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Lecture 3: Special contrast
enhancement methods in microscopy
Contrast enhancement methods using visible light (VIS)
 Darkfield microscopy
 Phase contrast microscopy
 Polarized light microscopy
 Differential interference contrast (DIC) microscopy
 Hoffman modulation contrast microscopy
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Darkfield microscopy
The oldest, simplest contrast method
 Image formation of darkfield microscopy:
 A condenser central stop: block incoming direct light
Generate a hollow cone of illumination
When no specimen on stage:
an oblique illumination with dark background
When there is specimen on stage:
 Oblique rays hit light scattering, granular objects
light is scattered away from propagation direction
enter objective lens cone of light
 Form bright dot image on a black background
 Suitable specimen:
unstained, non-light absorbing small particles
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Construction of a low magnification, low NA
darkfield microscope
 Condenser of Abbe type
 Condenser NA > objective lens NA
(Cond_NA usually 0.95)
 Objective lens NA < 0.75
(usually =< 40x)
If objective lens NA is too high, ordinary
condenser NA cannot match Obj_lens NA
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Low Mag, Low NA darkfield microscope
can be home-made
Darkfield microscope with low mag, ≤ 40x, low NA, < 0.75
does not need complicated optics, can be home-made easily
 An Abbe condenser with NA 0.95.
a swing-in top lens.
 A central-stop cut from carton
board at 22 mm.
 Tape the stop on the bottom surface
of the condenser.
 This central stop size works for
20x/0.5, 40x/0.75 objective lens
condenser central stop size for different Mag/NA obj lens
4x/0.1
10x/0.25
20x/0.4
40x/0.65
8-14 mm 16-18 mm 18-20 mm 20-22 mm
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Other types of darkfield microscope
 Darkfield microscope with high magnification, high NA obj-lens
 specially made high NA paraboloidal immersion condenser has to be used
Dry DF condenser
Narrow light cone
Paraboloidal oil DF condenser
Wide light cone
 Or objective lens NA is reduced by variable NA iris lens
 Reflected light darkfield microscope: used for material microscope.
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Usage of darkfield microscopy
Detecting microbes in rapid diagnosis
(field diagnosis)
Syphilis Spirochete
One of the oldest
disease with human
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leptospirosis
In south east Asia
water-born bacterial
Borrelia burgdorferi
spirochete
In north America
bacteria spread by tick
Lyme disease
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Phase contrast microscopy
 Amplitude objects:
capable of absorbing light, cause intensity change of visible light
 Phase objects:
Do not interact with light strongly
Light passes through, little absorbing
little intensity change
Diffracted light is slightly delayed
by ¼ λ (phase difference)
 Phase object image formation
Original ray (O) and diffracted light (D) reach BFP of objective
interfere with each other, resulting particle wave P on image plane
P wave has 1/20 λ phase difference and no visible intensity difference compared
to original wave O
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Development of phase contrast microscope
Fritz Zernike, a Dutch physicist found a way to enhance the contrast in 1934.
 Shift direct light ¼ λ distance ahead to make total ½ phase difference between direct
and diffracted light.
 Constructive or destructive interference occur on image plane.
 Visible light intensity change on the image.
2. Obj. phase ring
Dr. Zernike won Nobel prize
to speed up direct ray
for Physics in 1953
1. Condenser phase stop
surround by a transparent ring
to separate direct ray apart
Figure Adopted from Zernike
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Phase contrast optics
Condenser phase annulus
(phase-stop)
Objective lens
Phase plate
(phase ring)
Phase-stop and
phase ring overlay
Seen at Objective lens back focal plane (BFP)
Condenser turret
With phase, DIC optics
Thinned Ring with
light-attenuation coating
Aligned rings
PH2: 10-20x objective lens
PH3: 40-100x obj. lens
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Results of phase contrast
 Direct light is ½ λ ahead of deviated light with similar intensity.
 Constructive or destructive interference occur at image plane effectively:
D ¼ λ delay
No phase contrast
D ½ λ delay
Constructive interference
D ½ λ delay
Destructive interference
 The method of advancing direct light or O-wave is called positive phase contrast
in which high RI region looks darker.
(Negative phase contrast retard deviated light, in which high RI region looks brighter)
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Interpreting phase contrast images
 Concept of optical path length(OPL)
 Refractive index n and specimen thickness T together determine: OPL = n.t;
 Optical thickness difference between two parts of the specimen:
∆OPL (or OPD) = (n2-n1)t
Ex. Cell culture: cell thickness 5 µ, n1=1,36, media: n2=1,335
∆OPL=5.(1.36-1.335)=125 nm (about 1/4 λ of green light)
(If not enhanced by phase contrast, 1/4 λ phase change is not visible.
If thinner than 5 µ, even phase contrast enhancement is not effective enough)
Phase contrast enhance the boundary where refractive index differ sharply
In Positive phase contrast: structure with higher RI looks darker.
Cell nuclei, nucleoli, mitochondria, ribosomes looks darker than media.
Pinocytic vesicles, vacuoles, lipid droplet, looks brighter than media, it is not thinner though
The darker region is not necessarily the thick region, it is region with bigger OPL
In Negative phase contrast: D-ray is further delayed 1/4 λ, effect is same but opposite
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Pros and cons of phase contrast microscopy
 Advantage:
View live cell without staining with high image quality becomes possible,
which made a big impact on biomedical research field.
 Disadvantage:
Reduced resolution:
Objective cone of light not fully filled, objective aperture not fully utilized: about 10% NA used.
 Phase halo around large, low spatial frequency structure like entire cells, nuclei.
 Shade-off: in large, extended structure, intensity is not uniform, increase towards center.
 Not works well with very thin specimen, poor depth of field and z-resolution.

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Polarized Light Microscopy
 Polarized light and cross-polarization
Electromagnetic wave is formed by oscillation of electrical field (and magnetic field)
Most light source emit light with electric field vibrating randomly all planes perpendicular to
its propagation: they are unpolarized light.
If vibration is restricted in one plane, the light is plane polarized light
EM wave has both electric and
magnetic field
Polarized light
Only electric field is described in
polarization
 Plane polarized light cannot pass polarizer perpendicular to its
vibration direction
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Birefringent material for polarized light microscopy
 Birefringence material (double refraction)
 Distinct optical axis: axes have different Reflective index
 Light pass through it subjects to different RI
 Structure of birefringent material
Regular repeated structures: crystalline or paracrystalline
 Common birefringent material:
 Anisotropic crystal like quartz, calcite, some plastic polymer
 Bio-birefringent material:



DNA, collagen fiber, muscle fibrils
uric acid crystal
structure with beta-pleated sheet: amyloid fibrils, Ig-light chain
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Birefringent material for polarized light microscope
Interaction of light with Birefringence material
 Rays oblique to axis is divided:


Ordinary ray: same speed
Extraordinary ray: slowed down.
phase retardation.
 Wave perpendicular to axis
E ray slower but same direction as O
 Rays parallel to optical axis
E and O rays are the same
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Configuration of microscope
• Polarizer: to provide polarized light
• Analyzer (another crossed polarizer)
• *A 360 degree rotate stage is useful (helps to
identify beam direction)
• *An accessary retardation plate (λ plate) helps
enhance contrast (change interference color)
*not necessary but helpful
Specimen observation, image formation
• When there is no specimen:
dark background
•
When there is birefringent specimen:
Light divided, retardated and recombined
Interference occurs, visible intensity change
Max intensity at a particular rotation angle
Newton’s color (polarization color)
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Birefringent specimen under polarized light
Amyloid fibrils
Congo Red staining
Brightfield microscopy
Dull brick-red color
in bright field microscopy
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Amyloid fibrils
Congo Red staining
Polarized light microscopy
Apple green birefringence
in polarized light microscopy
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Differential interference contrast microscopy (DIC)
Image formation in DIC
7. Analyzer allows elliptical polarized light to pass
6. Obj. Nomarski remove shear, combine two phase
changed wavefronts to elliptical polarized light
5. Objective lens accepts parallel ray from specimen,
focus them to obj BFP, interaction
4. Sheared rays pass through specimen, diffracted,
optical path changed and wavefront distorted.
3. Condenser project sheared rays parallel apart
2. Condenser Nomarski divides beam (shear)
The divided rays reach condenser FFP
1. Polarizer produce linear polarized light
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PL_MIC + DIC optics
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How DIC form images of cells
DIC Contrast formation
A and C: no OPD, no contrast enhancement.
B: an apparent change of OPD, contrast enhanced.
DIC enhances contrast where the shear of two rays show a big change rate of
optical path difference (OPD).
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DIC image formation and
Introduction of bias retardation
Bias retardation translation
http://microscopy.fsu.edu/primer/techniques/dic/dicintro.html
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DIC and its usage
 Advantage of DIC
 High resolution, use full objective aperture; no restriction from condenser
 Shadow-cast, psudo-3D profile of the structure
 Good depth of field and z-resolution, optical sectioning is possible
 View slightly stained specimen is possible
 Determine orientation of phase gradient
 Disadvantage of DIC
 Qualitative, not quantitative, not true thickness but optical path length
 Birefringent specimen cannot be used
 Polystyrene plastic cell culture dish, bottle, chamber cannot be used
 Even the mounting media has to be under scrutiny: not use PVA which a
popular component in mounting media
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Example of DIC images
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DIC VS. Phase contrast (1)
Phase contrast: halo effect at
edge, less details visible.
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DIC image: no halo effect, more
intracellular details, sharp edge
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DIC VS. Phase contrast (2)
Phase contrast: thick specimen
Poor image quality and depth
of field
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DIC: perform well in thick specimen
Optical section effect
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DIC VS. Phase contrast (3)
Phase contrast: poor z-resolution
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DIC: good z-resolution reveals
intracellular structures
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Hoffman modulation contrast
Inverted microscope with Hoffman modulation
P1: rotatable
polarizer
P2: polarizer
with slit
Modulator:
3-zone ND
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Re-map phase gradient to intensity gradient
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Hoffman contrast image formation
Polarizer and modulator alignment, adjustment
Align p2 with
modulator
P2 light
Micrograph of cell culture
by Hoffman contrast
P2 dark
Contrast control effects of P2
by rotating P1
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Image formation mechanism in
Hoffman modulation contrast
 Does not change phase of light
(the P-wave is 1/20 λ delayed measured at BFP,
same as phase specimen in ordinary bright
field microscopy)
 The specimen’s phase gradient
is re-mapped to different zone
(on objective lens’s BFP)
 Image is formed on image plane
with different intensity
Phase gradient has direction, rotate specimen help achieve better result
when direction of the gradient is unknown
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Comment on Hoffman contrast
 Better resolution than phase contrast.
 Bypass birefringent restriction from DIC.
 Cheaper to implement than DIC.
 Through gradual phase gradient to gradual intensity gradient, an image with 3D
relic is formed and depth detection is easier
 Newer on the market: If want add to existing system, need buy accessories and
modify existed objective lens. Extra cost need.
 http://micro.magnet.fsu.edu/primer/techniques/hoffman/hoffmanintro.html
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DIC VS. Phase contrast Vs. Hoffman modulation contrast
(1) mechanism
 All work with phase specimen.
 All use RI difference (phase difference) between structures and surrounding.
Convert RI difference to image amplitude difference for contrasting.
 Enhance contrast by different mechanisms:
DIC
Convert deep OPD over pairs of ray shear divided by Nomarski prism into polarization
difference, filter away linear polarized rays, and finally produce intensity difference.
The shear is small, usually smaller than obj-lens resolution: enhancement is very localized
Phase contrast
enhance contrast by speed up direct ray to stretch small phase differences existed in the
specimen, making the difference big enough for interference, yield intensity difference
Hoffman modulation contrast
Re-map (modulate) phase gradient into different intensity zones to enhance contrast
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DIC VS. Phase contrast Vs. Hoffman modulation contrast
(2) Usage
 Phase contrast
 Simple, easy to configure, cheap, time-honored
 Phase halo, not good for too thin, too thick specimen
 Low resolution: 1/10 of the objective lens NA is used
 DIC
 Full resolution of the lens, high image quality, good for both
thick and thin specimen
 Not suitable for birefringent specimen or container with
birefringent material.
 Expensive, difficult to setup and align the optics
 Hoffman contrast
 avoid phase halo with higher resolution, bypass birefringent
restriction
 Not widely available, image quality is moderate
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