light microscopy course differential interference contrast dic...- roman number (i, ii, or iii) has...
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Light Microscopy course
Light Microscopy Course – DIC
Slide 1
Differential Interference Contrast DIC
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Slide 2
DIC – differential interference contrast
Light Microscopy Course – DIC
- developed in the mid-1950s
by Georges (Jerzy) Nomarski (1919-1997), a Polish optics theoretician working in France at CNRS
For a detailed biography see: http://micro.magnet.fsu.edu/optics/timeline/people/nomarski.html
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Slide 3
DIC – probe differential by interference = contrast
Light Microscopy Course – DIC
- Differential = (minute) difference between two different values of something (given in Δ or d) over a certain (small) range (gradient or slope =Δy/Δx)
- Used in many different contexts (Math, Physics,
Engineering, Biology….)
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Slide 4
DIC – probe differential by interference = contrast – example for differential
Light Microscopy Course – DIC
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Slide 5
DIC – probe differential by interference = contrast – example for differential
Light Microscopy Course – DIC
Δx1=10 Δx2=30
Δy1=0
Δy2=-0.6
diff1= Δy1/Δx1=0/rad(10)=0
diff2= Δy2/Δx2=-0.6/rad(30)=-1.15
diff1= ?
diff2= ?
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Slide 6
DIC – probe differential by interference = contrast – example for differential
Light Microscopy Course – DIC
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Slide 7
DIC – differential by interference = contrast
Light Microscopy Course – DIC
Interference is a phenomenon in which two waves superpose to form a resultant wave of greater, lower or same amplitude. (Wikipedia)
https://machinesdontcare.wordpress.com/2011/02/05/interference-patterns/interference-fringes-04/
Wikipedia
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Slide 8
DIC – differential by interference = contrast The principle of DIC - interferometry
Light Microscopy Course – DIC
Classical double-path interferometer with a real reference sample
adapted from Peter Evennett
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Slide 9
DIC – differential by interference = contrast
Light Microscopy Course – DIC
No shift
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Slide 10 Light Microscopy Course – DIC
30 degrees slower
DIC – differential by interference = contrast
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Slide 11 Light Microscopy Course – DIC
90 degrees slower
DIC – differential by interference = contrast
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Slide 12 Light Microscopy Course – DIC
150 degrees slower
DIC – differential by interference = contrast
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Slide 13 Light Microscopy Course – DIC
180 degrees slower
DIC – differential by interference = contrast
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Slide 14
The principle of DIC - interferometry
Light Microscopy Course – DIC
Common path interferometer, lateral shearing interferometer
Used for DIC
adapted from Peter Evennett
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Slide 15
DIC – differential by interference = contrast
Light Microscopy Course – DIC
From ZEISS-campus website
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Slide 16
DIC – differential by interference = contrast
Light Microscopy Course – DIC
adapted from Peter Evennett
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Slide 17 Light Microscopy Course – DIC
DIC – introducing retardation bias
Blue beam retarded by λ/4 – 90 degrees, no further retardation
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Slide 18 Light Microscopy Course – DIC
λ/4 bias + further λ/6 retardation of blue beam
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Slide 19 Light Microscopy Course – DIC
λ/4 bias of blue + λ/6 retardation of red beam
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Slide 20 Light Microscopy Course – DIC
Without bias With λ/4 bias of blue beam
grey: no retardation, dark: blue retarded, light: red retarded
Resultants of retardation of beam by 60 deg (λ/6)
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Slide 21 Light Microscopy Course – DIC
Interference at analyzer
Polariser
Analyser
Note: shift between effective (interfering) beam components by λ/2 (180 degrees)
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Slide 22 Light Microscopy Course – DIC
Without bias With λ/4 bias λ/2 shift at analyser
Resultants of retardation of beam by 60 deg (λ/6)
grey: no retardation, dark: blue retarded, light: red retarded
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Slide 23
DIC – with retardation bias
Light Microscopy Course – DIC
adapted from Peter Evennett
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Slide 24
The principle of DIC – how does it work
Light Microscopy Course – DIC
Condenser
Objective Specimen
adapted from Peter Evennett
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Slide 25
The principle of DIC – how does it work
Light Microscopy Course – DIC
Specimen Objective
Condenser
Beam splitter
Beam combiner
adapted from Peter Evennett
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Slide 26
The principle of DIC – how does it work
Light Microscopy Course – DIC
Specimen Objective
Condenser
Front focal plane of condenser
Back focal plane of objective
adapted from Peter Evennett
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Slide 27
The principle of DIC – how does it work
Light Microscopy Course – DIC
Specimen Objective
Condenser
Wollaston prism
Wollaston prism
adapted from Peter Evennett
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Slide 28
The principle of DIC – how does it work
Light Microscopy Course – DIC
Specimen Objective
Condenser
Polariser
Wollaston prism
Wollaston prism
Analyser
adapted from Peter Evennett
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Slide 29
The Wollaston prism – how does it work
Light Microscopy Course – DIC
- Remember birefringence in a Calcite crystal:
- two spots out of one - But: one looks to be
further away (ie had a retardation introduced in its path)
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Slide 30
The Wollaston prism beam splitter – how does it work
Light Microscopy Course – DIC
Wikipedia
direction of polarisation
Shear = spatial separation of the two beams
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Slide 31
The Wollaston prism – beam combiner
Light Microscopy Course – DIC
Wikipedia
direction of polarisation
orientation of analyser
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Slide 32
Problem with objective Wollaston prism
Light Microscopy Course – DIC
Specimen Objective
Condenser
Front focal plane of condenser
Polariser
Analyser
Back focal plane of objective
adapted from Peter Evennett
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Slide 33
Wollaston versus Nomarski-modified prism
Light Microscopy Course – DIC
Back focal plane
o .
o .
Nomarski prism Wollaston prism adapted from Peter Evennett
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Slide 34
The principle of DIC – how to set it up
Light Microscopy Course – DIC
- Köhler!!!!!
vvvvv
vvvvv
vvvvv
vvvvv
Condenser Objective
Illuminated Field Diaphragm
Primary image
Lamp collector
Eyepiece lens
Filament
- Insert and cross polarizer and analyzer
- Köhler!!!!! - Insert and cross polarizer and
analyzer - Insert objective Nomarski
prism
- Köhler!!!!! - Insert and cross polarizer and
analyzer - Insert objective Nomarski
prism - Swing in corresponding
condenser prism
- Köhler!!!!! - Insert and cross polarizer and
analyzer - Insert objective Nomarski
prism - Swing in corresponding
condenser prism - move objective prism for
optimal bias Polarizer
Analyzer
adapted from Peter Evennett
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Slide 35
Examples of DIC – what it is good for
Light Microscopy Course – DIC
!Unlabeled human RBCs in buffer on uncoated glass cover slip. Zeiss Axiovert 200M, 100x / 1.4 oil DIC.
axis of shear
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Slide 36
Examples of DIC – what it is good for
Light Microscopy Course – DIC
Zebrafish keratocytes speed of cells: ~ 13.5um/min; 3min 30sec movie Zeiss Axiovert 200M, 100x 1.4 oil DIC
axis of shear
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Slide 37
Examples of DIC – what it is good for
Light Microscopy Course – DIC
C. elegans embryos Gunar Fabig, MTZ axis of shear
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Slide 38
Examples of DIC - summary
Light Microscopy Course – DIC
- Good for thin or thicker specimen - labeled or unlabeled specimen
- Highlights gradients in optical path differences, not absolute optical path values
- For contrast in DIC shape of an object is more
important than the absolute phase shift produced by the specimen
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Slide 39
Examples of DIC – limitations
Light Microscopy Course – DIC
- Samples must be placed in an non-bi-refringent environment (no plastic dishes, no plastic lids!)
- Samples must be placed in an non-bi-refringent environment (no plastic dishes, no plastic lids!)
- dependent on perfectly set up Koehler illumination and strain free optics
- Samples must be placed in an non-bi-refringent environment (no plastic dishes, no plastic lids!)
- dependent on perfectly set up Koehler illumination and strain free optics
- some objectives are especially suited for polarization and DIC:
- labeled with “DIC” or labels in red letters. - all other objectives still work too, but there
might be quality problems
- Samples must be placed in an non-bi-refringent environment (no plastic dishes, no plastic lids!)
- dependent on perfectly set up Koehler illumination and strain free optics
- some objectives are especially suited for polarization and DIC:
- labeled with “DIC” or labels in red letters. - all other objectives still work too, but there
might be quality problems - contrast only achieved in direction of shear
between the two beams!
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Slide 40
The principle of DIC – lets practice
Light Microscopy Course – DIC
1) Köhler your microscope carefully!
Wikipedia
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Slide 41
The principle of DIC – lets practice
Light Microscopy Course – DIC
2) Insert and adjust polarizer and analyzer – crossed polars
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Slide 42
The principle of DIC – lets practice
Light Microscopy Course – DIC
3) If microscope has a De Senarmont compensator, put it into its position of zero bias
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Slide 43
The principle of DIC – lets practice
Light Microscopy Course – DIC
4) Put in correct objective prism into back focal plane (BFP) - related position of objective
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Slide 44
The principle of DIC – lets practice
Light Microscopy Course – DIC
5) Swing in correct condenser prism in front focal plane - Roman number (I, II, or III) has to correspond to the number
on objective prism used
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Slide 45
The principle of DIC – lets practice
Light Microscopy Course – DIC
6) check BFP (using Betrand lens or telescope) for image of blurred cross or move objective prism into position were background is darkest (maximum extinction)
from olympusmicro.com
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Slide 46
The principle of DIC – lets practice
Light Microscopy Course – DIC
7) turn objective prism or compensator for optimal bias
from olympusmicro.com
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Slide 47
The principle of DIC – lets practice
Light Microscopy Course – DIC
Use either cheek cells or diatoms
from photomacrography.net