Microscopy

 
                                               
       
       
       
       
       
       
       
       
                                       
 

Exercise 1:  Image Orientation

The following exercise will provide you with practice in image orientation.

Materials:
           
Microscope
            Letter e slide

Procedure:
1. Prepare the microscope for viewing as described above.
2.
Hold the letter e slide up and note the orientation of the letter.
3. 
Draw the letter as you see it with your eye.
4. Now place the slide on the microscope and find the letter e using the 4x   objective. 
5.
View the e and draw its orientation as seen through the microscope.
6.
Now use the 10x to view the letter e.

Questions:
1. How did the orientation of the e differ under the microscope from that of your unaided eye? 

2. If you were viewing a living specimen (for example, an amoeba) that was moving towards the top of the slide,
       what direction is it moving on the slide?

Exercise 2:  Field of View

The field of view, or FOV, is the circular, illuminated area that you see when looking through the ocular of the microscope.  Knowing the size of the field of view allows you to determine the size of the specimen you are examining.

Materials:
            Microscope
            Plastic ruler

  Procedure:
1. Record the magnification of each objective in Table 1.1.
2.
Record the magnification of the ocular in Table 1.1.
3. Calculate the total magnification for the 4x objective by using the following formula:

                             Mag total =  (Mag objective) x  (Magocular )

4. To measure the diameter of the FOV of the 4x objective, place the plastic ruler on the stage and measure the
      diameter of the circular area that you see through the microscope in  millimeters (mm).  Record this measurement
       in Table 1.1.
5. Determine the FOV of the higher objectives by using the following formula:

                                               

6. Determine the radius for each objective in Table 1.1(remember that the radius is half of the diameter).
7. Determine the area of the FOV for each objective using the following  formula:

                                          Area =                  

 

Table 1.1

 

 

Objective Magnification

Ocular

Magnification

Total

Magnification

FOV

Diameter (mm)

FOV

Radius

(mm)

FOV

Area

(mm2)

1

 

 

 

 

 

 

 

2

 

 

 

 

 

 

 

3

 

 

 

 

 

 

 

 

Questions:
1. Why was the plastic ruler unsuitable to measure the FOV of the higher objectives?

2. Which objective provides the largest field of view?

3. How much more area do you see with the 4x objective than with the 40x objective?

4. Why would it be difficult to find an objective starting with the 40x objective?

   

Exercise 3:  Depth of Field

Depth of field is the thickness of the object that is in sharp focus.  Depth of field varies with different objectives.

Materials:
           
Microscope
            Fiber slide

  Procedure:
1. Place the intertwined fiber slide on the stage of your microscope.
2. With each objective, you should be able to determine that there are three colored fibers (red, yellow, and blue).
3. Using the 40x objective, slowly move the fine adjustment know so that only one fiber is in focus at a time.
4. Determine the order of the fibers from top to bottom.

Questions:
1. Can all three fibers be focused at once under the 40x objective?

2. Can all three fibers be focused at once under the 4X objective?   

3. Which objective provides the greatest depth of field?  Why?

   

Exercise 4:  Dry Mount Preparation

  Biological specimens are typically placed on a glass slide for viewing.  A drop of water is added to the specimen, and a thin piece of glass or plastic called a cover slip is laid on top.  The cover slip presses the specimen in a thin plane to allow for more precise focusing.

Materials:
            Microscope
            Glass slide
            Cover slip
            Medicine dropper
            Hair

Procedure:
1. Clean a glass slide with lens paper.
2. Pull a single hair from your head and lay it across the middle of the slide.
3. Place the slide on the stage and view using the 4x objective.

Questions:
1. What is the diameter of your hair?
2. 
Do you have split ends?