Using the Microscope

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Using the Microscope
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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

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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

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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 ?

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• 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. 

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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.

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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.

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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 ?

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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.

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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 ?

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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.

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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 ?

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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.

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Work Cited

• Lubey, Steve. Lubey's Biohelp! Using the
  Microscope. Aug. 26, 2005http//www.borg.com/lub
  ehawk/mscope.htm
• Microscope image. http//www.hc-sc.gc.ca/ewh-semt/
  contaminants/person/impact/index_e.html