FOLDER 1.4 Proper Use of the Light Microscope continued next page This brief introduction to the proper use of the light micro-scope is directed to those students who will use the micro-scope 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 advan-tage. Despite the availability of today’s fine equipment, rel-atively 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 appropri-ate illumination in your microscope. The alignment steps necessary to achieve good Kler 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 cir-cular field that has a radius directly proportional to the numeric aperture of the objective. As you close the con-denser diaphragm, its outline will appear in this circular field. For most stained materials, set the condenser di-aphragm to cover approximately two thirds of the objec-tive 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). FIGURE F1.4.1 • Diagram of a typical light microscope. This drawing shows a cross-sectional view of the microscope, its operating components, and light path. (Courtesy of Carl Zeiss, Inc., Thornwood, NY.) eyepiece final image exit pupil (eyepoint) real interme-diate image exit pupil of objective specimen condenser diaphragm field diaphragm light source focusing control light source tube objective auxiliary condenser lens stage condenser diaphragm condenser stage control field diaphragm KÖHLER ILLUMINATION THROUGH THE MICROSCOPE IMAGING BEAM PATH ILLUMINATING BEAM PATH the ﬂuorescence in situ hybridization (FISH) procedure, is extensively used in the clinic for genetic testing. For example, a probe hybridized to metaphase chromosomes can be used to identify the chromosomal position of a gene.
The FISH procedure is used to simultaneously examine chromosomes, gene expression, and the distribution of gene products such as pathologic or abnormal proteins. Many specific ﬂuorescent probes are now commercially available and are used clinically in screening procedures for cervical cancer or for the detection of HIV-infected cells. The FISH procedure can also be used to examine chromosomes from the lymphocytes of astronauts to estimate the radiation dose absorbed by them during their stay in space. The frequency of chromosome translocations in lymphocytes is proportional to the absorbed radiation dose.
Autoradiography makes use of a photographic emulsion placed over a tissue section to localize radioactive material within tissues.