Leica DME Microscope Use Compound microscopes will be used in this lab. Microscopes are expensive pieces of precision equipment, susceptible to damage by careless handling. Please use them with care. It is likely that you have already used microscopes before. However, we ask that follow the procedures as written here, even if they may vary from your past experience. Setting up a Microscope for Use Putting Away a Microscope 1. Remove the dust cover, fold it and put it aside. 1. Remove your slide. 2. The compound microscope is usually stored in its storage position, with the eyepieces pointed backwards toward the arm. This is not the position with which it is to be used. Gently rotate the entire binocular head into its working position. This may require that you temporarily loosen (and then retighten) a screw holding the head onto the microscope. 3. Turn off the light and allow it to cool. 3. Plug in the microscope. Make sure that the cord is positioned where it will not get in the way. 6. Make sure that the stage is clean. 4. Turn on the light source in the base. Adjust the illumination for your comfort. 8. Wrap the electrical cord. 2. Rack down the stage. 4. Clean the lenses (eyepieces and objectives) with lens paper ONLY. It is especially important to remove all oil, if the 100X oil immersion lens was used. Do not use any other paper, a handkerchief or your fingers as they could damage the lenses. 5. The lowest power objective should be in place for microscope storage. 7. Return the stage mechanism to its central position. 9. Replace the dust cover. A. Components Compound microscopes magnify thin specimens mounted on microscope slides. They are ideal for observing unicellular or very small organisms, cells, and cell structures. Below is a list of parts of a compound microscope, and the definition or function of each. Base: the bottom of the microscope, which supports the entire instrument. Illuminator or light source: the light source is usually built into the base of the microscope, and directs light through the condenser to the specimen. Alternatively, the light source may be separate, and be directed toward the condenser with a mirror. The intensity of the light can be adjusted using the rheostat (light) control knob on the left side of the base. Arm: the frame that supports all components above the base. Revolving nosepiece or turret: a revolving disc-shaped support or frame for the objective lenses. Objective lenses: the primary optical system which produces a magnified image of the specimen. There are typically four objective lenses attached to the nosepiece: a 4X scanning objective, a 10X low power objective, a 40X high power (dry) objective and a 100X oil immersion objective. The magnification of each objective is engraved on its side. Ocular lens or eyepiece: the secondary optical system that you look through. The ocular lens further magnifies (10X) the image and brings the light rays to a focal point. A binocular microscope has two ocular lenses and a monocular microscope has one ocular lens. They sit on the adjustable binocular body. 1 Leica DME Microscope Use Stage: the flat surface upon which the slide with your specimen is placed. Most microscopes have a stage finger assembly to hold the slide on the stage. The entire mechanism including the slide moves horizontally across the stationary stage (left/right and forward/back) using two stage adjustment knobs situated under the stage (variably on the left or right side, in front of the focusing knobs). be adjusted by turning it to increase or decrease the size of the hole that light passes through. Condenser: the lens located below the stage, which focuses light (from the illuminator) through the specimen being observed. Most microscopes have a moveable condenser allowing its distance from the specimen to be adjusted using the condenser knob and condenser alignment screw. Fine adjustment or fine focusing knob: the smaller knob towards the back of the instrument that is used to make small adjustments in the height of the stage for final focusing on a specimen. It is the only focusing knob used with high power objectives. Iris diaphragm: a unit below the condenser that controls the amount of light directed to the specimen. The diameter of the diaphragm can Coarse adjustment or coarse focusing knob: the large knob towards the back of the instrument that is used to significantly raise or lower the stage, when you first focus on a specimen at low power. It is never used when high power objectives are in place. Note: If the microscope does not appear to be functioning properly, do not attempt to fix it. Bring the problem to the immediate attention of your instructor. It is never good to force a part of the microscope to move. 2 Leica DME Microscope Use B. Köhler Illumination C. Calibration Köhler illumination is the alignment of the image-forming light path and the illumination light path of the microscope. In this process the condenser is centered and focused, thereby providing an evenly illuminated field of view and more importantly maximum resolution of the specimen. Microscopes must be calibrated so accurate measurements can be made. To calibrate a microscope both an ocular and a stage micrometer are used. Directions: Position focusing eyepiece to the appropriate number for your eyesight on the left eyepiece. Ocular micrometer (O) - a very small ‘ruler’ engraved on a piece of glass and placed inside one of the ocular lenses • The ruler’s magnification does not change when switching between the objective lenses. • At each objective, the value (in µm) of an ocular tick mark (division) must be determined. • Stage micrometer (S) - a very small ‘ruler’ engraved on a piece of glass then mounted on a slide • The ruler’s magnification does change when switching between the objective lenses. • The ‘ruler’ is usually 1 to 2 mm (1000 to 2000 µm) across. • Each tick mark (divisions) on the ruler is 10 µm (0.01mm) from the next tick mark. Position a stained slide on the stage. Focus the specimen using the 10x objective and comfortable illumination intensity, adjusting focusing eyepiece as needed. Close the field diaphragm (black lever below the stage and condenser) until about ¼ of the field of view is illuminated. Partially close the aperture diaphragm (on condenser). Use the condenser height adjustment knobs to position the condenser so the diaphragm image is in focus. Use the two condenser centration screws to position the condenser the diaphragm image in the center of the field of view. Fully open the field diaphragm. 0.01 mm tick mark 1000 µm 1 mm = 10 µm tick mark Directions (complete the steps below for 4X, then 10X, and finally 40X): Focus on the stage micrometer (S). Rotate the ocular lens until the ocular micrometer (O) is exactly superimposed on S. Adjust the aperture diaphragm for appropriate contrast (also dependent upon intensity of stain and thinness of sample). Adjust S so that its first tick mark lines up precisely with O’s first tick mark. Your microscope is now set to maximize resolution of the specimen. Look for another tick mark (as far from the beginning as possible) where S and O line up precisely. Note: The microscope is now set to maximize resolution of the specimen. If you adjust the condenser height to gain contrast or adjust light intensity you will sacrifice the resolution capability. Use the aperture diaphragm and /or the illumination intensity to adjust contrast. Count the number of tick marks between these two tick marks for both O and S. See examples 1 and 2 on following page. 3 Leica DME Microscope Use Example 1: Example 2: 70 tick marks for S 30 tick marks for O 10 um S = 23 um for each O tick mark (Ocular division) 4