1. Always carry the scope upright.
2. Using the dissecting scope diagram, find the parts of your scope.
3. Plug in the lamp, use the outlet on the opposite side of the bench.
4. Adjust seat height. Place the dissecting scope on the bench in front of you and adjust your seat height so you are looking comfortably into the eyepieces without stretching your neck up or bending over too much.
5. Only slides can be placed directly on the stage. For fresh materials, work on a damp piece of towel paper or in a Petri dish.
6. Keep the microscope clean. Immediately wipe up all spills with a damp paper towel. Use cotton swabs and lens cleaner to clean the eyepieces.
7. Adjust thebinocular head by loosening the locking screw, turning the head, and then tightening the screw again.
8. Adjust the light.
a. For opaque objects, you must use reflected light (from above). Place lamp in one of the twin ports at the top of the arm.
b. For thin specimens which light will pass through, you can use either transmitted light (from below, port at back of base) or reflected light (from above). Then at the sides of the base, turn the knob to determine whether the reflector or mirror gives you a better view of your specimen. To get even light over your specimen, slide the swing in/out lens, at the right side of the base, to its best position. This lens is most useful when you are using lower magnifications.
c. The reostat on the lamp adjusts the light intensity.
9. Adjusting the eyepieces. Start by roughly focusing and then making the following adjustments. This is an important step to reduce fatigue and eyestrain.
a. Interocular distance: The eyepieces can be moved closer together or farther apart to accommodate the distance between your eyes, which varies from person to person.
You may also want to experiment with how close you want your eyes to the eyepieces. Sometimes it helps to move back a bit.
b. Focusing the 2 eyepieces separately: The vision in your two eyes is probably not identical.
i. Look through the right eyepiece (close left eye or simply ignore what it sees or put your hand over it). Focus with the focusing knob.
ii. Now look through the left eyepiece, closing the right eye. Focus using the focusable eyepiece tube (it should have a knurled ring which you can turn). Do not use focusing knobs.
9. Magnification changed by rotatingthe magnification knob.
10. Magnification is calculated by multiplying the magnification of the eyepieces' lenses by the increment marked on the right hand magnification knob. (e.g. 10X times 1.5X = 15X)
Additional suggestion.
When studying microscope slides, it is a good idea to examine them with the dissecting scope before using the compound scope
BH-2 AND CH-2 OLYMPUS COMPOUND MICROSCOPE
I. SETTING UP THE SCOPE
1. Carry the scope with both hands, grasp the arm with one hand and support the base with the other. Keep the scope upright at all times!! Avoid abrupt motions or any impact. Always handle the microscope with care.
2. Using the compound scope diagram, find the parts of the scope.
3. Adjust the binocular head. Loosen the clamping screw to move the head. Then gently tighten the screw so the head doesn't fall off.
4. Plug in the scope, using the outlet on the opposite side of the bench. This is so the scope doesn't accidentally get pulled off the bench.
5. Adjust seat height. Ideally, you will not be stretching your neck or bending your back excessively to look through the eyepieces.
6. Light intensity. Most of the Olympus scopes have a light switch on the base of the scope in front. Turn the light on. On the right side of the base, you will find the sliding voltage adjustment (reostate) which sets the brightness. For starters set it at low and then move it to about the middle.
7. Place specimen on the stage ONLY when the 10X (or 4X) objective are in position so that you don't ram the slide into the objective lens. The objective lenses are the mostexpensive partof the scope. These objectives are approximately parfocal so that you should be able to move from a lower to higher power objective with very little focusing. In practice it means move to a higher power objective only when the specimen is in focus with the lower power objective.
8. Focus on specimen using the 10X objective-Position area of interest on slide above path of light. Use coarse and then finefocus knobs and adjust light intensity.
9. Focus the condenser near the slide. Both the condenser and the condenser height adjustment knob are substage.
10. Adjusting the eyepieces. This is an important step to reduce fatigue and eye strain.
a. Interpupillary distance: The eyepieces slide closer together and farther apart to accommodate the distance between your eyes. Make this adjustment while looking at the tissue section. Sometimes it helps to move your head back slightly so that your eyes are further away from the eyepieces. You will have to experiment to find the best position for you.
b. Focusing eyepieces: The vision in your two eyes is probably not identical.
i. Look through the right eyepiece (close left eye or simply ignore what it sees or put your hand over it). Focus with the focusing knob.
ii. Now look through the left eyepiece, closing the right eye. Focus using the eyepiece itself (using knurled ring which you can turn).
II. BRIGHT FIELD ALIGNMENT (Koehler illumination)
1. Bright field illumination. The substage condenser has a plate which rotates displaying different numbers. The number (which shows in front) should always be in the zero position for bright field.
2A. CH-2 OLYMPUS SCOPE
a. Substage condenser. The substage condenser focuses the light on your slide. There is a knob (condenser height adjustment knob) which drives the condenser up and down. Move it so the condenser is all the way up, then back off slightly. The substage condenser should remain in this position.
2B. BH-2 OLYMPUS SCOPE
a. Adjust the condenser.
i. Close down the lamp iris diaphragm. It is at the base of the scope surrounding where the light exists. When looking through the eyepieces, you will see a lighted polygonal spot surrounded by darkness.
ii. Focus the condenser up and down (only a few mm) until the image of the polygonal lamp irisedge is sharp when viewed through the eyepieces. The sharpest point will be just when the edge changes from blue to red.
iii. Center the lamp iris using the condenser centering screws. These are the two screws, near the front, under the stage.
iv. Open the lamp iris progressively. As you widen it, also continue to center the field of view. When the polygonal image of the iris at the margin of the field of view, slightly increase the diameter until the polygonal image is just outside the field of view.
v. The amount of opening of the lamp iris will depend on which objective you use. It will be wider for low power objectives and smaller for high power ones.
3. Light adjustment.
a. Always make your initial light adjustment with the rheostat.
b. Use the condenser iris diaphragm, below the condenser, to get more contrast of objects which are not pigmented or stained. Closing down the condenser iris reduces resolution.
c. Never lower the condenser itself to get more light. The best optics are obtained with the condenser nearly all the way up.
d. In general, the light should not be too bright. Bright light produces light scatter and a fuzzy image and will tire your eyes.
4. Now switch to the 40X objective and practice adjusting the light, something you will have to do each time you change objectives. Only focus with fine focus knobs.
III. PHASE CONTRAST ALIGNMENT
Phase -contrast microscopy was invented by Zernike as a way of enhancing the very slight contrast present in specimens which are transparent but have parts which differ in their index of refraction which affects the speed at which light travels through the specimen. Precise alignment is required for proper set up.
1. Open the lamp iris all the way.
2. Swing the phase objective of your choice into the light path. Note its magnification.
3. Rotate the ring containing the phase stops (10,20, 40 and 100) to correspond with the magnification of the phase objective that you have chosen. Only objectives with a phase ring can give phase-contrast.
4. Focus on the specimen and adjust illumination with the rheostat. Do not try to close down the substage iris diaphragm. When you are using phase, a spring keeps it in the open position.
5. Your field of view should be a light silvery grey, if it isn't, let your instructor know. The phase rings in your scope may need to be recentered.
IV. OIL IMMERSION
The 100X objective is an oil immersion objective. With immersion oil, it achieves its highest resolving power.
1. With a 40X objective focus and center what you are observing on the slide.
2. Turn the objective nosepieces to a position half way between the 40X and 100X objectives.
3. Place a drop of immersion oil on top of the area to be examined. Then carefully swing the 100X objective in position over your specimen so that the front lens of the objective will be joined to the coverslip with a drop of oil.
4. Once a coverslip has immersion oil on it, you must remove the oil before using another objective. For fresh mounts, you will have to prepare another slide.
5. Clean the 100X objective lens and prepared slides with a cotton swab or lens tissue with lens cleaner.
V. CLEANING THE MICROSCOPES
1. Clean up all spills immediately with a damp towel or Kimwipe. Do not allow water or other liquids on any part of the microscope.
2. Use 2 cotton swabs for each lens or eyepiece. Add one drop of lens cleaner to a swab to gently clean the lens. Start in the center of the lens and wipe in a spiral motion moving outwards towards the edge of the lens. Then, with the second swab, dry the lens by repeating the spiral motion. Clean all lenses at the beginning and end of each lab. Too much lens cleaner can loosen lenses.
VI. CALCULATING SPECIMEN SIZE
1. Magnification is calculated by multiplying objective magnification (4X, 10X, 40X, or 100X) by eyepiece lens magnification (usually 10X). e.g. 40X times 10X = 400X.
2. Measure specimen with the eyepiecemicrometer. To get the size in micrometers, multiply the number of units measured by the factor determined for the objective used. You will find the factor for each objective on the Calibration of Ocular Micrometer (10X eyepiece) chart taped on the front base of your scope. e.g. for a 40X objective 1 unit =2.5 um; therefore, 5 units measured X 2.5 um = 12.5 um.
VII PUTTING AWAY THE MICROSCOPES INTO THE CABINETS
1. Switch off lamp.
2. Click 10x or lower power objective into place.
3. Unplug microscope and secure cord around microscope body.
4. Loosen the headpiece and turn it backwards so that it faces towards its arm. Retighten the headpiece or it could fall off in transporting.
5. Carry with both hands, hold upright and put back in its designated cabinet.
VIII. MICROSCOPE DOS AND DON'TS
1. Always use a coverslip. It reduces mess on the front lens of the objective and will give you better resolution.The only time it is permissible to not use a coverslip is with a low power objective (4X and 10X.)
2. Focusing
i. Never use the coarse focus knob to focus specimens with 40X and 100X objectives.
ii. Never lower the body tube using the coarse adjustment without observing from the side to make sure the objective does not run into the slide.
iii. Never force the focusing knobs when you reach the limit of their range.
iv. Never twist the two coarse adjustment knobs in opposite directions simultaneously.
3. Eyepieces. Do not remove it.
Ellie Duffield 1/2/07