Microscope Principles Study KEY Describe the function

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					                           Microscope Principles Study Questions KEY

1.      Describe the function of the following components of the microscope, noting their location
        on a microscope image and the microscope you are using: (textbook, page 1, 2, 6)
        a.     Aperture (iris) diaphragm (open aperture diaphragm on your microscope to 1/3 open)
               microscope component that regulates the angle of light presented to the
               specimen. Located at the base of the condenser, changes the diameter of the
               opening through which the source light rays must pass to enter the condenser

        b.      Condenser, substage (move the condenser of your microscope to the lowest position)
                microscope component that gathers and focuses the illumination light onto the
                specimen for viewing; located beneath the stage

        c.      Eyepiece (ocular)
                microscope lens or system of lenses located closest to the viewer’s eye; produces
                the secondary image magnification of the specimen

        d.      Illumination system (light source) a tungsten or a tungsten-halogen lamp located
                in the base of the microscope; provides microscope system with light, often
                called source light, source rays

        e.      Mechanical stage (adjust the stage to its lowest position using the coarse adjustment
                knob)     microscope component that holds the microscope slide with the
                specimen for viewing. Stage is adjustable, front to back, side to side, to enable
                viewing of the entire specimen

        f.      Objectives (place the low power (10X) objective into the light path)
                the lens or system of lenses located closest to the specimen. The objective
                produces the primary image magnification of the specimen

2.      Define the following terms as they relate to microscopy: (textbook, page 1, 2)
        a.     Birefringent the ability of an object to refract light in two directions (also called
               doubly refractile)

        b.      Field of view (FOV) the circular field observed through a microscope’s ocular

        c.      Numerical aperture a number that indicates the resolving power of a lens system.
                Derived mathematically. The greater the NA, the greater is the resolving power
                of the lens system. (Brunzel, p 2, 4)

        d.      Resolution the ability of a lens to distinguish two points or objects as being
                separate; ability of lens system to reveal fine detail (Brunzel, p 2, 3)

CLS 426 Urine and Body Fluid Analysis: Student Lab Rotation                                        1
Microscope Principles Study Questions KEY
3.      In the clinical laboratory, compound brightfield microscopes consist of a „two lens system‟.
        Explain what this means and identify these components on the microscope you are using.
        This means the microscope has an EYEPIECE (or ocular lens) and an OBJECTIVE
        lens. The objective lens is located closest to the specimen and produces the primary
        image magnification and directs this image to the second lens system called the eyepiece
        or ocular lens. The eyepiece further magnifies the image. (Brunzel, p3)

4.      How is the total magnification of a specimen determined? Provide an example.
        Total magnification of a specimen is the product of the ‘two lens system’: the objective
        lens magnification times the eyepiece lens magnification. If the eyepiece lens
        magnification is 10X and the objective lens magnification is 40X, then the total
        magnification would be: (Brunzel, p 3)
        (10) X (40) = 400X

5.      What is the purpose of the microscope lens system? (textbook, page 3)
        The purpose of the lens system is to magnify an object for viewing with maximum

6.      Where is the correct position of the condenser? (textbook, page 6).
        Always at its upper most stop, but is slightly lowered only with the Köhler illumination.

7.      When the aperture diaphragm is properly adjusted, what is the effect on the magnified image
        we see? (textbook, page 6).
        We achieve maximal resolution, contrast and definition of the specimen.

8.      What is the best way to reduce the brightness of the image field? List a common mistake
        made when attempting to reduce the image field brightness and its effect on the image.
        (textbook, page 6)
        You should decrease the intensity of the light source (or place a neutral density filter
        over the light source).
        A common mistake is to adjust the aperture diaphragm to reduce the brightness. When
        this is done, resolution of the image is decreased.

9.      Match the following objective magnification to the correct „power‟ terminology and clinical

        objective magnification          „power‟ terminology         clinical use

          c-2      10X                   a. Oil immersion            1. Slide differential, gram stain

          b-3      40X                   b. High power dry           2. Enumerating casts in urine

         _a - 1   100X                   c. Low power dry            3. Enumerating RBC in urine

CLS 426 Urine and Body Fluid Analysis: Student Lab Rotation                                         2
Microscope Principles Study Questions KEY
10.     What is probably the greatest cause of harm to both the mechanical and optical components
        of a microscope? Name at least three ways in which this hazard can be removed or avoided.
        (textbook, page 9) Dust.
        To remove dust:
        a. use a grease-free brush (camel hair) or an air syringe
        b. use a dust cover
        c. place the microscope in a storage cabinet

11.     Immersion oil should be used only with the oil immersion lens. Explain how, when and why
        the oil immersion lens should be cleaned. (textbook, page 9)
        How: remove oil using dry lens paper
        When: when finished using the objective and before putting the microscope away
        Why: oil left on the surface of the lens can impair its optical performance

12.     Review the “do‟s and don‟ts” for routine use and maintenance of a microscope found on
        page 11 in your textbook.

13.     Briefly explain the key characteristics of the following types of microscopy:
        (textbook, page 1,2)
        a.      Brightfield microscopy type of microscopy that produces a magnified image that
                appears dark against a white or bright background

        b.      Phase-contrast microscopy type of microscopy in which variations in the
                specimen’s refractive index are converted into variations in light intensity or
                contrast. Areas of the specimen appear light to dark with haloes or varying
                intensity related tot eh thickness of the component being viewed. Thin, flat
                components produce less haloing and the best-detailed images. Phase-contrast
                microscopy is ideal for viewing low-refractile elements and living cells

        c.      Polarizing microscopy type of microscopy that illuminates the specimen with
                polarized light. Polarizing microscopy is used to identify and classify
                birefringent substances (ie substances that refract light in two directions) that
                shine brilliantly against a dark background

        d.      Fluorescence microscopy type of microscopy modified to visualize fluorescent
                substances. Fluorescence microscopy uses two filters: one to select a specific
                wavelength of illumination light (excitation filter) that is absorbed by the
                specimen; and another filter (barrier filter) to transmit the different longer-
                wavelength light that is emitted from the specimen to the eyepiece for viewing.
                The fluorophore (natural or added) present in the specimen determines the
                selection (wavelength) of these filters

CLS 426 Urine and Body Fluid Analysis: Student Lab Rotation                                     3
Microscope Principles Study Questions KEY

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