The Laboratory Microscope, its Parts and Use. Name Date Period

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1 Name Date Period 1.1 Introduction Microorganisms, as their name implies, cannot be seen with the naked eye. Although they had been observed as early as 1674 by Anton Van Leeuwenhoek (using a simple single-lens microscope), it was not until the development of the modern compound microscope that the real diversity of microorganisms became apparent. There are two basic categories of microscopes: light microscopes and electron microscopes. As the name implies, light microscopes use light waves to provide the illumination. Electron microscopes may be of either the transmission (TEM) or the scanning (SEM) type, and beams of electrons form the image. These can cost tens of thousands of dollars and can magnify tiny things like viruses a million times. For this reason, we have no electron microscopes to use for this lab, but your teacher will explain them to you and show you various electron micrographs, which are highly-magnified pictures taken with SEMs or TEMs. With electron microscopes, a specimen is carefully prepared and placed ina chamber, where a beam of electrons is focused on it using electromagnets. The main difference between SEMs and TEMs is that SEMs give the image of the surface of an object, and are typically a bit less powerful than TEMs. TEMs require a thinly sliced specimen that has been treated with heavy metal salts to provide contrast. These give a powerful, penetrative picture of the interior of tiny specimens, like cells. Table 1.1 Comparison of Microscope Types Compound Light Microscope Transmission Electron Microscope Glass lenses Electromagnetic lenses Illumination by visible light Illumination by beam of electrons Resolution around 200 nm Resolution about 0.1 nm Magnifies up to 2000x Magnifies up to 1,000,000 x Costs up to tens of thousands $ Costs up to hundreds of thousands $ This laboratory introduces you to the features, functions, and use of two types of light microscopes, the stereomicroscope and the compound light microscope. These microscopes use a series of two or more lenses to magnify an illuminated image. In microscopy, there are two things to keep in mind: Magnification is a measure of how big an object looks to your eye compared to life size. Microscopes also enhance the resolution of an image. Resolution is the ability to distinguish between two objects that are close together. Resolution is important because it amounts to clarity and ability to focus on detail. Making objects appear larger is useless if the image is fuzzy! 1

2 1.2 Stereomicroscope (Binocular Dissecting Microscope) The Stereomicroscope (Binocular Dissecting Microscope) allows you to view small threedimensional objects. Although dissecting microscope does not have as high a magnification as the compound microscope, the dissecting microscope does have several advantages: (1) It can be used to view whole specimens (whereas the compound microscope requires that a thin slice of the specimen be prepared). (2) The stereomicroscope does not require that the specimen be mounted onto a microscope slide. (3) The stereomicroscope is usually easier to focus than the compound microscope. (4) Light is not required to pass through the specimen to see it, so these microscopes are used to view entire (whole) specimens, and those that are thick or opaque. Some stereomicroscopes have lights on them, but this is only to enhance the viewing. Identifying the Parts: Draw label lines to the picture to identify each part, and match the part with its function, below. Eyepiece lenses Binocular head Focusing knob Magnification changing knob Stage Illuminator (optional) 1. Often built into the binocular head, this changes the magnifying power. This may be a zoom mechanism, or a rotating lens mechanism of different powers that clicks into place. Which type does your scope have? 2. Used to light a specimen, either from above, or below. 3. Holds two eyepiece lenses that move to accommodate for the space between different individual s eyes. 4. a large black of grey knob located on the arm; used for changing the focus by raising or lowering the eyepiece lenses. 5. the two lenses located in the binocular head. What is the magnification of your eyepieces? 6. Platform where the specimen in placed for viewing. 2

3 Focusing the Stereomicroscope 1. Place a coin (dime, nickel, penny, etc) on the center of the stage. 2. Adjust the distance between the eyepieces, if needed. You should be able to see the specimen as a 3-dimensional image, with both eyes. 3. Use the focusing knob to slowly bring the object into focus. 4. Turn the magnification changing knob to look at the specimen at a different size. 5. Now, view the specimen at the lowest magnification possible. What is the total magnification power with this lens? Sketch the object, at low power, as big as it appears, in the circle below: x Rule: Always write the total magnification you are using beneath the drawing you make! 8. Now rotate the magnification changing knob to the highest magnification. What is the total magnification power with this lens? 9. Draw a smaller circle, inside of the sketch you just drew, indicating the smaller portion of the specimen you can now see. 10. Does the area of your field of vision increase, or decrease when you turn to a higher magnification?. 11. Experiment with looking at various objects (your pencil tip, a dollar bill, a plastomount specimen, etc) at various magnifications until you are comfortable with using the stereoscope. 12. When you are finished, turn off the light and unplug your scope, wrapping the cord neatly around the base. Question: Which type of electron microscope, like the stereomicroscope, allows the viewing of specimens in 3-dimension?. 1.3 Use of the Compound Light Microscope The Compound Light Microscope uses two sets of lenses and light to view an object. The two sets of lenses are the ocular lenses (in the eyepiece) and the objective lenses (suspended above the specimen). Illumination is from below the specimen and the light must pass unobstructed through the slide in order to see the image. 1. Turn on your microscope light and look through the eyepiece. You should see a circle of light. What is this called?. 3

4 2. What is the magnification of the ocular lens in the eyepiece?. 3. You may have a pointer in your eyepiece. If you turn the eyepiece, the pointer (arrow) will rotate. This is for pointing out interesting things on the slide to your partner. Does your eyepiece have such a pointer?. a. Identifying the Parts of the Light Microscope Draw label lines to the picture to identify each part, and match the part with its function, below. Eyepiece Body Tube Revolving Nosepiece Coarse Adjustment knob Fine Adjustment knob Arm Objective lenses Stage Clip Diaphragm Stage Light Source Base 1. where you look through to see the image of your specimen. 2. the part that holds the eyepiece and connects it to the objectives. 3. revolving device that holds the objectives. 4. (low, medium, high, oil immersion) the microscope may have 3 or more of these attached to the nosepiece; they vary in length (the shortest is the lowest power or magnification; the longest is the highest power or magnification). 5. part of the microscope that you carry the microscope with. 4

5 6. large, round knob on the side of the microscope used for focusing the specimen; it moves the stage or the upper part of the microscope. 7. small, round knob on the side of the microscope used to fine-tune the focus of your specimen after using the coarse adjustment knob. 8. large, flat area under the objectives; it has a hole in it (aperture) that allows light through; the specimen/slide is placed on the stage for viewing. 9. shiny, clips on top of the stage which hold the slide in place. 10. underneath the stage, it controls the amount of light going through the aperture source of light usually found near the base of the microscope; the light source makes the specimen easier to see 12. bottom part of the microscope that rests on the lab table. Questions: 1. Try dimming or brightening the light using the diaphragm control lever, beneath the stage. What type of diaphragm does your microscope have? (hint: it works like the colored part of your eye that adjusts light through your pupil). 2. How many objective lenses does your microscope have? 3. What are their magnification powers, from lowest to highest? 4. Aside from the magnification printed on the lenses, what are two other ways you can tell the lenses apart? Table 1.1 RULES FOR MICROSCOPE USE: 1. The lowest power objective should be in position both at the beginning and end of microscope use. 2. Use only lens paper for cleaning lenses. 3. Do not tilt the microscope when viewing a slide. 4. Keep the stage clean and dry to prevent rust and corrosion. 5. Do not remove parts of the microscope. 6. Keep the microscope dust-free by covering it after use. 7. Report any malfunctioning. 8. Do not use the coarse focus knob when viewing under high power. You can break the lens doing this! b. Table 1.2 Total Magnification Calculations: Objective Ocular lens Objective lens Total magnification Scanning/low-power x x x Medium power x x x High power x x x 5

6 1.4. Making a Wet Mount Slide Practice with the Letter e 1. Use the pipette (dropper) to place a drop of water in the center of a clean slide. 2. Using scissors, cut a small, lowercase letter e from a piece of newspaper. 3. With forceps, carefully place your e in the drop of water. 4. Now, cover the water drop with a clean cover slip. The best way to do this is to hold the cover slip at a 45 angle to the slide (see picture) and move it over the drop. As the water touches the cover slip, it will start to spread. Gently lower the angle of the cover slip to allow the water to evenly coat the under surface, then let the slip drop into place. You should not just drop the cover slip onto the slide or air bubbles will get trapped. This makes the slide very difficult to study. If you do trap several air bubbles, remove the slip and try again. Now, place your slide on the stage, making sure your letter e is right side up, as it would be when reading. Make sure the e is centered over the opening in the stage. Focusing the Microscope 1. Turn the nosepiece so that the lowest power lens is in straight alignment over the stage. 2. Always begin focusing with the lowest power objective lens. 3. With the coarse-adjustment knob, lower the stage until it stops (as far from the objective as it will go). 4. Place a slide of the letter e on the stage, and stabilize it with the clips. Again, be sure that the lowest power objective is in place. 5. Then, as you look from the side, decrease the distance between the stage and the tip of the objective lens until the lens comes to an automatic stop or is no closer than 3 mm above the slide. 6. While looking into the eyepiece, rotate the diaphragm (or diaphragm lever) to give the optimal amount of light. 7. Slowly increase the distance between the stage and the objective lens, using the coarseadjustment knob, until the object - in this case the letter e - comes into view, or focus. 8. Once the object is seen, you may need to adjust the amount of light. To increase or decrease the contrast, rotate the diaphragm slightly. 9. Use the fine-adjustment knob to sharpen the focus if necessary. 10. Practice having both eyes open when looking through the eyepieces, as it greatly reduces eyestrain. View the letter under the microscope and answer the following questions: 1. In the circle at right, draw the letter e as it appears under the microscope. 2. What is the total magnification of the low-power lens? 3. Describe any change in the position of the e (hint: look at the e before looking thru the eyepiece, then look at it through the eyepiece). 6 x

7 4. What happens to the e as you move the slide to the right? 5. What happens if you push the slide away from you? Higher Powers Compound microscopes are parfocal; that is, once the object is in focus with lowest power, it should also be almost in focus with the higher power. 1. Bring the object into focus under the lowest power by following the instructions in the previous section. 2. Make sure that the letter e is centered in the field of the lowest objective. 3. Move to the next higher objective (medium power, 10x) by turning the nosepiece until you hear or feel it click into place. Do not change the focus knobs yet! Parfocal microscope objectives will not hit normal slides when changing the focus if the lowest objective is initially in focus. 4. If any adjustment is needed use only the fine-adjustment knob. Always use only the fineadjustment knob with higher powers. 5. Draw the e under medium power 6. Draw the e under high power Total magnification= x x 7. Describe any changes you observe in the e when you change to the high-power lens? 8. About how many times was the magnification increased when you changed from low-power to high-power? 9. How does this change the area of the slide included in the high-power field? 7

8 *CLEAN OFF YOUR SLIDE AND COVERSLIP WITH LENS PAPER AFTER USE! 1.5 Calculation of the Field of Vision 1. Remove all slides from stage. Place a transparent metric ruler on the stage beneath the low power (LP) objective of a microscope. 2. Focus the microscope on the scale of the ruler, and measure the diameter of the field of vision in millimeters (mm). Record this number. mm 3. Convert this measurement to micrometers (µm) by using the following equation: Diameter of field in mm x 1000 = LP diameter in µm lpm = low-power total magnification (from Table 1.2) = hpm = high-power total magnification (from Table 1.2) = 4. Calculate the diameter in µm of the field of vision under high power (HP) using the following formula: hpd = lpd x (lpm/hpm) diameter of the HP field of vision = µm 1.6 Cross hairs Place a drop of water on a clean glass slide. Cross two pieces of hair on the drop. Place a cover slip on the slide using the previously described procedure. View the slide under the microscope, focusing on where the hairs cross (intersect) and answer the following questions: 1. Draw what you see under the three different magnification powers. low-power power high power x x x 2. Do you notice any loss of resolution at high power? (Do both hairs focus in equally well or it one of the hairs slightly out of focus). Explain. 3. Did the slide have to be re-centered in your field of vision as you went from low to high power? Explain 8

9 References: Laboratory Manual: Human Biology, 11 th edition, Sylvia S. Mader 9