Name ________________________
Biology 211 Laboratory Fall 2001
CELL STRUCTURE: WEEK 5
OBJECTIVES:
1. To review how to use the light microscope to visualize cell structure.
2. Learn to adjust the light intensity, magnification, and contrast in a light microscope.
3. Observe cells under bright field and phase contrast optics.
4. Learn how fluorescent dyes can be used to visualize particular organelles.
5. Be able to identify cell components and organelles from electron micrographs.
BACKGROUND READING: Read Campbell 5/e Chapter 7.
ASSIGNMENT: Next week in lab there will be a quiz on the microscope and cell structure. Be able to demonstrate how to set up and focus a light microscope and locate particular cell structures either this week or next week. Be able to identify cellular organelles under the light microscope and in electron micrographs. Be able to distinguish between bright field optics, phase contrast optics, the fluorescence microscope, and the transmission electron microscope.
INTRODUCTION: There are two main types of cells, prokaryotic and eukaryotic .
Eukaryotic cells have a membrane bound nucleus; prokaryotic cells do not. The compound light microscope can be used to observe the presence of a nucleus in animal cells and vacuoles and chloroplasts in plant cells. However, the light microscope cannot distinguish most of the various organelles in the cells. Use of fluorescent dyes helps identify specific structures in the cell that can be visualized using a fluorescence microscope. Most of our knowledge of the fine structure of cells comes from the electron microscope.
PROCEDURES:
1. Review the use of the light microscope (bright field microscopy)

If you need to review the use of the light microscope, consult the available handout. Each student should set up and adjust their own light microscope. a. Prepare a wet mount slide of a small piece of a young Elodea leaf in fresh water.
Elodea is a multicellular eukaryotic plant found in freshwater ponds and lakes. b. Examine the slide using low power, focusing on the leaf surface. c. Select a cell with numerous chloroplasts for further study, and switch to high power. d. Carefully focus on the side and end walls of the cell. The chloroplasts appear to be only along the sides of the cell because the large, membrane bound central vacuole pushes the cytoplasm against the cell walls.
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e. Can you locate the cell nucleus? It may be hidden by the chloroplasts, but when visible, it appears as a faint gray lump on one side of the cell. f. Can you detect cytoplasmic streaming (movement) of the chloroplasts in this cell or a neighboring cell?
Demonstrate your ability to use the light microscope.
Once you have finished observing some of the Elodea cells, adjust the focus and the light intensity as well as you can. Have your instructor observe your slide and initial your handout. __________
Test and observe what happens when a drop of 10% saline solution (hypertonic saline) is added to the slide. ___________________________________________
___________________________________________________________________
___________________________________________________________________
2. Demonstration: An inverted microscope with phase contrast optics.

Your instructor will demonstrate the use of the inverted phase contrast microscope to groups of 3-4 students at a time. Bring your wet mount slide of the
Elodea to the demonstration.
Key differences of this microscope as compared with standard light microscope:

Inverted (objective lenses below, light entering from above).

No attached stage on this particular microscope-- mainly used for viewing cell culture plates.

Can be used for bright field microscopy--we will look at Elodea slide.

Also equipped for phase contrast microscopy. These optics allow you to view the distinct regions of cells or tissues without stains or visible pigments.

Use of phase requires use of special ocular lenses to calibrate the microscope by allowing you to visualize the phase rings for adjustment. We will view the
Elodea and unstained HeLa (cervical cancer) cells with phase contrast.
Observations
Elodea under phase contrast Elodea under bright field
HeLa under bright field HeLa under phase contrast
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Demonstration: The fluorescence microscope and fluorescent images.
One method of improving the contrast between organelles is by fluorescence microscopy. Fluorescence is the property of a molecule illuminated with one wavelength
(color) of light to emit a second, longer wavelength of lower energy.
A number of fluorescent probes have been developed that can be used to label specific organelles, surface structures and even ions that move into and out of the cells.
Part A. The fluorescence microscope: Examine the drawing of the fluorescent microscope. In the figure, identify some of the unique parts on the fluorescent microscope as compared to an ordinary light microscope.
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Part B. Fluorescent tags for cell structures. Give the function of each of the structures that are fluorescently tagged in the micrographs.
Nucleus________________________________________________________________
Mitochondria____________________________________________________________
Lysosomes______________________________________________________________
Endoplasmic Reticulum____________________________________________________
Select any two of the fluorescent images. Describe or illustrate the distribution of the fluorescent marker(s) in the cells.
Image: Image:
4. Demonstration: Electron micrographs of cells and organelles.
Because the wavelength of electrons is much smaller than that of light, the resolution attainable for transmission electron microscope (TEM) images is many orders of magnitude better than that from a light microscope. Thus, TEMs can reveal the finest details of internal structure.
A transmission electron microscope works much like a slide projector. A projector shines a beam of light through (transmits) the slide, as the light passes through it is affected by the structures and objects on the slide. These effects result in only certain parts of the light beam being transmitted through certain parts of the slide. This transmitted beam is then projected onto the viewing screen, forming an enlarged image of the slide. TEMs work the same way except that they shine a beam of electrons (like the light) through the specimen (like the slide). Whatever part is transmitted is projected onto a phosphor screen for the user to see. Images can also be captured in black and white on photographic film. In this portion of the lab, you will be observing colorized electron micrographs to learn more about cell structure and function.
Part A. Cell Function. Cells may have quite different structures depending on their
. functions.
Observe the colorized micrographs of different types of cells # 1-6 and complete the table
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Micrograph #7 is ____________________. Is this a cell from a plant or an animal?
________________________________. The circular region in the center of the cell is
________________. The dark staining material in this region is ___________________.
The area that is colorized pink and contains the other organelles is called _____________
______________________The red colorized organelles in the cytoplasm are__________
_____________________.
Micrograph #8 is ___________________. The green colorized area in the center of the cell is ______________________. The name given collectively to the membrane bound structures in the cell______________________. The green colorized boundary of the cell is called _____________________.
Micrograph #9 is ____________________. What is the large organelle in the center of the picture?___________________________ How many membranes surround this organelle? ___________________________How does the chromatin appear in this image? __________________________________________Where would you find the nucleolus?__________________
Micrograph #10 is_________________________. Describe the structure of the nucleus in this image_____________________________________________________________.
The blue colorized dots illustrate tagged molecules passing through the nuclear pores.
What is the function of the nuclear pores? _____________________________________
_______________________________________________________________________
What molecules must pass into the nucleus? ___________________________________
What molecules must pass out into the cytoplasm? _______________________________
Micrograph #11 is ___________________________. What is the function of the ribosomes? ______________________________________________________________
What is the function of the endoplasmic reticulum? ______________________________
_______________________________________________________________________
What is the difference between rough ER and smooth ER?
Micrograph #12 is _____________________________. Describe the appearance of the
Golgi apparatus in the image. _____________________________________________
_____________________________________________________________________
What is the function of the Golgi apparatus? _________________________________
Micrograph #13 is _____________________________. What is the function of this organelle? ___________________________________________________________
What are the different membranes and compartments of this organelle? ______________
_______________________________________________________________________
Micrograph #14 is _______________________________. Name and describe the different organelles you can see in the image.___________________________________
_______________________________________________________________________.
Which organelle is responsible for generating ATP? _____________________________
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Which organelle breaks down old cell components? _____________________________
Which structure is the site of protein sythesis? __________________________________
Which structure is responsible for adding carbohydrate groups to secreted proteins? ____
_______________________________________________________________________
Micrograph #15 is _______________________________. How many membranes cover these organelles? ________________ What are the different regions of this organelle? What cellular reactions occur in these organelles? ___________________
_____________________________________________________________________
Micrograph #16 is ________________________________. What part of the body would these cells be found in? The green colorized organelles are specialized structures called synaptic vesicles that contain signaling molecules. What would happen next to these vesicles?
________________________________________________________________
________________________________________________________________________
Micrograph #17 is _________________________________. The function of the region you are seeing in cross section is ____________________________________________.
The protein that makes up much of the rings you see is __________________________.
Polymers of this protein are called ___________________________________________.
Micrograph #18 is ________________________________. The dark areas are a type of cell junction called an adherens junction. The role of this junction is to tie together two cells of an epithelium and prevent molecules from leaking into inderlying tissues.
Acknowledgements:
Colorized electron micrographs:
By permission of Dennis Kunkel (http://www.denniskunkel.com)
Fluorescent micrographs:
Available on the Molecular Probes website
Bull sperm: Peter Sutovsky
Bovine pulmonary artery endothelial cells: Wan-Nan U. Chen, Chii-Shiarng Chen, Yu-
Zhong Zhang
HeLa cells with mitochondrial markers, endoplasmic reticulum markers and green fluorescent protein: Nancy Bachman
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