OBJECTIVES

1. Understand the theory and use of the compound microscope
2. Distinguish the difference between magnification and resolution.
3. View and understand the structures of a typical plant and animal cell.
4. Locate and identify the organelles that are visible with the light microscope.

This laboratory is to help you to understand the function of the bright field microscope and introduce you to the world of microscopic anatomy - studying biology from the perspective of the individual cell. The cell is the basic unit of life. Some cells are free-living, others become more highly specialized and form integrated aggregates, associating together to form more complex organisms. There are cells that look rather "simple" inside, others appear much "busier" and more complex. Some cells have plant-like characteristics, others are animal-like. There is no such thing as a "typical" cell which incorporates and displays all the various internal and external structures. One must examine a number of different cell types to appreciate the great diversity, and the great similarity of form.

Once you have familiarized yourself with the basic operation of the light microscope, review the basic organization of different cells and get a feel for how the cell has become adapted to a variety of biological roles.

I. THE LIGHT MICROSCOPE

For well over a century, the light microscope has been one of the most important instruments available to the biologist. This instrument is used to extend the range of our vision. Ordinarily, humans cannot see objects smaller than 0.1 mm in diameter, but the light microscope renders objects as small as 0.2 microns (1 micron = 10^-3mm) visible to our eye. The modern compound microscope is a precision instrument, designed to perform particular functions in a particular way. When the microscope is used correctly, it will disclose many structures that the beginning student will probably not see. For this reason, do not be surprised or skeptical if at first you fail to see many of the details in plant or animal cell. How much you see depends mostly on how well you adjust the instrument. Below are outlined some of the various parts of the instrument (see fig. 1-1) and their function, as well as brief instructions on how to use the instrument to achieve maximal results.

A. Terms Used in Microscopy

Resolution This refers to the ability of the microscope to distinguish as separate and distinct objects that lie in close proximity. Magnification is not the sole aim of a microscope. There is no upward limit to the magnification of a microscope, but there is a limit to useful magnification. One can have increase in magnification without an increase in visible detail. The basic limit in seeing objects is resolving power, not magnification, and the physical phenomenon of diffraction of light imposes limits on the resolving power. Attaining the maximum resolution depends primarily on the design of the objective lens (see numerical aperture below). An objective lens capable of utilizing a large angular cone of light coming from the specimen will have a higher resolving power than a lens limited to a smaller cone of light. Of course the resolvable detail must be magnified a sufficient amount in order to be seen - hence the relationship between magnification and resolution.

Numerical Aperture This is a measure of the angle of the maximum cone of light that can enter the objective lens. It is expressed by the equation: N.A. = n (sin q), where  n  is the index of refraction of the medium between the object and the objective and q  is one half the angle of the entering cone of illumination. The greater the N.A., the greater the resolving power.

Working distance This is the distance between the front of the objective, when it is focused on the specimen, and the specimen itself. When viewing thin preparations mounted on microscope slides and covered with a cover glass, it is the distance to the top of the cover glass.

Field of view The circular field you see when you look through the ocular lens. The field of view changes in size at different magnifications.

Depth of focus This refers to the thickness of the specimen which may be seen in focus at any one time. As you focus up and down on your specimen, you will notice that only a thin layer is in focus, not the entire thickness. The greater the N.A. and magnification, the thinner is the layer in focus at any one time.

B. Mechanical Components

1. The mechanical stage is the square, horizontal platform that serves as a shelf to support the material to be examined. The hole in the center of the stage serves to let the light from the illuminator and condenser shine upon the specimen. On the surface of the stage there is a special device to securely hold the specimen (i.e., the microscope slide) and, using the two knobs below the right side of the stage, one can move the specimen left and right as well as front and back.

2. There are two focusing knobs found on either side of the support arm. The larger ones are the coarse adjustment and the smaller are for fine focusing. Both of these move the stage up or down, depending on the direction turned, and serve to change the distance between the specimen and the objective lens.

C. Optical Components

1. The light source is located in the base of the microscope, and the light beam passes out through the illuminator lens. Light is controlled by a rotating switch located on top of the microscope base.

2. The light passes into the condenser, which concentrates the light onto the specimen. On the left side of the instrument, beneath the stage, is the condenser focusing knob, and it controls the position of the condenser relative to the specimen. Associated with the condenser is the iris diaphragm, which in your microscope serves to control the amount of illumination reaching the specimen. There is a lever which, when moved back and forth, serves to open and close the iris diaphragm, altering the amount of light passing through. A very common fault in microscopy is to close down the iris diaphragm too far; contrast improves, but there is considerable loss of resolution and other undesirable image artifacts are introduced. You will be instructed in the proper adjustment of this critical component.

3. Immediately above the specimen is the revolving turret which carries the objective lenses. These form the initial magnified image of the specimen and consists basically of a series of lenses of different sizes. There is the "scanning" objective which magnifies 4 times, a low power objective that magnifies 10 times, and a high power objective that magnifies 40 times; the oil immersion objective (probably will not be used in this course) magnifies 100 times. One begins all observations with the lower power objectives and works up to the high power one. They are changed by rotating the turret, usually in a clockwise direction.

4. The lens system next to the eye is called the ocular, and this further magnifies the image and projects it onto the retina of your eye. The ocular lenses (fitted loosely into your microscope) have a magnifying power of 10X. In a binocular microscope, the two ocular lenses can be adjusted to accommodate the distance between the eyes and their individual focusing.

D. Preparation for Viewing

1. Clean the lenses. Dust and dirt will accumulate on the ocular lens, therefore, each time you use a microscope, clean the lenses with lens paper. Also clean the objective lens since other students use the same instrument and may cause water and dirt to soil the objective lenses.

2. Make sure the 4X or 10X objective is in place. Raise the condenser to its upper position. Turn on the illuminator.

3.Place your slide on the stage with the specimen over the condenser lens. Focus the specimen with the coarse focus knobs (the larger ones).

4.With the specimen in focus, lower the condenser until the illuminator is in focus. You will see sharp images of the lint on top of the illuminator. Then move the condenser down very slightly to even out the illumination.

5.Remove the right ocular lens and look down the tube, keeping your eye several inches away. Open and close the iris diaphragm and notice its image down by the objective lens. Adjust this so that almost the entire area of the objective lens is illuminated; this provides optimum definition and resolution for the available lighting. If you close it down excessively, you can see there is increased contrast, but this will reduce resolution and image quality. Try it and see.

6. If you want higher magnification and resolution, swing in the high power objective. The objective lenses are parfocal, meaning the specimen is close to focus from one objective to another. You will need to turn the fine focus knobs slightly. You should not need to use the coarse focus. When you go from the scanning lens to the 10X lens, you may need to use the coarse focus knobs.

7. You must repeat step 5 each time you change objectives!

E. Physical Properties of the Objective Lenses

1. Examine each objective lens and record its magnification and numerical aperture in the table below.

2. Measure the diameter of the field of view of the 10X and 40X lenses by counting the number of microns seen in a stage micrometer, and record the result in the table below. Can you detect any relationship between magnification and field of view?

3. Obtain a slide with three colored threads that have been mounted one on top of another. Use your microscope to determine which color thread is on top and which is on the bottom. TOP: _____________ BOTTOM: _____________.
 

Common Name	Working Distance	Magnification	N.A.
SCANNING LENS	25 - 55 mm
LOW POWER	5 - 10 mm
HIGH POWER	0.15 - 0.6 mm
OIL IMMERSION	0.05 - 0.15 mm

F. Microscopic Dimensions

The metric system is used exclusively in science for making and reporting measurements of weight, volume and distance. The table below explains the various units used in microscopic measurements.
 

Definition	Description
meter (m)	=1,000 millimeters, 9.37 inches
millimeter (mm)	=1,000 µm , or the smallest unit on your ruler
micron or micrometer (µm)	=1,000 nm; animal cells average 50 microns in diam.; the resolving power of the light microscope is 0.2 µm.
nanometer (nm)	=10 Angstroms, used to measure size of large molecules resolving power of electron microscope is 0.1 nm

II. EUKARYOTIC PLANT-LIKE CELLS

A. Onion Epidermis

1. An onion scale is lined on either side by a single layer of cells called an epidermis. Obtain a piece of onion epidermis by bending a piece of onion until it snaps. The two halves will only be attached by the epidermis. Carefully peel off the transparent epidermis from the rest of the onion piece.

2. Lay the peel flat on a clean microscope slide in a drop of acetocarmine, making sure the tissue isn't folded back on itself. Add a coverslip and view under the low power objective.

3. Not all the organelles will be clearly visible. You should see the cell shape, the large central vacuole which is colorless, and the nucleus. Why don't you see the plasma membrane? How about mitochondria, or ribosomes?

4.The nucleus will stain reddish and the cytoplasm will be pink. You should be able to distinguish between the vacuole and cytoplasm. Where is the nucleus located within the cell? Can you see the plasma membrane now?

B. Elodea Leaf

1. Make a wet mount of a small leaf taken from near the tip of an Elodea shoot, placing the upper surface of the leaf uppermost on the slide.

2. Focus the leaf using low power, find a portion where the cells are clearly visible and switch to higher power. Due to the thickness of the cells, you will have to continually focus up and down to see all the parts. This is an example of the concept of the depth of focus. How many cell layers thick is the leaf?

3. The cells of Elodea are considerably more complex than the onion epidermal cells. You should be able to see the cell wall; and inside the cell wall is the cytoplasm, an almost transparent, colorless substance. The nucleus, with a nucleolus, is difficult to see, but it is located near the periphery of the cell. The vacuole occupies the center of the cell. All of the cell membranes are below the resolving power of the light microscope, as is the plasma membrane. The most obvious structures are the chloroplasts, small green bodies located in the cytoplasm.

4. Look closely at the chloroplasts in cells located near the mid-vein of the leaf. You should find cells in which the chloroplasts are moving around the perimeter of the cells. This movement is the result of cytoplasmic streaming or cyclosis. Are the chloroplasts in the same cell all moving in the same direction? Are the chloroplasts within different cells moving in the same direction?

5. Draw and label a typical Elodea cell as it appears under high power.

III. EUKARYOTIC CELLS

A.Amoeba

The Amoeba is a free-living, single cell, and all of the functions and activities we equate with the living state are carried out within this single cell. You may have heard these organism being referred to as "primitive", but after you examine the Amoeba and Paramecium, you may wonder what that term means when used in reference to unicellular organisms.

1. Using the eye dropper, obtain a small sample of the detritus from the bottom of the culture jar, place it on the slide and cover with a coverslip. Examine under low power to be sure you have at least one cell.

2. View the Amoeba under high power. What structural features can you observe? You will probably find the nucleus, food vacuoles, a variety of inclusions (many of these are crystalline waste material), and maybe even a contractile vacuole.

3. A healthy Amoeba is quite active, extending pseudopodia in all directions and creeping along the slide. This movement is accompanied by cytoplasmic streaming. Look carefully at an extending pseudopod; notice that the inner core (endoplasm) of the pseudopod is doing most of the streaming and is surrounded by a more rigid shell of cytoplasm (ectoplasm). When the endoplasm reaches the advancing tip of the pseudopod, what happens? Can you tell if the endoplasm is being "squirted" forward into the pseudopod, or is it being "pulled" forward?

B. Paramecium

This organism is also a single cell with some of the same features as Amoeba. They both move, eat and digest, excrete, remove water with a contractile vacuole, etc., but when you look at them, it seems hard to believe they are similar.

1. Add a small drop of culture water to a clean slide and mix in a drop of methyl cellulose with a toothpick (this viscous substance will slow down paramecia locomotion). Locate them with low power and then switch to high power (you will have to continually move the slide to keep them in the field of view).

2. Try to locate the various internal structures, such as the nucleus (actually there is a macronucleus and an associated micronucleus), food vacuoles, contractile vacuoles (there are two, with radiating canals), and cytoplasmic inclusions. Watch one of the contractile vacuoles for a few minutes. Do you see anything happening? What is the frequency of the event? . Are the two vacuoles operating in synchrony? Notice the large oral groove that tends to accumulate food particles for ingestion.

3. Do you see signs of cytoplasmic streaming? Does it contribute to the locomotion of the animal? If not, what does cytoplasmic streaming do? The entire cell is covered with cilia which beat in a coordinated sequence to propel the organism along. Does the paramecium always swim in the same direction?

C. Human Cheek Epithelial Cells

Unlike Amoeba and Paramecium, the cells of multicellular organisms do not have to perform all the functions of life - there is a "division of labor". As an example of the "simpler" cells of multicellular organisms you will look at cells that line the inside of your cheek. These cells are continually dying and being sloughed off, so you shouldn't mind making this small contribution to science.

1. Using a clean toothpick, gently scrape the inside of your cheek and stir the cells and a little saliva into a small drop of acetocarmine dye on the slide. Add a coverslip and locate a group of flat and spread-out cells under low power.

2. Switch to a higher power for a better examination of the cells. What are their shape?  Do you see: the plasma membrane, a central vacuole, chloroplasts? You should be able to see a nucleus and the nucleolus within it. What is the function of the nucleolus?

3. Draw and label a cheek cell in the space below.