General Biology Lab
​ Introduction to Microscopes

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Lab 4 - Introduction to Microscopes

PARTS OF THE COMPOUND MICROSCOPE

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Introduction to the Compound Microscope

   The compound microscope allows us to magnify objects up to 1000 times (with a 100X objective). We can calculate the magnification we are observing with the microscope by multiplying the magnification power of the objective, by the magnification power of the eyepieces. The magnification of the eyepieces is always 10X. There fore, If you are using the 40X objective, the object you are viewing under the microscope would be magnified 400 times more than with the naked eye. If we do the math, we see that 10X  times 40X equals 400X.  Compound microscopes are good for viewing up to 1 micrometer, or 100 nanometers (nm). 
   
   (Side note about EM)  In order to view even smaller objects, we would have to go to using a method that does not depend upon light. This is because the wavelength of visible light itself is only between 390 nm and 700 nm. At these small levels we can use electron microscopes since they utilize the wavelengths of electrons to visualize the specimen which are several orders of magnitude smaller than the wavelength of visible light. With electron microscopes we can visualize objects as small as 0.5 nm. 

 (Side note about EM)  In order to view even smaller objects, we would have to go to using a method that does not depend upon light. This is because the wavelength of visible light itself is only between 390 nm and 700 nm. At these small levels we can use electron microscopes since they utilize the wavelengths of electrons to visualize the specimen which are several orders of magnitude smaller than the wavelength of visible light. With electron microscopes we can visualize objects as small as 0.5 nm. ​

Proper Handling of a Compound Microscope

HANDLING:  Before using the compound microscope, it is important to know how to handle and care for it. When transporting the microscope, it is very important to make sure that you have a good grip on the scope and carry it with 2 hands at all times. One hand should be placed on the base of the scope while the other should be placed on the body (see diagram). The scopes are not necessarily heavy, but their center of mass is at the base, making it unstable if supporting by one hand alone. You must also be sure that any cords are wrapped up and secured before supporting so as to not trip over the cord or create a hazard for others around you to possibly trip and fall.

HOW TO USE A MICROSCOPE

Skipping or overlooking any steps could result in very costly damage to the objectives, eyepieces or other microscopic parts. Please take care to read instruction first, before doing each and every step. 

INSTRUCTIONS:

1. Place microscope gently on the counter at your workstation.
2. Ensure that the scope is away from any edges so that it will not fall or get knocked over.
3. Plug in the microscope.
4. Locate the ON/OFF switch and turn the microscope ON.
5. Locate the LIGHT LEVEL adjustment and turn the light down so you do not hurt your eyes with bright light. 
6. Use the COARSE FOCUS knob to move the stage all the way toward the bottom (base) of the scope (if it is not already there).
7. Once the stage is all the way down, you can safely rotate the NOSEPIECE to select different powered objectives.
8. Locate the 4X objective and make sure that that objective is pointing directly down toward the stage.  (Failure to do steps 6, 7, and 8 in order can result in the objective hitting the stage, causing breakage!)
9. Once you have prepared your slide, you will place it on the STAGE, securing it under the two metal clips. 
10. Locate the STAGE ADJUSTMENT knob and notice that this allows you to move the stage right and left and forward and back. You should try to center the specimen on your slide over the light source with the naked eye using the STAGE ADJUSTMENT knob. 
11. Now with the stage all the way at the bottom and the 4X objective pointed at the slide, use both eyes to view the slide. You will almost always not see anything but a blur at this point. 
12. While you continue viewing the slide through the eyepieces, use the COARSE FOCUS knob to slowly move the stage upward (toward the objective) until the slide comes into view.
13. Once the object on the slide is visible, you may then use the FINE FOCUS knob to focus the object further.
14. You MUST always start with the 4X objective when viewing a new slide. This is to prevent hitting the objective with the stage, and since the field of view is larger at 4X, finding your specimen or object will be much easier. Even seasoned scientists would have a very hard time finding a small object under 40X if they did not start st the 4X first. ​

INCREASING MAGNIFICATION
Only after performing all of the steps (1-14) first and in order, do you want to proceed with the instructions here.

• Once you have your object fully focused at 4X, make sure the object is directly in the center of our point of view. from this point YOU WILL NO LONGER BE USING THE COARSE FOCUS KNOB!
• Now you can rotate the NOSE PEICE carefully to ensure that you are going directly to only the NEXT HIGHEST magnification only (this would be the 10X). Skipping over a magnification objective may cause the objective to hit the stage, causing damage! 
• If you have followed the instructions closely, your specimen should be relatively in focus and in your new (smaller) field of view. You may now use the STAGE ADJUSTMENT knob and the FINE FOCUS (NOT THE COARSE FOCUS) to center and focus the object at this new magnification. *If you "lose your specimen" in the field of view, you must go down to the 4X objective and start over. "Fishing" for the object with the higher objectives can cause the objective the hit the stage, not to mention that it will be much easier to find the object with the larger field of view of the 4X objective. 
• In order to go to 40X, you will repeat steps 16 and 17 accordingly.

NOTES

--- Microscopes have a special property, called "being PARFOCAL" which means that once the object is focus and centered using the 4X, when you switch to the next highest objective, your specimen will be relatively centered and in focus. You should only have to make MINOR adjustments to the FINE FOCUS and the STAGE POSITION, in order to view your object at the higher objective. If you have difficulty, you must go back to 4X and recenter and refocus!

The field of view DECREASES as you INCREASE magnification.
The amount of light observed through the microscope gets dimmer (is reduced) as you increase magnification.
You should notice that the image you see is UP-SIDE-DOWN and REVERSED (the is INVERSION).
This means the object will appear to move in the OPPOSITE direct as it is being moved in reality.
You should notice that your DEPTH OF FOCUS decreases as magnification INCREASES. This means that with a relatively small movement of the fine focus knob, you could be out of the focal range. For looking at cells, you will notice you will see fewer layers of cells at higher magnifications.
You can measure the actual length of your field of view by viewing a ruler or standardized grid with millimeter demarcations using the 10 X objective. count the number of mm in your field of view, then convert to micrometers. (1000um = 1mm).

 

Procedure for Shutting Down and Putting Away Microscopes

1) Turn off POWER switch.
2) Make sure the stage is all the way toward the bottom (toward the base).
3) Make sure that the 4X objective is pointing down toward the stage. 
4) Wrap up and Secure Cord. 
5) Carry with 2 hands; one on the base and the other on the body.
6) Be aware of your surrounding and carefully place the scope in the proper place. 
7) Make sure you area is left clean.

The Anatomy of a Plant Leaf

Layers of the Leaf

Epidermis

The (upper) epidermis of plant leaves function to form a boundary between the plant and the external environment. The epidermis serves several functions:

1. protects against water loss
   
2. regulates gas exchange
   
3. secretes metabolic compounds
   
4. absorbs water and mineral nutrients in roots

The upper epidermis of the leaf produces a waxy layer, called the cuticle. The cuticle is a waxy, waterproof barrier thar protects against water loss. 

Palisades Cell Layer

The palisade s cell layer lies just beneath the protective upper epidermis. The palisades layer is where photosynthesis takes place within the leaf. 

This tissue is where 80% of the photosynthesis takes place in the leaf.  The palisade cells have many chloroplasts in their cytoplasm and the box-like shape and arrangement of these cells ensures they are packed tightly together.

Spongy Mesophyll

Below the palisade layer is the spongy layer, which contains cells that are more spread out, allowing for air pockets.The spongy mesophyll allows for gas exchange. Namely, carbon dioxide (CO2) which is needed for photosynthesis, and oxygen (O2) which is given off as a product of photosynthesis. 

This tissue contains large air spaces which are linked to the atmosphere outside the leaf through microscopic pores called stomata on the lower surface.  Spongy mesophyll cells also contain chloroplasts and photosynthesis occurs here too.  The air spaces reduce the distance carbon dioxide has to diffuse to get into the mesophyll cells and the fact that these cells have fairly thin cell walls which are coated with a film of water together means that gas exchange between air space and mesophyll is speeded up.

STOMATA

     Stoma (singular) or stomata (plural) are openings in the leaves of plants that allow for gas exchange.  Stomata are usually found on the under-surface of the leaf. A single stoma has a chloroplast, a cell wall, a vacuole and a nucleus.
    Carbon Dioxide that is needed for photosynthesis enters the plant through the stomata. Excess oxygen is released from the plant through the stomata as one of the waste products of photosynthesis. Stomata can also gain water from the atmosphere through open stomata and the plant can conserve water by closing stomata to prevent dehydration. 

via GIPHY

By Charles Tschirhart - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=28249312

The stoma pore is formed by a pair of cells known as guard cells. These adjust the size of the opening by opening or closing. Guard cells reside in the epidermis of leaves, stems and other organs that are used to control gas exchange. 

By Ali Zifan - Own work; Used information from:Campbell Biology (10th Edition) by: Jane B. Reece & Steven A. Wasserman.and [1]., CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=50023738

A microscopic view of the underside of a leaf. In this species (Tradescantia zebrina) the guard cells of the stomata are green because they contain chlorophyll while the epidermal cells are chlorophyll-free and contain red pigments.

By Zephyris - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=11721372

Cross-section of a Ligustrum Leaf (Found in Sunny Yards)

• The Epidermis of the Ligustrium leaf is 1 cell-layer thick. 

Cross-Section of a Nerium Leaf (Found in Dry Environments)

• Stomata are found in the leaves of plants, because this this is where photosynthesis takes place. ​

• The underside of leaves have more stomata than the upper surface because less evaporation will take place in the cooler, shady underside of the leaf.

• The Epidermis of the Nerium leaf is 3 cell-layers thick. 

Cross-Section of a Nymphaea Leaf (aqueous plants)

Nymphaea re plants that usually float on top of the water. One example of a nymphaea is the water lily. 

Tradescantia (Shady Environments)