In light microscopy, phase contrast is widely used for examinations of transparent unstained specimens. To achieve phase contrast, two components of bright-field microscopes have to be modified: 1. The
condenser has to be equipped with a ring-shaped aperture or mask (the condenser annulus), which is placed near the condenser aperture diaphragm. 2. A conjugate phase plate (or ring) is placed in the back focal plane
of the objective. The condenser annulus and the phase ring in the objective have to be optically aligned so that they are conjugate. With this arrangement, the specimen is illuminated by the apex of a cone of light.
The light beams which are diffracted by the specimen pass through the objective lens at various angles
dependimg on the relative refractive index and the thickness and superficial texture of the specimen.
The other light components, corresponding to the background, pass through the phase ring in the objective which produces an additional phase difference. Thus, the phase differences between the specimen, its details
and the background are amplified in the final image, so that minimal differences in refractive index are visible even in colorless specimens with low contrast and thickness. The light pathway in normal phase
contrast is shown in fig. 1.
Fig. 1: Simplified optical pathway for phase contrast microscopy
(modified from 5)
Alignment of condenser annulus (bright) and phase ring (dark)
1 = light source
2 = annular shaped light mask (condenser annulus)
3 = condenser
4 = specimen
5 = background light
6 = light bent by the specimen
7 = phase ring
8 = eyepiece with intermediate image
9 = eye
Depending on the configuration and properties of the phase ring in the objective, the natural phase shift within phase preparations (circa
λ/4) is amplified, so that the resulting final difference in phase of the specimen and the background is around one-half wavelength (λ/2,
positive phase contrast) or one wavelength (λ, negative phase contrast). In positive phase contrast, the specimen is visible with medium
or dark grey features, surrounded by a bright halo, and the background is of higher intensity than the specimen. In negative phase contrast, the contrast of these features is inverted.
In standard techniques, phase contrast is affected by several optical limitations which can be regarded as characteristic attributes: In
common phase contrast, the condenser aperture iris diaphragm (if fitted) has to be wide open to allow the transmission of light through
the condenser annulus. Therefore, the depth of field is lower than in bright-field illumination carried out in the usual mode when the
condenser aperture iris diaphragm is adequately closed; the smaller the condenser aperture the higher the resulting depth of field will be.
Moreover, in phase contrast images, the resulting contrast is determined solely by the optical design of the phase ring within the
objective and the optical density of the specimen and its surrounding medium. Since phase contrast is usually optimized for observations
of native cells in their natural environment and calculated for an amplification of λ/4-phase-shifts, the quality of traditional phase contrast
images will be reduced the more the natural phase difference deviates from λ/4. On the other hand, contrast and contour sharpness in
bright-field images can be influenced by the aperture diaphragm as well; they will also be intensified when the aperture diaphragm is
closed. Furthermore, phase contrast is associated with marginal haloing, especially in structures containing steep gradients in refractive
index leading to high phase differences such as those occurring at cell membranes, the edges of crystallizations and other phase boundaries.
All in all, the grade of contrast, the sharpness of fine contours, the intensity of halo artifacts and the depth of field cannot be influenced
by the user when phase contrast examinations are carried out in their conventional mode.
Therefore, “condenser aperture reduction phase contrast” has been developed in order to achieve fundamental improvements of the
global image quality in phase contrast microscopy. In particular, this new method can be carried out on two modes: concentric and
eccentric; it promises visible enhancements of the focal depth, optimizations of the image contrast, improved contour sharpness and loss
of haloing especially in observations of three-dimensional specimens with a high local thickness.
Principles of “condenser aperture reduction phase contrast“
In light microscopes designed for standard techniques (light pathway shown in fig. 2), the phase plate with its phase annulus (ring) is mounted in the back focal plane of the objective (plane A in fig. 2).
Fig. 2: Pathway of the illuminating and imaging light in a conventional composite microscope, shift of projection planes in condenser aperture reduction phase contrast
1 = light source
2 = collector
3 = field diaphragm (for Köhler illumination)
4 = condenser aperture diaphragm
5 = condenser
6 = specimen (green arrow)
7 = objective
8 = back focal plane of the objective
9 = intermediate image
10 = eyepiece
11 = eye
A = projection plane of the condenser aperture
diaphragm in conventional illumination
B = specimen plane, projection plane of the field
diaphragm in conventional illumination
A´ = projection plane of the condenser aperture diaphragm
in condenser aperture reduction phase contrast
B´ = projection plane of the field diaphragm
in condenser aperture reduction phase contrast
3´ = position of an additional condenser diaphragm
The light annulus and the aperture diaphragm in the condenser are both projected into the same plane: the back focal plane of the
objective (A). Therefore, the aperture diaphragm must be wide open when phase contrast is carried out since the illuminating light will
be stopped immediately when the aperture diaphragm is closed. The field diaphragm necessary for Köhler illumination is projected into the specimen plane (plane B in fig. 1).
In condenser aperture reduction phase contrast, the optical
design of the condenser is modified so that the condenser aperture
diaphragm is no longer projected into the objective´s back focal plane, but into a separate plane situated circa 0.5 – 2 cm below (plane
A´ in fig. 5), i.e. into an intermediate position between the specimen plane (B) and the objective´s back focal plane (A). The field
diaphragm is no longer projected into the specimen plane (B), but shifted down into a separate plane (B´), so that it will no longer act as
a field diaphragm.. The optical size of the condenser annulus has to be adjusted to match the size of the objective phase ring by moving
the position of the condenser up and down. The optimal position of the condenser is monitored with a phase telescope to ensure that
condenser annulus and phase ring are conjugate. When the vertical position of the condenser is changed in tiny steps, the character of
the resulting phase contrast image can be modified with fine nuances. This effect seemed to be comparable with various nuances in
phase contrast achievable with an old-fashioned Heine condenser. The field diaphragm should be in a wide open position because the image can be
distorted when this diaphragm is closed.
An additional iris diaphragm can be mounted into plane 3´ separated from the aperture diaphragm. This diaphragm should be moveable in all
(horizontal and vertical) directions.
As a result of these modifications in the optical design, the illuminating light passing through the condenser annulus is no longer stopped
when the condenser aperture diaphragm is closed. In this way, the condenser iris diaphragm can work in a similar manner to bright-field
illumination, and the visible depth of field can be significantly enhanced by closing the condenser diaphragm. Moreover, the contrast of
phase images can now be regulated by the user. The lower the condenser aperture, the higher the resulting contrast will be. Importantly,
halo artifacts can be reduced in most cases when the aperture diaphragm is partially closed, and potential indistinctness caused by spherical or chromatic aberration can be mitigated
These positive effects can be enhanced furthermore if the second diaphragm situated in plane 3´ is partially closed. When this diaphragm
is moved into an off-centre position, oblique illumination will occur so that the three-dimensional relief of the specimen will be
accentuated. This relief effect seems to be comparable with the “relief phase contrast“ mode.
According to our own measurements, the visible depth of field is approximately doubled when the width of the condenser aperture
diapgragm is reduced. By closing the additional diaphragm, the depth of field can be quadrupled in concentric or ecccentric illumination.
The marginal haloing, which can be regarded as a typical artifact in most phase contrast images, can be mitigated or completely
eliminated by closing the condenser aperture iris diaphragm. Peripheral scattered light components are reduced in the same manner as
closing the usual field diaphragm. Also the contrast of the phase image can be well enhanced with the help of the aperture diaphragm.
In transparent specimens, existing three-dimensional reliefs can be visualized in an improved manner when “condenser aperture
reduction phase contrast“ is carried out in oblique illumination mode (the eccentric variant) using the second iris diaphragm in an off-centre position.
In aperture reduction phase contrast, the brightness of images is lower than in conventional phase contrast, because the area of the
illuminating light beams is more reduced (about -1.0 or -3.0 EV). Therefore, higher light intensities are necessary for observations and
photomicrographs. In the eccentric mode, the background can sometimes appear with variable brightness, as the specimen is illuminated
from one direction by oblique light beams. Similar effects are also known from interference contrast.
All relevant enhancements of image quality which are achievable by this new method are demonstrated in the figures 2 and 3.
Fig. 3: Alum crystallization, prepared without cover slip,
horizontal field width: 0.4 mm, specimen thickness: 0.018 mm
left: normal phase contrast
center: aperture reduction phase contrast, concentric mode
right: aperture reduction phase contrast, eccentric mode
Fig. 4: Leucaemia, blood smear, cover slip preparation,
aperture reduction phase contrast, eccentric mode
horizontal field width: 0.12 mm (left), 0.06 mm (right)
Piper, J.: Aperture reduction phase contrast - an attractive tool for improved imaging in phase contrast microscopy (in German)
(submitted: 05.01.2009, accepted: 21.01.2009)
Mikrokosmos 98 / 4, 249-254, 2009
Piper, J.: Condenser aperture reduction phase contrast – a new technique for improved imaging in light microscopy
(submitted 24.03.2010, accepted 09.04.2010)
Journal of Advanced Microscopy Research (JAMR)
Copyright: Joerg Piper, Bad Bertrich, Germany, 2010