Optical materials characterization

© 2014University of Illinois Board of Trustees. All rights reserved.

Optical materials characterization

Julio Soares

Frederick Seitz Materials Research Laboratory University of Illinois at Urbana-Champaign

Why optical characterization?

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Optics Communications, Vol 284(9), 2376-2381 (2011)

2


2


2


2


2


3


4

library.thinkquest.org


• The photoelectric effect

The Nobel Prize in Physics 1921

Albert Einstein

"for his services to Theoretical Physics, and

especially for his discovery of the law of the

photoelectric effect"

"for his work on the elementary charge of

electricity and on the photoelectric effect"


Robert A. Millikan

The Nobel Foundation


5


Black body radiation, which lead to quantum physics…


Max Planck

"in recognition of the services he rendered

to the advancement of Physics by his

discovery of energy quanta"


0.000

0.005

0.010

0.015

0.020

0.025

0.030

0 10,000 20,000 30,000

r(n

)

wavenumbers (1/cm) Copyright 2001 B.M. Tissue

Classical (Rayleigh-Jeans) Quantum (Planck)

6



Charles Kuen Kao

Willard S. Boyle

George E. Smith

transmission of light in fibers for optical

communication and invention of the CCD

2009

contributions to the quantum theory of optical

coherence and the development of laser-based

precision spectroscopy

Roy J. Glauber

John L. Hall

Theodor W. Hänsch

2005

development of methods to

cool and trap atoms with laser

light

Steven Chu

Claude Cohen-Tannoudji

William D. Phillips

1997

1981

contribution to the development of

laser spectroscopy

Nicolaas Bloembergen

Arthur Leonard Schawlow

discovery and development of

optical methods for studying

Hertzian resonances in atoms

Alfred Kastler

1966 fundamental work in the field of

quantum electronics, which has led to

the construction of oscillators and

amplifiers based on the maser-laser

principle

Charles Hard Townes

Nicolay Gennadiyevich Basov

Aleksandr Mikhailovich Prokhorov

1964

Frits (Frederik) Zernike

demonstration of the phase contrast

method, especially for his invention of

the phase contrast microscope

1953

Sir Chandrasekhara

Venkata Raman

discovery of the

Raman effect

1930 Robert Andrews Millikan

Albert Einstein

1923 1921

the photoelectric effect

Niels Henrik David Bohr

investigation of the structure of

atoms and of the radiation

emanating from them

1922

Albert Abraham Michelson

for his optical precision

instruments and the

spectroscopic and

metrological investigations

carried out with their aid

1907

Max Karl Ernst

Ludwig Planck

discovery of

energy quanta

1918

1908

method of reproducing

colours photographically

based on the phenomenon

of interference

Gabriel Lippmann

7

Light – matter interaction

8

• Interrogating the material properties with light. – Transmission/Reflection spectroscopy (identification, structure, concentration,

speed, etc.)

• Spectrophotometry, FTIR

– Ellipsometry (dielectric function, layer thickness, carrier density, etc.)

– Raman (identification, structure, phase, crystallinity, stress, drug distribution,

diagnosis, etc.)

– PL/PLE (electronic structure, carrier life-time, defect levels, carrier concentration, size

distribution, etc.)

– SFG (surface/interface chemistry, identification, orientation, etc.)

– Modulation spectroscopy (electronic structure, carrier life-time, defect levels,

internal electric fields, thermal properties, mechanical properties, etc.)

• PR, ER, TDTR, FDTR

– Photocurrent/Photovoltage (electronic structure, energy band profile, defect

states, etc.)

• Quantum efficiency, solar cell and detector characterization

Optical methods for materials characterization

9

15 15

What is measured?

The transmission, and reflection of light as a function of the incident photon

energy.

Basic principle: The absorption, reflection

and transmission of light by

a material depends on its

electronic, atomic, chemical

and morphological

structure.

Transmission, Reflection, Absorption

10

16 16 Transmission, Reflection, Absorption

UV-VIS-NIR

FTIR

11

Raman Spectroscopy

17 17

Ground state

Excited

states

Virtual state

Donnor

levels

Acceptor

levels

Vibrational states

Impurities

IR UV/VIS

Transmission, Reflection, Absorption

12

18 18

Instrumentation:

Spectrophotometry (UV-VIS-NIR)

Transmission

Specular reflectance

Diffuse reflectance

13

19 19

Instrumentation:


14

20

-10-8 -6 -4 -2 0 2 4 6 8 10

De

tect

or

volta

ge

Time

DL = nl => constructive interference

DL = (n+1/2) l => destructive interference

Instrumentation:

The FTIR uses a Michelson interferometer with a moving mirror, in place of a diffraction grating or prism.

detector

fixed mirror

moving mirror

IR source

Beam splitter

sample

Fourier Transform IR spectroscopy (FTIR)


Albert A. Michelson

"for his optical precision instruments and the

spectroscopic and metrological

investigations carried out with their aid"


15

21

DL = nl => constructive interference

DL = (n+1/2) l => destructive interference

Instrumentation:

The FTIR uses a Michelson interferometer with a moving mirror, in place of a diffraction grating or prism.

-10-8 -6 -4 -2 0 2 4 6 8 10

De

tect

or

volta

ge

Timedetector

fixed mirror

moving mirror

IR source

Beam splitter

sample



Albert A. Michelson

"for his optical precision instruments and the

spectroscopic and metrological

investigations carried out with their aid"


15

22

Spectrum formation:

. dtetSI ti

nn 2)()(

-10-8 -6 -4 -2 0 2 4 6 8 10

De

tecto

r vo

lta

ge

Time0.0 0.4 0.8 1.2

Inte

nsity

Frequency


16

23

An example of an interferogram and its corresponding FTIR spectrum.

1000 1500 2000 2500 3000 3500 4000

0.0

0.5

1.0

1.5

2.0

Ab

sorb

ance

Wavenumber (cm-1)

Polystyrene

Frequency (Hz)

Am

plit

ude

The signal is detected as intensity

vs. time (interferogram).

The Fourier transform of the

interferogram gives the spectrum,

as intensity vs. wave number.


17

24 24

Instrumentation:


18

25

Advantages:

•Multiplexing (all wavelengths

are measured simultaneously).

•No chromatic aberrations or

artifacts.

•Higher throughput.

•Improved S/N ratio.

Disadvantages:

•More complex system.

•Linearity of detectors (working

closer to saturation levels).

•Stray light scattering at low

signal level.

FT vs. Dispersive optics spectrometers


19

26 26

450 500 550 600 650

0.1

0.2

0.3

0.4

Absorp

tion

Wavelength (nm)

4ml Au/.1ml Hg

3ml Au/1ml Hg

2ml Au/2ml Hg

1ml Au/3ml Hg

.5mM solutions

Using absorption to determine Au/Hg

concentration in water solutions

As Au relative concentration rises, the absorption

peak shifts toward shorter wavelengths, increase in

intensity, and its FWHM decreases. The intensity

increase follows Beer-Lambert’s law.


0.1 0.2 0.3 0.4 0.50.0

0.1

0.2

0.3

0.4

0.5

Ab

so

rptio

nAu concentration (mM)

Abs = K l c = a l

Beer-Lambert Law

20

27 27 Spectrophotometry (UV-VIS-NIR)

25000 22500 20000 17500 15000 1250054

55

56

57

58

T(%

)

n (cm-1)

14000 13000

56.0

56.5

57.0

57.5

58.0

400 500 600 700 800l (nm)

12500 13000 13500

0

10000

20000

30000

40000

50000

60000

Slope = 50.99 m

peak ind

ex / 2

n (

m

/cm

)

n (cm-1)

peak positions

linear fit

14000 16000 18000 20000 22000 240000

10000

20000

30000

peak ind

ex / 2

n (

m

/cm

)

n (cm-1)

peak positions

linear fit

Slope = 2.76 m

Using transmission interference fringes to

determine thickness

Two sets of interference fringes are present on the

spectrum, corresponding to each film that composes

the system.

21 𝜆𝑚 = 𝑚

𝜈 = 2𝑛𝑑 sin 𝜃

3 m

Excitations in materials

•Plasmons

22

Plasmons are quanta of collective motion

of charge-carriers in a gas with respect of

an oppositely charged background. They

play a significant role on transmission and

reflection of light.


Phys. Today, 64, 39 (2011)

http://juluribk.com

29 29

0k

xk

Plasmonic crystal Brillouin zone from the transmission spectra measured for

many different angles of incidence.

G

X

M

G X M Optics Express 13, 5669 (2005)

200 nm

3 m


23

30

FTIR can be used to identify

components in a mixture by

comparison with reference

spectra.


J. of Archaeological Sci. 39 (2012), 1227

Discovery of beeswax as binding

agent on a 6th-century BC Chinese

turquoise-inlaid bronze sword

Wugan Luo, Tao Li, Changsui Wang,

Fengchun Huang

Fingerprinting:

http://www.clockhours.com 24

31

Fingerprinting:

FTIR can be used to identify

components in a mixture by

comparison with reference

spectra.


Complementary characterization

techniques, like XRD can provide

conclusive evidence for the

identification.

J. of Archaeological Sci. 39 (2012), 1227

25

32 32

Strengths:

•Very little to no sample preparation.

•Simplicity of use and data interpretation.

•Short acquisition time, for most cases.

•Non destructive.

•Broad range of photon energies.

•High sensitivity (~ 0.1 wt% typical for FTIR).

Spectrophotometry (UV-VIS-NIR) and FTIR

26

33

Complementary techniques:

Raman, Electron Energy Loss Spectroscopy (EELS), Extended X-ray

Absorption Fine Structure (EXAFS), XPS, Auger, SIMS, XRD, SFG.

Spectrophotometry (UV-VIS-NIR) and FTIR

Limitations:

• Reference sample is often needed for quantitative analysis.

• Many contributions to the spectrum are small and can be buried in the background.

• Usually, unambiguous chemical

identification requires the use of

complementary techniques.

• Limited spatial resolution.

27

34

www.bobatkins.com

Guimond and Elmore - Oemagazine May 2004

Polarization

28

http://www.bobatkins.com/photography/

35 35

What is measured?

The changes in the polarization state of light upon reflection from a mirror

like surface.

Ellipsometry

29

36 36

Basic principle:

The reflected light emerges from the surface elliptically polarized, i.e. its p

and s polarization components are generally different in phase and

amplitude.

s

pi

R

Re ~

~

)tan( D

f

Ellipsometry

30

37

)(

,

,

,,,

~ isp

rspi

i

sp

r

sp

sp eE

ER

ff

ff

isp

bc

sp

ab

isp

bc

sp

absp

sp

err

errR

nn

nnr

2,,

2,,

,

22,111,2

22,111,2,

12

~~1

~~~

cos~cos~cos~cos~

~

+

+

+

iknn +~a

b

c

d fb

D

D

ir

s

p

s

pi

R

R

R

Re

~

~

)tan(~

~

)tan(

fa

fc

bbnd

fl

cos~2

2211 sin~sin~ ff nn

Ellipsometry

31

38

400 500 600 700 800

0

2

4

6

8

10

12

14

60

80

100

120

140

160

180

Model fit

Experiment

D

(

de

gre

es

)

Wavelength (nm)

D (d

eg

ree

s)

SiO2 thickness 2.19 ± 0.04 nm

c 2 4.31

SiO2 21.9 Å

Si

Ellipsometry

Applications

Film thickness

32

39 39 Ellipsometry

Composition

Surface roughness

Film thickness

Band gap energy

Applications

Ellipsometric (l) and D(l) spectra of Cd1-xZnxS thin

films deposited under the different concentration of

ammonia: 0.19, 0.38, 0.56, and 0.75 M. The solid

lines represent the best fit to the theoretical model. (a)

and (b) were measured under 68° and 72°,

respectively.

Jpn. J. Appl. Phys. 49 (2010) 081202

33

40 40 Ellipsometry

Composition

Surface roughness

Film thickness

Band gap energy

Applications

Parameters of the Cd1xZnxS thin films as a function of

different ammonia concentrations obtained from SE analysis.

[NH4OH] (M)

Thickness (nm)

Roughness (nm)

ZnS (%) Band-gap (eV)

0.19 42.12 23.77 99.7 3.49

0.38 73.79 7.15 45.5 2.52

0.56 50.89 5.94 32.3 2.45

0.75 18.59 4.54 5.2 2.43

Jpn. J. Appl. Phys. 49 (2010) 081202

34

41 41 Ellipsometry

Composition

Surface roughness

Film thickness

Band gap energy

Optical constants

(dielectric function)

Applications

Jpn. J. Appl. Phys. 49 (2010) 081202

Refractive index (n) and extinction coefficient (k) as a function

of wavelength of Cd1-xZnxS thin films under different ammonia

concentrations.

Plots of (αhν)2 versus photon energy

h for Cd1-xZnxS thin films under

different ammonia concentration.

35

42 Ellipsometry

Optical Hall Effect 36

43 43 Ellipsometry

Applications

Electrical properties

From the fit for the C-face sample:

Two graphene layers with distinctly different properties:

a bottom p-type channel with Ns=(5.5±0.4)x1013 cm-2 and

=1521±52 cm2 V-1 s-1

a top p-type channel with Ns=(3.4±0.6)x1014 cm-2 and

=18±4 cm2 V-1 s-1

From electrical dc Hall effect measurement:

p-type conductivity with Ns=(3.0±0.5) x1013 cm-2 and

=3407±250 cm2 V-1 s-1

37

44 44 Ellipsometry

Applications


From the fit for the Si-face sample:

A p-type channel with Ns=(1.2±0.3)x1012 cm-2 and =794±80

cm2 V-1 s-1

From electrical dc Hall effect measurement:

p-type conductivity with Ns=(1.9±0.2) x1012 cm-2 and

=891±250 cm2 V-1 s-1

The correspondence between electrical

and OHE data is very good.

38

45 45 Ellipsometry

Applications


m* = (0.19−0.08√B) m0

m* = 0.035 m0

Top layer:

Bottom layer:

m* = 0.03 m0

C-face:

Si-face:

39

46 46 Ellipsometry

Stregths: – Fast.

– Measures a ratio of two intensity values and a phase.

• Highly accurate and reproducible (even in low light levels).

• No reference sample necessary.

• Not as susceptible to scatter, lamp or purge fluctuations.

• Increased sensitivity, especially to ultrathin films (<10nm).

– Can be used in-situ.

DC Comics

40

47 47

Limitations:

– Flat and parallel surface and

interfaces with measurable

reflectivity.

– A realistic physical model of the

sample is usually required to obtain

useful information.


PL, Modulation spectroscopies, X-Ray Photoelectron Spectroscopy,

Secondary Ion Mass Spectroscopy, XRD.

Ellipsometry

Neil Miller

41

http://www.filmschoolrejects.com/author/neil

Raman Spectroscopy Sir Chandrasekhara Venkata Raman

The Nobel Prize in Physics 1930 was awarded to Sir

Venkata Raman "for his work on the scattering of light and

for the discovery of the effect named after him".


48 48 Raman spectroscopy

42

Raman Spectroscopy


Ground state

Excited state

Virtual states

Vibrational states

Resonance

Raman

Ra

yle

igh

sca

tterin

g

An

ti-S

tokes

Sto

ke

s

An

ti-S

tokes

Sto

ke

s

Photon energy

Scattere

d inte

nsity

Raman shift

What is measured?

The light inelastically scattered by the

material.

Basic principle:

The impinging light couples

with the lattice vibrations

(phonons) of the material,

and a small portion of it is

inelastically scattered. The

difference between the

energy of the scattered light

and the incident beam is the

energy absorbed or

released by the phonons.

IR

43

http://www.physik.tu-berlin.de/institute/IFFP/richter/new/research/surface-phonons.shtml


•Phonons

•Molecular vibrations

44

Phonons are the quanta of Collective

lattice vibrations

Raman spectroscopy

51

Raman Spectroscopy


1000 1500 2000 2500 3000

1000 1500 2000 2500 3000

FTIR

Raman

Wavenumber (cm-1)

Inte

nsity

Raman shift (cm-1)

PolyetherurethaneLike the FTIR, Raman

spectroscopy measures the

interaction of photons with

the vibration modes of

materials, but the physical

processes behind the two

techniques are fundamentally

different and so are the

selection rules that apply to

each.

The two techniques are

complementary, rather than

equivalent.

45

52

Raman Spectroscopy

Molecular and crystalline structure characterization


1000 2000 3000

Ra

ma

n in

ten

sity

(a

rb. u

nits

)

Raman Shift (cm-1)

Diamond

C Nanotube

Coal (Rock)-norm

Graphite

coal (wood)

Raman is sensitive to the

atomic structure of the

material.

Physics Reports, 409 (2005), 47

46

53

Raman Spectroscopy

The AAPS Journal 2004; 6 (4),

32

Distribution of ingredients in a pharmaceutical

tablet

Renishaw, Inc.

Chemical composition & component identification

Components distribution at micron & sub-micron scale


47

< C

> C

Presence of N vacancies

yields poor crystallinity

Substitutional C fills N

vacancies improving the

crystallinity

C incorporates interstitially

causing a degradation of

the crystal lattice

54

Raman Spectroscopy

Molecular and crystalline structure characterisation


48

55

Raman Spectroscopy

Phase transition monitoring

Stress measurements

Mapping the Raman peak position of a

micro indentation in a silicon wafer.

Renishaw, Inc.


49

A complete Raman mapping of phase transitions in Si under indentation C. R. Das, H. C. Hsu, S. Dhara, A. K. Bhaduri, B. Raj, L. C. Chen, K. H. Chen, S. K. Albert, A. Raye and Y. Tzengc

Raman spectroscopy

50

Raman spectroscopy is able to clearly distinguish areas of

differing numbers of layers in thin graphene sheets.

Graphene monolayer, bilayer and other multiple-layer regions

identified

57

Raman Spectroscopy


K.S. Novoselov et al., Science 306, 666 (2004).

51

FS-MRL

Oriented CNT between two

electrodes

58

Raman Spectroscopy


52

Primary Strengths:

• Very little sample preparation.

• Structural characterization.

• Non destructive technique.

• Chemical information.

• Complementary to FTIR.

Super Optical Powers

59

Raman Spectroscopy


53

Primary Limitations:

• Expensive apparatus (for high

spectral/spatial resolution and

sensitivity).

• Weak signal, compared to fluorescence.

• Limited spatial resolution.

Kryptonite DC

Comics


FTIR, EELS, Mass spectroscopy, EXAFS, XPS, AES, SIMS, XRD, SFG.

60

Raman Spectroscopy


54

Raman Spectroscopy


Ground state

Excited

states

Virtual states

Donnor

levels

Acceptor

levels

Vibrational states

Impurities

Luminescence Raman scattering

Excitation

55

Acceptor

levels

Raman Spectroscopy


Ground state

Excited

states

Virtual states

Vibrational states

Impurities

Luminescence Raman scattering

Excitation

55

Donnor

levels

63

Lifetime: Phosphorescence, fluorescence

Mechanism: Photoluminescence, bioluminescence,

chemoluminescence, thermoluminescence,

piezoluminescence, etc.

Trevor Morris

Luminescence

56

Radim Schreiber

64 64 Photoluminescence

http://uclagettyprogram.wordpress.com

Charles Hedgcock © University of Arizona, Tucson, AZ

http://www.evidentcrimescene.com

http://kevincollinsphoto.smugmug.com

57

65 65

What is measured?

The emission spectra of materials due to radiative recombination following

photo-excitation.

Basic principle:

The impinging light promote electrons from the less energetic levels to

excited levels, forming electron-hole pairs. As the electrons and holes

recombine, they may release some of the energy as photons. The emitted

light is called luminescence.

direct band gap indirect band gap

Conduction

band

Valence band

Photoluminescence

58

Peter Abbamonte et al., Proc. Nat. Acad. Sci. 105 (34)

Photoluminescence

59

Exciton describes the bound state of an electron-

hole pair due their mutual Coulomb attraction


•Excitons •Bound excitons

•Excitonic complexes


Davydov et al. Phys. Stat. Solidi (b) 230 (2002b), R4

Photoluminescence spectra of

InN layers with different carrier

concentrations.

1 - n = 6x1018 cm-3 (MOCVD);

2 - n = 9x1018 cm-3 (MOMBE);

3 - n = 1.1x1019 cm-3 (MOMBE);

4 - n = 4.2x1019 cm-3 (PAMBE).

Solid lines show the theoretical

fitting cures based on a model of

interband recombination in

degenerated semiconductors. As

a result, the true value of InN band

gap Eg~0.7 eV was established.

60

http://www.ioffe.ru/SVA/NSM/Semicond/InN/reference.html








InxGa1-xN alloys. Luminescence peak positions of catodoluminescence and

photoluminescence spectra vs. concentration x.

The plots of luminescence peak positions can be fitted to the curve

Eg(x)=3.48 - 2.70x - bx(1-x) with a bowing parameter of b=2.3 eV

Ref.1 - Wetzel., Appl. Phys. Lett. 73, 73 (1998).

Ref.2 - V. Yu. Davydov., Phys. Stat. Sol. (b) 230, R4 (2002).

Ref.3 - O’Donnel., J. Phys .Condens. Matt. 13, 1994 (1998).

Hori et al. Phys. Stat. Sol. (b) 234 (2002) 750

61







69

Conduction band

Valence band

FE D

A (Ao,X)

(Do,X)

GaAs

(D+,X)

Photoluminescence Photoluminescence

62

70 70

Using PL to determine the width and quality of InGaAsN/GaAs quantum wells.

3nm InGaAsN QW

5nm InGaAsN QW

9nm InGaAsN QW

Photoluminescence

63

71

Strengths:

• Very little to none sample

preparation.

• Non destructive technique.

• Very informative spectrum.


64

72

Limitations:

• Often requires low temperature.

• Data analysis may be complex.

• Many materials luminescence

weakly.


Ellipsometry, Modulation

spectroscopies,

Spectrophotometry, Raman.


65

73 73

What is measured?

The reflectance variation with temperature.

hn0 hn0

Basic principle:

The dielectric function of a material, and thus its reflectance, is a function of

temperature. By modulating the material’s temperature, we can measure the

variation in its reflectivity, caused by that modulation. With time resolved

measurements, we can calculate the thermal conductivity of the sample.

Time-domain thermoreflectance

66

74 74

Instrumentation:

• fs Ti:Sapphire laser operating in the

range 700-800 nm.

• A variable delay line in the pump

beam path for time resolution.

• Scanning sample stage.

• Cryostats.

• Magnetic field.

Applications of TDTR include:

Spatially resolved thermal and elastic

properties of materials:

•Thermal effusivity (LC) and conductivity.

•Mechanical properties (by determination of

the speed of sound).

J. Appl. Phys., Vol. 93, 793 (2003).


67

75 75

The lowest thermal conductivity so far observed

for a fully dense solid. (48 mWm-1K-1)


68

76 76

The lowest thermal conductivity so far observed

for a fully dense solid. (48 mWm-1K-1)


68

77 Time-domain thermoreflectance

69

78 Time-domain thermoreflectance

69

79 79

Strengths:

– Thermoconductivity over a broad range can be measured.

– Can measure interface thermal conductance.

– Spatial resolution.

– Measurements can be made over a wide range of

temperatures.

– Fast.


70

Science 315, 342 (2007)

80 80

Limitations:

– Samples need coating with an Al

thin film.

– Alignment can be tedious.

– Thermal conductivity cannot be

measured for films much thinner

than the thermal penetration

depth.


3w method, Raman spectroscopy, Nanoindentation.


71

Optical microscopy

72

Optical microscopy

"Classical" Optical Microscopy

Image Formation

•In the optical microscope, light from the microscope

lamp passes through the condenser and then through

the specimen (assuming the specimen is a light

absorbing specimen).

• Some of the light passes both around and through the

specimen undisturbed in its path (direct light or

undeviated light).

•The direct or undeviated light is projected by the

objective and spread evenly across the entire

image plane at the diaphragm of the eyepiece.

•Some of the light passing through the specimen is

deviated when it encounters parts of the specimen

(deviated light or diffracted light).

73

Optical microscopy

74

Optical microscopy

Objective Type

Spherical Aberration

Chromatic Aberration

Field Curvature

Achromat 1 Color 2 Colors No

Plan Achromat 1 Color 2 Colors Yes

Fluorite 2-3 Colors 2-3 Colors No

Plan Fluorite 3-4 Colors 2-4 Colors Yes

Plan Apochromat 3-4 Colors 4-5 Colors Yes

75

Optical microscopy

Bright field Phase contrast Dark field Polarizing 76

Optical microscopy

Difference interference contrast 77

Optical microscopy

Bright field Phase contrast Dark field Polarizing

Fibers in bright field and dark field

78

Optical microscopy

Fluorescence 79

Optical microscopy

Specimen Type

Imaging Technique

Transmitted Light Transparent Specimens

Phase Objects Bacteria, Spermatozoa,

Cells in Glass Containers, Protozoa, Mites, Fibers, etc.

Phase Contrast Differential Interference Contrast (DIC)

Hoffman Modulation Contrast Oblique Illumination

Light Scattering Objects Diatoms, Fibers, Hairs,

Fresh Water Microorganisms, Radiolarians, etc.

Rheinberg Illumination Darkfield Illumination

Phase Contrast and DIC

Light Refracting Specimens Colloidal Suspensions powders and minerals

Liquids

Phase Contrast Dispersion Staining

DIC

Amplitude Specimens Stained Tissue

Naturally Colored Specimens Hair and Fibers

Insects and Marine Algae

Brightfield Illumination

Fluorescent Specimens Cells in Tissue Culture

Fluorochrome-Stained Sections Smears and Spreads

Fluorescence Illumination

Birefringent Specimens Mineral Thin Sections

Liquid Crystals Melted and Recrystallized Chemicals

Hairs and Fibers Bones and Feathers

Polarized Illumination

Contrast-Enhancing Techniques for Optical Microscopy

http://micro.magnet.fsu.edu

80

http://micro.magnet.fsu.edu/

Optical microscopy

Reflected Light

Specular (Reflecting) Surface Thin Films, Mirrors

Polished Metallurgical Samples Integrated Circuits

Brightfield Illumination Phase Contrast, DIC

Darkfield Illumination

Diffuse (Non-Reflecting) Surface Thin and Thick Films Rocks and Minerals

Hairs, Fibers, and Bone Insects

Brightfield Illumination Phase Contrast, DIC

Darkfield Illumination

Amplitude Surface Features Dyed Fibers

Diffuse Metallic Specimens Composite Materials

Polymers

Brightfield Illumination Darkfield Illumination

Birefringent Specimens Mineral Thin Sections

Hairs and Fibers Bones and Feathers

Single Crystals Oriented Films

Polarized Illumination

Fluorescent Specimens Mounted Cells

Fluorochrome-Stained Sections Smears and Spreads

Fluorescence Illumination

http://micro.magnet.fsu.edu

Contrast-Enhancing Techniques for Optical Microscopy

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http://micro.magnet.fsu.edu/

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Resolution

He defined the resolving power of an optical instrument as the distance

between two point sources for which the center of one Airy disk coincides

with the first zero of the other:

d 0.61lNA

Rayleigh criterion (light emitting particle)

Based on the Airy discs formation Abbé

derived a theoretical limit to an optical

instrument resolution.

Arch. Microskop. Anat. 9, 413 (1873)

Near-field Scanning Optical microscopy Optical microscopy

Abbé criterion (illuminated particle) 𝑑 ≈𝜆

𝑁𝐴𝑐𝑜𝑙 + 𝑁𝐴𝑜𝑏𝑗≈

𝜆

2 𝑁𝐴

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Optical microscopy

Rayleigh criterion (light emitting particle)

Abbé criterion (illuminated particle) 𝑑 ≈𝜆

2 𝑛 sin 𝜇

𝑑 ≈0.61 𝜆

𝑛 sin 𝜇

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Confocal microscopy

• In confocal microscopy, due to the

constrained light path provides an

increased contrast, allowing for resolving

objects with intensity differences of up to

200:1.

• The in plane resolution, in confocal

microscopy, is also slightly increased (1.5

times) while the resolution along the

optical axis is high.

•These improvements are obtained at the

expense of the utilization of mechanisms

for scanning either by moving a specimen

or by readjustment of an optical system.

Scanning application allows to increase

field of view as compared with conventional

microscopes.

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Confocal microscopy

The relation of the first ring maximum amplitude to the amplitude in the

center is 2% in case of conventional point spreading function (PSF) in a

focal plane while in case of a confocal microscope this relation is 0.04%.

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Confocal microscopy

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Confocal microscopy

Anna-Katerina Hadjantonakis; Virginia E Papaioannou

BMC Biotechnology 2004, 4:33

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Confocal microscopy

Strengths:

– Optical sectioning (0.5 m).

– three-dimensional images.

– Improved contrast (200:1).

– Better resolution (1.5x).

– Field of view defined by the

scanning range.

Limitations:

– Image is scanned, resulting in slower

data acquisition.

– High intensity laser radiation can

damage some samples.

– Cost (typically 10x more than a

comparable wide-field system).

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Beyond confocal microscopy

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Nature Reviews Genetics 4, 613 (2003)

(courtesy of Brad Amos MRC, Cambridge)

Biophysical J. 75, 2015 (1998)

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• Sample is illuminated with a

patterned light source.

• The pattern interact with

structures finer than the

diffraction limit;

• The pattern projection itself is

diffraction limited.

• The big advantage of HR-SIM is

again its ease of-use.

• Uses common

• Fixation and labeling

procedures are similar to normal

fluorescence microscopy,.

• Dye labeling must be stable

enough to survive the

acquisition of multiple images.

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PALM (photo-activated localization microscopy)

Widefield PALM Widefield PALM

Biophysical Journal 91, 4258 (2006)

Nature Methods 6, 689 (2009)

Nature Protocols 4, 291 (2009)

Other similar echniques:

STORM (Stochastic optical

reconstruction microscopy)

FIONA (Fluorescence Imaging with

One Nanometer Accuracy)

SHReC (Single Molecule High

Resolution Colocalization)

SPDM (Spectral Precision Distance

Microscopy)

And many variants.

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Near-field microscopy: resolution beyond the diffraction limit!

The near field microscope resolves objects much smaller than the

wavelength of light by measuring at very short distances from the sample

surface, through sub-wavelength apertures.

Basic principle:

The principle of operation of the NSOM

was devised by Synge in 1928 †.

•The sample is kept in the near-field

regime of a sub-wavelength source.

•If z<<l, the resolution is determined

by the aperture, not wavelength.

•The image is constructed by scanning

the aperture across the sample and

recording the optical response.

† Philos.Mag. 6, 356 (1928).

Near-field Scanning Optical microscopy Near-field scanning optical microscopy (NSOM)

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Instrumentation:

Near-field Scanning Optical microscopy Near-field scanning optical microscopy (NSOM)

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Strengths:

– Powerful, very attractive.

– No sample preparation.

– Non destructive technique.

– Sub diffraction limit

resolution (50 nm).

Near-field scanning optical microscopy (NSOM)

CBS Broadcasting Inc.

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Requirements and limitations:

– Careful alignment required.

– Very low throughput.

– Slow data acquisition.

– Limited to fairly flat samples (20 m).

– Interaction between tip and sample

may make analysis difficult.


AFM, SEM,TEM, Confocal microscopy.

Near-field scanning optical microscopy (NSOM)

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It is Important to use complementary techniques!

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