The Principles of Clinical Cytogenetics - Extra Materials - Springer

Fundamentals of Microscopy 83
Fig. 2. Cut-away view depicting the light path of a brightfield microscope. (Reprinted with permission of
Olympus America, Inc.)
A variety of filters are available for improving the image contrast of cytogenetic specimens. “Green
glass” filters are the least expensive option and will improve the contrast range of G-banded chromosomes.
Green interference filters absorb all but a single wavelength to a narrow band of green wavelengths,
but are more expensive. Because the optics of some microscopes rely upon the use of
monochromatic green light to produce quality images, investment in a green interference filter may
be required. Interference filters are easily identified by their partial reflective quality and unusual tint
(often orange) when viewed at an angle.
Field Diaphragm, Condenser, and Condenser (Aperture) Diaphragm
The field diaphragm, condenser, and condenser (aperture) diaphragm gather and focus the microscope
light, passing it through the specimen and into the objective lens. These components play an
important role in image contrast and resolution.
As light passes through a specimen, the light rays bend or diffract from their original path. It is
important to understand that the smaller structures of a specimen diffract light to a greater degree
relative to the diffraction of larger structures. To obtain well-resolved images, a microscope must
gather as many of these highly diffracted light rays as possible for viewing (1).
The process of seeing an image begins with the field diaphragm, which is used to control the
area of specimen illumination. From here, light passes into the condenser, through the specimen,
and into the small opening (aperture) of the objective lens. The path of light through a brightfield
microscope is depicted in Fig. 2. The numerical apertures (NAs) of the condenser and objective are