Exercise 1.1 - The Bright Field Microscope

LEVEL I


Figure 1.6 Nikon S-Cb


Figure 1.6a Nikon Binocular Microscope

Refer to Figure 1.6 and Figure 1.6a for the names of the various components of a Nikon binocular microscope.

Materials

Procedure

  1. Pick up a microscope from the cabinet by placing one hand under the base and the other on the arm of the microscope. Most microscope damage is due to careless transport. It is important that you carry the microscope securely, with two hands, and in an upright position. Remember that you are handling $1,000 of precision instrumentation.

  2. Place the microscope in front of you, unwind the power cord and plug it in. The microscope is normally provided in its storage position, that is, with its eyepieces pointed back over the arm. This takes less room in a cabinet, but is not the position for which it was designed to be used. If your instructor approves, slightly loosen the screw holding the binocular head and rotate the entire binocular head 180°. Carefully (and gently) tighten the screw to prevent the head from falling off.
    You will notice that all parts of the microscope are now conveniently located for your use, with an uninterrupted view of the stage, and substage. The focus controls are conveniently at arms-length.

  3. Note the magnification power and the numerical aperture of the lenses which are on your microscope's nose-piece. These values are stamped or painted onto the barrels of the objectives. Record the magnification power and numerical aperture of each lens in the space provided below.

    Magnification (x#) Numerical Aperture (NA)








    Enter the numerical aperture of the condenser __________
    Enter the magnification of the oculars and whether they are normal or widefield __________
    Your maximum resolution will depend upon the highest effective numerical aperture of the system. The highest value is normally given by the 100X, or oil immersion lens.
    Indicate the numerical aperture of the 100X lens __________
    Indicate the numerical aperture of the condenser __________
    The numerical aperture for an air interphase = 1.0
    The numerical aperture for oil interphase = 1.3 - 1.5
    The maximum effective numerical aperture is the lowest of those listed. It depends on the angle and thus on maximum positioning of the condenser. Using the lowest NA value from above as the working numerical aperture, calculate the limit of resolution for your microscope, assuming violet light with a wavelength of 400 nm.

    From Equation 1.1b, the limit of resolution = 0.61 x / NA, and therefore, the calculated value for your microscope is:

    Limit of resolution = ____________________ µ

  4. Obtain a prepared microscope slide with the letter e. Place the slide on the stage and ensure that it is locked in place with the slide holder.
    Rotate the condenser focusing knob to move the condenser to its highest position of travel. Although there is an ideal location for the condenser, the correct position of the condenser will vary slightly for each objective. Unless directed otherwise, it will not be necessary to move the condenser during any of the intended uses in this course.
    If, however, you wish to find the ideal location, focus the microscope on any portion of a slide, and then simply close down the condenser aperture and move the condenser until you have a sharply focused view of the condenser aperture (usually with a slight blue hazy edge). If you do this, you can then open the aperture until it just fills the field of view (different for each objective). This is the correct location and use of the condenser and aperture and the condenser should not be moved from this position. Never use the condenser aperture for control of light intensity. Control of light intensity is the purpose of the variable rheostat (dimmer switch, or voltage regulator) on the light source. 6

  5. Turn on the microscope by rotating the dimmer switch and adjust the light intensity to a comfortable level. Be sure that the condenser aperture is open if you have not set it as directed in the previous paragraph (slide the condenser diaphragm lever back and forth to check).

  6. Looking down into the microscope, adjust the eyepieces to your interpupillary distance and diopter. The Nikon microscope is equipped with a knob between the eye tube extensions for this adjustment. Many microscopes simply require pushing the eye tubes together or apart directly. Move the eye tubes back or forth until you see one uniform field of view.
    The first time you use the microscope, adjust the eyepieces for your personal comfort. Note that modern microscopes have HK (high eye point) eyepieces and consequently you need not remove eyeglasses if you are wearing them. Quite the contrary, they should be worn to prevent eyestrain while you constantly shift from looking through the microscope to reading the lab manual.
    Begin by focusing the microscope on any object within the field of view. 7


    All subsequent uses of the same microscope will involve use of the coarse and fine focus adjustments, without reference to the procedures in step 2. That is, step 2 need only be performed once at the beginning of your lab. It may, of course, be checked periodically if desired, and will need to be readjusted if someone else uses your microscope.

  7. Optional:

    Familiarize yourself with the operation of any tension adjustment options or pre-set devices that may be attached to the microscope. 8

    Unless otherwise instructed, do not use the preset device!

    If asked to use the device, refer to the specific directions from the manufacturer. 9

  8. Always begin focusing the microscope with the 10X magnification. Even if you are going to use the 100X, it is more efficient to begin with the 10X and then move up to the power desired. The objective lenses are parfocal, which means that if one is focused, each of the others is approximately in focus when revolved into position.
    With the slide from Step 4 in place, rotate the coarse focus control until the slide is as close to the 10X objective as possible. Move the stage manipulators until a portion of the slide is directly under the objective and focus carefully on the object in view. After adjusting the focus at 10X, center the object to be viewed, and rotate the nosepiece to the next highest magnification. Use the fine focus control only once the 40X or 100X objectives are in place.
    Manipulate the fine focus to obtain the sharpest image. During use of the microscope, one hand should remain on the fine focus as constant readjustment will be called for. Use the other hand to manipulate stage movements.
    Note that the microscope is typically designed so that one revolution of the fine focus knob raises or lowers the microscope stage 0.2 mm. This permits direct readings on the fine focus knob scale to 0.002 mm (2 microns) and can be used to determine the thickness of materials being examined.

  9. Return to the 10X objective and move the slide around until you locate the letter e in the view. Note the orientation of the letter e on your slide and in the field of view.

  10. To use the 40X objective, center the object you wish to view (the 40X will have a smaller field of view) and rotate the objective turret (referred to as the nosepiece) to bring the 40X objective into position. Is there any change in the orientation of the letter e?

    Do not rotate the turret in such a manner as to bring the 100X into position.

  11. Draw the image of the letter e at 10X.

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    Drawing of letter e (10 X magnification)

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu