Laboratory Exercise # 1: Microscope Purpose: To learn how to maintain and use a binocular microscope. Procedure: 1. Review the parts of the microscope by reading and performing the steps listed below under the titles of "Pathway of Light" and "Focusing of the Microscope". 2. Perform the calculations for Total magnification and Resolving power. Materials: Microscope, “e” slide and Blood smears (frog,bird, etc.) Pathway of Light: 1. Light source - on/off switch and intensity knob. The Rheostat knob controls the intensity of the light coming from the bulb. 2. Condenser - device found above the light source that converges the light beams into a point so that they can enter the specimen. a. Knob - raises and lowers the condenser, so that the point of light entering the specimen can be varied. For most smears the condensers should be all the way in the up position. b. Iris Diaphragm Lever/Control - works like the iris of your eye, a muscle that controls the amount of light entering the eye. It is found within the condenser and a lever is provided so the amount of light entering the specimen from the condenser can be varied. For oil magnification this lever should be positioned to allow maximum light to the specimen. 3. Stage - where the slide is placed and has a central opening to allow light from the condenser to pass through the specimen. a. Slide clip - pulling the slide clip towards you allows for the placement of the slide, flat on the stage. b. Stage adjustment knobs - this is a double knob found below the stage that allows for movement of the slide. 1) What does the lower knob do? ____________________ 2) What does the upper knob do? ____________________

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4. Objectives - after the light passes through the specimen it then is picked up by the objectives that are housed within the revolving nosepiece. a. Revolving nosepiece - allows for the movement of different power objectives into position over the specimen. b. Objectives - magnify the image that is received. There are four objectives housed on the revolving nosepiece. List the magnification of each and it's designation. 1) _______________________ 2) _______________________ 3) _______________________ 4) _______________________ 5. Body tube - light from the objective after being magnified travels through the body tube and is directed to the ocular by being reflected off a mirror. 6. Ocular - there are two eyepieces or oculars, therefore this microscope is called a binocular scope. The oculars also have the ability to magnify the image by 10X.

Focusing the Microscope: Using the "e" slide. 1. Look through the oculars using both eyes. You should see only one image, if not adjust the intrapupillary distance. **Can't see anything! Make sure your light source is on. a. Intrapupillary Distance Adjustment: Adjust the oculars by sliding them either closer together or further apart until you see one field of view when using both eyes. 2. Observe the "e" slide before you place it on the stage of the microscope. Record what you see here:

3. Place the "e" slide on the stage using the stage clip to hold it in position.

4. Rotate the 4X objective into position over the slide. 5. Use the Course Adjustment Knob (gives approximate focus), while not looking through the oculars to bring the stage as close as possible to the objective. This means turning the knob towards you.

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6. Looking through the oculars focus the image using the Course Adjustment knob and slowly turning it away from you. The image should be in almost perfect focus. 7. Any fine-tuning of the focus is accomplished with the Fine Adjustment Knob (Gives exact focus) while looking through the oculars. 8. Sketch your view of the letter "e" as seen under 4X in the space provided below. Make sure the letter is in the center of the field of view.

4X Sketch

9. This microscope is parfocal meaning that when you switch to a different objective the image should remain in perfect focus. a. Using the revolving nosepiece move the 10X objective into position over the slide. b. Use the fine adjustment only, if necessary. c. Sketch what you observe of the letter "e". Don't move the stage any!!

10X Sketch

d. Move the 40X objective into position. Without moving the stage, again sketch what you observe of the letter "e".

40X Sketch e. What happens to the field of view as you increase the power of the objective? ______________________________________ 10. Remove the "e" slide from the stage and replace it with a prepared slide. 11. Focus with the 10X objective in place on the edge of the coverslip. You should see a fuzzy line when it is in focus. 12. Using the stage adjustment knobs, slide into the center of the coverslip and refocus.

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13. Move the revolving nosepiece until it rests between the 40X and the 100X objectives. Place a drop of immersion oil onto the slide and rotate the 100X objective into the oil. Don't move the stage while you are putting on the oil! 14. Increase the light intensity and complete focusing with the fine adjustment knob only. Sketch what you observe below.

Important Calculations: 1. Total Magnification: Remember the image is magnified twice, once by the objective and second by the ocular. Calculate the total magnification for each objective. Total Magnification = Ocular power X Objective power a. Scanning objective (4X):

_____________________

b. Low power objective (10X):

_____________________

c. High power objective (40X):

_____________________

d. Oil immersion objective (100X):

_____________________

2. Resolving Power: a numerical measure of the resolution of the lens. The smallest distance between two objects that the microscope is able to distinguish as two distinct points. a. RP = wavelength of light / 2NA b. Wavelength of light = the distance between two troughs of the light wave, we will be using the average wavelength of visible light or .55 m c. NA = the light concentrating power of the objective. This is listed on the side of the objective after the magnification power.

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d. Calculate the resolving power for each objective. 1) RP of 4X objective = ____________________ 2) RP of 10X objective = ____________________ 3) RP of 40X objective = ____________________ 4) RP of 100X objective = _________________ Questions: 1. Define the following terms that are important to microscopy: a) Field of view b) Parfocal c) Resolving power d) Binocular e) Numerical aperture f) Brightfield g) Wavelength of light 2. The student should be able to locate each part of the microscope and give its function. Arm

Base

Body Tube

Condenser

Condenser knob

Course adjustment knob

Fine adjustment knob Iris diaphragm lever/control

Light source

Objective

Rheostat

Ocular

Revolving nosepiece Stage

Stage clip

Stage Adjustment knobs

3. If two points of a sample on a slide are 1.15 um apart and you are using the 40X objective with and N.A. of .85, will you observe two points or a blob?

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4. What is the total magnification of the specimen if you are using a 75X objective?

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