination
Savile Bradbury has contributed this easy way into the fascinating Rheinberg
Rheinberg illumination has three advantages:

 It’s informative
 It’s attractive
 It’s cheap!
Read below on how this picture was made.

Related to dark-ground illumination, which was described on a previous

Club Web page, is the technique of illumination which was invented by
Julius Rheinberg and first published in the Journal of the Royal
Microscopical Society in 1896. In its simplest form, all the direct light
entering the objective is of one colour whilst the light diffracted or
scattered by the object itself has a different, contrasting colour. This
gives very dramatic and colourful effects, and is equally easy to set up
for almost any microscope. It works well with objectives up to about a
magnification of ×40 and and a numerical aperture of c. 0.65. With more
effort, the technique can be made to work with even higher aperture
objectives. In its simplest form, Rheinberg uses a stop of two different
colours which is fitted into the front focal plane of the condenser.
Usually, the filter holder is located sufficiently close to this plane to
serve well. The coloured stop has a central disc of the dark colour, say
blue or very deep red, which is intended to form the background. This
disc should be truly centred and of such a size that it colours all the direct light which enters the objective. Its size may be found
by focusing the objective on a slide with the substage condenser diaphragm wide open. Remove the microscope eyepiece and
look at the back focal plane of the objective, which will be entirely filled with light. Close the condenser iris diaphragm until its
edge can just be seen encroaching at the edge of the objective.
A phase-contrast centring telescope helps greatly with this, as it magnifies the image of the objective’s back focal plane.
Alternatively, the image of the back focal plane of the objective may be observed in the exit pupil (Ramsden circle) of the
eyepiece. To see it clearly here, however, a magnifier is essential. When the condenser diaphragm has been adjusted, remove the
condenser and measure the diameter of the opening in the condenser diaphragm.

This dimension (plus a little extra to allow for errors in the making and fixing of the stop) is the
diameter required for the central coloured stop. A disc of this size may be cut out of coloured
gelatine and stuck centrally onto a disc of glass or plastic cut to fit in the filter holder. If you are
lucky enough to obtain a book of little samples of coloured gelatins as supplied by firms such as
Lee Filters, who specialise in making gelatins for theatrical lighting, you are fortunate indeed!

Having fixed the central disc, the remainder of the outer part may be filled in with the secondary,
lighter colour. The appearance of such a disc is shown in on the left
If you intend examining objects which have a pronounced structure at right angles (as, for
example, in a longitudinal section of wood, as shown above) then a very pleasing result is
obtained with an alternative type of stop, which I call a ‘quadrant stop’.

This is made as follows. Find the diameter of the circle which would supply all the direct light for
objective you wish to use (using a phase telescope as described above) but instead of using a
colour for this make it absolutely opaque with stuck-on black paper or by spinning a disc of black
enamel onto the base disc.

After this is hard or has dried then divide the outer part of the disc into four quadrants and fix two colours of gelatine to these.
Opposite segments are of the same colour and lighter colours serve best here. Yellow or orange and green or light blue make
good pairings. When completed, the disc should look like Fig 2.


Phase Contrast

Phase Contrast Microscope

Application in Microscopy; Advantages and Disadvantages

The Phase Contrast Microscope opened up an entire new world in the field of microscopy.

Up until this discovery, scientists were limited to bright field

illumination and did not have the ability to view live

Now, phase contrast observation is a standard feature on

almost all modern microscopes.

To the right is a view through the eyepiece of a standard

phase contrast microscope showing a hemocytometer with

History and Background Information

Frits Zernike, a Dutch physicist and mathematician, built the first phase contrast
microscope in 1938.

It took some time before the scientific community recognized the potential of Zernike’s
discovery; he won the Nobel Prize in 1953 and the German-based company Zeiss began
manufacturing his phase contrast microscope during World War II.
Zernike experimented with the speed of the light path directed onto a specimen; he
discovered interference patterns resulting in the image appearing darker or lighter.

He developed a system using annuli or rings, placed in the lens and beneath the lower
condenser of a compound microscope, to cause interference in light patterns. Zernike
manipulated the rings and light source, ultimately reducing the light wavelength by a ½
phase. Each magnification setting, whether 10x or 100x, needs an analogous annulus ring in
the light condenser.

The quality of observation, specifically the contrast and resolution of a specimen or particle
(P), is contingent on the relationship between the surround (S) waves (also known as un-
diffracted or zeroth-order) and diffracted (D) spherical wavefronts. The primary equation
behind phase contrast is P=S+D.

This process enabled him to separate zeroth-order (S) from diffracted light (D), increasing
the definition and visibility of images/particles (P).

In essence, Zernike’s approach and adjustments to illumination changed the way we use
microscopes and view specimens.

Phase Objects
Phase contrast is most useful in observing transparent, colorless and/or unstained
specimens referred to as “phase objects.”

Since these objects do not absorb light, they could not be seen with any detail before
Zernike’s discovery.

The new system took advantage of both direct and diffracted light to increase the quality
and definition of transparent samples.

Most importantly, many phase objects are living biological samples; the potential
implications and uses of phase contrast within the world of microscopy should not be

Today, two main types of phase contrast are positive and negative. Since the observed
particles are usually thin and transparent, these polar contrasts provide strikingly different

Positive phase contrast reveals medium to dark gray images on a lighter grey

background; these images often have a bright halo along the edge of the sample.

Negative phase contrast is the opposite. The specimen appears lighter with a dark
background; they also have a dark halo outlining the image.

Applications in Microscopy
The possible applications of Zernike’s phase contrast microscope in microscopy are evident
in the fields of molecular and cellular biology, microbiology and medical research.
Specimens that can be observed and studied include live microorganisms such as protozoa,
erythrocytes, bacteria, molds and sperm, thin tissue slices, lithographic patterns, fibers,
glass fragments and sub-cellular particles such as nuclei and organelles.

The advantages of the phase contrast microscope include:

 The capacity to observe living cells and, as such, the ability to examine cells in a
natural state
 Observing a living organism in its natural state and/or environment can provide far
more information than specimens that need to be killed, fixed or stain to view under a
 High-contrast, high-resolution images
 Ideal for studying and interpreting thin specimens
 Ability to combine with other means of observation, such as fluorescence
 Modern phase contrast microscopes, with CCD or CMOS computer devices, can
capture photo and/or video images
In addition, advances to the phase contrast microscope, especially those that incorporate
technology, enable a scientist to hone in on minute internal structures of a particle and can
even detect a mere small number of protein molecules.

Disadvantages and limitations of phase contrast:

 Annuli or rings limit the aperture to some extent, which decreases resolution
 This method of observation is not ideal for thick organisms or particles
 Thick specimens can appear distorted
 Images may appear grey or green, if white or green lights are used, respectively,
resulting in poor photomicrography
 Shade-off and halo effect, referred to a phase artifacts
 Shade-off occurs with larger particles, results in a steady reduction of contrast
moving from the center of the object toward its edges
 Halo effect, where images are often surrounded by bright areas, which obscure
details along the perimeter of the specimen
Modern advances and techniques provide solutions to some of these confines, such as the
halo effect.

Apodized phase contrast utilizes amplitude filters that contain neutral density films to
minimize the halo effect. Essentially, this is attempting to reverse the definition achieved
through phase contrast annuli, but the halo effect can never be eliminated completely.
The pros that phase contrast has brought to the field of microscopy far exceed its
limitations. This is easily seen with the myriad of advances in the fields of cellular and
microbiology as well as in medical and veterinary sciences.

The phase contrast microscope opened up an entire world of microscopy, providing
incredible definition and clarity of particles never seen before.

These transparent specimens could not be explored because they do not have the capacity
to absorb light.

Zernike found a way to manipulate light paths through the use of strategically placed rings
and his system is a staple of most modern microscopes.

Although there are a few disadvantages, such as shade-off and halo distortions, phase
contrast provides highly detailed, well-contrasted images.

The most important breakthrough is the ability to observe living particles in a natural state.


Phase Contrast Microscopy

Most of the detail of living cells is undetectable in bright field microscopy because
there is too little contrast between structures with similar transparency and there is
insufficient natural pigmentation. However the various organelles show wide variation
in refractive index, that is, the tendency of the materials to bend light, providing an
opportunity to distinguish them.

A culture of Amoeba proteus or a fresh suspension of Nagleria gruberi make good

practice specimens.


Highly refractive structures bend light to a much greater angle than do structures of
low refractive index. The same properties that cause the light to bend also delay the
passage of light by a quarter of a wavelength or so. In a light microscope in bright
field mode, light from highly refractive structures bends farther away from the center
of the lens than light from less refractive structures and arrives about a quarter of a
wavelength out of phase.
Light from most objects passes through the center of the lens as well as to the
periphery. Now if the light from an object to the edges of the objective lens is retarded
a half wavelength and the light to the center is not retarded at all, then the light rays
are out of phase by a half wavelength. They cancel each other when the objective lens
brings the image into focus. A reduction in brightness of the object is observed. The
degree of reduction in brightness depends on the refractive index of the object.

Applications for phase contrast microscopy

Phase contrast is preferable to bright field microscopy when high magnifications

(400x, 1000x) are needed and the specimen is colorless or the details so fine that color
does not show up well. Cilia and flagella, for example, are nearly invisible in bright
field but show up in sharp contrast in phase contrast. Amoebae look like vague
outlines in bright field, but show a great deal of detail in phase. Most living
microscopic organisms are much more obvious in phase contrast.

Figure. (a) organelles are nearly invisible in bright field although they have different
refractive indexes; (b) light is bent and retarded more by objects with a high refractive
index; (c) in phase contrast a phase plate is placed in the light path. Barely refracted
light passes through the center of the plate and is not retarded. Highly refracted light
passes through the plate farther from center and is held back another one quarter
wavelength.; (d) The microscope field shows a darker background (in this case the
cell cytoplasm has a higher refractive index than the contractile vacuole), with the
organelles in sharp contrast.
Using phase contrast

Phase contrast condensers and objective lenses add considerable cost to a microscope,
and so phase contrast is often not used in teaching labs except perhaps in classes in the
health professions and in some university undergraduate programs. This is unfortunate
since the images obtainable in phase contrast mode can be very dramatic.

To use phase contrast the light path must be aligned. An element in the condenser is
aligned with an element in a specialized phase contrast lens. This usually involves
sliding a component into the light path or rotating a condenser turret. The elements are
either lined up in a fixed position or are adjusted by the observer until the phase effect
is optimized. Generally, more light is needed for phase contrast than for
corresponding bright field viewing, since the technique is based on a diminishment of
brightness of most objects.