VM8054 Veterinary Histology

Exercise 2

Microscopes and Specimen Preparation

Author: Dr. Thomas Caceci
Where the telescope ends, the microscope begins. Which of the two has the grander view?

—Victor Hugo (1802-1885)
Les Miserables (1862)

Objectives for this Exercise
Appendix: Electron Microscopes


It's impossible to overestimate the importance of the microscope as a basic tool in the life sciences. Microscopes of many types are used in clinical diagnosis, teaching, and research. It's safe to say that you will probably use a microscope at least once in every day of your professional life. It's important that you learn the basics of how this instrument works, and how to use it properly.


The Compound Light Microscope

The "basic" instrument is the compound light microscope. Early microscopes (for example, the first ones made by Anton Van Leeuwenhoek in the 17th Century) had only one lens, and were "simple" ones; but all modern instruments have at least two lenses, and thus are "compound." The basic operating principle of the compound microscope is that the image formed by the first lens is magnified by the second, thus providing a much greater magnification than could be attained using only one.


Parts of the Microscope

The compound light microscope has several major parts, and these are common to all instruments, regardless of make or model. A schematic diagram of a "generic" microscope (left) shows these and their relationship to each other. The body (sometimes called the stand) holds the rest of the parts, and in some cases it incorporates filters and/or photographic equipment. The stage is the movable part that sticks out from the body, and onto which the slide is placed. In most modern instruments the stage moves up and down to achieve focus, and the lenses remain fixed in place, though this is not always the case; in some (especially older scopes) the stage is fixed and lenses move. Below the stage is the illumination system, which consists of a light source and a substage condenser. In some instruments the light source is located at the back and a mirror reflects illumination into the condenser (as shown); in others the illuminator is built into the base of the stand.

The condenser is a set of lenses which take the diffuse illumination from the light source and refract it into a coherent and relatively narrow beam. If the specimen is to be uniformly illuminated, and to be given maximum brightness, the diffuse light from the light source has to be concentrated, otherwise much of its intensity will be wasted by lighting the bottom of the stage and not the specimen. Incorporated into the substage condenser assembly is an iris diaphragm. There may be a second diaphragm (called the field diaphragm) placed between the light source and the condenser; this is sometimes omitted from low-priced scopes.

There's a great tendency on the part of beginning microscopy students to close these diaphragms down, in order to limit the light entering the condenser, but this should be avoided. BOTH THE CONDENSER and FIELD DIAPHRAGMS SHOULD BE FULLY OPEN WHEN USING THE MICROSCOPE. Stopping them down will result in greatly decreased resolution (see below for the reason why) and you may not be able to see your subject. More will be said about resolution and proper use later.

Modern microscopes usually mount anywhere from three to five objective lenses in a rotating turret called the nosepiece. These are the lenses which form the primary image of the object, and which provide most of the magnification. More than any other consideration, the quality of these lenses determines the quality of the microscope, and the more expensive instruments put most of their price into the glass. The objectives will be inscribed with a set of numbers, including their magnification, and a number (typically less than 1.0, but occasionally 1.2 or 1.3) called the numerical aperture (NA). The NA is of great importance in the overall resolution of the lens. The higher the NA, the better the resolution, as we shall see later on.


Resolution and Magnification

The primary image formed when the specimen is placed at the front focal point of the objective lens passes through the tube of the microscope. In older instruments, this was quite literally a tube, and straight up and down. Nowadays, the "tube" is in fact a prism, which reflects light internally and allows the use of angled eyepieces, which are far more comfortable to look through than the old up-and-down tube was. Nevertheless, the older term is still used to denote the optical path of the primary image, and the "tube length" has been standardized on modern instruments to a total of 160 mm. The primary image is secondarily magnified by the ocular lenses, which sit in the eyepieces.

THE FINAL MAGNIFICATION OF AN IMAGE IS THE PRODUCT OF THE INDIVIDUAL MAGNIFICATIONS OF THE OBJECTIVE AND THE OCULAR LENSES:

MAGfinal= MAGobj X MAGocl

The most important consideration in selecting a microscope is its resolution. Resolution is the ability of an instrument to discriminate between two points on a specimen. In the best grades of light microscopes, it's about 0.2 microns (mm). If two distinct points are separated by this distance or more, they can be resolved as such.

The formula for the resolution of a microscope is:

R = / 2 NA

Where is the wavelength of the illumination and NA is the numerical aperture of the objective lens. You should note several things about this equation. First of all, it should be obvious that the higher the NA, the better the resolution will be, because R will be smaller. Secondly, it should also be obvious that the shorter the wavelength the better the resolution. Achieving high resolution is a matter of getting the NA as high as possible and the illumination wavelength as low as possible. Note also that magnification has nothing to do with resolution. You do not get better resolution at high power, unless the NA of the lens is higher.


Effect of Illumination on Resolution

Wavelength has been mentioned, and this brings us to the second point about proper use of the light microscope.

THE ILLUMINATION SYSTEM SHOULD BE TURNED UP TO FULL BRIGHTNESS, and THE BLUE FILTER SHOULD ALWAYS BE USED.

Resist the tendency to turn the illumination down, and to leave the filter out of the instrument. The usual reason students do this is that the light is "too bright." But turning the illumination intensity down affects more than brightness: it also affects resolution, and degrades it. The explanation lies in the nature of the illumination system's bulb and the way it produces light.

Virtually all light microscopes use incandescent tungsten filament bulbs to produce light. These are simply small versions of the ones used in homes. They function by passing a current through a tungsten wire, and heating it. As in any light bulb, the hot filament glows. But the glow is indiscriminate: since heat and light are really the same thing, electromagnetically speaking, the filament emits wavelengths from the far infrared (i.e., heat) to the violet, and therefore encompasses most of the visible spectrum. In the visible range of light, the longest wavelengths are those at the red end of the spectrum, and those of the blue/violet end are the shortest.

Now, when the current flowing through the wire is reduced, the emission spectrum shifts towards the longer wavelengths: proportionally more infrared red and yellow wavelengths are produced than are blue ones. This shift causes a loss of resolution, because of the relationship expressed in the equation given above. To achieve maximum performance the filament should be white-hot, and the blue filter inserted to screen out most of the red and yellow long-wavelength light. In addition to increasing the resolution, the blue filter is necessary for proper rendition of "normal" colors with most biological stains, especially hematoxylin/eosin.


Controlling Illumination Level Without Affecting Resolution

The proper way to reduce illumination intensity without affecting resolution is to use neutral density filters. These are sold in photographic supply stores in varying sizes for a few dollars, and are rated in "stops," like a camera lens. A "one-stop ND filter" will reduce the light coming through by 50% without affecting wavelength. A pair of filters (a "one-stop" and a "half-stop") will provide a wide range of intensity control and still allow you to get the performance from your microscope for which you paid.

To use your instrument effectively, take the following steps:

  1. Turn the light all the way on.
  2. Insert the blue filter.
  3. Place a slide on the stage, and focus it as best you can with the low power lens.
  4. Close down the illuminator diaphragm about half-way, until you see a circle of light.
  5. Move the condenser assembly up and down until the edges of the diaphragm are sharp and clear.
  6. Open the condenser diaphragm all the way to fully illuminate the field; if there is a coarse graininess to the field background, move the condenser down slightly to eliminate it.

The condenser diaphragm should be open all the way, all the time. Generally, you'll move the condenser up for the high power lenses and down for the lower power ones.


Specimen Preparation for Light Microscopy

What you're actually looking at on the stage of your microscope is a section, a slice of wax- or plastic-embedded tissue. Tissue selected for microscopy is removed from an animal and fixed by immersion in (usually) formaldehyde. Fixation is necessary to prevent deterioration from microbial attack and autolysis. Following this step, the fixed tissue is washed, dehydrated in a series of increasingly concentrated solutions of ethanol, and embedded in wax. The wax impregnated block of tissue is chucked into the holder of a microtome, a device which (as the name implies) cuts very thin slices off the block. "Very thin" in this case means somewhere on the order of 2 to 7 mm thick. You can see that this is, for all intents and purposes, two-dimensional, even though it's cut from a three-dimensional object.

The section is collected on a glass slide, and the wax removed by dissolving it in xylene. Water is then replaced by rehydration (back down through the alcohol series) and the tissue is stained. Staining is needed for contrast, because unstained tissue has much the same refractive index as glass, and would otherwise be invisible. The stain imparts colors to the tissue, permitting differentiation of structures. The most common stain is hematoxylin/eosin, which is really a combination of two dyes. Hematoxylin comes from the Peruvian logwood tree (Hematoxylin campechianum), and has an affinity for acidic materials. It binds to nucleic acids, and stains nuclei well. Eosin, the other component, is a dye derived from coal tar, and originally used as a fabric dye. Its name comes from "eos," the Greek word for "dawn," and the dye has a rosy hue like the sun at dawn on a clear morning (Homer sings of the "...rosy-fingered dawn..." in the Iliad and the Odyssey). There are thousands of other stains, but only a few dozen are commonly used. In the course of lab exercises, as new stains are mentioned, their properties and staining affinities will be discussed.

After staining has been completed, the now colored section is again dehydrated (back up through the alcohol series, like a party-goer on a Saturday night) and finally covered with a glass coverslip cemented in place with a clear material such as Canada Balsam or "Permount." It's dried and ready to examine in the microscope.

To see some examples of specific stains, click here.


Lab Exercise List