Transcript Polarized Light

VII
Contrasting Techniques
From Brightfield to Plas-DIC
December 2008
Rudi Rottenfusser
0 Units
50 Units
100 Units
C ONTRAST
50 Units
50
50 – 100 / 50 + 100 =
Brightness of Specimen - Brightness of Background
Brightness of Specimen  Brightness of Background
50
-0.33
Illumination Techniques Overview
Transmitted Light
Incident Light
• Brightfield
• Oblique
• Brightfield
• Oblique
•
•
•
•
•
•
•
•
•
•
Darkfield
Modulation, Varel Contrast
Phase Contrast
Polarized Light
DIC (Differential
Interference Contrast)
• Fluorescence - not any
more > Epi !
Darkfield
Not applicable
Not any more (DIC !)
Polarized Light
DIC (Differential
Interference Contrast)
• Fluorescence (Epi)
Example
Brightfield
• For stained or naturally absorbing samples
• True Color Representation
• Proper Technique for Measurements
•Spectral
•Dimensional
Brightfield
Best Resolution when Condenser NA matches Objective NA!
Minimum Contrast!
d
Objective
Specimen
Resolution
(minimum resolved distance
between 2 details):
dmin 
Condenser
dmin
λ
2  NAObjective
Getting more contrast in the microscope:
“Dropping” the condenser
Bad Idea!
• No more separation of controls for
field size and aperture angle
• Higher contrast, but at the cost of NA
• Scattered light enters the objective
• Condenser not in proper position >
spherical/chromatic aberrations
Getting more contrast in the microscope:
“Stopping down” the condenser
(reducing the size of aperture diaphragm)
•Increases contrast
•Increases depth of field
•Reduces resolution
dmin 
λ
2  NAObjective
Condenser Aperture
matches Objective
dmin 
NAObjective
λ
 NACondenser
Condenser Aperture
stopped down
Effect of Aperture Diaphragm
NA Condenser
= NA Objective
Paramecium bursaria
Condenser diaphragm open
Condenser Diaphragm almost closed
Paramecium bursaria
Different Staining Techniques
Indian Ink Staining
Feulgen Staining
Silver Staining
Contrasting Techniques
Going more into details
 Brightfield
• Oblique
• Darkfield
• Phase
• Varel
• Hoffman
• Pol
• DIC
• Plas-DIC
Getting more contrast in the microscope:
Oblique Illumination
(moving the aperture diaphragm sideways)
•Increases contrast
•Increases depth of field
•3-D effect
•Slightly reduces resolution
Low NA
Objective
Darkfield
Iris Diaphragm
High NA
Objective
Required conditions:
Illumination Aperture must be
larger than objective aperture
I.e. direct light must bypass
observer
Darkfield
Highest contrast
Detection of sub-resolution details possible
No staining necessary
Central Darkfield via “hollow cone”
Oblique Darkfield via Illumination from the side
Excellent technique to detect traces of contaminants
Not useful for Measurements (sizes exaggerated)
Paramecium bursaria
Darkfield
Polarized Light
Phase Contrast
(Frits Zernike 1934)
- “Halo” effect > Reduced resolution
+ No staining necessary
+ Good Depth of Field
+ Easy alignment
+ Orientation independent
+ Repeatable setup
+ Works with plastic dishes
+ New positive / negative Phase Contrast
Required Components
for Phase Contrast:
1.
Objective with built-in
Phase Annulus
2. Condenser or Slider
with Centerable Phase
Ring for illumination
(Ph0, 1, 2 or 3)
Required Adjustment:
Superimpose Phase Ring of
condenser over (dark)
phase plate of objective
(after Koehler Illumination)
•Illumination bypasses
Specimen > no phase shift
•Illumination passes
through thin part of
Specimen > small phase
retardation
•Illumination passes
through thick part of
Specimen > larger phase
retardation
Phase Shifts:
Cells have higher n than water. Light moves slower in
higher n, consequently resulting in a phase retardation
Phase shift depends on n and on thickness of specimen
detail
Intermediate Image
Phase Contrast
Imaging Path
Diffraction
Orders
Non-diffracted wave
(shifted by l/4)
Phase Plate
Non-diffracted wave
Diffracted wave
(shifted by -l/4)
Objective
Specimen
Condenser
λ  520nm
{
Condenser
Phase Ring
4. Non-diffracted and diffracted light are
focused via tube lens into intermediate
image and interfere with each other; ¼+¼=
½ wave shift causes destructive interference
i.e. Specimen detail appears dark 
Tube Lens
Objective
Specimen
Condenser
3. Affected rays from specimen, expressed by
the higher diffraction orders, do not pass
through phase ring of objective
>¼ wave retarded 
2. Objective Phase Ring 
a) attenuates the non-diffracted 0th Order
b) shifts it ¼ wave forward 
1.
Illumination from Condenser Phase Ring
(“0” Order) > meets phase ring  of
objective
More Information in Phase Contrast
Positive and negative Phase
Contrast in one Objective
Objectives:
LD Plan-Nefluoar 20x Ph1 Ph2Korr
LD Plan-Neofluar 40x Ph1 Ph2Korr
MDCK cells (dog)
R. Nitschke and F. Kotsis,
Life Imaging Center, Freiburg
Positive Phase Contrast
Negative Phase Contrast
Sales Training Oct. 2006
Observation
Paramecium bursaria
Brightfield
Condenser diaphragm open
Phase Contrast
Rhipidodendron
Phase Contrast
Cochliopodium
Phase Contrast
Lyngbya Bacteria
Phase
Contrast
Thin Phase Object
in plastic vessel
Varel
Contrast
Neurons
Varel Contrast (1996 - Zeiss)
For unstained (live) specimens
Combination of oblique illumination and
attenuation of non-diffracted light
No “Halo”-effect as in Phase Contrast
Complementary technique to Phase
(easy switchover)
Simulated 3-D image (similar to DIC)
Less resolution than DIC
Works with plastic dishes
Back Focal Plane of
Varel / Phase Objective
Required Components for Varel:
1. Objective with Varel- and Ph ring
2. Slider or Condenser with specific
Varel 1 or Varel 2 ring sector
Brightfield / oblique
Darkfield
“Varel”
Movable Ring Sector (Varel Ring)
Modulation Contrast (Hoffman)
For unstained (live) specimens
Simulated 3-D image (similar to DIC)
No Halo-effect (as in Phase Contrast)
Usable with plastic dishes
Less resolution as DIC
Note: Modulation Contrast Objectives are not
recommended for fluorescence; due to potential
damage of modulator and uneven illumination
Modulation Contrast
3% transmittance
Required Components
for Modulation
Contrast:
Specially Modified Objective
(With Built-in Modulator)
Modified Condenser with offaxis slit (double slit with
polarizer)
Polarized Light
One starts out usually by crossing two polarizers
(polarizer and “analyzer”) in a microscope.
The specimen is located between them.
Only birefringent particles (e.g. crystals) become
visible, when they are rotated via rotating stage.
Isotropic components will remain dark.
Polarized Light looks sometimes just like
Darkfield because edges become visible due to
“edge birefringence”.
Polarized Light
Analyzer
Birefringent
Material
Polarizer
Polarized Light
Analyzer
Polarizer
Analyzer
Birefringent
Material
Polarizer
Polarized Light
Analyzer
Analyzer
Polarizer
Birefringent
Material
Polarizer
Polarized Light
Analyzer
Birefringent
Material
Polarizer
Polarized Light
Analyzer
Birefringent
Material
Polarizer
Polarized Light
Polarizer 2
(Analyzer)
When Polarizers are
crossed, only items that
rotate the plane of
polarization reach the
detector.
Wave plate adds color
Specimen
Polarizer 1
Birefringent Material
Background
Brightfield
Polarized Light
Color of
sample and background
modified by wave plate
Pol + Red I
Required / Recommended
Components:
• Polarizer (fixed or rotatable)
• Analyzer (fixed or rotatable)
• Strain-free Condenser and Objective
• Rotating, centerable Stage
• Wave plate and/or Compensator
• Crossline Eyepiece
Birefringence
• The numerical difference between the maximum and minimum
refractive indices of anisotropic substances. nγ - nα.
• Birefringence may be qualitatively expressed as
• low (0 - 0.010),
• moderate (0.010 – 0.050)
• high (>0.050)
• extreme (>0.2)
• Birefringence may be determined by use of compensators, or
estimated through use of a Michel-Lévy Interference Color Chart.
Optical Path  n  d
Optical Path Difference  nObject  d - nBackground  d  (no - nb   d
•An excellent introduction to this chart is provided at McCrone’s website
http://www.modernmicroscopy.com/main.asp?article=15
LOW
< 0.010
Moderate
0.010 – 0.050
High
> 0.050
3rd
Order
Red
2nd
Order
Red
1st
Order
Red
1st
Order
Red
2nd
Order
Red
3rd
Order
Red
alpha
gamma
1st Order Red Plate
550 nm Retardation
Sensitive Tint
Field of
View
50
I/Io
40
30
20
10
0
0
100
200
300
400
500
600
700
800
900
1000
Retardation (nm)
1100
1200
1300
1400
1500
1600
1700
1800
Orthoscopy / Conoscopy
•
Analyzing minerals is based on such morphological and optical features as form,
cracks, color, pleochroisms, and their characteristic interference colors.
•
Orthoscopy and conoscopy are the most important techniques in classical transmitted
light polarization microscopy. With their different ways of examining, they provide
different options, e.g. in mineral diagnosis in geological microscopy.
•
In orthoscopy, each pixel corresponds to a dot in the specimen.
•
In conoscopy, each pixel corresponds to a direction in the specimen. This technique
requires the use of the highest objective and condenser aperture possible.
•
Conoscopy is used when additional information about the specimen is necessary for
analysis. It provides interference images that can be seen through the eyepiece and
enables differentiation according to 1 or 2 axes and with compensator λ (λ-lamda,
Red I), according to 1-axis positive/negative or 2-axis positive/ negative.
•
A Bertrand lens in the light path makes visible the interference or axial image in the
back focal plane of the specimen.
Some Types of Birefringence
• Intrinsic or crystalline
(Quartz, Calcite, Myosin Filaments, Chromosomes, Keratin, Cellulose Fibers)
• Form or Textural (Plasma membranes, Actin filaments, microtubules)
• Edge (resulting from diffraction at edges of objects embedded in a
medium of different refractive Index)
• Strain (resulting from mechanical stress e.g. glass, plastic sheets)
• Circular –also known as- Optical Rotation
(sugars, amino acids, proteins)
Light as an electromagnetic wave
The wave exhibits electric (E) and magnetic (B) fields whose amplitudes oscillate as a
sine function over dimensions of space or time.
The amplitudes of the electric and magnetic components at a particular instant or
location are described as vectors that vibrate in two planes perpendicular to each
other and perpendicular to the direction of propagation.
At any given time or distance the E and B vectors are equal in phase.
For convenience it is common to show only the electric field vector (E vector)
of a wave in graphs and diagrams.
Polarized Light
y
E
z
x
E
E
Ey
Ex
l
l
Polarized Light and Birefringence
Polarized Light and Birefringence
Interface with
birefringent Material
ng = higher refractive index > slower wave
na = lower refractive index > faster wave
How to create circularly polarized light
Linear
polarizer
¼ wave plate
Circularly Polarized Light
5
x
E
z
4
3
2
1
5
4
3
2
1
l
E
y
Sénarmont Compensator*
E
x
z
E
y
E
x
¼ wave plate, located before
analyzer, is oriented with its
birefringence parallel to the
polarizer
E or analyzer. Therefore,
there will be no effect on the
polarized beam.
Birefringence produced by
specimen (occurring at 45˚), will
be converted by ¼ wave plate into
circular polarized light which can
pass through the analyzer.
By rotating the analyzer, it is
possible to introduce “bias”
birefringence because it will not be
parallel to ¼ wave plate any more.
* 1st described by de Sénarmont in 1840
DIC Principle
9 Image
8 Tube lens
7 Analyzer
7a Wave Plate)
6 Wollaston Prism Slider
5 Objective
4 Specimen
3 Condenser
2 Wollaston Prism
1 Polarizer
(F.H.Smith, 1952)
DIC
(Nomarski/Allen 1969)
Differential Interference Contrast
Changes GRADIENTS into brightness differences
Eye-pleasing 3-D Image appearance
High Contrast and high resolution
Control of condenser aperture for optimum contrast
Great for “optical sectioning” due to small depth of field
Color DIC by adding a wave plate
Best contrast / resolution via different DIC sliders
Orientation-specific > orient fine details perpendicular to DIC prism
Requires strain-free elements, not for birefringent specimens
Wollaston Prism
y
E
x
E
z
Ey
Ex
l
Polarized beam, under 45˚ to prism,
gets split into “ordinary” and
“extraordinary” beam
DIC
Observing local differences in phase retardation
IR-DIC
IR increases depth of field – useful for thick tissues
Achieve Contrast in Electrophysiology applications
Special Objective and Polarizer recommended
Requires IR filter for transmitted light
For heat protection, special filter combination
Special Filter Arrangement
for IR-DIC
100.00
100.00
80.00
80.00
60.00
60.00
40.00
20.00
RG 9
Calflex
30
0
33
0
36
0
39
0
42
0
45
0
48
0
51
0
54
0
57
0
60
0
63
0
66
0
69
0
72
0
75
0
78
0
81
0
84
0
87
0
90
0
93
0
96
0
99
0
0.00
-20.00
RG 9 = IR Filter
Calflex = Heat reflecting filter
40.00
20.00
0.00
-20.00
PlasDIC
Rainer Danz, 2004 (patented 2006)
Most important before
injection: sharp image
of
zona pellucida,
tip of injection pipette
and oolemma
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Homogeneous
Exit Pupil at Back
Focal Plane when
both Wollaston
prisms are in place
Conventional DIC
(Nomarski-Principle):
Observing
Back Focal Plane
between
crossed Polarizers
(Only DIC Slider of
objective in place!)
Note:
-1
Both prisms
in place:
Observing
Back Focal Plane
between
crossed Polarizers
(Only DIC Prism of
condenser in place!)
0
+1
Condenser,
Specimen,
Objectives are
between polarizers,
therefore they
must not produce
any birefringence
for optimal results
in DIC!
-1 are0oriented
+1at 45o in the microscope. Polarizers are East-West,
Note: The fringes in the back focal plane
Analyzers South-North. It is impractical to “draw” a prism cross-section under 45o to the drawing surface…
Observing Back Focal
Plane between crossed
Polarizers:
FrançonYamamoto
Analyzer
.
BFP
-1
0
Conventional
DIC
+1
Objective
Objective
Condenser
Slit
Polarizer
o
Note: The fringes in the back focal plane are actually oriented at 45 in the microscope.
Polarizers are East-West, Analyzers South-North. This display takes into account that it is
o
impractical to “draw” a prism cross-section under 45 to the drawing surface…
ZEISS Plas-DIC
Analyzer
.
BFP
Observing Back Focal
Plane between crossed
Polarizers:
-1
0
Conventional
DIC
+1
Objective
Polarizer
Objective
Condenser
Slit
o
Note: The fringes in the back focal plane are actually oriented at 45 in the microscope.
Polarizers are East-West, Analyzers South-North. This display takes into account that it is
o
impractical to “draw” a prism cross-section under 45 to the drawing surface…
Sinc Function
Contrast = f (Slit Width)  sinc D 2pl
1
Contrast
0.75
0.5
0.25
l/4 Optimal
Condition !
Slit Width as it is projected into
Back Focal Plane (BFP) of Objective
0
-0.25
0
1/4
1/2
3/4
Distance between 0 and 1st order of birefringence
1
Required Components for Plas DIC
1) Nosepiece with receptacles for DIC sliders (AxioObserver or Axiovert 40 CFL)
2) Slit diaphragm PlasDIC
for condenser or slider
3) Objective for available
Plas-DIC Sliders
4) The right PlasDIC slider
for each objective
5) Fixed Analyzer Slider or Analyzer in Cube
Note:
PlasDIC and Analyzer sliders should be removed during fluorescence
imaging. They will reduce the intensity substantially if left in place!
PlasDIC - Advantages
The first polarizationoptical interference
contrast designed for
plastic vessels
High optical resolution, close to regular DIC, at
least equal to *Hoffmann Modulation contrast
Patent No. DE 10219804
Cost effective (no special objectives, no special
condensor, no second prism)
Excellent relief and three dimensional impression,
large depth of field, great for work with
manipulators and multiple probes
Plastic dishes, Ph objectives, birefringent specimens
have no effect on image quality
Very simple handling: no centering or change of diaphragm
Easily upgradable: takes customers budget into account
*Hoffmann Modulation contrast: Also very good contrast, but most
users don´t know how to optimize the settings, which are much
more complicated to establish.
68
Comparison of Contrast Methods
Phase
VAREL
Contrast thick
specimen
__
+
Contrast thin specimen
++
Resolution
Hoffmann
PlasDIC
DIC
+
+
++
-
+
+
+
+
__
+
+
++
Optical Sectioning
capability
__
_
+
+
++
Depth of focus in living
cells
__
+
+
++
+
Homogenity field of
view
++
_
+
+
+
Reproducibility of
setting
_
_
_
+
+
Plastic Vessels
+
+
(+)
++
_
Price
l
ll
llll
lll
lllll
69