DENDROLOGY
THE COMPOUND AND DISSECTING MICROSCOPES
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Obiectives
The most important tools in your study of
wood anatomy will be the compound and the dissecting microscopes.
Although
for most of you this exercise will be review, you should take the time
to read about the care and use of these microscopes before beginning
the
other exercises. You will obtain much more from all the exercises that
follow if you first familiarize yourself with the "tools of the
trade".
Indeed, this is the only way to proceed.
General Description
The conventional compound
microscope used in biology consists of a light source, a condenser
lens with an adjustable aperture, specimen slide for trans-illumination
specimens, an objective lens and an ocular lens plus appropriate
mechanical
fixtures for focusing and mounting specimens. This
exercise purposesly uses an unfamiliear microscope to test your
understanding
of the basic components and functions of all microscopes. Once
you
understand the functional reason of each microscope component, it won't
matter who manufactured the microscope, you will be able to evaluate
any
microscope by your criteria! So shoddy microscope sales people
won't
be able to sell you shoddy microscopes!
The illumination
system is designed to fill both the condenser and objective
apertures,
if Kohler illumination is maintained. Objectives are designed to
produce
a real image of the specimen of a quality which approaches the
theoretical
limit imposed by the wave-nature of light. Oculars are designed to
produce
(a) a virtual image which may be observed visually or (b) a real image
which can be projected onto a screen or photographic plate. Efficient
use
of the microscope and interpretation of images required consideration
of
the following factors:
-
1. Magnification
-
2. Resolving power
-
3. Depth of field
-
4. Contrast
-
5. Practical errors in lens design and
manufacture
Magnification,
when
the image is viewed directly, is the ratio of the tangent of the angle
subtended at the eye by the virtual image compared to the tangent of
the
angle subtended at the eye by the object when viewed at 25 cm from the
eye. This value is approximately the product of the nominal objective
and
ocular magnifications; e.g. 40X objective with 10X ocular = 400 total
magnification.
In the case of a projected image, magnification is simply the ratio of
the linear dimensions of the image to the corresponding dimensions of
the
object. If the image is projected a distance of 25 cm from the ocular
the
magnification will be, as above, the product of the nominal
magnifications
of the objective and ocular. At other projection distances this
value
is multiplied by the projection distance in cm divided by 25. Critical
use demands the selection of a magnification such that the finest
details
of the image (determined by the resolving power of the microscope) can
be resolved by the retina or photographic plate. Magnification which
exceeds
this is known as "empty magnification" since it reveals no additional
detail.
Objectives are usually designed so that persons with normal vision can
see all that can be seen by using a 10X or a 15X ocular.
Resolving
power
(STEP By STEP) is defined as the
least distance between points in the object (d) that can be
distinguished
in the image. For a perfect lens with a circular aperture, the
resolving
power is given (according to Rayleigh' s criterion for
"distinguishability")
by:
d = 0.6 l
/ (n* sin(q))
where d is resolvable distance given in
the same units of length as l
; l is
the
wavelength of light; n is the index of refraction of the material in
which
the specimen is embedded and q
is the half angle of the cone of light accepted by the lens when this
angle
is measured at the specimen.
This expression for resolving power
applies
to any kind of lens (telescope, camera, microscope, eye, etc.) but it
assumes
certain conditions of illumination that are not automatically met when
a lens is used as a microscope objective. To satisfy these conditions
the
cone of light illuminating the specimen must equal the cone of light
accepted
by the lens.
For a lens used at a single set of
object
and image distances (as in a microscope) the quantity (n sin q)
is a constant known as the numerical aperture (N.A.) of the lens., Most
microscope objectives have the values of the N.A. engraved on the lens
barrel along with the focal length and/or the magnification of the
lens.
The resolving power defines a limiting accuracy with which lateral
dimensions
of a specimen can be measured.
Depth of field, D, is the
thickness
of the specimen, measured along the optical axis, which is in sharp
focus
for any one set of conditions. It is given approximately by the formula
D = 2r*cot(q).
2r is the diameter of the Airy disc, see below, and
q is as defined above. Note
that
for practical purposes d of the Abbe equation is equal to 2r of
the
depth of field equation. As the aperture angle q,
increases, the depth of field decreases to a limiting value of about
0.5
microns with the oil immersion objective .
Depth can be measured with the compound
light microscope. Knowing the depth of field associated with particular
lenses, which is a small fraction of the thickness of a typical
biological
specimen, makes possible the "opt ical sectioning" of specimens.
CAUTION --
Beginners
often restrict the aperture of the microscope by closing the
condenser of the microscope because of a subjective impression that
they
can see more. They also tend to increase the apparent depth of field by
forcing the eye to accommodate for failure to use the fine focusing
adjustment.
These "techniques" do not enhance available information and often
result
in eye strain and headaches.
Contrast depends on the signal
to
noise ratio and upon the physiology of vision. In this case the signal
is the image of interest and the noise is background of light
associated
with out-of-focus images, light scattered by dirt on optical surfaces,
etc. Detectable contrast depends upon the ability of the eye (or of any
other measuring devise) to make fine distinction in intensity and color
on resolvable areas of the image. In general the greater the noise the
greater the signal intensity required to produce a detectable image.
Texts
in human physiology should be consulted on this point. Nearly all
histological
and cytological techniques have been developed to compensate for the
fact
that living cells in visible light display very little contrast
.
Practical errors. While the best
of the modern microscope lenses approximate very closely the
theoretical
limits of a "perfect lens," most introduce compromises in design and
defects
in manufacture. By convention the resulting errors are classified as
follows:
-
1. Airy disc, circle of
confusion.
It is an inherent property of waves that the image of a point cannot
itself
be a point, but must be a small circle, the Airy disc. Thus a point
cannot
be distinguished from a small circle which is called the circle of
confusion.
Any two points which fall within the radius (not the diameter) of the
circle
of confusion cannot be resolved as separate points. The Airy disc is
the
circle of confusion of the image. For the normal eye at normal viewing
distance from a print or a photograph, the circle of confusion is
between
0.1 and 0.2 mm and any print with circles a confusion less than this
will
appear sharp.
-
2. Chromatic aberration.
This
aberration is the inability of the lens to bring rays of different
wavelength
to the same focus. Modern Achromatic lenses are designed to correct
this
aberration for blue and yellow wavelengths of visible light. The more
expensive
Apochromatic lenses are designed to correct this aberration for all
visible
light wavelengths.
-
3. Spherical aberration.
This
aberration arises from the inability of a lens to bring those rays
close
to the axis to the same focus as those more distant from it, and
effectively
limits numerical apertures to a very small angle. Aplanatic lenses are
designed to correct this aberration, thus making higher numerical
apertures
possible.
-
4. Astigmatism.
This aberration
arises from imperfection in the radial symmetry of the lens such that
the
image is distorted along one diameter of the lens. Most good lenses
have
sufficient quality that this aberration is minimal.
The above errors are interrelated so that
typically when one defect is present other types of defects will also
be
found. The effect of these errors upon the quality of an image must be
understood before the results of observations and measurements can be
interpreted.
The best lens (and most expensive) one can buy is an Aplanatic
Apochromatic
lens. Most good quality research microscopes are equipped with such
lenses.
Specif ic Description
Two types of light microscopes (as
distinct
from electron microscopes) will be employed during our laboratory
sessions.
Most often we' ll use standard binocular compound microscopes; those
available
in this laboratory have a built-in light source, and provide
magnifications
of 100, 400, or 1000 diameters, depending upon which of the three
objective
lenses of the revolving nosepiece is in place under the main tube. The
ocular (eyepiece) magnifies 10X, the objective lenses 10, 40, and 100X,
respectively, hence the overall magnification is the product of the
magnification
provided by the ocular and that provided by the objective lens.
The 10X magnifying objective lens
should
be used first, and only then if additional magnification is required
should
one shift to the 40X (high-dry) objective lens. These lenses are
essentially
parfocal, which means that only little focusing is necessary in
shifting
from one objective lens to another. The 100X objective is an oil
immersion
lens , which means that a clear image can be obtained only if a special
type of oil (immersion oil) is used to bridge the space between the
bottom
element of this lens and the cover glass and between the top of the
condenser
lens and the bottom of the specimen slide. If you have not used one
previously,
please ask the laboratory instructor to show you how. After using
the oil immersion lens it is essential that both the condenser
and
objective lenses be thoroughly cleaned!
CAUTIONARY
NOTES:
-
1. When you return the
microscope
to the cabinet be certain that either of the two lower-magnification
objective
lenses is in place, and that these are at least a centimeter above the
stage.
-
2. Unless the surfaces of the
cover
glass, the bottom lens of the objective, and the condenser lens are
absolutely
clean the image will be blurred; therefore it is a good plan to wipe
these
surfaces with lens paper before (and after) using the microscope.
-
3. The light microscopes
you will
be using are equipped with phase optics. Phase contrast
microscopy
is a useful technique for enhancing the contrast of unstained
biological
material. These optics utilize a special phase ring, inserted below the
condenser, in combination with an objective containing a phase annulus.
(This 40X objective has a red ring about its barrel and should not be
used
for bright field microscopy.) Since you will be using bright
field
optics almost exclusively in this laboratory it is necessary to learn
how
to properly remove the phase ring from the condenser. This is
accomplished
by grasping the phase ring insert at the bottom on the condenser with
thumb
and forefinger while holding the condenser firmly with the other hand.
Gently pull the phase ring insert out of the condenser making sure that
the condenser stays intact.
REMEMBER : THESE
OPTICS ARE DELICATE AND EXPENSIVE. CAUTION SHOULD BE TAKEN NEVER TO
DROP
ANY OF THE MICROSCOPE COMPONENTS OR TO PLACE ANY LENS SYSTEM IN SUCH A
WAY THAT ITS SURFACE CAN BE SCRATCHED.
To insert the phase ring insert after
you
have finished the current exercise reverse the above procedure. BE SURE
TO REPLACE THE PHASE RING INSERT BEFORE RETURNING THE MICROSCOPE TO THE
STORAGE CABINET.
Kohler illumination procedure:
To obtain the optimum resolution of the
light
microscope precise control of the light path should be obtained. The
accepted
method for this is termed Kohler illumination after August Kohler who
developed
the theory and procedure in 1894. This procedure should be preliminary
to each exercise session of this laboratory. Click on each link
to
obtain additional help.
NOTE : When
the
objective lens is changed it is necessary to adjust the condenser
diaphragm
to 80% illumination as in steps 10-13.
BE SURE TO
FOLLOW
EARNST ABBE'S ADVICE TO AVOID EYE STRAIN AND SUBSEQUENT HEADACHES!
The other type of microscope that we'
ll
use occasionally is the dissecting binocular or stereoscopic
microscope.
Illumination units which can be used either above or below the stage
are
available. These microscopes (or at least some of them) have zoom
facilities,
which provide a continuous range of magnification. This type of
microscope
is used at relatively low magnification for dissection, and to show the
three-dimensional nature of what is observed.
Exercises:
1. Obtain a
compound
light microscope and review its component parts.
2. Work through the Kohler illumination
procedure until you can establish this in less than 1 minute.
3. Use the stage micrometer to
calibrate
the mechanical stage micrometer for your microscope. The stage
micrometer is a vernier scale.
Verify your calibration
at all magnifications.
Materials
1. Olympus
Student Microscope Instruction Manuals
2. Fake
field diaphragms and lens paper
3. Stage
micrometer