Exercise 1.1 - The Bright Field Microscope
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.
- Binocular Microscope
- Microscope slide with letter e
- 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
- 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.
- 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.
||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.
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.
|Indicate the numerical aperture of the 100X lens
|Indicate the numerical aperture of the condenser
|The numerical aperture for an air interphase =
|The numerical aperture for oil interphase =
||1.3 - 1.5
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
- 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
- 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).
- 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
- Find a suitably contrasty location in the center of
the field of view and close your left eye. Using the coarse
and fine adjustments, focus until you obtain a sharp image
with your right eye only!
- Now close your right eye and adjust the focus of the
left eyepiece by rotating the diopter adjusting ring located
on the left eyepiece. Do not readjust the focus of the left
eye with the coarse or fine adjustments of the microscope -
use the adjustment ring on the eye tube.
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.
Familiarize yourself with the operation of any tension
adjustment options or pre-set devices that may be attached
to the microscope.
- Coarse Adjustment Tension: The coarse adjustment may
be eased or tightened by the adjusting ring. If the rotation
of the coarse focus knob is too loose, turn the adjusting
ring counterclockwise. Too much tension may be adjusted by
turning clockwise. Avoid excessive rotation as it will place
undo stress on the internal gears. Adjust the tension so
that the stage will remain stationary after focusing but can
be moved with relative ease by turning the coarse adjustment
knob. Some microscopes require turning the two coarse
adjustment knobs in opposite directions, while others
require the use of a screwdriver. Be sure to check with your
instructor or the manufacturer's directions before adjusting
- Preset Device: On the Nikon S-Cb, the right-hand
focus knob has a preset lever on its drum. When the lever is
turned clockwise, it will lock the stage so that it can not
be moved closer to the objective. That is, the stage can be
moved away from the objective, but not closer. The preset
device is used to rapidly change slides, but has a distinct
disadvantage for the neophyte, since it can be locked and
the operator might proceed to force the focus. Forcing the
focus will result in immediate and costly damage to the
Unless otherwise instructed, do not use the preset device!
If asked to use the device, refer to the specific
directions from the manufacturer.
- 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
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.
- 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.
- 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.
- Draw the image of the letter e at 10X.
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 -- firstname.lastname@example.org