Light Microscopy: Instrumentation and Principles

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Light Microscopy Instrumentation and Principles

• David R. Caprette, Ph.D.

Compound Microscope, circa 1751
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Compound Light Microscope
oculars (eyepieces)
objective lenses
stand
stage
coarse focus
condenser
fine focus
field lens (light source)
illuminator control
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Refraction
Light bends when it strikes a surface at an angle
...just as a vehicle changes direction when it
strikes a soft shoulder.
Muddy shoulder
Glass
Air
This wheel slows first
Incident wave
Hard pavement
The part that hits first slows first.
Perpendicular axis
The wave is compressed and bent at the boundary
of the slowing medium (i.e., the glass).
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Magnification and Orientation I
Actual position
Apparent image
Dashed lines are perpendicular to the lens surface
A biconvex lens bends light in the same direction
coming and going
Light path
Frog gastrula, saggital section arrow indicates
yolk plug
fast
slow
fast
Direction of movement
Apparent direction
down
focal point
up
Actual object
Projected image
left
right
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Magnification and Orientation II
Why doesnt a simple magnifying lens produce an
inverted mirror image?
With the object closer to the lens than the focal
point, the light rays diverge, giving the viewer
the illusion that he/she is seeing a larger
object, farther away, in the same orientation.
object
focal point
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Resolution
0.61l
Resolution (d)
n sin a
l wavelength of light n refractive index
0.2 µm
Scale resolved beyond theoretical limit
5 µm
2 µm
1 µm
0.4 µm
Scale resolved to 1 µm
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Empty Magnification
Final magnification using a simple lens system
(e.g., dissecting microscope
40x
100x
400x
Same images Final magnification using a compound
light microscope
40x
100x
400x
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Depth of Focus
Three dimensional volume in view changes with
magnification
Depth of focus at different magnifications
Scale 5 mm
Coverslip (1) 0.15 mm thick Typical wet mount
0.1 mm deep
volume of space in view
100x
40x
40x
400x
100x
1000x
Typical slide thickness 1 mm
400x
1000x
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Field of View and Light Intensity
Final magnification
100x
400x
1,000x
too bright
too dim
too dim
good
good
good
Apparent field (left) without adjusting brightnes
s (right) after compensating with intensity
control
Objective magnification
100x
40x
10x
Quantity of light
True field diameter
2 mm
0.5 mm
0.2 mm
Area
3 sq. mm
0.2 sq. mm
0.03 sq. mm
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Contrast
Three views of Paramecium caudatum (food vacuoles
contain stained yeast cells)
low contrast
optimum contrast
high contrast
A
C
(left) Bacillus thuringinensis with endospores
(A) bright field (B) phase contrast
(400x) (right) Pseudopodium of Chaos (Pelomyxa)
carolinensis (C) bright field (D) dark field
(100x)
B
D