The optical microscope is as important to the pathologist now as it was 100 years ago (and surprisingly still similar in function and use). Choosing an instrument of the correct quality, specification, construction and support is vital for obtaining good quality images, ensuring ease and comfort of use and maintaining the microscope over the career-life of the pathologist. The pathologist should have a fundamental knowledge of:
• How to set up a microscope to obtain the best images.
• How to keep the instrument in good condition.
The aim of this chapter is to give a practical pointer as to the type of microscope to purchase, what to look for, the questions to ask the supplier, and some tips on everyday use and care.
Everything in this guide refers to the upright or compound microscope. There are other types of microscopes, such as dissecting or operating microscopes, and most of the information discussed here is applicable to these as well. However, there are specific technical differences that will not be covered. This chapter will be focused on the basic brightfield technique used for examining stained specimens on glass slides.
1.1 Choosing a Supplier
Purchasing a microscope is much like any other large capital purchase, be it a television or a car. Although different models are available, it is better to purchase from the supplier that provides the best information, seems the most knowledgeable on the product and offers the most comprehensive after-sales support. Aspects to look for when purchasing a microscope should include the following:
• Installation and training: is this provided by the supplier?
• Length and level of cover of the warranty.
• Telephone/email support: is there a specialist at the supplier’s office who can be contacted for help or an area representative who can call or visit to assist?
1.2 Parts of a Microscope
Microscope head
There are four types of microscope heads:
• Monocular.
• Binocular.
• Trinocular.
• Ergonomic.
Monocular head microscopes are very low-cost instruments and are not comfortable for long-term use. Therefore, a binocular head is considered a minimum requirement. A trinocular tube is required if there is the need to add a camera to the microscope.
The ergonomic head allows users of different height to adjust the head angle in order to achieve the most comfortable and safe viewing angle for prolonged microscope use.
Microscope stand
The microscope stand nearly always has a coaxial coarse and fine focusing mechanism. The illumination system can be integrated within the stand or located in a lamp-house at the back of the microscope. More sophisticated stands, usually designed for clinical use, have a low-position stage and focusing controls, in order to guarantee reduced hand and wrist fatigue. Some stands are also equipped with an adjustable focus height safety lock, which can be set to avoid hitting the slide into the objective, thus destroying the specimen and potentially damaging the objective lenses.
There are two mechanically different versions of microscope stands, which require two different means of setting up the components. These are differentiated based on their type of illumination and are as follows:
• Koehler illumination microscopes
These clinical/research-grade microscopes generate an extremely even illumination of the sample, ensuring that an image of the light source (e.g. halogen lamp filament or LED light) is not visible in the resulting image.
• Critical illumination microscopes
These lower-cost systems rely on frosted glass diffusers. The major problem associated with this type of microscope is the evenness of illumination, as an image of the illumination source is visible in the resulting image.
Eyepieces
The microscope is invariably supplied with a pair of 10× eyepieces (other magnifications such as 15× are rarely offered as an option). The magnification power is recorded on every eyepiece, followed by another number. The designations might look like the following:
• 10×/20
• WF (widefield) 10/22
• WF 10×/22
The number (e.g. /20) following the power of magnification of the eyepiece (e.g. 10×) represents the field-of-view number, or field number, and it designates the size of the visual field (measured in millimetres) provided by any given objective. The larger the number, the larger is the field of view of the specimen. It is not recommended to purchase a microscope with a field of view less than 20 mm; ideally, a field of view of 22 mm guarantees a comfortable image with sufficient coverage of the specimen. Some of the eyepieces may also display an image of a pair of glasses. This indicates that they are high-eyepoint, designed to be comfortable when used wearing spectacles. Some of the eyepieces allow the insertion of a measurement scale (eyepiece scale, reticule or graticule).
Objectives
There are several different types and special variants of objectives. These can be broadly classified based on their qualities as:
• Achromatic: routine quality.
• Fluorite: research/clinical grade.
• Apochromatic: highest possible quality and resolution.
The quality of an objective depends on the degree of chromatic aberration correction, difference in spherical aberration and numerical aperture.
Understanding the specifications displayed on the objective is very important to ensure the correct use of the lens. The key elements of an objective and the identification of the technical points specified on the barrel are discussed below.
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