Introduction to the light microscope lab report

Microscopes and Cells

PRE-LAB ASSIGNMENT:

Students are expected to watch this video (which is also posted on Blackboard): https://www.youtube.com/watch?v= - b3Eejf4rDQ

AND read pages 1 to 4 before coming to the lab to complete the experiments.

Print this entire lab packet and bring it to the laboratory. Please provide a FULL lab report for this experiment following the “Lab Report Guidelines”.

Objectives:

After completing this laboratory assignment, students will be able to:

· Identify the parts of a compound microscope.

· Properly use a compound microscope for biological studies.

· Describe the features of specific cells.

· Determine characteristics shared by all cells studied.

Microscopes and Lenses:

Although cells vary in size, they’re generally quite small. For instance, the diameter of a typical human red blood cell is about eight micrometers (0.008 millimeters). To give you some context, the head of a pin is about one millimeter in diameter, so about 125 red blood cells could be lined up in a row across the head of a pin. With a few exceptions, individual cells cannot be seen with the naked eye, so scientists must instead use microscopes (micro- = “small”; -scope = “to look at”) to study them. A microscope is an instrument that magnifies objects otherwise too small to be seen, producing an image in which the object appears larger. Most photographs of cells are taken using a microscope, and these pictures can also be called micrographs. From the definition above, it might sound like a microscope is just a kind of magnifying glass. In fact, magnifying glasses do qualify as microscopes; since they have just one lens, they are called simple microscopes. The fancier instruments that we typically think of as microscopes are compound microscopes, meaning they have multiple lenses. Because of the way these lenses are arranged, they can bend light to produce a much more magnified image than that of a magnifying glass.

In a compound microscope with two lenses, the arrangement of the lenses has an interesting consequence: the orientation of the image you see is flipped in relation to the actual object you’re examining. For example, if you were looking at a piece of newsprint with the letter “e” on it, the image you saw through the microscope would be “ə." More complex compound microscopes may not produce an inverted image because they include an additional lens that “re-inverts” the image back to its normal state.

What separates a basic microscope from a powerful machine used in a research lab? Two parameters are especially important in microscopy: magnification and resolution.

· Magnification is a measure of how much larger a microscope (or set of lenses within a microscope) causes an object to appear. For instance, the light microscopes typically used in high schools and colleges magnify up to about 400 times actual size. So, something that was 1 mm wide in real life would be 400 mm wide in the microscope image.

· The resolution of a microscope or a lens is the smallest distance by which two points can be separated and still be distinguished as separate objects. The smaller this value, the higher the resolving power of the microscope and the better the clarity and detail of the image. If two bacterial cells were very close together on a slide, they might look like a single, blurry dot on a microscope with low resolving power, but could be told apart as separate on a microscope with high resolving power.

Both magnification and resolution are important if you want a clear picture of something very tiny. For example, if a microscope has high magnification but low resolution, all you’ll get is a bigger version of a blurry image. Different types of microscopes differ in their magnification and resolution.

Light Microscopes:

Most student microscopes are classified as light microscopes. In a light microscope, visible light passes through the specimen (the biological sample you are looking at) and is bent through the lens system, allowing the user to see a magnified image. A benefit of light microscopy is that it can often be performed on living cells, so it’s possible to watch cells carrying out their normal behaviors (e.g., migrating or dividing) under the microscope.

Student lab microscopes tend to be brightfield microscopes, meaning that visible light is passed through the sample and used to form an image directly, without any modifications. Slightly more sophisticated forms of light microscopy use optical tricks to enhance contrast, making details of cells and tissues easier to see.

Another type of light microscopy is fluorescence microscopy, which is used to image samples that fluoresce (absorb one wavelength of light and emit another). Light of one wavelength is used to excite the fluorescent molecules, and the light of a different wavelength that they emit is collected and used to form a picture. In most cases, the part of a cell or tissue that we want to look at isn't naturally fluorescent, and instead must be labeled with a fluorescent dye or tag before it goes on the microscope.

A confocal microscope is a specialized kind of fluorescence microscope that uses a laser to excite a thin layer of the sample and collects only the emitted light coming from the target layer, producing a sharp image without interference from fluorescent molecules in the surrounding layers.

Electron Microscopes:

Some cutting-edge types of light microscopy (beyond the techniques we discussed above) can produce very high-resolution images. However, if you want to see something very tiny at very high resolution, you may want to use a different, tried-and-true technique: electron microscopy.

Electron microscopes differ from light microscopes in that they produce an image of a specimen by using a beam of electrons rather than a beam of light. Electrons have a much shorter wavelength than visible light, and this allows electron microscopes to produce higher-resolution images than standard light microscopes. Electron microscopes can be used to examine not just whole cells, but also the subcellular structures such as organelles and compartments within them.

One limitation, however, is that electron microscopy samples must be placed under a vacuum in electron microscopy (and typically are prepared via an extensive fixation process). This means that live cells cannot be imaged.

In the image above, you can compare how Salmonella bacteria look in a light micrograph (left) versus an image taken with an electron microscope (right). The bacteria show up as tiny purple dots in the light microscope image, whereas in the electron micrograph, you can clearly see their shape and surface texture, as well as details of the human cells they’re trying to invade.

There are two major types of electron microscopy. In scanning electron microscopy (SEM), a beam of electrons moves back and forth across the surface of a cell or tissue, creating a detailed image of the 3D surface. This type of microscopy was used to take the image of the Salmonella bacteria shown at right, above.

In transmission electron microscopy (TEM), in contrast, the sample is cut into extremely thin slices (for instance, using a diamond cutting edge) before imaging, and the electron beam passes through the slice rather than skimming over its surface. TEM is often used to obtain detailed images of the internal structures of cells.

Electron microscopes are significantly bulkier and more expensive than standard light microscopes, perhaps not surprisingly given the subatomic particles they have to handle!

(Above information was adapted from Khan Academy: https://www.khanacademy.org/science/biology/structure-of-a-cell/introduction-to-cells/a/microscopy)

Please Note: Treat these microscopes with the greatest care!

Exercise 1: Basic Microscope Techniques

In this exercise, you will learn to use the microscope to examine a recognizable object, a slide of the letter e and crossed threads. Recall that microscopes vary, so you may have to omit steps that refer to features not available on your microscope. Practice adjusting your microscope to become proficient in locating a specimen, focusing clearly, and adjusting the light for the best contrast.

1. Obtain the following materials:

· Clear ruler  Blank slides  2 prepared slides: letter “e” & crossed thread

· Lens paper  Kimwipes®  Dropper bottle with distilled water

· Coverslips

2. Clean microscope lenses.

a. Each time you use the microscope, you should begin by cleaning the lenses. Using lens paper moistened with a drop of distilled water, wipe the ocular, objective, and condenser lenses. Wipe them again with a piece of dry lens paper.

3. Adjust the focus on your microscope:

a. Plug your microscope into the outlet.

b. Turn on the light. Adjust the light intensity to mid-range (if your microscope has that feature).

c. Rotate the 4X objective into position using the revolving nosepiece ring, not the objective itself.

d. Obtain the letter slide and wipe it with a Kimwipe® tissue.

i. Each time you study a prepared slide, you should first wipe it clean.

e. Place the letter slide on the stage and center it over the stage opening.

Please Note: Slides should be placed on and removed from the stage only when the 4X objective is in place. Removing a slide when the higher objectives are in position may scratch the lenses.

f. Look through the ocular and bring the letter into rough focus by slowly focusing upward using the coarse adjustments.

g. For binocular microscopes, looking through the oculars, move the oculars until you see only one image of the letter e. In this position, the oculars should be aligned with your pupils. In the margin of your lab paper, make a note of the interpupillary distance on the scale between the oculars.

h. Raise the condenser to its highest position, and fully close the iris diaphragm.

i. Looking through the ocular, slowly lower the condenser just until the graininess disappears. Slowly open the iris diaphragm just until the entire field of view is illuminated. This is the correct position for both the condenser and the iris diaphragm.

j. Rotate the 10X objective into position.

k. Look through the ocular and slowly focus upward with the coarse adjustment knob until the image is in rough focus. Sharpen the focus using the fine adjustment knob.

l. You can increase or decrease the contrast by adjusting the iris diaphragm opening.

m. Move the slide slowly to the right. In what direction does the image in the ocular move? _ Left _

n. Is the image in the ocular inverted relative to the specimen on stage? __Yes__

o. Center the specimen in the field of view; then rotate the 40X objective into position while watching from the side.

p. After the 40X objective is in place, focus using the fine adjustment knob.

q. The distance between the specimen and the objective lens is called the working distance. Is this distance greater with the 40X or the 10X objective? ___10X__

r. Compute the total magnification of the specimen being viewed. To do so, multiply the magnification of the ocular lens by that of the objective lens.

i. What is the total magnification of the letter as the microscope is now set? _400x__

Analysis Question 1
What would be the total magnification if the ocular was 20X and the objective was 100X (oil immersion)?

This is the magnification achieved by the best light microscopes.

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Total Magnification of a microscope can be calculated by multiplying the magnification of ocular and objective. So, 20 × 100 = 2000X

Therefore the total magnification will be 2000X.

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4. Measure the diameter of the field of view. Once you determined the size of the field of view for any combination of ocular and objective lenses, you can determine the size of any structure within that field. a. Rotate the 4X objective into position and remove the letter slide.

b. Place a clear ruler on the stage, and focus on its edge.

c. The distance between two lines on the ruler is 1 mm. What is the diameter (mm) of the field of view?

d. Convert this measurement to micrometers, a more commonly used unit of measurement in microscopy (1 mm=1,000 µm).

e. Measure the diameter for the field of view for the 10X and 40X objectives, and enter all three in the spaces below to be used for future reference.

4X = _ 4000Nm______ 10=__ 1000Nm_______ 40=__ 500Nm________

Analysis Question 2
What is the relationship between the size of the field of view and magnification?

As the magnification increases the field of view decreases (The field of view specifies how much of a specimen is visble in the eyepiece. Field of view and magnification are inversely related: the higher the magnification, the narrower the field of view, and vice-versa).

5. Determine spatial relationships. The depth of field is the thickness of the specimen that may be seen in focus at one time. Because the depth of focus is very short in the compound microscope, focus up and down to clearly view all the planes of a specimen.

a. Rotate the 4X objective into position and remove the ruler. Obtain the slide of crossed threads, wipe it with a Kim wipe, and place the slide on the stage. Center the slide so that the region where the two threads cross is in the center of the stage opening.

b. Focus on the region where the threads cross. Are both threads in focus at the same time? Yes

c. Rotate the 10X objective into position and focus on the cross. Are both threads in focus at the same time? Yes

Analysis Question 3
Does the 4X or the 10X objective have a shorter depth of field?

The 10X have a shorter depth of field because is zooms in more than the 4X that gives a larger depth of field.

d. Focus upward (move the stage up) with the coarse adjustment until both threads are just out of focus. Slowly focus down using the fine adjustment. Which thread comes into focus first? Is this thread lying under or over the other thread? Blue Over Red

e. Rotate the 40X objective into position and slowly focus up and down, using the fine adjustment only. Does the 10X or the 40X objective have a shorter depth of field? 40X has the shorter depth

Exercise 2: Viewing Prepared Slides

1. Using the Basic Microscope Techniques from Exercise 1, view a prepared slide of an Amoeba and one of a Paramecium.

a. Draw your field of view of each objective for each slide.

2. View a prepared slide of a cheek cell.

a. View the cells using the 4X and 40X objectives.

b. Draw your field of view for the 4X and 40X objectives.

c. Can you identify any organelles? If so, which ones? What is the function of the identified organelle(s)?

Exercise 3: Preparing a Slide of Elodea 1. Prepare a wet mount.

a. Remove a leaf of Elodea.

b. Place the leaf onto a clean slide.

c. Add a drop of water to the leaf.

d. Place a coverslip over the leaf.

2. View the cells using the 4X and 40X objectives.

3. Draw your field of view for the 4X and 40X objectives.

4. Can you identify any organelles? If so, which ones? What is the function of the identified organelle(s)?

Exercise 4: Proper Storage of the Microscope 1. Rotate the 4X objective into position.

2. Remove the slide from the stage.

3. Lower the stage all the way down.

4. Unplug the cord and wrap it around the base of the microscope.

5. Replace the dust cover.

6. Return the microscope to the cabinet using two hands; one hand should hold the arm, and the other should support the base.

7. These steps should be following every time you store the microscope.

8. Dispose of the Elodea slide according to the instructor’s directions.

9. Return all other materials to their original location.

Note: The results section of the lab report should include images from your field of view as well as answers to the questions asked throughout the exercises and the analysis questions