​Sarah Craig

Pre-Lab Questions

1. What is the source of energy for an electron microscope?
The source of energy for an electron microscope is a stream of electrons emitted. This is unlike the compound microscope, where the source of energy is light. With electrons as the source for illumination, a much greater magnification and resolution is achieved. Transmission electron microscopes transmit electrons through a thinly sliced sample while scanning electron microscopes create a three dimensional image. (Council-Garcia)

2. What type of light microscope is used to view large objects such as flowers?
A stereo microscope is used to view large objects such as flowers. This is because the stereo microscope uses reflected light to create an image. A magnified image is formed when the lenses gather and focus light that bounced off of the specimen. Therefore, the specimen does not have to be transparent as it does when working with the compound microscope, when light is transmitted through the specimen. The use of reflected light and the fact that the stereo microscope has two eye pieces instead of one creates a three dimensional image, thus the term stereo microscope. Because the specimen does not have to be thinly sliced and the image created is three dimensional, this microscope is best suited for larger objects.

3. What is the difference between transmitted light and reflected light?
Transmitted light and reflected light are very different from each other. A compound microscope works with light passing through a very thinly sliced specimen. Because the light passes through the specimen, it is called transmitted light. A stereo microscope works with light that has bounced off of the specimen, where it is then gathered in two lenses to make a three dimensional image. Because the light bounces off of the specimen, it is called reflected light.

4. Why is reflected light used with a stereo microscope?
Reflected light is used with a stereo microscope because it allows for there to be a three dimensional image and an opaque specimen. The disadvantages of transmitted light are that the specimen has to be translucent in order to observe it and that it can’t have too much depth to it. However, because light is reflected upon the specimen from a variety of angles (instead of just straight on as compared to the light transmission microscope), a three dimensional image is achieved.

Post –Lab Questions

Page 5

1. If an ocular lens has a magnifying power of 15X and the objective lens is 10X, what is the total magnification?
The total magnification is 150X. This is because one multiplies the magnifying power of the objective times the ocular lens. 15 x 10 = 150X

2. Identify the parts of the microscope with the following functions:
a. lens that further magnifies the image formed by the objective lens
The lens that further magnifies the image formed by the objective lens is the ocular lens. The ocular lens is also called the eyepiece, and it is where one is able to observe the magnified image of the specimen.
b. concentrates the light before it passes through the specimen
The condenser concentrates the light before it passes through the specimen. Positioned right below the stage, it condenses the light into a focused beam to be passed through the hole in the stage. An objective lens from 20X upward will need a condenser in order to properly view the specimen because the subject has to be illuminated evenly. If it is not, the image may be coarse, dark, or lacking in detail.
c. gives the microscope a firm, steady support
The base of the microscope gives itself a firm, steady support. The stage, on the other hand, gives the slide that the specimen is placed on (at least if it is a specimen small enough to be placed on a slide) proper support. The stage clips hold the specimen in place while viewing.
d. regulates how much light goes through the specimen
The iris diaphragm regulates how much light goes through the specimen. It functions similarly to the way the human iris functions. In some models of microscopes, the iris diaphragm may be a wheel with different sized holes to regulate the amount of light passing through. In the microscopes used during the lab, a small lever was used to dilate and contract the iris diaphragm. The iris diaphragm is needed to regulate light because cellular structures may be nearly transparent, and the less light there is the more contrast there is between the cellular structures and the background.
e. adjustment knob that gives the microscopist more precise control over the position of the lens
The adjustment knob that gives the microscopist more precise control over the position of the lens is the fine adjustment knob. While the coarse adjustment knob is larger, placed next to the body of the microscope, and  is used for more general focus of the microscope, the fine adjustment knob is smaller and used to focus on the finer details.
f. source of light
The illuminator, which is located directly below the condenser, is the source of light in the microscope. The specific source of light in the microscopes used during the lab was a light bulb, which was powered electrically and turned on and off using a switch. Some microscopes have a mirror instead. However, this does not create as precise an image as a light bulb because it could be maladjusted, throwing off the image, or dust could collect on it, causing dark spots a microscopist could mistake for a part of the specimen.
g. projects a magnified image of the specimen just beneath the ocular
The objective lens projects a magnified image of the specimen just beneath the ocular. The ocular lens then allows the on to observe the image produced by the objective lens.

Page 6-7

1. Using these observations, what is the relationship between working distance and magnification?
There is an inverse relationship between working distance and magnification; as magnification increases working distance decreases. Working distance is defined as the space between the objective lens and the specimen. It makes sense that as working distance increases, magnification decreases because the object is simply being viewed from farther away. Also, when the 4X lens was used to view the “e,” for example, the working distance appeared to be around an inch in length. However, when the 40X lens was used the working distance appeared to be around a centimeter in length.

2. Using these observations, what is the relationship between field of view and magnification?
There is an inverse relationship between field of view and magnification. The field of view is defined as how much of the specimen one can see. When magnification increases, one is zooming in upon a smaller and smaller area of the specimen in order to observe it in greater detail. Thus, as magnification increases the field of view decreases. When observing the letter e, the entire e could fit in the field of view when the total magnification was 40X. When it was 100X, however, the entire e could not fit in the field of view.

3. Using these observations, what is the relationship between depth of field and magnification?
There is an inverse relationship between depth of field and magnification. Depth of field is defined as the vertical depth of an image that remains in sharp focus under a particular magnification. The depth of field decreases because as magnification increases the structures of the specimen become comparatively farther and farther apart; it is like when somebody is able to focus on a water bottle and the wall behind it collectively if they hold the water bottle at arms length, but if they move the water bottle too close to their eyes they have to choose on focusing on the water bottle or the wall behind it. In the experiment, all of the threads were in focus at 40X total magnification. However, when the total magnification was set to 100X, only one string was able to be focused on at a time.

Page 8

1. If chloroplasts are typical of plant cells, explain why they are not present in onion cells.
Chloroplasts are not present in onion cells. This is because chloroplasts are organelles found in plant cells (as well as other eukaryotic organisms) to conduct photosynthesis. Onion cells do not need to carry out photosynthesis because an onion is a root found underground, where there is no sun to absorb radiant energy from, therefore photosynthesis cannot be carried out. However, the top part of the onion plant, the leaves that stick out of the ground, contain chloroplasts in order to carry out photosynthesis.

2. Your textbook describes many different types of cell organelles. Why do you think these numerous organelles are not visible in your stained slide of the onion epidermis?
Numerous organelles were unable to be seen in the experiment because they were simply to small to be seen with the light microscope. The acetocarmine was able to stain the chromosomes in the nucleus, which was the only organelle that could be seen because it was the largest organelle in the onion cell. An electron micrograph would have to be used in order to observe the other organelles in the onion cell.

Page 9

1. As you moved the specimen to the right of the stage, did the image move in the same direction?
As the specimen was moved to the right of the stage, the image moved in the same direction as well. This is because stereo microscopes often contain inverting prisms located between the objective lens and the ocular to flip the image, which is inverted by the objective lens, back to its original state.

2. Was this observation true of the compound microscope?
This observation was not true of the compound microscope. The compound microscope inverted the image and flipped it backwards, so that when the specimen was moved left it appeared to move right in the microscope, and when it was moved up it appeared to move down when looking through the ocular lens. This is because the objective lens in the compound microscope is a positive lens, it is thicker at the center than at the edge. This causes the image to be inverted, but it is necessary in order to achieve the highest magnification the most efficiently. This image is then relayed to the ocular lens, which is then observed by the researcher.

3. Did the image of the specimen appear in the same position as the specimen on the stage?
The image of the specimen appeared in the same position as the specimen on the stage. It was not inverted, as the image in the compound microscope was. This is because of the inverting prisms placed in many of the stereo microscopes which flips back the image the objective lens inverts to its normal position.

4. Was this observation true of the compound microscope?
This observation was not true of the compound microscope. Compound microscopes do not often contain inverting prisms in order to flip the image that the positive objective lens originally distorts.

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1. Based on your observations of the electron micrographs, which type of electron microscope is best for observing internal cell structure?
The transmission electron microscope is best for observing internal cell structure. This is because it functions similarly to the compound microscope in the sense that one is able to observe the inside of the cell. The specimen must be thinly sliced in order for the electron micrograph to work, and the electrons pass, or transmit, through it, thus displaying the internal structure of the cell.

2. Which electron microscope is best for observing surface features and overall appearance?
The scanning electron microscope is best for observing surface features and overall appearance. This is because it functions similarly to a stereo microscope. A stereo microscope detects reflected light coming from a specimen while a scanning electron microscope scans the specimen with a stream of electrons and forms an image by sensing the way the electrons bounce back from it. Thus, only the external environment of the cell is revealed. However, the images produced are three dimensional and extremely detailed.

3. Identify the appropriate microscope or microscopes.
a. uses light as an energy source
Both the compound and stereo microscopes use light as an energy source. The compound microscope uses transmitted light while the stereo microscope uses reflected light.
b. image is neither reversed nor inverted
While it is common for stereo microscopes and electron microscopes to not have reversed nor inverted images because they have inverting prisms, inverting prisms can be placed in any microscope, including compound ones. The compound microscopes used in the experiment did not contain these prisms, however.
c. may view living material
All types of microscopes except for electron microscopes could view living material because light does not harm them while the preparation process for an electron microscope is harmful to cells.
d. largest depth of field
The stereo microscope has the largest depth of field because it often has the lowest magnification of all types of microscopes and it relies upon reflected light, which gives it a three dimensional image.
e. must use fixed or dead material
Electron microscopes must use fixed or dead material because the specimens observed are often so small any movement would cause drastic distortion in the image.
f. uses a beam of electrons as a source of illumination
The transmission electron microscope sends a beam of electrons through a specimen in order to illuminate it.
g. can observe the internal structure of a nucleus
A transmission electron microscope can observe the internal structure of a nucleus because it is able to observe minute details as electrons pass through a cell. A compound microscope that uses the transmission of light may also do this, but not as precisely because the magnification is not as high as the electron microscope.
h. highest resolving power
The electron microscope has the highest resolving power because it detects small changes in the path of electrons rather than relying upon a simple lens.
 
Sources
http://biology.unm.edu/ccouncil/Biology_203/Summaries/Microscopes.htm

http://www.micrographia.com/tutoria/micbasic/micbpt02/micb0200.htm

http://www.microscopyu.com/articles/stereomicroscopy/stereointro.html

http://www.nightlase.com.au/education/optics/lenses.htm

http://www.colorado.edu/physics/phys1230/phys1230_fa01/topic39.html