Download The Microscope Microscopes were invented during the latter part of

The Microscope Microscopes were invented during the latter part of the 1600's and by the end of the 1700's had undergone dramatic improvements in their construction and the quality of the images they produced. The use of microscopes opened a whole new world previously unseen, eventually resulting in challenge of the theory of "spontaneous generation" (a.k.a. abiogenesis) as well as leading to development of the Cell Theory and the Germ Theory of Disease. The simple, single­lens apparatus of Leeuwenhoek led to the more complex compound microscope, and finally to the sophisticated instruments commonly used in today's laboratories. It is important to remember that a microscope is a delicate instrument and that its proper care and use are quintessential to making meaningful observations as well as prolonging the useful life of an expensive piece of equipment. Objectives:
· Learn the proper names and functions of the main parts of the optical (light) microscope
· Practice proper care and use of the light microscope
· Be able to describe, in correct order, the steps in the process of bringing a specimen into focus
· Become reasonably proficient in use of the microscope for observation of biological specimens Prior to class read through the laboratory exercise and answer the questions about proper care and use of the microscope. This practice will make you familiar with the purposes of the exercise as well as acquaint you with the terms and activities you will encounter. (Note: your instructor may collect your answers to the questions as part of the lab session or use them as a source for a lab quiz). Materials: Compound microscope Slides and coverslips Pipettes Toothpicks, flat Methylene blue stain solution Dissecting (Stereo) Microscope Distilled water Prepared slides (letter "e" and colored threads) Disinfectant (10% bleach solution) Forceps (“tweezers") Colored Pencils Lens paper Care of the Microscope: In spite of its size and weight a microscope is a precision instrument and must be handled and used with care. The microscope is quite costly and each student is responsible for its proper handling and use for each lab session. Following is a list of handling practices each student is expected to exercise whenever the "scope" is in use. 1. Transport: Carry the microscope with both hands ­ one under the base supporting the weight of the instrument and the other grasping the arm to balance it. 2. Lens Care: Use only designated lens paper to clean any and all glass surfaces. 3. Electrical cord: Do not allow the electrical cord to hang off the table or present a "trip" hazard or interfere with the movement of people or materials.
Page 1 of 9 ver. 1.6 4. Inspection: Each time you take a microscope from the cabinet inspect it to confirm that all of the parts are functioning properly. All of the adjustment knobs should operate smoothly ­ DO NOT force any of the adjustments. Notify the instructor of any malfunctions or damage to the scope. Parts of the Microscope and their Functions: Locate each of the following parts on a microscope and label the photographs provided with this exercise (pp. 17 and 18). Include the proper name of each part and its function for each of the components described below. 1. Ocular Lenses: (a.k.a. eyepieces) are located at the top of the Body Tube. Some scopes have two ocular lenses (binocular) others have only one (monocular). On most teaching microscopes the ocular lens has a magnification of 10X. Most binocular microscopes permit adjustment of the distance between ocular lenses. A Diopter adjustment ring allows one to bring both eyes into clear focus by providing a means to adjust the focus of the left eye to match that of the right. 2. Body Tube: supports the nosepiece at one end and the ocular lenses at the other. 3. Arm: provides a means to support the microscope when it is carried and supports the upper parts of the microscope. 4. Nosepiece: a revolving support for three to five objective lenses of different magnifying powers. The nosepiece is used to rotate the desired objective lens into place. 5. Objective lens: the lens located on the end of the body tube closest to the specimen. Most teaching microscopes will have from three to four objective lenses of different magnifying powers located on a nosepiece. Most objective lenses include the following: Scanning lens: 4X, red, for scanning the slide to locate the specimen more quickly Low Power lens: 10X, yellow, used to observe the specimen at lower magnifications High "Dry" lens: 40X, blue, used to make observations at higher magnification (no oil) Oil Immersion lens: 100X, white, used for maximum magnification and requires the use of immersion oil to be focused properly. 6. Coarse­adjustment focus knob: used with low power objective lenses, used to bring the specimen into initial focus. 7. Fine­adjustment focus knob: used to bring the specimen into final focus. 8. Condensor: a set of lenses that produces a cone of light focused on the specimen. 9. Iris Diaphragm: controls the amount of light available to view the specimen, usually adjustable via a lever or special knob. 10. Field Diaphragm Lever: (located on the base under the condensor of some scopes) adjusts the diameter of the field of view. Ordinarily this should be wide open. 11. Light Source: a lamp, or in some cases a mirror that serves as source of light to illuminate the specimen. 12. Base: the flat bottom part of the frame that sits on the table and supports the rest of the instrument. 13. Stage: the flat surface that supports the microscope slide during observation. Most modern microscopes have a mechanical stage with knobs that provide easy precise movement of the microscope slide left­to­right and forward or back.
Page 2 of 9 ver. 1.6 14. Stage Clips: spring­loaded devices hold the microscope slide in place during observation. Resolution: (a.k.a. resolving power) Resolution refers to the capacity of a microscope lens system to accurately distinguish between two separate entities that lie close to each other. As a rule of thumb the maximum resolution for a good light microscope observing a good specimen is approximately 0.2 micrometers (0.2 microns, or 0.2 um). Resolving power is influenced by the wavelength of light and the numerical aperture (light wave bending) of the lens being used. It is calculated by the following formula: Resolving Power (R.P.) = Wavelength of light (in nm) 2 X the numerical aperture of the objective lens The use of a blue filter provides light of relatively short wavelength (violet/blue light) and improves the resolving power of the microscope. Other factors influencing resolution include the quantity of light, diffraction of light by air (see oil immersion) and contrast in the specimen (e.g. unstained versus stained). Magnification versus Resolution Magnification refers to creating an enlarged image of the specimen being observed. One or more lenses work together to create an enlarged image of the specimen, usually expressed by a number in combination with an “X” (“times”, e.g. 10X, “ten times actual size”). Total Magnification is calculated by multiplying the magnifying power of the objective lens times the magnifying power of the ocular lens (eye piece), e.g. 10 X 10 = 100X = total magnification. Resolution refers to the capacity of a microscope lens system to accurately distinguish between two separate entities that lie close to each other. It is responsible for the clarity of the magnified image. Operation of the Microscope ­ Low Power Objectives 1. Rotate the nosepiece to bring the scanning (lowest power) objective lens into place over the stage. 2. Always begin focusing with the scanning (4X) or low power (10X) objective in place. 3. Using the coarse focus knob lower the stage to provide sufficient clearance to put a slide in place. Check to see that the condensor is all the way up. 4. Using the coarse focus knob bring the specimen into focus. Adjust the iris diaphragm until the amount of light coming through provides the clearest image. Also, adjusting the condensor may provide additional resolution depending on the specimen. 5. Using the Fine focus knob bring the image into sharpest focus. 6. Keeping both eyes open while looking through the objective lenses will reduce eyestrain. With a little practice this habit will become part of your technique in proper use of the microscope.
Page 3 of 9 ver. 1.6 Operation of the Microscope ­ High Power Objectives Modern light microscopes are parfocal, meaning that once a specimen is in focus under low power it is very close to being in focus when the nosepiece is rotated to bring the next higher power lens into place. 1. Center the specimen, or the image of interest, in the field of view of the low power objective. 2. Rotate the nosepiece to bring the next higher power objective into place. The objective lens will be very close to the slide (a short working distance), but it should not touch the slide if the microscope is in good working order. 3. If the image is not in sharp focus, using only the Fine adjustment knob, sharpen the focus as needed. Note the difference in the image now occupying the field of view. As the magnification is increased more light will be needed. This is accomplished by opening the iris diaphragm with its lever until the light is optimal for the situation. 4. When your observations are complete rotate the nosepiece until the scanning objective (lowest power, shortest lens) clicks into place, then remove the slide. Using the Oil Immersion Lens At higher magnifications (e.g. 1000X) using the light microscope the image becomes blurred due to bending of the light by air. The use of immersion oil (special high quality mineral oil) to displace the air between the oil objective lens and the specimen greatly reduces distortion of the image and improves resolution at high magnification. The objective lens is literally immersed in the oil. The oil immersion lens is used primarily for special situations such as observing very small entities like bacteria. Ordinarily it is not necessary for the observations made during activities for this class. 1. The specimen is first brought into focus using the High Dry objective lens. 2. The High Dry objective is rotated out of position to allow access to the slide, and a small drop of immersion oil is then placed directly on the slide over the area to be observed. 3. The Oil Immersion lens is then carefully rotated into place. The FINE ADJUSTMENT knob is used to bring the oil immersion image into sharp focus. In order to avoid slide breakage or damage to the lens ONLY the Fine Adjustment knob should be used to focus using the oil immersion lens due to the extremely close working distance. 4. Immersion oil can get quite messy. It is very important to use lens paper to remove all oil from the oil immersion objective lens, other lenses, the condensor and stage. Making a habit of following the procedures described above will improve both the efficiency and enjoyment of your experiences using the light microscope. Procedures: Observations using the light microscope 1. Get a microscope from the storage cabinet and carefully carry it to the table where you will be working. 2. Inspect the microscope as described above. Report any malfunctions, etc. Focal Plane: Get a "3­colored Thread" slide from the lab cart and observe it as noted in the procedures above. The threads are layered one on the other. Use the fine focus knob to bring each thread in and out of focus. This process demonstrates the focal plane of the
Page 4 of 9 ver. 1.6 lens system in use. By focusing "up and down" through the threads use the focal plane to determine the sequence of the layering of the colored threads from top to bottom. What is the order of the threads on the slide you observed? Which color was on… top? _______________________ middle? ____________________ bottom ?____________________ Inverted Image: 1. Get a "Letter e" slide from the lab cart and place it on the microscope stage with the label readable. 2. In the space provided below draw the letter e as it appears when simply looking directly at the slide without the microscope. 3. Observe the letter e through the microscope, and draw its appearance in the second space. 4. What is different about looking directly at the slide or observing it through the microscope? _________________________________________________________ 5. While looking in to the microscope use the mechanical stage knobs to move the slide around. How does the movement of the image compare to the direction the slide is moved? _____________________________________________________________ Real Cells: Observation of some of yours! 1. Prepare a wet mount of cheek cells as follows:
· Use a clean dry microscope slide to make the wet mount
· Use a flat toothpick to gently remove some of the mucous and cheek cells from the inside of your cheek (they come off really easy… remember we're not trying to draw blood!)
· Place the cheek cells (and accompanying saliva) on a slide
· Place one very small drop of Methylene blue on top of the cells/saliva
· Carefully lower a cover slip down onto the drop.
· Place the slide on the microscope stage and bring the cells into focus. They will be very tiny on low power, but will become more distinct as you graduate to the high dry objective lens (remember to increase the amount of light as you increase the magnification)
· In the space below draw a few of the cells and label the following: cytoplasm, nucleus, and plasma membrane. Be sure to always include the identity of the specimen being drawn, what it was stained with and the total magnification at which it was observed. (remember, total magnification is calculated by multiplying the magnifying power of the objective times the magnifying power of the ocular (eye piece). Colored pencils come in handy for many drawing, also.
· When you have completed your observations of the cheek cell slide, place it in the beaker of bleach solution to be disinfected.
Page 5 of 9 ver. 1.6 Putting the Microscope Away: Taking good care of the equipment is an important part of laboratory activities. Always take a few minutes to go over the following check list to prepare the microscope for storage:
· Remove the microscope slide from the stage
· Clean the lenses with lens paper to assure removal of oil, dust, etc.
· Rotate the scanning objective into position
· Adjust the rheostat to its lowest intensity
· Secure the electrical cord taking care to not damage lamp, condensor, etc.
· Adjust the mechanical stage to its most compact size
· Replace the dust cover
· Return the scope to its correct position in the cabinet Also, carefully clean all slides and return them to their proper place Parts of the Stereomicroscope (a.k.a. dissecting scope) and their Functions: Follow these instructions when using the Leica Zoom 2000 Stereomicroscope 1. Always use the dissecting microscope on a hard, stable surface 2. Plug the power cord into a suitable grounded electrical outlet using a three­pronged plug. 3. Select the light switch appropriate for the specimen. The Zoom 2000 is equipped with three types of illumination: ­ Transmitted light to view translucent specimens lighted from beneath ­ Reflected light to view opaque specimens ­ Combination of both to develop contrasting condition on any variety of specimens 4. Place a specimen on the circular stage directly below the center of the stereomicroscope body. While looking through the eyepieces with both eyes, without moving you head, slowly adjust the distance between the eye piece tubes until you see a complete circle of light. Eyepiece tubes are inclined at the proper angle for comfortable viewing, and do not require individual focusing. They are factory adjusted for normal or corrected­to­normal (via eye glasses, contacts) vision. 5. Turn the zoom control knob until the lowest number on the knob is opposite the index mark on the body. Now, while looking thorough the eyepieces, bring the specimen into sharp focus by turning the focusing knobs. The distance is about four inches from the specimen to the bottom of the microscope body when focus is obtained. Hint: in practice it is always best to focus the zoom model microscopes at the highest power and then turn the zoom control knob to the lower powers. This will keep the specimen in close focus at any magnification. A slight turn of the focus knob may be needed to sharpen the focus.
Page 6 of 9 ver. 1.6 Laboratory Questions: 1. What are three things that should be checked when the microscope is removed from the cabinet and before it is put away at the end of the lab session? a. ____________________________________________________________ b. ____________________________________________________________ c. ____________________________________________________________ 2. How does one determine the total magnification of an image being observed with a compound a microscope? _________________________________________________________________. 3. When using the 40X objective what is the total magnification of the image you are viewing? ___________________________________. 4. What practice(s) help prolong the life of the lamp bulb? _________________________________________________________________. 5. Describe the proper way in which a microscope should be carried from one place to another. ________________________________________________________. 6. The microscope lenses (and all other glass surfaces) should be cleaned using only… ___________________________________________________________. 7. The small lever on the condensor controls the ____________________, which regulates the amount of light passing through to the specimen. 8. In what century was the first microscope considered to have been invented? (careful about your terminology) ______________________________________. 9. What happened to the intensity (brightness) of the light in the field of view when you increased the magnification by moving the next objective into place? _________________________________________________________________. What should be done to make the image brighter and sharper as the magnification is increased? ______________________________________________________. 10. In the spaces provided below draw, with as much detail as possible, at least two examples of the microscopic critters you observed in the pond water samples.
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3 4 13 12 11 10 5 9 6 8 7 Leica ATC 2000 Microscope Write the number of each microscope part in the space provided by its respective name and/or function. ____ Base; provides sturdy support for instrument ____ Mechanical Stage Adjustment Knobs; moves slide to locate specimen ____ Condenser / condensing lens; focuses condensed beam of light on specimen ____ Fine Focus Knob; for fine focusing and use with high power lenses ____ Rheostat ("dimmer")/Power Switch; Turns light on / adjusts light intensity ____ Coarse Focus Knob; for use with low power and high­dry lenses ____ Iris Diaphragm Adjustment Lever; adjusts the amount of light on the specimen ____ Spring­loaded Slide Holder; holds microscope slide in place ____ Stage; supports microscope slide during observation ____ Objective Lens; enlarges specimen image, produces "real image" ____ Arm; supports lens systems ____ Revolving Nose Piece; supports objective lenses in place, allows change of lenses ____ Ocular lenses ("eye piece"); magnifies real image producing "virtual image" ____ Sub­stage Illuminator; built­in light source for illuminating specimen on slide Page 8 of 9 ver. 1.6 1 2 3 6 5 4 7 9
Leica ZOOM 2000 Stereomicroscope (a.k.a."Dissecting Scope") Write the number of each microscope part in the space provided by its respective name and/or function. ____ Focusing Knob; brings specimen into focus ____ Base; supports instrument ____ Objective Lens System; enlarges specimen image ____ Zoom Lens Adjustment Knob; zooms in or out on specimen ____ Ocular (eye piece) Lenses; provides binocular "stereoscopic" (3D) view of specimen ____ Above­stage Illuminator; illuminates specimen from above ____ Sub­stage Illuminator; illuminates specimen from below ____ Sub­stage Illuminator Switch ____ Above­stage Illuminator Switch Page 9 of 9 ver. 1.6