Which part of the microscope should not be used when using a high power objective lens?

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).

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Short introduction to focusing a compound microscope
Reason 1: protect the objectives from damage
Reason 2: danger of loss of focus
Reason 3: faster to find the focus
Reason 4: Different specimen thickness causes loss of focus
When you are (!) allowed to use coarse focus for high power

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.

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.

With the exception of stereo microscopes (which generally only have one focus knob), compound microscopes have a coarse and a fine focus. The coarse focus knob raises and lowers the stage quickly, the fine focus knob does this slowly. As a matter of fact, you will not see the movement of the stage. The coarse focus knob should only be used with the 4x low power objective in place. The fine focus know can be used with all objectives, including the 4x, but the effect will not be visible with this latter one (you will have to turn the knob many times, which is inconvenient).

Short introduction to focusing a compound microscope

Using the coarse focus, lower the stage. Be careful that you turn it into the correct direction. Usually this means that you have to turn the focus towards you.
Rotate the low power 4x into position.
Use the coarse focus to raise the stage all the way up until it blocks. The objective at the highest position, the 4x objective should of course not touch the slide.
Close the condenser and looking through the eyepiece lower the stage using the coarse focus until you see a clear image. Only rotate the coarse focus into one direction and stop when you see a clear image. If you are all the way down and still have not found a clear image, then you are either looking at a place of the slide that contains no specimen or the concentration is too low. Often dust, air bubbles etc. can help you find the focus.
Only when in focus, switch to the next higher objective and then only use the fine focus. Re-center the slide as well./li>
If you want to change the slide, you have to start again at point 1.

There are several reasons why you should use the coarse focus knob only with the 4x objective and not with the high power objectives.

Reason 1: protect the objectives from damage

If you focus the high power objectives with the coarse focus knob, then you risk slamming the objective into the slide, risking the damage of both. Some high power objectives are spring-loaded, with a front-part that retracts when touched by the slide. This is a protective mechanism.

Reason 2: danger of loss of focus

The coarse focus raises and lowers the stage to quickly, that it is inevitable that you lose the focus when you use high-power objectives. One purpose of focusing is to look at the different layers of a specimen. By turning the fine focus, you can “section” through the specimen. The coarse focus does not allow you to do this.

Reason 3: faster to find the focus

Last, it is simply faster to start with the 4x objective and then work your way up. The 4x objective also allows you to center the slide better, because it gives you a better overview. The 4x also has the greatest depth of field and this means that the whole specimen (top and bottom parts) are in focus. If you start with a high power objective and then start focusing, then you don’t know if you are focusing at the actual specimen or on the dust which is on the top of the cover glass.

Reason 4: Different specimen thickness causes loss of focus

If you look at a specimen under high power and then directly exchange the slide, you run the risk of losing focus again. The thickness of the mounting medium is not always the same and also the position of the specimen in the mounting medium can be different. Using the coarse (and even the fine focus) will take longer than starting again with the 4x objective and the coarse focus.

When you are (!) allowed to use coarse focus for high power

Some better microscopes have a focus lock lever. This lever blocks the stage at the pre-determined position. You set up the focus starting at 4xy and work your way up to 100x oil. You then engage the lever and the position will be remembered. You can then use the coarse focus to lower the stage, insert a new slide, and without changing the objective use the coarse focus (!) to raise it up again until the stage blocks (this position was remembered). This is an efficient method that allows you to quickly observe one slide at high magnification after another. Why not just change the slide by pulling it out horizontally? The reason is that if there is immersion oil on the slide, then objective should be lowered into the oil from the top and not from the side, to prevent smearing of the immersion oil all over the place.

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.