One of the oldest and most basic tools of the biologist is the microscope. In order to fully understand microscopy you must consider two things: resolution and contrast. Resolution is the ability to distinguish between two points. In the case of microscopy, resolution is determined by the design of the lenses and is inversely related to the wavelength of radiation used by the microscope. Contrast is the difference in intensity between different parts of an image and is enhanced by using dyes or stains on the tissue to be examined or by adjusting the settings on the microscope itself. Tissue must be carefully prepared to enhance contrast, yet maintain resolution. You will use a conventional binocular light microscope to look at your own cheek cells and a specialized light microscope that uses phase contrast optics to examine the living cells of your cultures.

Binocular Light Microscope

The microscope that you are probably most familiar with is the light microscope. As the name implies, visible light is passed through the specimen that has been embedded, sectioned and stained on a glass slide. Embedding a specimen in a solid substance such as paraffin wax supports the tissue so that it can be thinly sectioned or sliced. Fresh specimens can also be observed using the light microscope without embedding but usually staining is required to enhance contrast or allow identification of specific components of the specimen.

Familiarize yourself with the parts of the microscope. Light microscopes with two eyepieces or ocular lenses are called binocular; a monocular microscope has a single eyepiece. The ocular lens on the microscopes you are using magnify ten times or 10 x. Further magnification is obtained through the use of several objective lenses located on a revolving nosepiece. The objective lens is the most important component of the microscope and magnifies an image from 4x or 10x (scanning objectives) to 100x (oil immersion objective). Total magnification of an image takes into account both the ocular and objective magnification. The fine and coarse focus knobs on both sides of the base of the microscope adjust the distance between the stage and the objectives. The coarse adjustment knob should only be used with the scanning and low power objectives. Using the coarse focus knob with the higher power objectives risks ramming the objective through the slide and damaging both the slide and the very expensive objective. The fine focus knob is used with all objectives. The microscopes you are using are parfocal. Once an area of interest is identified with the scanning or low power objective, switching to a higher power objective requires only a minimal adjustment with the fine focus knob in a parfocal microscope. The amount of light passing through the slide is controlled via the condenser-iris diaphragm. Adjust the diaphragm to reduce glare and allow maximum contrast and better viewing of the specimen. Your microscopes have either a mechanical stage or fixed stage. A mechanical stage has controls located beneath the stage platform that move the stage in x and y planes. Microscopes with a fixed stage require manipulation of the slide by a very steady hand.

Here are some pointers for using a light microscope.

1. Care and Maintenance: Handle the microscope with care. Carry the scope by placing one hand under the base and the other hand on the arm above the stage. Never touch the glass surface of lenses with your hands. If the image appears cloudy or smudged, clean the lenses with lens paper only. Do not clean with any fluid other than lens cleaner. Even water can damage the delicate lenses.

2. Look at the slide using your eye first. Notice the shape of the section and any contrast in the tissue identified as differences in staining intensity. This will allow you to more easily orient yourself while examining the slide using the microscope. But remember, the microscope image is reversed and upside-down.

3. Place the slide on the stage, specimen side up. Position the area you want to view directly over the illuminated center of the stage. Viewing from the side, move the coarse adjustment so that the slide and the low power (either 4x or 10x) objective are nearly touching. Use caution here. Do not ram the slide through the objective! Setting the slide and objective close together means that you will only need to focus by moving the adjustment knob. Be sure you know which direction to turn the knob before proceeding.

4. Remember to adjust the light coming through the condenser by adjusting the diaphragm. Viewing should be comfortable for you, without too much light and glare.

5. Once you have an area of interest in the field of view, change to the objective with the next highest magnification making sure that the longer lens does not hit either the stage or slide. If you have not touched the stage or the focus knobs, the image should come easily into focus using only the fine focus knob. Readjust the condenser and diaphragm.

6. Once you are through with the slide, return to the lowest power objective and remove the slide. Never remove or change slides while a high power objective is in place.

7. When you are through, make sure the lowest power objective is in place and turn the lamp off. Wrap the cord around the base of the microscope and store on the side bench.

Phase Contrast Inverted Microscope:

The phase contrast microscope is a light microscope with a special set of optics that enhance contrast by altering or defracting the path of light coming through the specimen. Since living cells are transparent (bags of water), they are difficult to see with regular optics such as those in the student microscopes. Phase contrast optics are routinely used to study living material, such as cells and tissues in culture.

You will also notice another difference between the student microscopes and the phase contrast scope: the objectives are under the stage and the light source is on top. This is an inverted scope. Most vessels or containers used to grow cells in vitro are too thick to fit between the objective and stage of a regular microscope. In addition, you have to focus on cells on the bottom of the culture vessel all the way through the lid and intervening space. Without a special long focal length objective, this is impossible. To circumvent this, turn the basic design of the scope upside down. The objectives are now located right next to the surface of the vessel containing the living cells. I will bring the inverted scope into lab but either Shea or I will help you use the scope. So, don't touch this scope unless we are helping you!

Slides:

Fresh Tissue: The tissues in the prepared slides that you will examine today have been chemically fixed, processed, embedded in paraffin wax and thinly sectioned using a microtome. However, it is fairly easy to observe fresh tissue prepared as a thin smear of cells. You will prepare a wet mount of the squamous epithelial cells lining the inside of your cheek.

Scrape the inside of your cheek using the flat blade of a toothpick. Smear the scrapings onto a clean, dry slide. Add a drop of water to the smear and carefully cover with a coverslip. Blot off any excess water around the coverslip with absorbent paper such as a Kimwipe. Look at your cells under the microscope. How difficult is it to view a transparent specimen?

Now prepare a second slide but instead of adding a drop of water, add a drop of the stain methylene blue. Notice how staining the cells enhances contrast and allows you to see cellular membranes better. Always use a coverslip when preparing a wet mount to protect the objective lens from the water or stain.

Prepared Slides: Choose one of the prepared slides of tissue and examine it with the microscope. The microscopic study of prepared pieces of tissues is called Histology. Histological slides are used to examine the normal structure of tissues and cells and are used to detect disease or pathological conditions such as cancer. In addition, special staining techniques can provide a great deal of information about the function of a tissue and what kind of proteins exist in the tissue or within the cells of a tissue. The tissue has to undergo a series of steps called processing before a slide can be made. To prevent the tissue from decaying and keep the cells, their contents (nucleus, cytoplasm, etc.) and the components outside the cells, or extracellular matrix, intact, the tissue has to be treated with a preservative. The preserved tissue is then embedded in wax that supports the tissue for cutting. The tissue is cut into thin sections about one cell thick so that light can pass through the tissue and cellular components can be seen with the microscope. Finally, the slide with the section of tissue is stained with dyes to enhance the contrast between different cells or components of the tissue.

There are four basic types of tissues in the body. If you have a slide of the skin you will see two types: epithelial and connective. Epithelial tissue is lining tissue and will be found all over the body. Epithelium always has a "free edge", i.e. one side of the tissue is open, while the other side is next to connective tissue. Epithelial cells are closely opposed, or side-by-side, and control the transport of substances into the body or organ. Examples of where you find epithelial tissue include the skin, the lining of all blood vessels, the lining of the intestinal tract, and the surface of all organs. Most epithelial tissue has to replaced quite often; the cells are constantly abraded, die or are exposed to influences outside of the body. This means that epithelial cells are constantly multiplying by the process called mitosis to replace those cells that are lost. For a cell to make a copy of itself it must go through it's life cycle and replicate it's DNA prior to dividing into two new daughter cells. If there is a mistake in the DNA or in the copying of the DNA, that mistake or mutation will be copied, and each new daughter cell will contain the mutation. The faster a cell goes through it's life cycle, the greater the risk of accumulating mutations. Cancer is caused by mutations in specific regions of DNA (called tumor suppressor genes and oncogenes). Because epithelial tissue must be constantly replaced, tumors or cancers of epithelial origin are common. More than one million new cases of skin cancer are diagnosed annually, making it the most common form of cancer. Cancers of epithelial origin are commonly found in the lung, colon, breast, and prostate.

The other type of tissue you can clearly see in a histological slide of the skin is connective tissue. Connective tissue can bind other tissues together, can be fluid such as blood, and can be supportive such as bone and cartilage. Unlike epithelial tissue, connective tissue is never exposed to the outside environment. Like epithelial tissue, however, connective tissue is found everywhere in the body. Cancers of connective tissue include sarcomas and cancers involving cells of the blood.

The other two types of tissues of the body include nervous and muscle. Examine slides of nervous tissue and muscle tissue to get an appreciation of the different types of cells all of the tissue types contain. When you design your cytotoxicity experiment, decide which type of tissue you would like to examine. For example, are you interested in the effect of caffeine on the brain? You would choose nervous tissue as your experimental model. How about the effect of alcohol on the body? Choose tissue of the liver. Realize, however, that organs are composed of multiple tissue types and you will have a mixture of tissues and cell types in your culture model.