The microscope is at tool that allows us to see the structures and tissues that comprise organisms in very fine detail. Often it is possible to understand function of a structure on the basis of its microscopic morphology. For example, seeing the orientation of muscle cells in the body wall of an earthworm allows one to understand how it moves and burrows. You will also see strong evidence in support of the cell theory.
The total magnification that you see can be calculated. Find the magnification imprinted on the ring around the ocular lens--it is probably 10x. Then find the magnification imprinted on the objective lens that you are using--it is probably either 4x, 10x, or 40x. Multiply the magnification of the ocular lens times that of the objective lens; this is the total magnification that you see. The quality of the microscope is in its objective lenses. If they are awful, magnifying what they see by the ocular lens will make no improvement. It is important that you do not get the objective lenses wet. Dry them promptly if water or stain is transferred from the slide to the lens.
Letter e. Hold this slide up to the light and you will see a small piece of typewriter paper under the middle of the cover slip. It has an e typed on it. Start with the low power objective (the shortest) in place. Center this piece of paper in the light coming through the stage of your microscope and focus the e with the large, coarse focusing knob. You will see immediately that the e is upside down and backwards, just the reverse of the way you oriented it on the stage. The microscope lenses are responsible for this reversal. With the e centered, raise the magnification to medium power. The focus is nearly correct; recenter the e and sharpen the focus, again with the coarse focusing knob. Now raise the magnification to high power. Adjust the focus with the smaller, fine focusing knob only. Never use the coarse focusing knob, when you are using high power, because you can easily crunch the objective through the slide�a costly mistake. Always begin the process at low power and then raise the magnification of the objective lenses. You will have noticed that the higher the magnification of the objective lens, the smaller and dimmer the field of view. On high power, you will need to maximize the brightness of the light source and regulate the iris diaphragm.
Bolting silk. This is a small square of fine silk fabric. Examine it with low power, and then raise the magnification. On high power use the iris diaphragm to improve the resolution. You see much more detail when the light is passed through a very tiny aperture. Remember this fact when you are looking at nearly transparent organisms in future labs. With the fine focussing knob raise and lower the stage to get a sense of the thickness of the fabric.
Millimeter rule. Put the transparent millimeter rule on the stage of the microscope. Observe the length of the diameter in millimenters of the field of view at each available magnification. Fill in the following chart:
| objective | magnification | diameter (mm) |
| low power | ||
| medium power | ||
| high power |
The Cell Theory
In 1665, Robert Hooke used the word cell, meaning little rooms, to describe the small cavities separated by walls in cork, which is the bark of a tree. Matthias Schleiden, a botanist, published (1838) his conclusion that all plants are made of cells; in the following year (1839), Theodor Schwann extended the observation to animal tissues and proposed a cellular basis of all life. The pathologist Rudolf Virchow added an important extension of the theory in 1858 that all living cells arise from pre-existing living cells; there is no spontaneous creation of cells from nonliving matter. As you look with the microscope at tissues representing the 5 or 6 kingdoms of organisms, you will confirm the validity of the cell theory. It seems odd that something so obvious to us with modern technology had to be discovered and proposed as a theory.
Animal cells--cheek. Take a clean microscope slide, and place a small drop of iodine stain on it. Use a sterile wooden stick, and rub the tip gently across the inside of you cheek. Cells that were about to slough off have been collected on the end of the stick. Swirl the end of the stick in the iodine on the slide to transfer and stain the cells. Place a cover slip on the preparation, and view with the microscope; remember to start with the low power objective in place. The cells are irregular; you can see flat surfaces where they met other cells in your cheek. The boundary of a cell, the plasma membrane, is so thin you can only see where the cell ends. The nucleus is stained an orangish brown and is near the center in each cell. Tiny dots on the surface are probably bacteria.
Plant cells--onion. Place a drop of iodine stain on a clean microscope slide. Take a layer of onion, and use a scalpel tip to peel the very thin layer of cells that line its inner curvature. The peel held between your finger tip and the scalpel should be like extremely thin cellophane. Place the peel in the iodine on the slide, and put a cover slip over it. View the preparation with the microscope. These cells are quite regular in shape, and there is a thick cell wall surrounding them. Since the cells secreted the wall material, the plasma membrane of the cell is inside the wall. Nuclei are stained as in the animal cells, but they are at the side of the cell. The center of each plant cell is occupied by a large transparent organelle called the central vacuole. You cannot see it, but you can see the result of its presence: the nucleus is off to the side.
Cell dimensions: Use the information in the chart above, in which you determined the diameter of the field of view at each magnification, to measure approximately the average diameter of a cheek cell and the length and width of an onion cell.
Protista. If cultures of the single-celled organisms are available, put a drop on a new slide, and gently place a cover slip on top. Examine with the microscope.
What exactly can you view under the microscope at different magnifications? Microscope field of view changes as magnification changes. In short, as magnification increases, the field of view decreases. When looking through a high power compound microscope it can be difficult to determine what you will see through the eyepieces at different magnifications.
The images below were created to help you determine how much of the field of view will be occupied by certain samples at different magnifications. The following four samples are illustrated to show the microscope field of view at 200x, 400x, 600x and 1000x magnification:
- Black squares = microbes (2-8µm)
- Blue rectangles = nanoplankton (10-20µm)
- Red circles = red blood cells (6-8µm)
- Orange rectangles = Large organisms (100µm)
It is a common misconception that at 1000x magnification items will be visible under the microscope that are not visible at 400x. This is not typically true - you can view the same samples at 400x that you will view at 1000x, they will just take up a greater portion of the microscope's field of view at 1000x.
If you are having trouble determining what your microscope field of view will be at a certain magnification, contact Microscope World and we will be happy to help.
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| Magnifying PowerA compound microscope has two sets of lenses. The lens you look through is called the ocular. The lens near the specimen being examined is called the objective. The objective lens is one of three or four lenses located on a rotating turret above the stage, and that vary in magnifying power. The lowest power is called the low power objective (LP), and the highest power is the high power objective (HP). You can determine the magnifying power of the combination of the two lenses by multiplying the magnifying power of the ocular by the magnifying power of the objective that you are using. For example, if the magnifying power of the ocular is 10 (written 10X) and the magnifying power of an objective is 4 (4X), the magnifying power of that lens combination is 40X. |
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Field of View (FOV)The field of view is the maximum area visible through the lenses of a microscope, and it is represented by a diameter. To determine the diameter of your field of view, place a transparent metric ruler under the low power (LP) objective of a microscope. Focus the microscope on the scale of the ruler, and measure the diameter of the field of vision in millimeters. Record this number.
When you are viewing an object under high power, it is sometimes not possible to determine the field of view directly. The higher the power of magnification, the smaller the field of view.
The diameter of the field of view under high power can be calculated using the following equation:
For example, if you determine that your field of view is 2.5 mm in diameter using a 10X ocular and 4X objective, you will be able to determine what the field of view will be with the high power objective by using the above formula. For this example, we will designate the high power objective as 40X.
Estimating the Size of the Specimen Under ObservationObjects observed with microscopes are often too small to be measured conveniently in millimeters. Because you are using a scale in millimeters, it is necessary to convert your measurement to micrometers. Remember that 1 μm = 0.001 mm.
To estimate the size of an object seen with a microscope, first estimate what fraction of the diameter of the field of vision that the object occupies. Then multiply the diameter you calculated in micrometers by that fraction. For example, if the field of vision’s diameter is 400 μm and the object’s estimated length is about one-tenth of that diameter, multiply the diameter by one-tenth to find the object’s length.