1.When an object is sharply focused and the slide is moved towards you, in which direction does the

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1 image upright or inverted? Name: Date: _ BIOLOGY EXPERIMENT:Class: Using a Compound Light Microscope II: Depth Perception, resolution, field of view MATERIALS: Compound light microscopecolor magazine clipping Water dropper bottlescissors SlidesScotch tape Cover slipsforceps 3 different color threadsprobe Clear plastic metric ruler Part 1: Determining the position and movement of objects: PROCEDURE: 1.Place the low power objective in viewing position. rm 2.Place a prepared slide of the letter "e" on the stage under the clips. Position it so that the letter is over the light coming up through the stage. 3.Before observing the letter through the microscope, make a note of its position on the slide. 4.w observe the letter under low power magnification. Focus on the letter by using the coarse adjustment knob. 5.Move the slide toward you while looking through the microscope noting the apparent movement of the letter. Move the slide from side to side, again noting the apparent movement of the letter. 6.It is important that you become familiar with the direction of movement in the field of vision; becoming adept at moving the slide in the proper direction will enable you to locate an object, and will save you time and frustration. Review: 1.When an object is sharply focused and the slide is moved towards you, in which direction does the 2.When an object is sharply focused and the slide is moved to the right, in which direction does the 3.Under the microscope, is the image in the same position as when viewed by the naked eye or is the

2 Part 2: Examining depth of field: The term depth-of-field refers to the vertical distance that is in focus at any one time. Higher magnification lenses have a smaller depth of field - that is, only a thin horizontal slice of your sample may be in focus at any one time. So, at high magnification, very small adjustments to the fine focus can quickly take your sample completely out of focus. PROCEDURE: Prepare a wet mount slide of 3 different colored threads. Cut a 1 cm piece of each color thread and cross them one on top of the other like this: b.which color thread is in the middle? _ c.which color thread is on the bottom? Place the slide on the stage and focus using scanning magnification. The center of your field should be at the point where the three fibers cross each other. Answer the questions below. 1.Focus up and down using your fine focus knob. Under scanning magnification, are all three fibers in focus at the same time, that is, are they all in the same focal plane? 2.Can you see the entire length of a fiber? 3.Can you easily tell which fiber is on top and which is on the bottom? w rotate your nosepiece so that the low powered objective clicks into place. Answer these questions. 4.Focus up and down using your fine focus knob. Under low magnification, are all three fibers in focus at the same time, that is, are they all in the same focal plane? 5.Can you see the entire length of a fiber? 8. Do you see more detail in each fiber? 5. At which magnification is there a greater depth of field?low Power Scanning Power

3 w rotate your nosepiece so that the high powered objective clicks into place. Answer these questions. 4.Focus up and down using your fine focus knob. Under high power magnification, are all three fibers in focus at the same time, that is, are they all in the same focal plane? 6. Can you see the entire length of a fiber? 8. Do you see more detail in each fiber? 5.At which magnification is there a greater depth of field?low Power High Power Make a drawing of the three threads. Describe their appearance when viewed under a microscope. THREAD: (8) Low power magnificationhigh power magnification TAKE THE THREADS FROM YOUR SLIDE AND TAPE THEM IN ORDER, INSIDE THIS CIRCLE:

4 Part 3: Examining resolution & high definition Resolution As two small objects are moved closer to each other, a point is reached where the eye is unable to distinguish the objects as separate entities, and only a single object is observed. The smallest distance at which two points can be seen separately is called the resolving power of the lens, PROCEDURE: 1.Clean off your slide from Part II and make a new wet mount with the magazine picture. rm 2.Select a picture from a magazine that has several bright colors. Choose a square that has both light and dark tones, but not black.base 3.Cut out a 1 cm square from the picture that has a variety of colors. Make sure to use your metric ruler and be precise. 4.What color or colors does your magazine picture have that you can see with the naked eye? Record the colors and describe what you see. 5. How are the colors spread out (distributed)? 6. Next, observe the magazine picture, starting on low power and scan the image. Then switch to high power and observe the colors. Sketch exactly what you see with the microscope below. PIECE OF COLOR PHOTOGRAPH FROM MAGAZINE: (8) Low power magnificationhigh power magnification

5 7. Do the colors appear different under the microscope? Describe in detail the similarities and differences you see: TAKE THE PIECE OF MAGAZINE FROM YOUR SLIDE AND TAPE IT INSIDE THIS CIRCLE: Part 4: Field of View: Estimating the size of microscopic objects The term field-of-view refers to how much of the horizontal area of your sample you can see at any one time. The field of view decreases with increased magnification. As you increase the magnification, you are looking at progressively smaller subsections of your sample. That is why you need to be sure to center what you are viewing before increasing the magnification. Microscopic measurements are generally made in micrometers (urn). It is possible to make a good guess of the size of a cell in the microscope if you know the diameter of the entire field which is visible when you look in the microscope. Metric conversions: I meter (m) = 100 centimeter (cm)s = 1000 millimeters (mm) = 1,000,000 micrometers (^m) (1 mm = 1000 micrometers). Equations for conversions: To convert meters to centimeters:multiply by 100 To convert meters to millimeters:multiply by 1000 To convert centimeters to millimeters:multiply by 10

6 To convert centimeters to meters:divide by 100 To convert millimeters to meters:divide by 1000 To convert millimeters to centimeters: divide by 10 PROCEDURE: 1. Place the small clear plastic ruler under the microscope, right side up with 5 cm mark over the stage opening (see figure below). It should be facing you on the stage. nn Stage Opening1 Stage Base 2.Look through the eye piece (for this lab, keep the objective on IPX^. 3.Move the clear plastic ruler on the stage of the microscope in such a way that the metric edge of the ruler is visible along the diameter (center) of the field. Draw a diagram showing the appearance of the ruler in the microscope field which is provided below. Low Power Field Ruler 4. Estimate the number of millimeters that are visible to the closest tenth. This is the diameter of the field in millimeters. Convert this diameter of the field to micrometers. Diameter of the low power field in millimeters (mm): Diameter of the low power field in micrometers ( xm): 5. Assuming that the low power objective is 10X and the high power objective is 40X, images will be magnified 4 times more with high power than with low power. Because objects will appear four times larger the high power field will be approximately 'A the size of the lower power field. Indicate below the approximate diameter of the high power field. Diameter of the high power field in micrometers ( im):

7 Diameter of the high power field in micrometers (um): 6. Calculate the approximate fields of view in micrometers for your microscope (1 mm = 1000 micrometers). te that the field size shrinks as the magnification increases. 4X =micrometers 10X = 40X = 100X = micrometers micrometers micrometers Procedure using a stage micrometer: A stage micrometer has lines on it which are exactly 0.01 mm (10 urn) apart. Place a micrometer on the stage of your microscope. View the lines under both low power and high power to determine the diameters of the low power and high power fields in micrometers. 6. What is the difference between magnification and resolution? magnification: resolution: Please use what you have learned in the above exercises to complete this Table. In the Table, draw in arrows to show how an increase or decrease in magnification will change the five listed components. Use an up arrow (^>) to indicate an increase, a down arrow (4<) to indicate a decrease, or a horizontal arrow (^ ^) to indicate no change. Field of View Depth of tield Resolution Working Distance Li^ht._ Intensity - r" x ^ ^T r- jjhow does it change" "^ithincreasing _*^ magnification ~--^_ ji ft- *t - -- >t ~T^ - Conclusions: 1.State the relationship between magnification and resolution i.e. what happens to resolution (clarity of image) as magnification increases? 2.State the relationship between magnification and depth of field (depth of field is how easily something moves into and out of focus and coincides with the movement of the body tube)? 3.State the relationship between magnification and width of the field? 4.Which magnification would you use when you are searching for an object?

8 Answer true or fabe to each of the statements On high power, you should use the coarse adjustment knob. The diaphragm determines how much light shines on the specimen. The high power objective has a greater magnification than the scanning objective. The fine focus knob moves the stage up and down visibly. Images viewed in the microscope will appear upside down but not backward. If a slide is thick, only parts of the specimen may come into focus. The type of microscope you are using is an electron microscope. For viewing, microscope slides should be placed on the ocular lens. To switch from low to high power, you must rotate the revolving ear. The total magnification is determined by adding the ocular to the objective lens. *When you are finished, do the following for lab station or lose points: Place your microscope on scanning power. Turn the coarse adjustment until the nosepiece is all the way down. Wrap cord around base. Throw the pieces of hair in trash. Clean out your sink well. CAREFULLY dry slide and cover slide and return to tray.