USE AND CARE OF THE MICROSCOPE

Written by Jose Mascorro, Asst. Professor of Anatomy

A. Introduction:1,2 The light microscope is an instrument designed to magnify small objects. To simply magnify an object is not sufficient, as the object must be magnified with sufficient resolution, or the ability to see very fine details. Student microscopes cost less than $1500, can magnify up to 1,000 X, and are quite adequate for studying histological material. Research microscopes cost thousands of dollars, but they face magnification limitations because they are optical systems that utilize glass lenses and photons to produce the desired image. Magnification without sufficient resolution produces images that are blurred and without value, i.e., empty magnification. Conversely, the electron microscope utilizes electrons and electromagnetic fields to form images and can easily magnify a specimen by more than 100,000 X. It can be stated that the purpose of any microscope is to magnify and visualize small things as clearly as possible. For general information, refer to the section on Light Microscopy on pages 1-2 in Color Textbook of Histology by Gartner and Hiatt. The student should become familiar with the basic parts of the microscope. Light Source (illumination): Photons of visible light serve as the illumination source in an optical system. It is necessary to illuminate the specimen before an image can be formed. Condensers: The substage condenser serves to focus the light rays onto the specimen, thus making it possible for the optical system to produce an image of the specimen. Objectives: The objective lens rests immediately above the specimen and serves as the information-gathering lens of an optical system. It is the most important lens of the microscope since it forms the image. Student microscopes usually are equipped with objectives that magnify to different degrees: 4X for initial viewing at low magnification; 10X for medium magnification; 40X (dry lens) for high magnification; 100X (oil immersion) for very high magnification. The oil immersion rarely is used for the purposes of this course. Specimen Stage: The stage is a platform that serves to support and carry the specimen slide. Stage controls allow the specimen to be moved along the horizontal and vertical planes. Iris Diaphragm: Just as cameras have an iris diaphragm to vary the amount of light entering the camera and ultimately the contrast in the negative, so do microscopes. The diaphragm is used to achieve the best contrast by opening or closing it to the proper size.

Eyepieces: Each of two eyepieces serve as projection lenses that transfer the formed image to the human eye for final visualization and interpretation. The eyepieces should be set to match the interpupillary distance of the operator so that the specimen can be viewed comfortably with both eyes. B. Use of the Microscope:2,3 Lamp Illumination. Connect the microscope to a wall electrical outlet and turn the appropriate dial/switch to produce the initial illumination. The lamp illumination on/off switch is variable and is used to produce the amount of light desired. Mounting Specimen. Place a histological slide on the stage condenser, making certain that the slide cover glass faces upwards (otherwise a focused image cannot be obtained because of the thickness of the cover glass). Initial Examination, Focusing, and Adjustments. Traverse the specimen stage with appropriate controls until the specimen is located and focused. Rotate the revolving nosepiece holding the objectives to the 4X lens. Use this low setting to briefly view the specimen and make all necessary adjustments, listed below. Interpupillary Distance Adjustment. Two images are seem when viewed through the eyepieces; Make the right and left images merge into one by sliding the eyepieces in or out, depending upon your individual interpupillary distance. Eyepiece Diopter Adjustment: The two eyepieces must be individually focused; First, look into the right eyepiece (with left eye closed) and focus the image with the microscope focus knob and then look into the left eyepiece (with right eye closed) and focus the image with the adjustable diopter ring at the base of the eyepiece. Adjustment of Iris and Field Diaphragms. This iris diaphragm is designed for acquiring a good image with sufficient contrast rather than for adjusting illumination (this is done with the variable on/off switch). The diaphragm can be stopped down (closed) to a size that produces the best contrast. The field diaphragm is used to align the specimens illuminated area relative to the microscope viewfield. If this aperture is opened to widely, extraneous light will enter the viewfield, thus reducing image quality and contrast. Objective Selection. Once all adjustments are made, the specimen is ready to be viewed critically. Rotate the revolving nose piece to the increasing 4X, 10X, and 40X (dry) magnifications, stopping at each step in order to study fine specimen details.

_______________________________________________________________________ 1 Basic Histology, L. Junqueira, J. Carneiro, R. Kelley, Basic Histology, 9th ed., Appleton & Lange, Stamford, CT., pp. 3-4. 2 Introduction to Light Microscopy Page ,http://nsm.fullerton.edu/skarl/EM/Microscopy/IntroLightMicro.html 3 Biological Microscope ALPHAPHOT 2", 95(89.9C)H-E-7S, Nikon Corporation, Tokyo, Japan.