Microscope Measurement (1)

8/11/2019 Microscope Measurement (1)

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The Microscope• That gizmo pictured to the

left is a BIG deal. It literallyopened up worlds of organismsand information toscientists. It's importance inthe history of medicine andour understanding of diseaseshould not be underestimated.That gizmo is a compound

light microscope.

For you, the biology student,it is perhaps the mostimportant tool for you tounderstand. You should beable to :1. name all of its parts and

describe the function of each2. explain how to carry thething, properly prepare aslide, & focus correctly3. calculate totalmagnification4. estimate the size of a

specimen being observed



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 lenslow 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



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 theactual object.

resolution \rez-e-loo-shen\ n 1. clarity, sharpness 2. the ability of a microscopeto show two very close points separately

OK, well. There are a few other tidbits about the compound microscope youshould remember :

1. Why is called a "compound" light microscope ?"Compound" just refers to the fact that there a two lenses magnifying thespecimen 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 ?



3. How do you carry one of those things ?With two hands, one holding the arm & the other underthe base. Kinda like a football. (They're expensive, wedon't want to drop 'em.)

4. What about focussing ? How do you do that ?Here's what I suggest. Once you have your slide inplace on the stage, make sure the low power objective(the shortest objective lens) is in position & turn thecoarse focus until the lens is at a position closest to the

stage. Set the diaphragm to its largest opening (where itallows the most light through). Then, while lookingthrough the ocular, begin to slowly turn the coarsefocus. Turn slowly & watch carefully. When thespecimen is focussed under low power, move the slideso that what you want to see is dead-center in your fieldof view, & then switch to a higher power objective. DONOT touch the coarse focus again --- you will breaksomething ! Once you are using a high power objective,focus using the fine focus knob ONLY. Be sure to centeryour specimen before switching to a higher power

objective or it may disappear.



MICROSCOPIC MEASUREMENTS Estimating Specimen Size

The area of the slide that you see when you look through a microscope iscalled the "Field of View". If you know how wide your field of view is, youcan estimate the size of things you see in the field of view. Figuring out thewidth 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 wouldusually go) and focusing under low power, we can measure the field of viewin millimeters. Through the microscope it would look something like whatyou see here on the left. The total width of the field of view in this exampleis 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 wetend to use MICROmeters. To convert from millimeters to micrometers,move the decimal 3 places to the right. Our 1.3 mm estimate becomes1300 micrometers.

Now we can get the ruler out of the way, prepare a slide, focus, andestimate the size of things we see ! (Exciting, ain't it ?)

For example, if something we were looking at took up half of the field ofview, its size would be approximately 1/2 x 1300 micrometers = 650micrometers. If something appeared to be 1/5 of the field of view, we wouldestimate its size to be 1/5 x 1300 = 260 micrometers.



MICROSCOPIC MEASUREMENTSCalculating Specimen Size

Because the high power objective is so close to the stage, we can'tmeasure the width of the field of view under high powerdirectly. The ruler just doesn't fit between the objective & thestage. No problem. We can use the width of the field of view underlow power (which we measure using the steps above) and therelationship between the low & high power magnifications to

mathematically calculate the width of the field of view under highpower.

First of all memorize this :

When switching from low to high power, the area in the field of viewgets smaller & darker. (You see a smaller area of the slide underhigh power.) This is why centering what you want to see prior toswitching to high power is so important.

The fraction of the area seen under high power is the same as theratio of the low & high power magnifications.

For example : if the low power objective is 20x and the high powerobjective is 40x, then under high power we will see 20/40 or 1/2 ofthe area of the slide we saw under low power.



Example #1:

ocular power = 10xlow power objective = 20xhigh power objective = 50x

a) What is the highest magnification you could getusing this microscope ?b) If the diameter of the low power field is 2 mm,what is the diameter of the high power field of viewin mm? in micrometers ?

c) If 10 cells can fit end to end in the low power fieldof view, how many of those cells would you seeunder high power ?



ANSWER to Example #1:


a) What is the highest magnification you could get using this microscope ? 500xOcular x high power = 10 x 50 = 500. (We can only use 2 lenses at a time, notall 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 mmThe ratio of low to high power is 20/50. So at high power you will see 2/5 of thelow power field of view (2 mm). 2/5 x 2 = 4/5 = .8 mmin micrometers ? 800 micrometersTo convert mm to micrometers, move the decimal 3 places to the right (multiplyby 1000). .8 mm x 1000 = 800 micrometersd) If 10 cells can fit end to end in the low power field of view, how many of thosecells would you see under high power ? 4 cells.We can answer this question the same way we go about "b" above. At highpower we would see 2/5 of the low field. 2/5 x 10 cells = 4 cells would be seenunder high power.



Example #2:


The diagram shows the edge of a millimeter ruler viewed underthe 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 theapproximate size of each cell ?



ANSWER to example #2:


The diagram shows the edge of a millimeter ruler viewed under the microscope with thelenses 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 - 3800micrometersEach white space is 1 mm. We can see approximately 3 1/2 (or so) white spaces. That isequivalent to 3.5 mm, which converts to 3500 micrometers. Any answer in the range abovewould be OK.b) What would be the width of the field of view under high power ?875 micrometersThe ratio of low to high power for this microscope is 10/40 or 1/4. So, under high power wewill see 1/4 of the low power field of view. 1/4 x 3500 micrometers (from "a" above) = 875micrometers.c) If 5 cells fit across the high power field of view, what is the approximate size of each cell

?175 micrometersIf 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.



Example #3:

ocular = 10x

low power objective = 20x

high power objective = 40x

The picture shows the low power field of view for themicroscope 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 ?



ANSWER to Example #3:

ocular = 10xlow power objective = 20xhigh power objective = 40x

The picture shows the low power field of view for the microscope with the lenses listedabove.

a) What is the approximate size of the cell in micrometers ?500 micrometersFirst, we have to visualize how many of those cells could fit across the field --- about 4. So2 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 micrometersThe ratio of low to high power for this scope is 20/40, or 1/2. So we will see 1/2 of the lowpower 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.



Work Cited

Lubey, Steve. Lubey's Biohelp! – Using

the Microscope. Aug. 26, 2005

http://www.borg.com/~lubehawk/mscope.htm

Microscope image. http://www.hc-sc.gc.ca/ewh-

semt/contaminants/person/impact/index_e.ht

ml


http://www.hc-sc.gc.ca/ewh-semt/contaminants/person/impact/index_e.html











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Microscope Measurement (1)