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Microscopy:Techniques for Exploring Cells.

INTRODUCTION

If there's any one piece of equipment that people associate with biology, it's the microscope. Like peanut butter and jelly, biologists and microscopes go together and there are few disciplines in biology where at least occasional use of a microscope is not required. For many biologists, it was that first peek through a microscope that interested them in science.

PROCEDURE:

1- Remove your assigned microscope from its cabinet. To carry the microscope, grasp it with one hand by its ARM (the curved part) while supporting the BASE with your other hand. Do not tip the microscope upside-down (the upper lens may drop out). Gently place it on your laboratory bench with the EYEPIECE facing you.

2- The arm supports the BODY TUBE; the upper end of which holds an EYEPIECE or

OCULAR LENS. At the lower end you'll find the NOSEPIECE and three or four cylindrical OBJECTIVE LENSES.

3- Identify the microscope STAGE. Slides are placed on the stage over a hole near center stage. Depending on the type of microscope, the slide may be secured by SLIDE CLIPS) or held by a MECHANICAL STAGE. A SUBSTAGE CONDENSER or ROTATING LIGHT-ADJUSTMENT DISK is located below the stage. If your microscope has a light-adjustment disk, the disk can be rotated to control the illumination level transmitted through the slide (it has differing-sized holes to admit light). If a substage condenser is provided, an IRIS DIAPHRAGM ARM and CONDENSER EIGHT ADJUSTMENT knob control the light intensity and focus. A SUBSTAGE LAMP or MIRROR directs light through your slide.

4- Located on the arm, near the body tube, or on the base are the FOCUS ADJUSTMENTS (the location of these knobs varies considerably from one brand of microscope to another). There are two focusing knobs (a FINE- and a COARSE ADJUSTMENT). Depending on the microscope, either the stage or the body tube will move in a vertical direction when these knobs are rotated (move one to see how your microscope functions). In either case, the coarse adjustment moves the stage or body tube further per revolution than does the fine adjustment knob. The fine adjust knob is usually smaller than the coarse and may have lines etched into its surface.

EXERCISE 1b Magnification.

PROCEDURE:

1- Most student microscopes allow specimens to be viewed in a magnification range of about 40 to 430 times actual size. The power you select is determined by the product of the magnifications of the ocular and objective lens. The magnification of your ocular (eyepiece) lens is usually etched on the top surface, but may be engraved on the lens shaft (lift the lens from the body tube to read the magnification). The magnification of most eyepieces is usually 10X. Determine the magnification of your ocular lens and record it in the report section.

2- View the microscope from the side, while turning the coarse adjustment knob so that the objective lenses (at the bottom of the body tube) move away from the stage. Rotate the objective lenses until the shortest of them can be easily viewed. When rotated, the lenses occasionally will "click" into place and provide a slight resistance to further movement. Several numbers are engraved on each lens. One is followed by an "X" (the magnification power; most likely a number around 3.5 or 4.0 for the short lens). Enter the magnification of the short objective lens (also known as the SCANNING OBJECTIVE) in the results section. The other numerals you'll see are the focal length (followed by "mm") and numerical aperture ("N.A."). Manufacturing product identifications may also be engraved on the lens. Although these numbers are of importance in purchasing a microscope, they need not concern you.

3- Rotate the objectives to view the second longest of the lenses (the LOW POWER OBJECTIVE). Record its magnification as before. Do the same for the third lens (the HIGH-DRY OBJECTIVE). If your microscope has a fourth (an OIL IMMERSION LENS), record its magnification.

4- Determine the TOTAL MAGNIFICATION for each combination of lenses by multiplying the power of the ocular by that of each objective. If, for example, the power of your ocular lens is 10X and that of the objective is 3.2X, the total magnification for this combination is 32X (10 X 3.2). these values in the Results Section.

EXERCISE 1c Use and Characteristics of the Microscope.

PROCEDURE:

1- If you have not already done so, turn the coarse adjustment knob so that the distance between the stage and objective lenses is at a maximum. Then rotate the scanning objective over the hole in the stage.

2- Obtain a slide provided by your instructor Examine it to find out where the specimen(s) are located under the COVER SLIP.

3- Look from the side, while you turn the coarse adjustment to position the objective as close as possible to the specimen (do not let it touch the slide). NOTE: If the objective can touch the slide, then the scanning objective is not selected (try again).

4- Turn the lamp on and look in the eyepiece. You won't see much other than a circular disk of light since the specimen is out of focus. If the light is off-center or half-moon in shape, either the objective or rotating light adjustment disk is not fully "clicked in". Adjust the illumination to a comfortable level by turning the light adjustment disk (or moving the iris diaphragm arm). First-time users usually adjust their microscopes for far too much light. Cut back on the illumination since excess light washes out details of your specimen (like over-exposing a photograph) and can cause eye strain if slides are viewed for more than a few minutes.

5- If you see a stationary hair or needle, that's a pointer (not a crack in the lens). You may also see moving objects. Quick-moving lines are your eye lashes (back away a little) while small mobile dots are "floaters" within your eyeball (don't panic; they're normal, but you probably have the illumination too high). If you wear glasses, you may be more comfortable using the microscope them removed (I know I am, and there's less chance you'll scratch your eyeglass lenses).

6- If there are non-moving dots (smears, lines, etc), the eyepiece and/or objective lenses are dirty.

CAUTION: If the lenses are dirty they MUST be cleaned with LENS PAPER. Do NOT clean them with a handkerchief, finger, or anything else. Since microscope lenses are made of a soft glass, cleaning them with anything other than lens paper may damage the instrument. If your microscope needs cleaning, use the lens paper provided for that purpose. If it's incrusted with deposits, use the cleaning solution available from your instructor.

7- While looking in the microscope, first focus with the coarse adjustment knob. Whenever the coarse adjustment is used for focusing, always turn it so that the objective moves away from the slide. If you need to re-focus, look from the side while you decrease the objective-slide distance. This makes it less likely you'll damage the lens or slide while using higher- objectives (unlike the scan power, the other objectives can driven into the slide). Rotate the knob slowly or you may go past the specimen. If you have difficulty locating them, try re-positioning the slide on the stage and focusing again (do not pass go. do not collect $200.00).

8- Once your specimen is more-or-less in focus, switch to the fine adjustment. adjusting the light to coax more detail from your microscope. Aren't they pretty?

9- Continue to observe the specimen(s) while moving the slide slightly to the left, then right. Then move the slide slightly up and down. Record what you have seen in the results section. This phenomenon is known as IMAGE REVERSAL and is common to all compound microscopes. Although annoying at first (especially when trying to follow a mobile organism), you will get used to it.

10-Find an interesting area of the slide and move it as close to the center of the field of view as possible and then re-focus for a sharp image. Without touching the focus or moving the slide, rotate the objectives so that the low power lens (the second shortest) is in position. Look in the eyepiece. If you were careful when centering the pair and your focus was sharp before switching to low power, the pair should be clearly visible and apparently much larger. Microscopes that retain their focus when switched to a higher magnification are said to be of PARFOCAL design. Make some minor fine focusing adjustments, re-center, and then switch to high-dry. The area of interest should still be more-or- in focus (you'll probably have to increase the illumination at this point). If your microscope has an oil immersion lens, do not switch to that at this time.

CAUTION: To avoid damaging the slide and/or lens, never use the coarse adjustment with the high-dry or oil immersion objectives.

11-Look from the side of the microscope and you'll find that the high-dry lens is nearly touching the glass slide (hence the caution). Switch to low power, and then scan. Notice how the distance between the objective lens and slide decreases with increasing power. This separation is the WORKING DISTANCE for the objective.

12-Remove the slide from the stage, and then place it back on the stage. Select the high power objective, rotate the coarse adjustment to bring the lens close to the slide, and then attempt to focus and find an interesting specimen. After a short time, it should be apparent that beginning with a power other than scan is a waste of time. Not only is it more difficult to focus at higher powers without first adjusting under scan, it is also considerably harder to locate items of interest on the slide. This is because, like the working distance, the FIELD OF VIEW decreases with higher magnification and you are looking at a smaller portion of the slide. Therefore, to save time and frustration, always start with the scan and work your way up.