Chapter 1: The Microscope - Endnotes
For absolute maximum resolution, oil should be used between
the condenser and the slide as well, although in practice
this is rarely done.
Blue has an additional advantage in that the eye
perceives light blue as white . that is the reason for
adding bluing to white laundry, to make it look brighter.
green light, is nearly as good for resolution as blue and is
more pleasing to the eyes. we can withstand longer and more
intense observations with green light (note the color of
computer monitors and most automobile dash lights). if a
green filter is available, replace the blue filter with the
green -- be sure to remove the blue -- and note any
Since the electrons are shot down the column, the electron
source is known as the electron gun, and the electrons are
drawn off of the tungsten wire by a low voltage gun cap,
then accelerated by the high voltage anode.
It should be noted that for most routine cell biology, a
resolution of about 30 Å is all that is required.
Electron microscopes will give resolutions of around 5-7 Å,
and the top research microscopes are beginning to approach
the theoretical limit of just above 1 Å. as we approach
this theoretical limit, however, we enter the can't see
the forest for the trees syndrome, or in more perspective,
can't see the forest for the chlorophyll molecules syndrome.
The cost of the instrument increases exponentially with
increased resolution and extremely high resolution
electron microscopes are used for research on electron optic
theory, rather than for biology. The one advantage of high
voltage EM for biologists is the ability to view thicker
specimens, even hydrated living cells.
The directions are given for the Nikon Model S-Cb, but will
apply to most microscopes, provided allowances are made for
variations in manufacturer design.
Exceptions to this occur when you are not attempting to use
maximum resolution and are willing to trade resolution for
some other purpose, such as ease of operation, or to
compensate for the lack of a rheostat.
The Nikon S-Cb is provided with coaxial, coarse and fine focus
knobs, both of which are located near the base. Clockwise
rotation of either of the focus knobs by the operator lowers
the microscope stage. other microscopes may have two separate
focus knobs, and may alter the position of the upper lens tubes
and/or the objectives rather than the stage.
On the Nikon S-Cb models, the most confusing and potentially
damaging part of the microscope is the tension adjustment device
for the coarse adjustment, and the preset device. incorrect
adjustment of these will result in certain damage to the focus
adjustment of the microscope, necessitating complicated repair.
For the Nikon microscopes equipped with a pre-set device, the
lever should remain in its counter-clockwise position -- be sure
to check this position periodically during the use of the
microscope. this device has proven to cause enough problems that
many laboratories are resorting to removing the lever from the
microscope, to prevent damage to the focusing gears caused by
Type A immersion has a lower viscosity than Type B and is much
preferred. Some laboratories will mix A and B to obtain
intermediate viscosities. In any case, the oil should be a
non-drying and with low fluorescence.
If the image observed with the 40X appears dull and with a
milky halo to it, the first thing to suspect is oil on either
the front of the lens or the slide. the second thing to check
is the presence of oils and/or make-up from the eye lids on the
The average is statistically more valid, since there may be
differing concentrations within various areas of the slide. A
blood smear will definitely have variations since it will be
normally a gradient from one end of the slide to the other,
due to the means of preparing the smear.
If desired, whole blood may be substituted, diluted 1:200
with saline prior to use.
The coverslip used for the hemacytometer is especially thick,
in order to prevent sagging in the middle. A regular microscope
coverslip should never be used as a substitute, as it will yield
incorrect volumes, often varying with each count.
Most bright field microscopes come with or can be easily fitted
with a dark field stop. If your microscope does not have the disk
supplied, try taping an ordinary dime into the filter holder below
the condenser, and moving the condenser to a position where light
can move in a ring around the coin. For many microscopes, this is
an economical method of producing a dark field microscope.
Alternatively, copy the image of a darkfield disk, size to
fit your filter holder and cut it out of cardboard, construction
paper or aluminum foil. Place it or tape it in the filter holder,
or directly onto the condenser. The important feature is to keep
the light moving in a cone around the periphery of the lens, and
not through the center of the lens.
Plastic plates are preferred for this purpose, since the plastic
has a more uniform thickness than ordinary glass petri plates.
Special glass plates are available with ground optically flat
glass in the bottom, but these are both expensive and fragile.
Return to Table of Contents
Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org