Using the Microscope

1. Using the Microscope

2. The Microscope • That gizmo pictured to the left is a BIG deal.  It literally opened up worlds of organisms and information to scientists.  It's importance in the history of medicine and our understanding of disease should not be underestimated. That gizmo is a compound light microscope. For you, the biology student, it is perhaps the most important tool for you to understand.  You should be able to : 1. name all of its parts and describe the function of each 2. explain how to carry the thing, properly prepare a slide, & focus correctly 3. calculate total magnification 4. estimate the size of a specimen being observed

3. What the parts do! 1.the lens you look through, magnifies the specimen ocular (eyepiece) 2. supports the microscope base 3. holds objective lenses nosepiece 4. magnify the specimen (2) high power objective lens low power objective lens 5. supports upper parts of the microscope, used to carry the microscope Arm 6. used to focus when using the high power objective fine focus knob 7. where the slide is placed stage 8. regulates the amount of light reaching the objective lens diaphragm 9. used to focus when using the low power objective coarse focus knob 10. provides light light source 11. hold slide in place on the stage stage clips

4. Important 'Scope Vocab : • magnification  \mag-ne-fe-'ka-shen\ n 1. apparent enlargement of an object 2. the ratio of image size to actual size A magnification of "100x" means that the image is 100 times bigger than the actual object. • resolution  \rez-e-loo-shen\ n 1. clarity, sharpness 2. the ability of a microscope to show two very close points separately • OK, well.  There are a few other tidbits about the compound microscope you should remember : 1. Why is called a "compound" light microscope ? "Compound" just refers to the fact that there a two lenses magnifying the specimen at the same time, the ocular & one of the objective lenses. 2. If two lenses are always magnifying the specimen (see #1), how do you figure out the total magnification being used ? You multiply the power of the ocular and the power of the objective being used. • total mag. = ocular x objective • For example, if the ocular is 10x and the low power objective is 20x, then the total magnification under low power is 10 x 20 = 200x. Easy, ain't it ?

5. 3. How do you carry one of those things ? With two hands, one holding the arm & the other under the base.  Kinda like a football. (They're expensive, we don't want to drop 'em.) 4. What about focussing ? How do you do that ? Here's what I suggest.  Once you have your slide in place on the stage, make sure the low power objective (the shortest objective lens) is in position & turn the coarsefocus until the lens is at a position closest to the stage.  Set the diaphragm to its largest opening (where it allows the most light through).  Then, while looking through the ocular, begin to slowly turn the coarse focus.  Turn slowly & watch carefully.  When the specimen is focussed under low power, move the slide so that what you want to see is dead-center in your field of view, & then switch to a higher power objective.  DO NOT touch the coarse focus again --- you will break something !  Once you are using a high power objective, focus using the fine focus knob ONLY.  Be sure to center your specimen before switching to a higher power objective or it may disappear. 

6. MICROSCOPIC MEASUREMENTS Estimating Specimen Size • The area of the slide that you see when you look through a microscope is called the "Field of View".  If you know how wide your field of view is, you can estimate the size of things you see in the field of view. Figuring out the width of the field of view is easy --- all you need is a thin metric ruler. • By carefully placing a thin metric ruler on the stage (where a slide would usually go) and focusing under low power, we can measure the field of view in millimeters.  Through the microscope it would look something like what you see here on the left.  The total width of the field of view in this example is less than 1.5 mm.  A fair estimate would be 1.3 or 1.4 mm. (Relax, it's an estimate). • Now millimeters is a nice metric unit, but when we use a MICROscope we tend to use MICROmeters.  To convert from millimeters to micrometers, move the decimal 3 places to the right.  Our 1.3 mm estimate becomes 1300 micrometers. • Now we can get the ruler out of the way, prepare a slide, focus, and estimate the size of things we see ! (Exciting, ain't it ?) • For example, if something we were looking at took up half of the field of view, its size would be approximately 1/2 x 1300 micrometers = 650 micrometers.  If something appeared to be 1/5 of the field of view, we would estimate its size to be 1/5 x 1300 = 260 micrometers.

7. MICROSCOPIC MEASUREMENTS Calculating Specimen Size • Because the high power objective is so close to the stage, we can't measure the width of the field of view under high power directly.  The ruler just doesn't fit between the objective & the stage.  No problem.  We can use the width of the field of view under low power (which we measure using the steps above) and the relationship between the low & high power  magnifications to mathematically calculate the width of the field of view under high power. • First of all memorize this : • When switching from low to high power, the area in the  field of view gets smaller & darker.  (You see a smaller area of the slide under high power.)  This is why centering what you want to see prior to switching to high power is so important. • The fraction of the area seen under high power is the same as the ratio of the low & high power magnifications. • For example : if the low  power objective is 20x and the high power objective is 40x, then under high power we will see 20/40 or 1/2 of the area of the slide we saw under low power.

8. Example #1: • ocular power = 10x low power objective = 20x high power objective = 50x • a) What is the highest magnification you could get using this microscope ? b) If the diameter of the low power field is 2 mm, what is the diameter of the high power field of view in mm? in micrometers ? c) If 10 cells can fit end to end in the low power field of view, how many of those cells would you see under high power ?

9. ANSWER to Example #1: • ocular power = 10x low power objective = 20x high power objective = 50x • a) What is the highest magnification you could get using this microscope ? 500x Ocular x high power = 10 x 50 = 500. (We can only use 2 lenses at a time, not all three.) b) If the diameter of the low power field is 2 mm, what is the diameter of the high power field of view in mm ? .8 mm The ratio of low to high power is 20/50. So at high power you will see 2/5 of the low power field of view (2 mm). 2/5 x 2 = 4/5 = .8 mm in micrometers ? 800 micrometers To convert mm to micrometers, move the decimal 3 places to the right (multiply by 1000). .8 mm x 1000 = 800 micrometers d) If 10 cells can fit end to end in the low power field of view, how many of those cells would you see under high power ? 4 cells. We can answer this question the same way we go about "b" above.  At high power we would see 2/5 of the low field.  2/5 x 10 cells = 4 cells would be seen under high power.

10. Example #2: • ocular power = 10x low power objective = 10x high power objective = 40x • The diagram shows the edge of a millimeter ruler viewed under the microscope with the lenses listed above.  The field shown is the low power field of view. • a) What is the approximate width of the field of view in micrometers ? b) What would be the width of the field of view under high power ? c) If 5 cells fit across the high power field of view, what is the approximate size of each cell ?

11. ANSWER to example #2: • ocular power = 10x low power objective = 10x high power objective = 40x • The diagram shows the edge of a millimeter ruler viewed under the microscope with the lenses listed above.  The field shown is the low power field of view. • a) What is the approximate width of the field of view in micrometers ? 3500 - 3800 micrometers Each white space is 1 mm. We can see approximately 3 1/2 (or so) white spaces.  That is equivalent to 3.5 mm, which converts to 3500 micrometers. Any answer in the range above would be OK. b) What would be the width of the field of view under high power ? 875 micrometers The ratio of low to high power for this microscope is 10/40 or 1/4.  So, under high power we will see 1/4 of the low power field of view.  1/4 x 3500 micrometers (from "a" above) = 875 micrometers. c) If 5 cells fit across the high power field of view, what is the approximate size of each cell ? 175 micrometers If 5 cells fit in the high power field of view (which we determined is 875 micrometers in "b"), then the size of 1 cell = 875/5 = 175 micrometers.

12. Example #3: • ocular = 10x low power objective = 20x high power objective = 40x • The picture shows the low power field of view for the microscope with the lenses listed above. a) What is the approximate size of the cell in micrometers ? b) What would be the high power field of view ? c) How many cells like the one in the picture could fit in the high power field of view ?

13. ANSWER to Example #3: • ocular = 10x low power objective = 20x high power objective = 40x • The picture shows the low power field of view for the microscope with the lenses listed above. • a) What is the approximate size of the cell in micrometers ? 500 micrometers First, we have to visualize how many of those cells could fit across the field --- about 4.  So 2 mm (the width of the field) / 4 = .5 mm, which converts to 500 micrometers. b) What would be the high power field of view ? 1000 micrometers The ratio of low to high power for this scope is 20/40, or 1/2.  So we will see 1/2 of the low power field under high power.  1/2 x 2 mm = 1mm, which converts to 1000 micrometers. c) How many cells like the one in the picture could fit in the high power field of view ? 2 cells Again the ratio of low to high power is 20/40, or 1/2.  If we can see 4 cells across the low field of view we will see 1/2 as many in the high field of view.  1/2 x 4 = 2 cells.

14. Work Cited • Lubey, Steve. Lubey's Biohelp! – Using the Microscope. Aug. 26, 2005http://www.borg.com/~lubehawk/mscope.htm • Microscope image. http://www.hc-sc.gc.ca/ewh-semt/contaminants/person/impact/index_e.html