Transmitted light contrasting techniques: BF, DF, PC, pol, DIC · 2) DIC is higher resolution because it can utilize the instrument at full numerical aperture (a) without the masking

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 .


Phase shift: speed of light,

thickness and r.i. of sample


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


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.


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


! 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.


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.


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



Extraordinary ray

Ordinary ray

DIC uses birefringence


the O and E rays

Extraordinary ray

Ordinary ray



DIC light extinction in absence of

light matter interactions


the O and E rays

Extraordinary ray

Ordinary ray



Polarized light

Analyzer

No transmission

DIC and generation of phase

shifted light

Extraordinary ray

Ordinary ray



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


Phase contrast versus DIC

No halos seen in DIC images.

Human

Erythrocytes

PC

DIC

Green algae

genusHeLa cells



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.


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)



(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.


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


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”

[email protected] [email protected]

Documents

Transmitted light contrasting techniques: BF, DF, PC, pol, DIC · 2) DIC is higher resolution because it can utilize the instrument at full numerical aperture (a) without the masking