The Principles of Clinical Cytogenetics - Extra Materials - Springer

Fundamentals of Microscopy 81
INTRODUCTION
The Fundamentals of Microscopy
From: The Principles of Clinical Cytogenetics, Second Edition
Edited by: S. L. Gersen and M. B. Keagle © Humana Press Inc., Totowa, NJ
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Christopher McAleer
Microscopy is an essential technique for clinical cytogenetic analysis and is an integral part of the
cytogenetics laboratory. Computer imaging (see Chapter 7) is a newer technology that has also
become an essential component of most cytogenetics laboratories over the last decade.
There are many variables involved in obtaining good microscope images. By themselves, the individual
processes might have little impact on a specimen image, but together they can have a significant
influence on the final product. It is key to recognize that each step in microscopy tends to build
upon those before it. Achievement of the best image quality thus requires a full understanding and
application of many principles. This chapter will explore a process that many take for granted.
BRIGHTFIELD MICROSCOPY
The brightfield microscope is arguably the most important piece of equipment in the cytogenetics
laboratory. Knowledge of its component parts and their proper use is fundamental. The basic components
of a brightfield microscope are illustrated in Fig. 1.
The Transmitted Light Source and Power Supply
A transmitted light source is found in the base of a microscope, often in the rear, but occasionally
in the front of the scope. The bulb housings of many microscopes will automatically center the light
bulb, but some microscopes might require the bulb to be manually centered and focused.
The power supply for transmitted light sources is typically located within the base of a microscope,
but might be an external unit. In either instance, the intensity of the microscope light is usually
controlled by regulating the bulb voltage through a rheostat.
Even illumination and precise control of the light supply are important for good microscope images
and are critical for the proper function of computer imaging systems. Proper alignment of a bulb
and use of dispersion filters are requirements for illumination that is free of shadows. The quality and
age of a light bulb, the setting of the microscope rheostat, and the quality of the power supplied to the
microscope can quickly degrade images if they do not permit a stable supply of light.
Microscope Filters for Brightfield Microscopy
In general, the resolution of a microscope image improves with the quantity of focused light passing
from the smallest details of a specimen into the objective lens. In addition, the color of light plays
an important role in the resolving capacity of a microscope. Resolving power is the ability to visually
distinguish two separate objects that lie in close proximity. As the distance between two such objects
approaches the wavelength of light, the ability to see separate points becomes highly dependent on