The Principles of Clinical Cytogenetics - Extra Materials - Springer

84 Christopher McAleer
measurements of their ability to gather highly diffracted light, thus a measure of a microscope’s
resolving potential.
How the condenser is used to illuminate the specimen will influence the number of light rays
passing into an objective lens. A properly adjusted condenser will illuminate the small structures of a
specimen from many different angles. This increases the likelihood of producing light rays with an
angle of diffraction that will pass through the small opening of an objective and, thus, be present for
viewing.
Microscope condensers come in a variety of NAs, ranging from 1.4 to less than 1.0. Because the
operating NA of an objective lens cannot be greater than that of the condenser, these components
should have similar NA values.
Condensers require correction for two basic groups of optical imperfections (aberrations) that are
created as light passes through any lens. Monochromatic aberrations are those that can occur with
any wavelength of light, whereas chromatic aberrations are problems unique to a specific range of
wavelengths. A microscope is equipped with one of three of condensers: abbe, aplanatic, or aplanatic/
achromatic. These differ in their ability to correct optical aberrations. Condensers are generally labeled
with the type of optical correction they make and their NA.
Abbe condensers are the most basic type of condenser and are not corrected for either type of
optical aberration. They are not recommended for use in the cytogenetics laboratory.
Aplanatic condensers are corrected solely for monochromatic aberrations and rely upon green
light for the greatest degree of correction (2). The performance of aplanatic condensers is highest
when a monochromatic green interference filter is used.
Aplanatic/achromatic condensers are corrected for both types of optical aberration and do not
require the use of green light for the correction (2).
The aperture diaphragm of a condenser is adjusted to achieve a balance between the resolving
power of the microscope and image contrast. When the aperture diaphragm is completely open, the
small structures of a specimen are illuminated by light from the greatest number of angles and resolving
capacity is at its highest. Unfortunately, the details of these structures are so well illuminated that
they lose the “shadowing” or contrast variation that give such structures perspective.
As the aperture diaphragm is closed, the structures of the specimen are illuminated from fewer
angles, resulting in a loss of resolving power, but an improvement in the “shadowing” or contrast of
the image. Considering this, the aperture diaphragm should be set to produce a suitable balance
between image contrast and resolution. Many microscope manufacturers recommend setting the condenser
aperture between 66% and 75% open to achieve the best balance.
Köhler Illumination
Centering and focusing the condenser (Köhler illumination) are crucial for optimum image quality.
The process begins by closing the field diaphragm so that light traveling through the condenser
can be visually centered and focused. Once this is achieved, the field diaphragm is opened so that the
light illuminates the specimen just beyond the field of view. Finally, the aperture diaphragm is adjusted
to generate the desired image contrast.
The Phase-Contrast Condenser
Phase-contrast microscopes use a special condenser and objective lens to increase the contrast
range of a specimen by darkening areas of greater density and lightening areas of lesser density.
Phase contrast is often used for visualizing living or other unstained cells, but can be used successfully
to increase the contrast range of G-banded specimens. A microscope equipped for phase contrast
makes use of a special condenser and a phase objective lenses. Proper use of a phase-contrast
microscope requires achievement of Köhler illumination, followed by an adjustment to align the
phase components of the microscope. Of key importance is selection of a phase condenser setting
that matches the “Ph” number of the phase objective.