HISTOLOGY - BIOL 0509

LAB INTRODUCTION II

BASIC MICROSCOPY

ARTIFACTS:

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 components
2. 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 section
4. tears in section
5. air bubbles
6. 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 between them?

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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 symbol)

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 = 1.3)

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 the specimen,

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 objective
l = 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 mm

For oil immersion,

R = 0.61 . 550nm/1.31 = 256 nm = 0.000000256 m = 0.26 mm

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 index.

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 improve.

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.

These are,

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 achromatic objectives.

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 objectives.

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 objective.

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 below.

1. Phase contrast microscopy - takes advantage of phase differences in the light beam that are caused by different refractive indexes of components within a tissue.

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 cells.


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.

ELECTRON MICROSCOPY

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.