Metric Measurement and Microscopy PDF

Metric Measurement and Microscopy Learning Outcomes 2.1 The Metric System Use metric units of measurement for length, weight, volume, and temperature. 2.2 Microscopy Describe similarities and differe...

Metric Measurement and Microscopy Learning Outcomes 2.1 The Metric System Use metric units of measurement for length, weight, volume, and temperature. 2.2 Microscopy Describe similarities and differences between the stereomicroscope (dissecting microscope), the compound light microscope, and the electron microscope. 2.3 Stereomicroscope (Dissecting Microscope) Identify the parts and tell how to focus the stereomicroscope. 2.4 Use of the Compound Light Microscope Identify and give the function of the basic parts of the compound light microscope. List, in proper order, the steps for bringing an object into focus with the compound light microscope. Describe how the image is inverted by the compound light microscope. Calculate the total magnification and the diameter of field for both low- and high-power lens systems. Explain how a slide of colored threads provides information on the depth of field. 2.5 Microscopic Observations Name and describe the three kinds of cells studied in this exercise. State two differences between human epithelial onion epidermal cells. cells and Examine a wet mount of Euglena and pond water. Contrast the organisms observed in pond water. Introduction This laboratory introduces you system, which biologists use to measure the size of cells and to the metric cell structures. This laboratory also examines the features, functions, and use of the compound light microscope and the stereomicroscope (dissecting microscope). Transmission and scanning electron microscopes are explained, and micrographs produced using these microscopes appear throughout this lab manual. The stereomicroscope and the scanning electron microscope view the surface and/or the three-dimensional structure microscope can view only object. The compound light microscope and the transmission electron of an If a subject was sectioned lengthwise for viewing, the interior of the extremely thin sections of a specimen. projections at the top of the cell, called cilia, would appear in the micrograph. A lengthwise cut through any type of specimen is called a longitudinal section (ls). On the other hand, if the subject in Figure 2.1 was sectioned crosswise below the area of the cilia, you would seeother portions of the interior of the subject. of specimen is A crosswise cut through any type called a cross section (cs). Figure 2.1 Longitudinal and cross sections. cell. b. A longitudinal section a. Transparent view of a at the top of the cell. c. A cross a. The cell b. Longitudinal C. Cross section would show the cilia cut is made. section section shows only the interior where the Laboratory 2 Metric Measurement and Microscopy 2-1 2.1 The Metric System The metric system is the standard system of measurement in the sciences, including biology, chemistry, and physics. It has tremendous advantages because all conversions, whether for volume, mass (weight), or length, can be in units of ten. Refer to Appendix B, page B-1, for an in-depth look at the units of the metric system. Length Metric units of length measurement include the kilometer (km), meter (m), centimeter (cm), millimeter (mm), micrometer (um), and nanometer (nm) (Table 2.1). The prefixes milli- (103), micro- (10T), and nano (10") are used with length, weight, and volume. Table 2.1 Metric Units of Length Measurement Unit Meters Centimeters Millimeters Relative Size Kilometer (km) 1,000 (10) m 100,000 cm 1,000,000 mm Largest Meter (m) m 100 cm 1,000 mm Centimeter (cm) 0.01 (1073) m 10 mm Millimeter (mm) 0.001 (103) m 0.1 cm 1.0 mm Micrometer 0.000001 (107) m 0.0001 (10T) cm 0.001 (107) mm Nanometer (nm) 0.000000001 (107) m 0.0000001 (107) cm 0.000001 (10) mm Smallest Volume Two metric units of volume are the liter (1) and the milliliter (ml). One liter = 1,000 ml. Temperature There are two temperature scales: the Fahrenheit (F) and Celsius (centigrade, C) scales (Fig. 2.4). Scientists use the Celsius scale. 2.2 Microscopy of Because biological objects can be very small, we often use a microscope to view them. Manydevices. kinds electron microscope, are effective magnifying instruments, ranging from the hand lens to the follows. electron microscopes A short description of two kinds of light microscopes and two kinds of Light Microscopes (dissecting lenses to magnify the object. The stereomicroscope Light microscopes use light rays passing through three dimensions at low magnification. The compound light microscope) is designed to study entire objects in higher magnification than microscope is used for thinly sliced sections of objects under examining small or the ocular lenses The term compound refers to the use of two sets of lenses: that of the stereomicroscope. from below, and visible located near the eyes and the objective lenses located near the object. Ilumination 1s To improve contrast, the microscopist through opaque portions. light passes through clear portions but does uses stains or dyes that bind to ex not cellular res and absorb light. Photomicrographs, also called light micro- microscope (Fig. 2.5a). graphs, are images produced by a compound light Figure 2.5 Comparative micrographs of a lymphocyte. (light micrograph) of a lymphocyte shows less detail a. A photomicrograph A lymphocyte is a type of white blood cell. c. A scanning electron micrograph (SEM) of a lymphocyte shows the than a (b) transmission electron micrograph (TEM) (c) Steve Gschmeissner/Science Source cell surface in three dimensions. (a) Michael Ross/Science Source: (b) CNRI/Science Source, lymphocyte erythrocyt 3.000x 2,150x b. Transmission electron micrograph (TEM).Photomicrograph or light micrograph (LM) 5,000x Scanning electron micrograph (SEM) 14 Laboratory Metric Measurement and Microscopy 2-6 Electron Microscopes Electron microscopes use beams of electrons to magnify the object. The beams are focused on a photographic plate by means of electromagnets. The transmission electron microscope is analogous to the compound light microscope. The object is ultra-thinly sliced and treated with heavy metal salts to improve contrast. Figure 2.5b is a micrograph produced by this type of microscope. The scanning electron microscope is analogous to the dissecting light microscope. It gives an image of the surface and dimensions of an object, as is apparent from the scanning electron micrograph in Figure 2.5c. The micrographs in Figure 2.5 demonstrate that an object ismagnified more with an electron microscope than with a compound light microscope. The difference between these two types of microscopes, however, is not simply a matter of magnification; it is also the electron microscope's ability to show detail. The electron microscope has greater resolving power. Resolution is the minimum distance between two objects at which they can still be seen, or resolved, as two separate objects. The use of high-energy electrons rather than light gives electron microscopes a much greater resolving power since two objects that are much closer together can still be distinguished as separate points. Table 2.2 lists several other differences between the compound light microscope and the transmission electron microscope. Table 2.2 Comparison of the Compound Light Microscope and the Transmission Electron Microscope® Transmission Electron Microscope Compound Light Microscope 1. Glass lenses 1. Electromagnetic lenses 2. Illumination due to beam of electrons 2. Illumination by visible light 3. Resolution _ 0.1 nm 3. Resolution ~ 200 nm 4. Magnifies to 1,000,000X 4. Magnifies to 2,000X tens of thousands of dollars 5. Costs up to hundreds of thousands of dollars 5. Costs up to 2.3 Stereomicroscope (Dissecting Microscope) The stereomicroscope (dissecting microscope, Fig. 2.6) allows you to view objects in three dimensions at low magnifications. It is used to study entire small organisms, any object requiring lower magnification, and opaque objects that can be viewed only by reflected light. It is called a stereomicroscope because it produces a three-dimensional image. Identifying the Parts After your instructor has explained how to carry a microscope, obtain a stereomicroscope and a separate illuminator, if necessary, from the storage area. Place it securely on the table. Plug in the power cord, 2-7 Laboratory 2 Metric Measurement and Microsconv  yourinstructor wide variety of stereomicroscope styles, and and turn on the illuminator. There is a of style, the following features should be present: will discuss the specific style(s) available to you. Regardless move to accommodate for the various distances between 1. Binocular head: Holds two eyepiece lenses that different individuals' eyes. the binocular head. What is the magnification of your 2. Eyepiece lenses: The two lenses located on knurled knob to Some models have one independent focusing eyepiece with a eyepieces? called the fixed eyepiece. allow independent adjustment of each eye. The nonadjustable eyepiece is Figure 2.6 Binocular dissecting microscope (stereomicroscope). the text material. Lecia/Microsystems Label this stereomicroscope with the help of 2000 Leica  3. Focusing knob: A large, black or gray knob located on the arm; used for changing the focus of both eyepieces together. 4. Magnification changing knob: A knob, often built into the binocular head, used to change magnification in both eyepieces simultaneously. This may be a 200m mechanism or a rotating lens mechanism of different powers that clicks into place. 5. Illuminator: Used to illuminate an object from above; may be built into the mieroscope or separate, Locate each of these parts on your stereomicroscope, and label them on Figure 2.6. 2.4 Use of the Compound Light Microscope As mentioned, the name compound light microscope indicates that it uses two sets of lenses and the and the objective light to view an object. The two sets of lenses are the ocular lenses located near eyes clear portions but does lenses located near the object. Illumination is from below, and the light passes through examine small or thinly sliced sections of not pass through opaque portions. This microscope is used to objects under higher magnification than would be possible with the stereomicroscope. 17 Laboratory 2 Metric Measurement and Microscopy 2-9 Identifying the Parts the table. Identify the place it securely on Obtain a compound light microscope from the storage area, and text material. them in Figure 2.7 with the help of the following parts on your microscope, and label Figure 2.7 Compound light microscope. with the help of the Label this microscope Compound light microscope with binocular head and mechanical stage. text material. OLecia/Microsystems ATC 2000 of the ocular lenses on your microscope? What is the magnifying power 1. Eyepieces (ocular lenses): 2. Viewing head: Holds the ocular lenses. 3. Arm: upper parts and Supports provides carrying handle. objectives. 4. Nosepiece: Revolving device that holds 5. Objectives (objective lenses): objective lenses and is used to scan the whole slide. the shortest of the a. Scanning objective: This iS It is a number followed by an x. What is The maghification is stamped on the housing of the lens. your microscope? the magnifying power of the seanning objective lens on Metric Measurement and Microscopy 2-10 18 Laboratory 2  b. Low-power objective: This lens is longer than the scanning objective lens and is used to view objects in greater detail. What is the magnifying power of the low-power objective lens on your microscope? C. High-power objective: If your microscope has three objective lenses, this lens will be the longest. It is used to view an object in of the high-power objective even greater detail. What is the magnifying power lens on your microscope? d. Oil immersion objective (on microscopes with four objective lenses): Holds a 95x (to 100x) lens and is used in conjunction with immersion oil to view objects with the greatest magnification. Does your microscope have an oil immersion objective? If this lens is available, your instructor will discuss its use when the lens is needed. 6. Stage: Platform that holds and supports microscope slides. A mechanical stage is a movable stage that aids in the accurate positioning of the slide. Does your microscope have a mechanical stage? a. Stage clips: Clips that hold a slide in place on the stage. b. Mechanical stage control knobs: Two knobs that control forward/reverse movement and right/left movement, respectively. 7. Coarse-adjustment knob: Knob used to bring object into approximate focus; used only with low-power objective. 8. Fine-adjustment knob: Knob used to bring object into final focus. 9. Condenser: Lens system below the stage used to focus the beam of light on the object being viewed. control lever: Lever that controls the amount of light passing through the Diaphragm or diaphragm condenser. 10. Light source: An attached lamp that directs beam a of light up through the object. microscope that rests on the table. 11. Base: The flat surface of the Rules for Microscope Use microscope: Observe the following rules for using a low) should be in position at both the beginning and 1. The lowest power objective (scanning or use. the end of microscope 2. Use only lens paper for cleaning lenses. 3. Do not tilt the microscope because the evepieces could fall out, or wet mounts could be ruined. stage clean and dry to prevent rust and corrosion. 4. Keep the 5. Do not remove parts of the microscope. by covering it after use. b. Keep the microscope dust-free malfunctions. 7. Report any Microscope-Lowest Power Focusing the Compound Light straight alignment over that the lowest power objective on your microscope is in 1. Turn the nosepiece sO the stage. power objective on your microscope (4x [scanning] or 2. Always begin focusing with the lowes! 10x [low power)). lower the stage (or raise the objectives) until it stops. 3. With the coarse-adjustment knob, 19 Laboratory 2 Metric Measurement and Microscopy 2-11  it with the clips. (If your microscope has a letter e on the stage, and stabilize 4. Place a slide of the of the slide arms on the stage, and insert the slide.) Center the e ns has a mechanical stage, pinch the spring below the stage (if your microscope best you can on the stage or use the two control knobs located look from the side, decrease mechanical stage) to center the e. the is in place. Then, as you 5. Again, be sure that the lowest-power objective lens comes to an automatic stop or lens until the distance between the stage and the tip of the objective is no closer than 3 mm above the slide. give the 6. While looking into the eyepiece, rotate the diaphragm (or diaphragm control lever) to appropriate amount of light. the stage and the objective lens 7. Using the coarse-adjustment knob, slowly increase the distance between until the object in this case, the letter e comes into view, or focus. 8. Once the object is seen, sor imny need to adjust the amount of light. To increase or decrease the contrast, rotate the diaphragm slightly. the focus if necessary. 9. Use the fine-adjustment knob to sharpen this greatly reduces eyestrain. through the eyepiece, as 10. Practice having both eyes open when looking Inversion down and reversed. Inversion refers to the fact that microscopic image is upside Observation: Inversion the slide (with the unaided eye, not looking through the eyepiece). on 1. Draw the letter e as it appears when you look through the eyepiece. 2. Draw the letter e as it appears 3. What differences do you notice? image appear to move? 4. Move the slide to the right. Which way does the to move? 5. Move the slide toward you. Which way does the image appear Microscope-Higher Powers Focusing the Compound Light lowest power, it should in focus with the parfocal; that is, once the object is Compound light microscopes are also be almost in focus with the higher power. power by following the instructions in the previous section. 1. Bring the object into focus under the scanning the field of the lowest objective. in 2. Make sure that the letter e is centered power [40x]) by turning the nosepiece until Move to the next higher objective (low power [10x] or high not "hit" change the focus; parfocal microscope objectives will 3. into place. Do not you hear it click is initially in focus. (If you are on low the focus if the lowest objective normal slides when changing to step 4.) before going on power [10x], proceed to high power [40x] only the fine-adjustment use only the fine-adjustment knob. (Note: Always use 4. If any adjustment is needed, do not use the coarse-adjustment knob.) knob with high power, and around the portion of the letter that you are now the right, draw a circle drawing of the letter e to letter e will not disappear because your microscope is parcentric 5. On a seeing with high-power magnification. The the center). (the focus remains near finished your observations of this slide (or any slide), rotate the nosepiece until 6. When you have then remove the slide. lowest-power objective clicks into place, and the Total Magnification is calculated by multiplying the magnification of the ocular lens (eyepiece) by the Total magnification lens is imprinted on the lens casing. the objective lens. The magnification of magnification of Metric Measurement and Microscopy 2-12 20 Laboratory 2 Field of View A microscope's field of view is the circle visible through the lenses. The diameter of field is the length of the field from one edge to the other. Depth of Field When viewing an object on a slide under high power, the depth of field (Fig. 2.8) is the area-from top to bottom- -that comes into focus while slowly focusing up and down with the microscope's fine-adjustment knob. Observation: Depth of Field  2.5 Microscopic Observations When a specimen is prepared for observation, the object should always be viewed as a wet mount. A wet mount is Figure 2.9 Preparation of a prepared by placing a wet mount. drop of liquid on a slide or, if the mate- rial is dry, by placing it directly on the slide and adding a drop of water or stain. The mount is then covered with a coverslip, as illustrated in Figure 2.9. Dry the bottom of your slide before placing it on Add drop of liquid or the stage. a. b. Lower coverslip slowly dry object and liquid. Onion Epidermal Cells Epidermal cells cover the surfaces of plant organs, such as leaves. The bulb of an onion is made up of fleshy leaves.