The Principles of Clinical Cytogenetics - Extra Materials - Springer

Fundamentals of Microscopy 85
Immersion Oil
As light rays enter and exit a lens, they can reflect (refract) off of its outer and inner surfaces. This
occurs because the lens and air each have a different refractive property (refractive index).
Most microscopes are engineered to reduce the incidence refraction to that occurring between the
condenser and microscope slide and between the specimen and objective lens. Immersion oil can be
used to greatly reduce the remaining refraction by removing the air from these spaces, filling them
with a substance that has a nearly identical refractive index. As a result, more light rays will exit one
surface and pass into the next than would occur if air occupied these spaces, thus increasing the
resolving capacity of the microscope.
A question that is often raised is whether oiling a condenser will actually improve the image
quality to the extent that it is worth having to clean an oiled condenser. The highest operating NA of
an unoiled condenser and objective lens is slightly less than 1.0, as the refractive index of the air (1.0)
prevents a higher operating NA (3). If the NA of a condenser or objective lens is less than 1.0, no
benefit results from applying immersion oil.
Even with higher NAs, the loss might not be of practical significance. Remembering that resolving
capacity must be balanced with image contrast, the act of applying immersion oil increases the
operating numerical aperture, but also lowers image contrast. In turn, closing the aperture diaphragm
improves image contrast but also lowers the resolving capacity of the microscope. Thus, the application
of immersion oil to a condenser might only be beneficial when a specimen can be viewed at a
reasonable contrast while the aperture diaphragm of the condenser is nearly wide open.
Immersion oil comes in several types that vary in viscosity and fluorescent properties. Each is
formulated to have a refractive index of 1.5150 ± 0.0002 at 23°C (essentially the refractive index of
glass).
Non-cover-slipped microscope slides should be cleaned of immersion oil at the completion of
microscopic analysis. Immersion oil can cause fading of unprotected G-banded chromosomes if left
on the slides for prolonged periods of time.
The Microscope Stage and Coordinate Location
The microscope stage provides a flat, level surface for the microscope slide and a means of affixing
the slide to the stage. Controls on a mechanical stage allow the microscope slide to be moved
along x- and y-axes. Mechanical stages usually have a coordinate grid on each axis to precisely identify
the location of an object on the slide. The microscope stage can also be moved in an up-and-down
manner (z-axis) by using the coarse and fine focus controls.
Coordinate Location
Recording accurate coordinates is essential for documentation of cytogenetic findings. In most
instances, notation of the x and y coordinates are used for this purpose.
VERNIER GRIDS AND ENGLAND FINDERS ®
When a metaphase is to be relocated at a microscope other than that used for the original analysis,
a system of coordinate conversion between the two microscopes needs to be employed.
Microscopes of the same manufacturer and model can often have their stages aligned so that the
coordinates of one scope can be used at another. Vernier grids or England Finders ® allow for easy
conversion of coordinates between similar microscopes whose stages cannot be aligned or when the
microscopes are made by different manufacturers. This technique provides a printed grid whose value
is read at one microscope and then simply relocated at the second.
Microscope Slides, Cover Slips, and Mounting Media
The microscope slides, cover slips, and mounting media play a significant role in the contrast and
resolution of an image. Microscope slides and cover slips should be made from high-quality glass to