Histology A Text and Atlas With Correlated Cell and Molecular Biology (6th Ed.) (Ross)

Histology A Text and Atlas With Correlated Cell and Molecular Biology (6th Ed.) (Ross)

This brief introduction to the proper use of the light microscope is directed to those students who will use the microscope for the routine examination of tissues. If the following comments appear elementary, it is only because most users of the microscope fail to use it to its fullest advantage. Despite the availability of today’s fine equipment, relatively little formal instruction is given on the correct use of the light microscope. Expensive and highly corrected optics perform optimally only when the illumination and observation beam paths are centered and properly adjusted. The use of proper settings and proper alignment of the optic pathway will contribute substantially to the recognition of minute details in the specimen and to the faithful display of color for the visual image and for photomicrography. Köhler illumination is one key to good microscopy and is incorporated in the design of practically all modern laboratory and research microscopes. Figure F1.4.1 shows the two light paths and all the controls for alignment on a modern laboratory microscope; it should be referred to in following the instructions given below to provide appropriate illumination in your microscope. The alignment steps necessary to achieve good Köhler illumination are few and simple: • Focus the specimen. • Close the field diaphragm. • Focus the condenser by moving it up or down until the outline of its field diaphragm appears in sharp focus. • Center the field diaphragm with the centering controls on the (condenser) substage. Then open the field diaphragm until the light beam covers the full field observed. • Remove the eyepiece (or use a centering telescope or a phase telescope accessory if available) and observe the exit pupil of the objective. You will see an illuminated circular field that has a radius directly proportional to the numeric aperture of the objective. As you close the condenser diaphragm, its outline will appear in this circular field. For most stained materials, set the condenser diaphragm to cover approximately two thirds of the objective aperture. This setting results in the best compromise between resolution and contrast (contrast simply being the intensity difference between dark and light areas in the specimen)...

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