Phase Contrast Optics. Abb é Theory. Designed optics for amplitude objects Absorb light without change in phase of light waves Based on assumption of no difference in index of refraction between specimen and background. Criterion for Resolution.
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Phase Contrast Optics
Theory & Appl. Light Microscopy
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Zernike Phase Vector Diagram
For propagation of light through phase object
S
S = incindent wave
P = particle wave
P = phase shift of ray through specimen
(S = U, undiffracted (0-order) ray
P
Length of P = amplitude specimen/amplitude medium =
transmission ratio
Theory & Appl. Light Microscopy
Calculate P by vector addition
D
U + D = P
By the law of sines
U
P
D = of all diffracted orders of light from specimen
U = undiffracted light
P = resulting specimen light, produced by interference between U and D in image formation
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Theory & Appl. Light Microscopy
U + D = P
U = P
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In microscope
At image plane
In specimen
D'
D'
D
D
P'
U
U'
U'
P
U' P'
Amplitude!
U = P
Theory & Appl. Light Microscopy
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Theory & Appl. Light Microscopy
Positive Phase
D
D'
U'
U
P
P'
U = P
U'> P'
Retard D relative to U (move D vector clockwise)
Negative Phase
D'
D
P'
U'
U
P
U = P
U'< P'
Advance D relative to U (move D vector counterclockwise)
Theory & Appl. Light Microscopy
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Popular among algae workers in Great Britain in 50s–60s
Theory & Appl. Light Microscopy
Anoptral Phase
D
No phase shifts on ring
D'
U
U'
P
P'
U = P
U'< P'
Produces delicate image against brown background
Theory & Appl. Light Microscopy
Theory & Appl. Light Microscopy
From: Rose & Pomerat (1960) J. Biophys. Biochem. Cytol. 8:423.
Theory & Appl. Light Microscopy
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' = optical path length
t = physical thickness
Can measure ', then calculate s = ('/t) + m
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(For most biological specimens, = 1.8 x 10-3 i.r./gm solute/100 ml)
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R = reference beam = U = P = A0
R
R
U
P
U'
P'
U' = 2 A0 1.4 A0
Interference between P and R produces P' 1.8 A0
(1.8/1.4)2 1.6
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Phase Contrast
Cheaper
Easier to set up
Uses less than full aperture of objective
Phase Halo — surrounds specimen and other changes in i.r.
Nomarski
More expensive
Fussy alignment
Uses full aperture — closet to theoretical limit
Shadow Effect — contrast greatest at shear direction maximum
Phase Contrast
Insensitive to birefringence in specimen or slides
Extremely large depth of field — sensitive to artifacts far out of plane of specimen
Doesn’t work well with stained specimens
Nomarski
Optics disrupted by birefriengence
Extremely shallow depth of field — useful for optical sectioning of specimen
Works well with stained specimens; optics can be adjusted to enhance contrast
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