BIO 137
MICROSCOPE
LAB
Cells phones silenced
and OUT OF SIGHT!!!
Copyright 2016
Dr. Mary Cat Flath
In anatomy &
physiology, we will
study anatomy by
using a
compound light
microscope
CARRYING THE
MICROSCOPE
• PICK UP THE MICROSCOPE WITH THE
SAME NUMBER AS YOUR TABLE NUMBER
• PICK SCOPE UP WITH BOTH HANDS
– ONE SHOULD BE PLACED
UNDERNEATH THE BASE
– ONE SHOULD BE HOLDING THE ARM
• GET POWERCORD IN FRONT OF SCOPE
• WALK CAREFULLY BACK TO YOUR SEAT
Copyright 2016
Dr. Mary Cat Flath
Identify the following
parts on your microscope
ANATOMY
OF THE
MICROSCOPE
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Dr. Mary Cat Flath
• Oculars or
• Condenser
Eyepieces (10x)
• Iris Diaphragm
• Head
lever
• Arm
• Sub-stage light
• Base
• Light Intensity
Knob
• Rotating
Nosepiece with
• Power Switch
• Objective Lenses • Coarse
Adjustment Knob
• Stage
• Fine Adjustment
• Mechanical
Stage/controlsCopyright 2016Knob
Dr. Mary Cat Flath
1
Oculars or
eyepieces (10x)
head
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Dr. Mary Cat Flath
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Dr. Mary Cat Flath
arm
base
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Dr. Mary Cat Flath
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Dr. Mary Cat Flath
Rotating nosepiece
with objective lenses
stage
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Dr. Mary Cat Flath
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Dr. Mary Cat Flath
2
Slide clip>
stage
stage
Condenser
Mechanical stage
controls
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Dr. Mary Cat Flath
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Dr. Mary Cat Flath
stage
Condenser with iris
diaphragm lever
Substage
light
Copyright 2016
Dr. Mary Cat Flath
Copyright 2016
Dr. Mary Cat Flath
Identify the following
parts on your microscope
• Oculars or
• Condenser
Eyepieces (10x)
• Iris Diaphragm
• Head
lever
• Arm
• Sub-stage light
• Base
• Light Intensity
Knob
• Rotating
Nosepiece with
• Power Switch
• Objective Lenses • Coarse
Adjustment Knob
• Stage
• Fine Adjustment
• Mechanical
Stage/controlsCopyright 2016Knob
Dr. Mary Cat Flath
Copyright 2016
Dr. Mary Cat Flath
3
Identify the following
parts on your microscope
Copyright 2016
Dr. Mary Cat Flath
• Oculars or
• Condenser
Eyepieces (10x)
• Iris Diaphragm
• Head
lever
• Arm
• Sub-stage light
• Base
• Light Intensity
Knob
• Rotating
Nosepiece with
• Power Switch
• Objective Lenses • Coarse
Adjustment Knob
• Stage
• Fine Adjustment
• Mechanical
Stage/controlsCopyright 2016Knob
Dr. Mary Cat Flath
Getting Started
Copyright 2016
Dr. Mary Cat Flath
Next Steps
• The stage should be at its lowest
position.
• The scanning power (4x)
objective lens (red) should be in
place.
• Use lens cleaner and lens paper;
clean all lenses
on scope carefully.
Copyright 2016
Dr. Mary Cat Flath
• PLUG THE ELECTRICAL CORD FIRST
TO THE BACK OF SCOPE AND THEN
INTO THE POWER SUPPLY
– MAKE SURE THE SCOPE IS OFF (O)
AND THE LIGHT INSTENSITY KNOB
IS DOWN
• TURN THE OCULARS TO FACE YOU BY
slightly UNSCREWING THE SILVER
SCREW ON THE HEAD AND HOLDING
THE HEAD WHILE TURNING OCULARS
AROUND 180O. Copyright 2016
Dr. Mary Cat Flath
Next Steps
• Turn scope on, and slowly
increase light intensity until light
is visible.
• Move the stage using mechanical
stage controls until light is
centered.
• Look through oculars to view the
circular visual field.
– Note that a “pointer” has been
placed in your right or left ocular.
Copyright 2016
Dr. Mary Cat Flath
4
MAGNIFICATION
AND
RESOLUTION
(Important Definitions)
Magnification
Scanning Low
High
Power
Power
Power
(red)
(Yellow) (blue)
Magnification
of Objective
Total
Magnification
Copyright 2016
Dr. Mary Cat Flath
Resolution
is the ability to
discriminate two close
objects as separate.
Human eye = 100 µ
CLM = 0.2 µ
Total Magnification
=
Magnification of
objective lens
x
Magnification of
ocular lenses (x10)
Magnification
Scanning
Power
(red)
Magnification 4x
of Objective
Total
40x
Magnification
Low
Power
(Yellow)
10x
High
Power
(blue)
40x
100x
400x
Copyright 2016
Dr. Mary Cat Flath
As magnification
increases,
resolution increases
5
Viewing the letter “e”
• Obtain a “letter e” slide and millimeter ruler.
• Make sure the stage is at its lowest position
and that the scanning objective lens is in
place.
• Open slide clip and place slide securely into
position, close clip. Move stage using
mechanical stage controls, so specimen is in
middle of the lighted field.
• Adjust inter-pupillary distance to fit your
eyes, if needed. Note distance.
Copyright 2016
Dr. Mary Cat Flath
WORKING
DISTANCE
AND
FIELD SIZE
(DIAMETER)
(Important Definitions)
Viewing the letter “e”
• While looking through both oculars, use coarse
adjustment knob to raise stage until you SEE
the letter “e”.
• Center the object in the middle of the field
using mechanical stage controls.
– What happens when you move stage to
right?___________________
– What happens when you move stage toward
you?___________________
– This is called “REVERSING”; all objects are
upside-down and flipped right to left.
• Use fine adjustment knob to sharpen the
appearance of the “e”.
• Put your pointer on the letter “e”, and call me
over to check your scope.
Copyright 2016
Dr. Mary Cat Flath
WORKING DISTANCE
IS THE VERTICAL
DISTANCE BETWEEN THE
BOTTOM OF THE
OBJECTIVE LENS AND
YOUR SLIDE
Use the millimeter ruler to
measure the scanning power
working distance
Magnification, Working
Distance
Working Distance l
Copyright 2016
Dr. Mary Cat Flath
Scanning
Power
(red)
Magnification 4x
of Objective
Total
40x
Magnification
Working
Distance
30 mm
Low
Power
(Yellow)
10x
High
Power
(blue)
40x
100x
400x
Copyright 2016
Dr. Mary Cat Flath
6
FIELD SIZE (OR
DIAMETER) IS THE
DISTANCE FROM ONE
SIDE OF THE FIELD TO
THE OTHER
Lay the millimeter ruler across
your field, to measure the
scanning power field diameter
FIELD DIAMETER IS
USED TO ESTIMATE
THE SIZE OF
OBJECTS
How big is your letter “e”?
_________
Magnification, Working
Distance, and Field Size
Scanning Low
High
Power
Power
Power
(red)
(Yellow) (blue)
Magnification 4x
of Objective
10x
40x
Total
40x
Magnification
100x
400x
Working
Distance
30 mm
Field
Diameter
4.5 mm
Copyright 2016
Dr. Mary Cat Flath
OUR MICROSCOPES ARE
PARFOCAL (Important Definition)
• Once initial focusing has been
completed using scanning power
• We can change objective lenses to
low power and then high power,
and the specimen should still be
focused
• However
– your light will have to be increased
– your object will need to be centered
– you will need to use fine adjustment
Copyright 2016
Dr. Mary Cat Flath
As magnification
increases,
resolution increases
As magnification
increases, light
decreases and you
will need to
increase your light
Increase light intensity
Open iris diaphragm
7
Viewing the letter “e”;
scanning to low power
• Using your rotating nosepiece,
move the low power objective lens
into place.
• Center your letter “e”, and use fine
adjustment to sharpen image.
• Measure the low power working
distance
• Measure the low power field
diameter
Copyright 2016
Dr. Mary Cat Flath
Viewing the letter “e”;
low power to high power
• Using your rotating nosepiece, move
the high power objective lens into
place.
– NEVER USE COARSE ADJUSTMENT KNOB
WHEN USING THE HIGH POWER LENS
• Can you still see the entire letter “e”?
• Measure the high power working
distance ________
• Calculate the high power field
diameter????? 2000µ divided by 4 = 500µ
Copyright 2016
Dr. Mary Cat Flath
USING THE ROTATING
NOSEPIECE,
MOVE THE SCANNING
OBJECTIVE LENS INTO
PLACE,
LOWER YOUR STAGE,
AND REMOVE LETTER “e”
SLIDE
Magnification, Working
Distance, and Field Size
Scanning Low
High
Power
Power
Power
(red)
(Yellow) (blue)
Magnification 4x
of Objective
10x
40x
Total
40x
Magnification
100x
400x
Working
Distance
30 mm
7 mm
Field
Diameter
4.5 mm
2 mm =
2000 µ
Copyright 2016
Dr. Mary Cat Flath
Magnification, Working
Distance, and Field Size
Scanning Low
Power
Power
(red)
(Yellow)
Magnification 4x
10x
of Objective
Total
40x
100x
Magnification
Working
30 mm
7 mm
Distance
Field
4.5 mm
2 mm =
Copyright 2016
Diameter
2000 µ
Dr. Mary Cat Flath
High
Power
(blue)
40x
400x
0.65 mm
2000µ ÷ 4
= 500 µ
Exchange your slide
with the person next
to you,
and repeat previous
steps of “viewing the
letter ‘e’,
until you feel
comfortable with the
procedure
8
Return letter “e”
slides to appropriate
box,
and obtain a
“crossed thread
slide”
Depth of Field
• Obtain a “crossed thread slide”, and
secure it on stage with stage clips.
• Center cross over light, and raise stage
until the crossed threads are visible
• Which color thread is on top? _____
• Which color thread is on middle?_____
• Which color thread is on bottom? _____
• Remember “reversing”.
Copyright 2016
Dr. Mary Cat Flath
Magnification, Working
Distance, and Field Size
Scanning Low
High
Power
Power
Power
(red)
(Yellow) (blue)
Magnification
of Objective
Total
Magnification
Working
Distance
Field
Diameter
Copyright 2016
Dr. Mary Cat Flath
Important Definitions
Total Magnification Calculation
Resolution
Working Distance
Field Size (Diameter)
Parfocal
Magnification, Working
Distance, and Field Size
Scanning Low
Power
Power
(red)
(Yellow)
Magnification 4x
10x
of Objective
Total
40x
100x
Magnification
Working
30 mm
7 mm
Distance
Field
4.5 mm
2 mm =
Copyright 2016
Diameter
2000 µ
Dr. Mary Cat Flath
High
Power
(blue)
40x
400x
0.65 mm
2000µ ÷ 4
= 500 µ
PRACTICE
MICROSCOPE
QUIZ
Copyright 2016
Dr. Mary Cat Flath
9
1.Name this piece of
equipment (be specific)
1. Compound Light
Microscope
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Dr. Mary Cat Flath
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Dr. Mary Cat Flath
2. NAME THESE.
2.
oculars.
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Dr. Mary Cat Flath
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Dr. Mary Cat Flath
3. Name
this.
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Dr. Mary Cat Flath
3. Arm.
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Dr. Mary Cat Flath
10
4. Rotating Nosepiece
4. Name this.
Copyright 2016
Dr. Mary Cat Flath
Copyright 2016
Dr. Mary Cat Flath
Magnification
Magnification
Scanning
Power
(red)
Magnification 4x
of Objective
Total
40x
Magnification
Low
Power
(Yellow)
10x
High
Power
(blue)
40x
100x
5. ____x
Scanning
Power
(red)
Magnification 4x
of Objective
Total
40x
Magnification
Low
Power
(Yellow)
10x
High
Power
(blue)
40x
100x
5. 400x
Copyright 2016
Dr. Mary Cat Flath
Copyright 2016
Dr. Mary Cat Flath
6. _____________
is the ability to
discriminate two close
objects as separate.
6. RESOLUTION
is the ability to
discriminate two close
objects as separate.
Human eye = 100 µ
CLM = 0.2 µ
Human eye = 100 µ
CLM = 0.2 µ
11
7. __________
____________ IS THE
VERTICAL DISTANCE
BETWEEN THE BOTTOM OF
THE OBJECTIVE LENS AND
YOUR SLIDE
8. Name one of two
ways to increase
light on your scope.
9. NEVER USE THE
_____________
ADJUSTMENT KNOB
WHEN USING THE
HIGH POWER LENS
7. WORKING
DISTANCE IS THE
VERTICAL DISTANCE
BETWEEN THE BOTTOM OF
THE OBJECTIVE LENS AND
YOUR SLIDE
8. Iris Diaphragm
Lever or Light
Intensity Knob
9. NEVER USE THE
_COARSE
ADJUSTMENT KNOB
WHEN USING THE
HIGH POWER LENS
12
Magnification, Working
Distance, and Field Size
Scanning Low
Power
Power
(red)
(Yellow)
Magnification 4x
10x
of Objective
Total
40x
100x
Magnification
Working
30 mm
7 mm
Distance
Field
4.5 mm
2 mm =
Copyright 2016
Diameter
2000 µ
Dr. Mary Cat Flath
High
Power
(blue)
40x
400x
0.65 mm
10.
______µ
Magnification, Working
Distance, and Field Size
Scanning Low
Power
Power
(red)
(Yellow)
Magnification 4x
10x
of Objective
Total
40x
100x
Magnification
Working
30 mm
7 mm
Distance
Field
4.5 mm
2 mm =
Copyright 2016
Diameter
2000 µ
Dr. Mary Cat Flath
High
Power
(blue)
40x
400x
0.65 mm
10. 500µ
Return crossed thread slides to
appropriate box;
Prepare scope for
storage
Woof, Good Luck
Studying, Folks!
Scanning Objective Lens in place
Lenses cleaned
Head turned 180o
Stage at its LOWEST position
Cord unplugged and placed in near
scope
Copyright 2016
Dr. Mary Cat Flath
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