The Principles of Clinical Cytogenetics - Extra Materials - Springer

88 Christopher McAleer
High-dry objectives typically have a very low tolerance to slide thickness (4). In addition, when a
high-dry lens is used on a cover-slipped specimen, a correction collar should also be present to allow
compensation for the additional thickness generated by the mounting medium and cover slip.
Infinity Correction
The symbol “∞“ engraved on an objective identifies it as an infinity-corrected lens. Infinity-corrected
objectives project parallel light rays into the microscope. These rays do not come into focus until they
pass through a special tube lens, where they are brought into focus at the back lens of the microscope
eyepiece. As the tube length for the objective is not fixed, a variety of features can be added to a
microscope without significantly impacting image magnification. Infinity-corrected lenses are typically
engineered for a specific microscope and are not usually interchangeable between microscopes.
Objective-Correction Collar
Objectives with a correction collar are often designed for use with materials that have a range of
refractive properties, as well as a variation in overall specimen thickness. For brightfield microscopy,
it is very important to ensure that a correction collar is adjusted appropriately for the specimen preparation.
Typically, correction collars are labeled with a scale that should be adjusted to the appropriate
cover-slip or plating material thickness. Often, the correction collar is left in a specific position for
cytogenetic analysis, as even a slightly misadjusted collar can result in a poorly resolved image.
The quality of fluorescent images can also be impacted by use of a correction collar and one is
sometimes used for controlling image contrast. Again, it is important to realize that the collar has been
designed to allow optimization of the resolving capacity of a lens for the plating materials and overall
thickness of the specimen. Although closing a collar might improve contrast, it might also lower the
resolving capacity of the lens (see the Fluorescence Microscopy section for additional information).
Optivar Lenses and Magnification Changers
A microscope could have additional lenses that increase the total magnification of the image. A
magnification changer or Optivar lens can appear as a rotating control or a sliding bar located between
the objective lenses and the eyepieces. Optivar lenses allow microscopes using a mid-range objective
(×63) with a high NA to increase the size of an image without the loss of resolution.
Use of Optivar lenses to increase the magnification of an image beyond that provided by the
objective should be done so with care, as image magnification that exceeds the resolving capacity of
a microscope will result in “empty magnification.”
Eyepieces
Microscope eyepieces increase the magnification of the microscope image and position the image
so that it can be seen by each eye. Eyepieces may also be engineered with a variety of features,
including those that correct chromatic aberrations (C, K), those that provide a wide field of view
(WF), and those that allow viewing from a greater than standard distance (H) (thus allowing the
microscopist to wear corrective eyeglasses) (5). Most eyepieces are adjustable so that the focus characteristics
of each eye can be optimized for the individual viewing an image. This allows the images
at each eyepiece to be brought into simultaneous focus, permitting individuals with vision deficits to
be able to use the microscope without the need for corrective lenses. Finally, cross-hairs may also be
present in an eyepiece to provide an indication of the image focus at the microscope compared to the
image focus at a photographic camera.
Beam Splitter
A beam splitter is present on microscopes capable of photomicrography or electronic image capture.
A beam splitter allows the light to be diverted between the eyes and the photographic port at
various relative intensities.