HISTOLOGY - BIOL 0509
LAB INTRODUCTION II
In examining slides of sectioned tissues with the light and electron
microscopes, one should be aware that some of the structures observed may not
be real, that is, they may be artifacts. Artifacts are the result of changes in
a tissues structure or the addition of "new structures" that are
usually the result of fixation, dehydration, embedding, sectioning, staining,
and/or section mounting techniques. Types of artifacts that are commonly
encountered are listed below.
REVIEW OF BASIC ARTIFACTS.
1. Swelling of tissue components2. Shrinkage of tissue components
Artifact types 1 and 2 are the result of poor fixation and/or dehydration
techniques, i.e. osmolarity of the fixative may be wrong, pH may be wrong, too
short a fixation time was used, and/or dehydration of the tissue was too rapid.
Swelling and shrinkage can sometimes result in rupture of membranes. This sort
of damage is particularly evident at the ultrastructural level.
3. wrinkles in section4. tears in section5. air bubbles6. dust
Artifact types 3, 4, 5 and 6 are usually the result of poor sectioning
technique or poor technique during mounting of sections. In some cases, poor
fixation and/or embedding can be responsible for tears or wrinkles in sections
by modifying fixed tissue consistency such that the tissue cannot be sectioned
without its tearing or wrinkling.
7. stain precipitate
This sort of artifact can result from use of old stain solutions, use of
unfiltered stain solutions, mistakes made during preparation of the stain, or
poor staining technique.
THE LIGHT MICROSCOPE:
We have gone over the use of your light microscopes during lab and your
laboratory handout has instructions that describe how to set-up your microscope
for viewing such that "proper Kohler illumination" is established. In
setting up "proper Kohler illumination" you are adjusting the
microscope illuminatin such that 1) all light passes through the centers of the
lenses and 2) the light beam is set at its smallest useful diameter thus
eliminating reflections of light off of internal components of the microscope.
The end result of your adjustments for "proper Kohler
illumination" is that you are able to view tissue sections at the highest
possible resolution that your microscope is capable of. This means that you
will be able to see the maximum amount of structure within the tissue that can
be seen with your microscopes.
The objective and ocular lenses are responsible for magnifying the image of
the specimen being viewed.
Total magnification = Objective magnification X ocular magnification
So for 10X objective and 10X ocular,
Total magnification = 10 X 10 = 100X (this means that the image being viewed
will appear to be 100 times its actual size).
For a 40X objective and 10X ocular,
Total magnification = 10 X 40 = 400X
Magnification is not of much value unless resolving power is high.
Resolution is a measure of the ability to distinguish 2 points as two
points. That is, when viewing something through a microscope, how close
together can two points be placed such that you can still see some space
We can't say much more about resolution without a few words about numerical
aperture (n.a. or NA). The value for numerical aperture measures to what extent
the light that passes through a specimen is spread out over and collected by
the objective lens. The light that passes through the specimen contains
information about what the specimen looks like, that is, about its structure.
If we consider the cone of light that originates from the specimen and
enters the objective lens, Numerical aperture can be defined as,
NA = n . sin m ( . is the multiplication
n = refractive index of substance between the specimen and the objective
lens (usually air, n = 1.0; quartz, n = 1.5; glass, n= about 1.5; water, n =
m = 1/2 the aperture angle (also called the
semiangle). The aperture angle is the angle described by the cone of light that
enters the objective lens after passing through the specimen. This angle will
depend on the curvature of the lens and also on how close the objective lens is
to the specimen when it is in focus.
So, for an objective with an aperture angle of 120 o with air between
specimen and objective lens,
NA = 1 . sin 60o = sin 60 o = 0.87
If oil with refractive index of 1.5 is used between the objective lens and
NA = 1.5 . sin 60 o = 1.5 (.87) = 1.31
Now, numerical aperture is important because it allows us to calculate the
resolving power of the objective. Remember, that's what we really were
interested in determining initially.
R = 0.61 . ( l / NA)
R = resolution of the objectivel = wavelength
of light (average value for white light ~ 550 nm).
NA = numerical aperture
So, for air situation,
R = 0.61 . 550nm/.87 = 386 nm = 0.000000386 m = 0.386
For oil immersion,
R = 0.61 . 550nm/1.31 = 256 nm = 0.000000256 m = 0.26
Thus, one can see that higher resolution is possible if the substance lying
between the specimen and the objective lens has a refractive index as close as
possible to that of the lens itself without exceeding the lens' refractive
It is important to realize that while both the ocular and objective lenses
are responsible for the final magnification on a compound microscope, ONLY the
objective lens is responsible for resolution.
The discussion above should demonstrate the importance of resolution. By
using the appropriate lenses I can create extremely high magnifications, say
5000X with the light microscope. However, magnification tells us nothing about
resolution. If resolution of objective lens is 0.3
mm, no matter how much I magnify the specimen
image, the resolution will remain the same. At 5000X, I will still only be able
to resolve points a minimum of 0.3 mm apart.
Points that are closer together may be visible, but the will be superimposed
and blurred, appearing as one fuzzy point. So nothing has been gained by
increased magnification. The amount of visible information available at 5000X
is the same as at lower magnifications of 1500X.
Using the mathematical equations given above and the values for maximum
numerical aperture attainable with the lenses of a light microscope it can be
shown that the maximum useful magnification on a light microscope is between
1000X and 1500X. Higher magnification is possible, but resolution will not
In addition to numerical aperture and an incorrect light path, there are 3
major lens defects that can affect the quality of the image in a compound
microscope and result in decreased resolution.
A. Chromatic aberration - caused by spherical a lens bringing different
wavelengths of light into focus at different levels. Thus, you get multiple
images superimposed on top of each other. This defect is corrected in
B. Spherical aberration - optical quality of image lessened due to the fact
that the center of lens has slightly different qualities than the edges. Both
spherical and chromatic aberration are corrected in apochromatic objectives.
C. Curvature of field - causes image to be in focus centrally, but out of
focus peripherally or vice versa. This defect is corrected in planar
The type of objective, magnification, numerical aperture, and even the best
cover slip thickness to use on your slides is listed on the side of an
There are a number of special types of light microscopy that can enhance
certain features of a specimen that is being examined. Some of these are listed
1. Phase contrast microscopy - takes advantage of phase differences in the
light beam that are caused by different refractive indexes of components within
Consider air, n=1.0; water, n=1.3; glass, n=1.5. Light travels fastest
through air and slowest through glass. Thus, if a light beam encounters (at the
same time) three different spaces of equal thickness that are filled with air,
water and glass, the beam will emerge first from the air filled space and last
from the glass filled space. The emerging light beams are said to be out of
phase with each other.
In the phase contrast microscope, the condenser and objectives are specially
made to detect the phase differences of light passing through different
components within a tissue specimen. The construction of the condenser and
objective lenses is such that these phase differences are made visible by
increasing the contrast between light waves of different phase. As a result,
components of cells that are normally of low contrast (clear or nearly clear),
are given higher contrast and, thus, made visible.
2. Polarizing microscopy - A polarizing filter (called the polarizer) is
placed below the condenser and allows only light vibrating in one plane to
reach the condenser. A second polarizing filter (called the analyzer) is placed
between the objective and ocular. If these two filters are oriented such that
their axes of light transmission are perpendicular, no light will pass through
the analyser to the ocular. So nothing will be seen. One use of polarizing
light microscopy is related to the fact that certain crystals found in or
associated with some cells can bend light waves because of their refractive
index. If some of the light waves that have passed through the polarizer are
bent into different planes as they pass through crystalline parts of the
specimen, then some of these light waves will be able to pass through the
analyser even if it is oriented at 90 degrees to the polarizer. This property
of crystals to bend polarized light waves is called birefringency. It is
important in identifying certain crystalline structures in or associated with
3. Interference or Nemarski interference microscopy. - this is another
method utilized to observe structures of different refractive index, but
similar optical density. It is not the same as phase contrast microscopy.
Nemarski interference microscopy requires 2 different light beams that are
recombined after passing through the specimen. Differences in phase between the
two beams are visualized as depth. The result is an image with depth (sort of
3-D). This type of microscopy is particularly useful for viewing living cells.
The function of this instrument is dependent on the fact that an electron
beam has many properties that are similar to a light beam.
In fact, a beam of electrons may be treated as either 1.) a beam of
particles or 2.) as a wave (i.e. like a light wave). As it turns out, both
properties are necessary in order for an electron microscope to work. The fact
that the effective wavelength of an electron beam is very much smaller than
that of the shortest visible light wave makes very high resolution possible
with this instrument (i.e. 5 - 20 A)
Recall that, R = 0.61 . (l/NA)
This means that very high useful magnification is possible since very small
distances between two points can be resolved. The highest magnification
commonly used with the electron microscope is 200,000X. However, higher useful
magnifications are possible.
Suffice it to say, that for the purposes of this course, we can consider the
electron microscope in relatively simple terms. An electron beam is produced by
inducing a high voltage between a cathode (-) and an anode (+). Electromagnets
are used to direct the path of this beam and also to act as magnetic lenses
that are responsible for magnification of the image of the specimen. As the
electron beam passes through the specimen, electrons are either unaffected,
scattered, or absorbed by the tissues of the specimen and various stains
(usually heavy metals) that have been applied to the tissues. The unaffected
electrons and many of the scattered electrons pass through the specimen and
then are focused by magnetic lenses on a fluorescent viewing screen. The number
of electrons hitting various parts of this screen determine how brightly these
parts fluoresce and thus form an image of the specimen on the screen that can
be examined by the person using the scope. In addition, the focused electrons
can be used to expose photographic film from which black and white pictures can
be printed. The photographs produced are actually more useful in interpreting
electron microscope images because they are permanent and of higher contrast
than the fluorescent image.