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Transmitted light contrasting
techniques: BF, DF, PC, pol,
DIC
Judith Lacoste, Ph.D.
McGill Systems Biology Program
Second Annual
Introduction to Light Microscopy
December 7th 2010
Light Microscopy
Sample
Image
Light source
! Bright Field (BF)
! Dark Field (DF)
! Phase Contrast (PC)
! Polarization (Pol)
! Differential interference contrast (DIC)
Transmitted Light
Sample
ImageLight source
Fluorescence
Why is transmitted light
microscopy important?
Bright Field (BF) Microscopy
Opaque
Thick
Chromophores
Sample
Image
Light source
Condenser
Objective
www.zeiss.com
! Wavelength,frequency and color
! Light matterinteraction:absorption,reflection.
Wave Nature of Light: !
wavelength
~490 ~520 ~640~570~550! (nm):
Chromophores
BF detects colorsLight source Sample Light-matter interactions
Light-matter interactions Light-matter interactions
! Amplitude or intensity
! Absorption, reflection,diffraction, scattering
BF detects intensity modulations
amplitude
Opaque
Thick
BF light path
www.zeiss.com
Objective
Image
Condenser
Sample
Light source
An important BF application:
histology H&E stain! Hemotoxylin: binds acidic components
(e.g. nucleic acids) and stains them
purple/blue.
! Eosin: binds basic structures such as
positive amino acids (e.g. collagen) and
stains them pink/magenta.
BF and Image Contrast! How the object of interest is perceived over its surrounding
! Magnification and resolution are irrelevant without contrast
! How many dark squares?
Claire Brown, McGill Imaging Facility
Image Contrast: intensity
Murray et al., J. Microscopy, 2007
Image Contrast Modulation
http://micro.magnet.fsu.edu/primer/java/contrast/intensity/index.html http://commons.wikimedia.org/wiki/Imag
e:Contrast_change_photoshop.jpg
Strategies for low contrast samples
Strategies to translate invisible optical effectsinto human-visible contrast:
! Minimize background intensity:
! dark field
! Detect phase variations:
! phase contrast
! Detect light polarity variations:
! Polarization
! DIC
Minimize background intensity
and oblique illumination
www.zeiss.com and J.Piper
Dark field microscopy detects small
amplitude modulationsBF
DF
DF implementation and light path
www.zeiss.com
Image
Sample
Objective
Light source
Condenser
Annular stop
DF provides more details
Less details More details
http://micro.magnet.fsu.edu/primer/techniques/darkfield.html
Bright field Dark field
Radiolaroians, small marine protozoans
Darkfield Images
Silkworm larva
spiracle and trachea
Mosquito Head
and ProboscisDiatom Arachnoidiscus
ehrenbergi
http://www.olympusmicro.com/primer/techniques/darkfieldgallery.html
Thin unstained objects
http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.html
! Cells in culture, bacteria, parasites, histological and cytologicalpreparations, platelets, urine sediments, vaginal smears, cytogenetic andbone marrow preparations, organelles.
! Barely exhibit any light absorption.
! Human vision in BF or DF barely detects them
! They do interact with light (diffraction and refraction), but the result is notdetectable by human vision: phase shift
Phase shift and BF microscopy
S wave
P wave
PHASE OBJECT
D wave:
Lower amplitude
!/4 phase retarded
Light going through a phase object creates S and D waves
S and D waves are added together in the focal plane to create the P wave
In BF, the P wave is undistinguishable from the illumination: same amplitude but phase retarded
P wave
Positive: Speed up the direct light using a high refractive index plate with an
etched ring physically reducing the distance the direct light has to travel.
Negative: Slow down the direct light using a high refractive index phase
plate with a raised ring physically increasing the distance the direct light
has to travel .
http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.html
Phase shift: speed of light,
thickness and r.i. of sample
http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.html
Positive phase contrast
Positive (thick/high RI objects look dark)
Ctenoid fish scale Chinese hamster
ovary (CHO) cells
Human erythrocytes
Optical Path Length (OPL) = thickness (t) x refractive index (n)
So contrast is related to both how thick a specimen is and also how
high the refractive index is.
The higher the refractive index the slower the light travels and the
longer the OPL.
Therefore, a thick area of the specimen with moderate RI may result
in the same contrast as a thinner area of the specimen with high RI.
http://micro.magnet.fsu.edu/primer/lightandcolor/refractionintro.html
Optical path length
PC detects phase shift: light path
www.zeiss.com
Sample
Objective
Light source
Condenser
Annular stop
S wave
D wave:
Lower amplitude
!/4 phase retarded
Image
Phase plate
http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.html
Implementing PC microscopy
! In general, withincreasing magnification:
! The phase plate ringgets smaller and thinner
! The condenser annuliget larger and wider
http://www.olympusmicro.com/primer/java/phasecontrast/phasemicroscope/index.html
The condenser annulus and the phase ring must be well aligned to
generate good phase contrast images.
Phase ring alignment
Phase contrast halos
http://www.olympusmicro.com/primer/anatomy/numaperture.html http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.html
Some of the first order diffraction – resulting from
diffraction around larger structures - passes through
the phase ring at the rear aperture plane of the
microscope objective.
These light rays are sped up with the incident light
causing constructive interference. This results in bright
halos in the image.
http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.html
Phase contrast halos
http://www.olympusmicro.com/primer/techniques/dic/dicphasecomparison.html
! Wavelength, frequency or color: BF
! Amplitude or intensity: BF or DF
! Phase or coherence or step: PC
! Polarity or direction of vibration: polarization microscopy and DIC
Properties of light
phase
amplitude
wavelength
Natural light vibrating in all directions,
perpendicular to the ray of light
Polarization microscopy
Sample
Image
Light source
Condenser
Objective
www.zeiss.com
Analyzer
Polarizer
Sample-
induced
light polarity
modulation
Pol microscopy implementation
http://www.olympusmicro.com/primer/techniques/polarized/polarizedintro.html
! Polarizer
! Between lightsource and sample
! Analyzer
! Between sampleand detector
! Cross-polarization
! Perpendicular toeach other to createdark background (nosample).
Polarizer and analyzer
Natural light
vibrating in all
directions,
perpendicular to
the ray of light
No incident light
Polarizer
Analyzer
(second polarizer)
Polarized light without any light
matter interactions
http://www.olympusmicro.com/primer/lightandcolor/images/polarizedlightfigure6.jpg
Polarized light microscopy
! The amount of light passing through crossed polarizers is determinedby the orientation of the analyzer with respect to the polarizer .
! For maximum level of extinction they should be cross polarized andthe image field of view will look dark.
Specimen altering light polarity
http://micro.magnet.fsu.edu/primer/lightandcolor/birefringenceintro.html
http://www.geocities.com/prasanth_p_jose/liquid_crystal.jpg
! Birefringence (double refractions):
! Decomposition of a ray of light into tworays (ordinary and extraordinary) whenpassing through anisotropic matter.
! Isotropic matter:
! Having the same optical properties in alldirections (e.g. homogenous material).
! Anisotropic matter:
! Having orientation-dependent opticalproperties (e.g.crystal, plant cellwall).
Birefringence by calcite crystals
http://micro.magnet.fsu.edu/primer/lightandcolor/birefringenceintro.html
! Birefringence is exhibited to a greater or lesser degree in allanisotropic crystals.
! The O and E rays travel through the material at different speed.
! They emerge the crystal as phase-shifted and separated rays.
Birefringence! When a polarizer is placed in front of the
crystal, one of the images disappears.
! When the polarizer is rotated, the other
image disappears.
! When the polarizer is oriented E-W only the
ordinary waves pass.
! When the polarizer is oriented N-S only the
extraordinary waves pass.
! When the polarizer is oriented at 45o partial
ordinary and extraordinary waves pass.
http://micro.magnet.fsu.edu/primer/lightandcolor/birefringenceintro.html
Birefringence by most biological
material
Birefringent material
Polarized light
Extraordinary ray
Ordinary ray
Phase shifted
Ordinary and
Extraordinary rays
! The O and E rays travel through the material at different speed.
! They emerge the crystal as phase-shifted rays and but they are notseparated rays.
O-E rays phase shift translated into
contrast amplitude
Phase shifted
Ordinary and
Extraordinary rays
Analyzer
(second polarizer)
Vectorial
recombination of
phase shifted rays
!The O and E waves being indifferent planes, they can’tundergo constructive/destructive interference tocreate contrast.
!Instead, the two waves arecombined vectorially and onlythe horizontal vector passesthrough
No transmission
Dinosaur Bone Alkalic Syenite
http://www.olympusmicro.com/galleries/polarizedlight/index.html
Polarization colors result from the
interference of the two components of
light split by the anisotropic specimen
and may be regarded as white light
minus those colors that are interfering
destructively.
Polarized light images
http://www.nikonsmallworld.com/gallery/year/2009/57
Bensoic Acid
Differential Interference
Contrast (DIC) – Nomarski Contrast! Technique popularized by Georges Nomarski in mid 1950’s.
! Birefrigent properties of modified Wollaston prisms translate phase
shifts into measurable amplitude differences.
Claire Brown, McGill Imaging Facility
Differential Interference Contrast Microscopy
Sample
Image
Light source
Condenser
Objective
www.zeiss.com
Prism
Prism
Analyzer
Polarizer
http://www.olympusmicro.com/primer/techniques/dic/dicoverview.html
DIC light path
Birefringent material:
Modified Wollaston (Nomarski prism)
Extraordinary ray
Ordinary ray
DIC starts with polarized light
Polarizer
Polarized light
Sample illuminated by
the O and E rays
Birefringent material:
Modified Wollaston (Nomarski prism)
Extraordinary ray
Ordinary ray
DIC uses birefringence
Sample illuminated by
the O and E rays
Extraordinary ray
Ordinary ray
Birefringent material:
Modified Wollaston (Nomarski prism)
DIC light extinction in absence of
light matter interactions
Sample illuminated by
the O and E rays
Extraordinary ray
Ordinary ray
Birefringent material:
Modified Wollaston (Nomarski prism)
Polarized light
Analyzer
No transmission
DIC and generation of phase
shifted light
Extraordinary ray
Ordinary ray
Birefringent material:
Modified Wollaston (Nomarski prism)
Phase shifted Ordinary
and Extraordinary rays
Analyzer
(second polarizer)
Vectorial
recombination of
phase shifted rays
No transmission
DIC Components
http://www.olympusmicro.com/primer/techniques/dic/dicoverview.html
DIC alignment1. Align the optical system for Köhler illumination.
2. Install the polarizer and analyzer and cross them so they are at 90o to one
another.
3. If the polarizer and analyzer are properly positioned a dark extinction cross
will appear in the objective rear aperture (a).
4. Install the objective prism and turn the adjustment knob until a single dark
interference fringe extends across the middle of the aperture at 45o (b).
5. Remove the objective prism and introduce the condenser prism into
position by rotating the condenser turret.http://www.olympusmicro.com/primer/techniques/polarized/polmicroalignment.html
DIC alignment
6. Once again, a single fringe should be present having the same
orientation as the fringe produced by the objective prism (b).
7. Typically there is no further alignment of the condenser prism.
8. Put the objective prism back in place and now the polarization cross
should be visible at the rear focal plane of the objective (c)
9. The condenser aperture should be adjusted to 75-80 percent of the full
aperture of the objective for maximum contrast with DIC.
http://www.olympusmicro.com/primer/techniques/polarized/polmicroalignment.html
DIC movies
Claire Brown, McGill Imaging Facility
Phase contrast versus DIC
No halos seen in DIC images.
Human
Erythrocytes
PC
DIC
Green algae
genusHeLa cells
http://www.olympusmicro.com/primer/techniques/dic/dicphasecomparison.html
Phase contrast versus DIC
2) DIC is higher resolution because it can utilize the instrument at full
numerical aperture (a) without the masking effects of phase plates or
condenser annuli (b). Can also use for z-sectioning, not the case for
phase imaging.
http://www.olympusmicro.com/primer/techniques/dic/dicphasecomparison.html
Phase annulus restricts a
significant portion of the
illumination that would
ordinarily traverse the
microscope optical train
Phase contrast versus DIC3) DIC can preserve direction for all points relative to a selected
point (azimuth contrast effect)
(a) DIC: Diatom aligned parallel to the shear axis !"good details
(b) DIC: Diatom aligned perpendicular !"lose some details
(c) Phase: Diatom aligned parallel !"azimuth effects are absent (no matter
if aligned parallel or perpendicular)
http://www.olympusmicro.com/primer/techniques/dic/dicphasecomparison.html
Phase contrast versus DIC
(c-d) Birefringent fiber exhibits
interference colours.
4) Phase Contrast is better suited for birefringent specimens. That
is ordered structures or samples on plastic.
Potato starch
granulesRayon fiber
(a-b) Carbohydrates in potato
starch grains are birefringent so
exhibit interference colours
Fibroblasts
polystyrene
(e-f) Fibroblasts have no
contrast when growing on
polystyrene.
http://www.olympusmicro.com/primer/techniques/dic/dicphasecomparison.html
PC
DIC
Parameter Phase Contrast DIC
Image Brightness
(Brightfield = 100 Percent)1.3 Percent 0.36 - 2.3 Percent
Lateral ResolutionCondenser Annulus
RestrictedSuperior
Axial Resolution
(Depth Discrimination)Poor Superior
Illuminating Aperture10 Percent of
Objective NAVariable
Azimuthal Effects No Yes
Halos and Shade-Off Yes No
Stained Specimens Not Useful Useful
Birefringent Specimens Useful Not Useful
Birefringent Specimen
ContainersYes No
Brightfield Image
DeteriorationSlight None
Cost Moderate High
Phase contrast versus DIC
Why transmitted light microscopy?
! Use bright field and a testslide to:
! Adjust Kohler illumination
! Evaluate objectivequalitative performance
! Adjust ocular/detectorparfocality
! Use the test slide to adjustDIC optics.
! Use TL to focus on thespecimen.
! Save the fluorescencesignal.
! Evaluate quality of sample.
! Monitor cell health
! Cell proliferation andmotility
http://www.microscopyu.com/articles/livecellimaging/livecellmaintenance.html
! Presented today:
! BF: simplest method, wavelength and intensity, for high contrast samples
! DF: easy, quick, inexpensive, for small amplitude modulation, high resolution
! PC: best for thin, unstained samples, shows intracellular details, halo problem
! Pol: best for molecularly ordered structures and birefringent samples
! DIC: high resolution, thin and thick samples, 3D-imaging, detects edges andintracellular details, glass only
! Other methods exist:
! Axial illumination
! Oblique illumination
! Rheinberg illumination
! Hoffman Modulation Contrast (HMC)
! Modulation Contrast Microscopy (MCM)
! New ones are being developed:
! PlasDIC from Zeiss
! Riveal contrast
TL contrast methods
!"#$%&'( )%#**&+,#-&./*%01%0&2&3-04*5-6&789%0#8" :;<(<:=
" No change regarding adaption of
micromanipulators
Relief contrast PlasDIC:
# Insensitive to birefringent
materials
# Suitable for plastic dishes and
glass bottom vessels
# Excellent relief display
# Ideal for thicker adherent cells,
oocytes, ICSI and transgenics
Relief Contrast PlasDIC – Perfect for Micromanipulation
!"#$%&'> )%#**&+,#-&./*%01%0&2&3-04*5-6&789%0#8" :;<(<:=
Relief contrast PlasDIC
PlasDIC Items Required:
1x PlasDIC Diaphragm
1x PlasDIC Objective
LD A-Plan
LD Plan Neofluar
1x PlasDIC Slider
1x Analyzer
P&C Cube
or
Analyzer Slider
or
Sénarmont Slider
PlasDIC Slider
PlasDIC Diaphragm
PlasDIC Objective
- LD A-Plan
- LD Plan-Neofluar
Analyzer
! Oblique illumination results in resolution better thandiffraction limit
! Two optical light paths! Morphology Mode
! For observation of general structure & morphology
! Increased resolution and light efficiency vs phase & DIC
! Full colour from unstained specimens (4 visualization modes)
! Simultaneous fluorescence
! High Contrast Mode! For observation of fine detail from scattering structures &
membranes
! All light not directly interacting with specimen is removed
! Full colour from unstained specimens (4 visualization modes)
! Simultaneous fluorescence
! Four visualization modes! Applied in real time
! Transformations enhance contrast & visibility in transparentsamples
Riveal contrast
?0#@5A#%"$
B#1%8"
www.youtube.com/quorumtechnologies1
THANK YOU!
" Colleagues and users:
" Cellular Imaging and Analysis Network (CIAN)
" Imaging Facility
" MIA Cellavie
" Canadian Cytometry Association members
" “Commercial faculty”