Bio 236: Plant Biology

Light Microscopy

Using a Dissecting Microscope

1. The oculars (eyepieces) may be adjusted by gently holding each eyepiece at the base and slowly pulling them sideways until the distance between them matches the distance between the pupils of your eyes. These distances match when a single clear image of the specimen is obtained. Locate the adjustable ocular (usually the left one) and vary its length until you have stereoscopic perspective with no planar distortion (= the image should be clear, with no field distortion to strain your eyes and make you feel woozy when peering down the eyepieces).
   
2. The magnification knob on the side of the microscope head allows you to increase or decrease magnification.
   
3. The focus knob brings the microscope head up and down the arm and focuses the image.
   
4. The American Optical (AO) Spencer teaching microscopes and the Olympus SZH stereomicroscopes each have an external light source, while the Olympus SZ40/45 microscope (this microscope is in the image analysis room, HH 104) has an internal light source (the on/off switch for the 40/45 microscopes is on the base). For the microscopes with external light sources, set the illuminator transformer to the "off" position before plugging it into an electrical outlet. Gradually increase the illumination by moving the adjustment knob one increment at a time.
   
5. The clear glass insert and the rotatingmirror in the base of the AO Spencer and SZ40/45 microscopes allow you to trans-illuminate (light from below) your specimen. Adjust the mirror, the intensity of the light, and/or the arms of the fiber-optic lamp (for the AO microscopes) to provide optimal illumination of your specimen. Use a transparent specimen to see how varying the mirror angle can illuminate you specimen to advantage. Sometimes you may wish to illuminate your specimen strictly from above. To do so, place a piece of black construction paper over the glass insert in the base or use the SZH microscopes. You might want to try this if you stain your hypocotyls for starch.
6. When finished, gradually move the illuminator adjustment knob to its lowest setting, then to the "off" position, and replace the dust cover.

Using A Compound Microscope

1. Inspect the objective ring of the microscope and familiarize yourself with the various objectives. Each AO microscope is equipped with four objectives, which are arranged as follows: 3.5X = black, followed by 10X = green, followed by 43X = yellow, followed by 97X (oil immersion) = red. Each B-MAX microscope has a 4X, 10X, 20X, and 40X objective, while one also has a 2X objective and another one has an oil-immersion objective lens (100X).
2. Before placing a slide on the stage of the microscope, make sure that one of the low-power objectives (3.5/4X or 10X) is in place and that the stage is cranked up as far as it will go. If not, then make the necessary adjustment using the coarse focus knob (large, middle knob).
3. Make sure both diaphragms are open, and place a practice slide on the microscope stage. Secure the slide with the stage clips.
4. With the voltage selector knob at 2.5, turn on your transformer (at brighter settings, the bulbs get "shocked", and so do your eyes). Gradually turn up the light.
5. For the AO microscope, place the ocular with the pointer into the tube that corresponds to your dominant eye. Determine which of your eyes is dominant as follows: hold your finger about 1 foot in front of you and focus on it with both eyes. Alternately shut each eye - the dominant one is the one that shows no background shift when the other eye is closed.
6. Focus the specimen, using the coarse focus, followed by the fine focus (lower/proximal) knob. Note that you can focus in only one direction with the coarse focus knob since you started with the stage all the way up.
7. Adjust the distance between the oculars, if necessary. Do this by sliding the ocular tubes back and forth until the images in both eyes are comfortably in register. (For the B-MAX microscopes, make sure the eye rests are oriented to comfortably fit the contours of your face.) To make the oculars par focal, shut your left eye and focus on the specimen with your right eye. Then, without touching the coarse or fine focus knobs, open your left eye and bring the same objective into focus by adjusting the focus knob at the base of the left ocular. (These steps often mean the difference between comfort and migraine!)
8. To adjust the condenser, proceed as follows:
   • close down the field diaphragm so that only a small circle of light passes through;
   • focus the condenser by making the edges of the field diaphragm as sharp as possible using the condenser focus knob (small upper, distal knob). This should be ~1/8th of a turn from the top excursion of the condenser. If the outline of the field diaphragm is not in the center of the field, please notify your lab instructor.
   • open the field diaphragm just to the limit of the field of view;
   • adjust field contrast by slightly closing the diaphragm on the substage condenser;
   • make any final adjustments of image brightness using the voltage selector of the illuminator.
9. Increase magnification simply by changing objectives. (Hold the nosepiece, not the objective when changing objectives.) To change from an objective to the next highest power objective, you should only have to make slight adjustments using the fine focus! When using this procedure to increase magnification, close down the field diaphragm to just the limit of the visual field of the objective in use.
10. If necessary, adjust or reset your substage diaphragm, following the steps in Part 8.
11. The 40X/43X objective will resolve almost all structures to be identified in this course. However, if you would like to examine a slide at higher power, proceed as follows:
    • center and focus the structure you wish to study in the field of the 40X/43X objective;
    • turn the nosepiece halfway between the 40X/43X and oil immersion objectives (93X or 100X);
    • place a small drop of oil on the slide, then swing the oil immersion objective into place;
    • only a slight turn of the fine focus should be needed to bring the sample into focus. Do not force the focus with either the 40X/43X or the oil immersion objectives, as slide or objective breakage may occur;
    • when you are finished with the oil immersion lens, turn the nosepiece halfway back towards the 40X/43X objective and clean off the oil immersion lens with lens paper. Remove the slide from the stage and clean it with a Kimwipe.

Reminders On Microscope And Slide Use

1. Treat your microscope and slides with respect and care. Pick up the microscope by holding the arm with your dominant hand and supporting the base with your other hand. Keep the microscope vertical while moving it, as this will reduce the possibility of losing or breaking parts that could fall off or out.
2. Do not remove any parts of the microscope, nor open ocular backs or objective backs. This allows dust and moisture in.
3. Use lens paper to clean external glass surfaces of the microscope. (No other tissue papers are suitable!) The Kimwipes on the lab benches are for cleaning the slides or the stage of the microscope. Do not use solvents for cleaning. Remember to wipe dry any microscope part with oil or any other liquid on it.
4. Avoid wetting or smearing the stage, as this will corrode it.
5. Do not force levers or knobs. If something feels funny or seems to be reluctant, STOP AND GET HELP!
6. Place each slide back into the slide box immediately after you remove it from the microscope stage: in other words, you should have only one slide out of the box at any one time. Do not leave slides on the bench top, as these slides invariably end up on the floor in many pieces. Please handle the slides carefully, as they are relatively expensive and we do not have extras of most of the slides.
7. Turn off the transformer, replace the microscope dust cover, and return the slide set to a desk drawer when finished.