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Using the Microscope

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
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
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 ?
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
coarse focus 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.
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.
MICROSCOPIC
Calculating Specimen Size
MEASUREMENTS
 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.
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 ?
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.
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 ?
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.
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 ?
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.
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/pe