Download Optical materials characterization

Optical materials characterization
Julio Soares
Frederick Seitz Materials Research Laboratory
University of Illinois at Urbana-Champaign
© 2014University of Illinois Board of Trustees. All rights reserved.
Why optical characterization?
2
Why optical characterization?
2
Optics Communications, Vol 284(9), 2376-2381 (2011)
Why optical characterization?
2
Why optical characterization?
2
Why optical characterization?
2
Why optical characterization?
2
Why optical characterization?
3
Why optical characterization?
4
Why optical characterization?
5
• 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"
The Nobel Foundation
The Nobel Prize in Physics 1923
Robert A. Millikan
"for his work on the elementary charge of
electricity and on the photoelectric effect"
The Nobel Foundation
library.thinkquest.org
Why optical characterization?
6
Black body radiation, which lead to quantum physics…
Classical (Rayleigh-Jeans)
Quantum (Planck)
0.030
0.025
r(n)
0.020
0.015
The Nobel Prize in Physics 1918
Max Planck
0.010
0.005
0.000
0
10,000
20,000
30,000
"in recognition of the services he rendered
to the advancement of Physics by his
discovery of energy quanta"
wavenumbers (1/cm)
Copyright 2001 B.M. Tissue
The Nobel Foundation
Why optical characterization?
7
2009
2005
Roy J. Glauber
John L. Hall
Theodor W. Hänsch
Charles Kuen Kao
Willard S. Boyle
George E. Smith
transmission of light in fibers for optical
communication and invention of the CCD
1981
Claude Cohen-Tannoudji
William D. Phillips
Nicolaas Bloembergen
Arthur Leonard Schawlow
development of methods to
cool and trap atoms with laser
light
1923 1921
1964
1997
contributions to the quantum theory of optical
coherence and the development of laser-based
precision spectroscopy
Steven Chu
1930
Robert Andrews Millikan
Albert Einstein
contribution to the development of
laser spectroscopy
Sir Chandrasekhara
Venkata Raman
the photoelectric effect
Charles Hard Townes
Nicolay Gennadiyevich Basov
Aleksandr Mikhailovich Prokhorov
fundamental work in the field of
quantum electronics, which has led to
the construction of oscillators and
amplifiers based on the maser-laser
principle
1953
Frits (Frederik) Zernike
1922
1918
Niels Henrik David Bohr
1966
investigation of the structure of
atoms and of the radiation
emanating from them
Alfred Kastler
discovery and development of
optical methods for studying
Hertzian resonances in atoms
demonstration of the phase contrast
method, especially for his invention of
the phase contrast microscope
discovery of the
Raman effect
1907
Max Karl Ernst
Ludwig Planck
discovery of
energy quanta
1908
Albert Abraham Michelson
for his optical precision
instruments and the
spectroscopic and
metrological investigations
carried out with their aid
Gabriel Lippmann
method of reproducing
colours photographically
based on the phenomenon
of interference
The Nobel Foundation
Light – matter interaction
8
Optical methods for materials characterization
9
• 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
states, etc.)
• Quantum efficiency, solar cell and detector characterization
profile, defect
Transmission, Reflection, Absorption
10
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.
15
Transmission, Reflection, Absorption
11
UV-VIS-NIR
FTIR
16
Transmission, Reflection, Absorption
Raman Spectroscopy
12
17
Impurities
Excited
states
Donnor
levels
Virtual state
Acceptor
levels
Vibrational states
Ground state
IR
UV/VIS
Spectrophotometry (UV-VIS-NIR)
13
Instrumentation:
Transmission
Specular reflectance
Diffuse reflectance
18
Spectrophotometry (UV-VIS-NIR)
14
Instrumentation:
19
Fourier Transform IR spectroscopy (FTIR)
20
15
The Nobel Prize in Physics 1907
Albert A. Michelson
"for his optical precision instruments and the
spectroscopic and metrological
investigations carried out with their aid"
Instrumentation:
The FTIR uses a Michelson
interferometer with a moving mirror, in
place of a diffraction grating or prism.
The Nobel Foundation
fixed mirror
DL = nl => constructive interference
DL = (n+1/2) l => destructive interference
moving mirror
Beam splitter
sample
detector
Detector voltage
IR source
-10 -8 -6 -4 -2 0 2 4 6 8 10
Time
Fourier Transform IR spectroscopy (FTIR)
21
15
The Nobel Prize in Physics 1907
Albert A. Michelson
"for his optical precision instruments and the
spectroscopic and metrological
investigations carried out with their aid"
Instrumentation:
The FTIR uses a Michelson
interferometer with a moving mirror, in
place of a diffraction grating or prism.
The Nobel Foundation
fixed mirror
DL = nl => constructive interference
DL = (n+1/2) l => destructive interference
moving mirror
Beam splitter
sample
detector
Detector voltage
IR source
-10 -8 -6 -4 -2 0 2 4 6 8 10
Time
Fourier Transform IR spectroscopy (FTIR)
22
16
.

I (n )   S (t )e 2int dt

-10 -8 -6 -4 -2 0 2 4 6 8 10
Time
Intensity
Detector voltage
Spectrum formation:
0.0
0.4
0.8
Frequency
1.2
Fourier Transform IR spectroscopy (FTIR)
23
17
An example of an interferogram and its corresponding FTIR spectrum.
Frequency (Hz)
Polystyrene
Amplitude
The signal is detected as intensity
vs. time (interferogram).
The Fourier transform of the
interferogram gives the spectrum,
as intensity vs. wave number.
Absorbance
2.0
1.5
1.0
0.5
0.0
1000
1500
2000
2500
3000
-1
Wavenumber (cm )
3500
4000
Spectrophotometry (UV-VIS-NIR)
18
Instrumentation:
24
Fourier Transform IR spectroscopy (FTIR)
19
FT vs. Dispersive optics spectrometers
Advantages:
Disadvantages:
•Multiplexing (all wavelengths
are measured simultaneously).
•More complex system.
•No chromatic aberrations or
artifacts.
•Higher throughput.
•Improved S/N ratio.
•Linearity of detectors (working
closer to saturation levels).
•Stray light scattering at low
signal level.
25
Spectrophotometry (UV-VIS-NIR)
26
20
0.4
0.3
Abs = K l c = a l
.5mM solutions
0.2
0.5
0.4
0.1
450
500
550
600
650
Wavelength (nm)
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.
Absorption
Absorption
Beer-Lambert Law
4ml Au/.1ml Hg
3ml Au/1ml Hg
2ml Au/2ml Hg
1ml Au/3ml Hg
0.3
0.2
0.1
0.0
0.1
0.2
0.3
0.4
Au concentration (mM)
0.5
Spectrophotometry (UV-VIS-NIR)
l (nm)
500
400
58
600
700
800
58.0
57.5
57.0
57
56.5
T(%)
56.0
14000
𝜆𝑚 = 𝑚 𝜈 = 2𝑛𝑑 sin 𝜃
peak index / 2n (m/cm)
21
13000
56
27
peak positions
linear fit
30000
20000
10000
Slope = 2.76 m
0
14000 16000 18000 20000 22000 24000
55
60000
22500
20000 17500
n (cm-1)
15000
12500
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.
peak index / 2n (m/cm)
54
25000
n (cm-1)
50000
peak positions
linear fit
40000
30000
20000
10000
Slope = 50.99 m
0
12500
13000
n (cm-1)
13500
Spectrophotometry (UV-VIS-NIR)
22
Excitations in materials
•Plasmons
Phys. Today, 64, 39 (2011)
http://juluribk.com
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
3 m
reflection of light.
Spectrophotometry (UV-VIS-NIR)
29
23

M

X

k0
G
200 nm

kx
3 m
M
G
X
Optics Express 13, 5669 (2005)
Plasmonic crystal Brillouin zone from the transmission spectra measured for
many different angles of incidence.
Fourier Transform IR spectroscopy (FTIR)
24
http://www.clockhours.com
Fingerprinting:
FTIR can be used to identify
components in a mixture by
comparison with reference
spectra.
Discovery of beeswax as binding
agent on a 6th-century BC Chinese
turquoise-inlaid bronze sword
Wugan Luo, Tao Li, Changsui Wang,
Fengchun Huang
J. of Archaeological Sci. 39 (2012), 1227
30
Fourier Transform IR spectroscopy (FTIR)
25
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
31
Spectrophotometry (UV-VIS-NIR) and FTIR
26
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).
32
Spectrophotometry (UV-VIS-NIR) and FTIR
27
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.
Complementary techniques:
Raman, Electron Energy Loss Spectroscopy (EELS), Extended X-ray
Absorption Fine Structure (EXAFS), XPS, Auger, SIMS, XRD, SFG.
33
Polarization
34
28
www.bobatkins.com
Guimond and Elmore - Oemagazine May 2004
Ellipsometry
29
What is measured?
The changes in the polarization state of light upon reflection from a mirror
like surface.
35
Ellipsometry
36
30
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.
f
~
Rp
iD
tan( )e  ~
Rs
Ellipsometry
37
31
fa
a
d
fb
b
c
fc
~
R p,s 
E
E
r
p,s
i
p,s
n~1 sin f1  n~2 sin f2
n~  n + ik
~
r12p , s
n~2,1 cos f1  n~1, 2 cos f2
~
n cos f + n~ cos f
2 ,1
1
1, 2
~
rabp , s + ~
rbcp , s e  2i
~
R p,s 
1+ ~
rabp , s ~
rbcp , s e  2i
2d ~

nb cos fb
e
i ( pr ,s  pi ,s )
~
Rp
iD
tan( )e  ~
Rs
l
~

Rp
tan( )  ~

Rs

r
i
D





2
Ellipsometry
32
38
Applications
21.9 Å
Si
SiO2 thickness
c 2  4.31
2.19 ± 0.04 nm
14
12
10
8
6
4
2
0
Model fit
Experiment

D
400 500 600 700 800
Wavelength (nm)
180
160
140
120
100
80
60
D (degrees)
SiO2
 (degrees)
 Film thickness
Ellipsometry
33
39
Applications




Composition
Surface roughness
Film thickness
Band gap energy
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
Ellipsometry
34
40
Applications




Composition
Surface roughness
Film thickness
Band gap energy
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
Ellipsometry
35
41
Applications





Composition
Surface roughness
Film thickness
Band gap energy
Optical constants
(dielectric function)
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.
Jpn. J. Appl. Phys. 49 (2010) 081202
Ellipsometry
36
Optical Hall Effect
42
Ellipsometry
37
43
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
Ellipsometry
38
44
Applications
 Electrical properties
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.
Ellipsometry
39
45
Applications
 Electrical properties
C-face:
Top layer:
m* = (0.19−0.08√B) m0
Bottom layer: m* = 0.035 m0
Si-face:
m* = 0.03 m0
Ellipsometry
46
40
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
Ellipsometry
47
41
Limitations:
– Flat and parallel surface and
interfaces with measurable
reflectivity.
– A realistic physical model of the
sample is usually required to obtain
useful information.
Neil Miller
Complementary techniques:
PL, Modulation spectroscopies, X-Ray Photoelectron Spectroscopy,
Secondary Ion Mass Spectroscopy, XRD.
42
Raman spectroscopy
Raman Spectroscopy
48
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".
The Nobel Foundation
Raman spectroscopy
Raman Spectroscopy
Anti-Stokes
Stokes
Scattered intensity
43
What is measured?
The light inelastically scattered by the
material.
Photon energy
Stokes
Rayleigh
scattering
Anti-Stokes
Raman shift
Excited state
Virtual states
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.
Vibrational states
Ground state
49
Resonance
Raman
IR
Raman spectroscopy
44
Excitations in materials
•Phonons
•Molecular vibrations
http://www.physik.tu-berlin.de/institute/IFFP/richter/new/research/surface-phonons.shtml
Phonons are the quanta of Collective
lattice vibrations
45
Raman spectroscopy
Raman Spectroscopy
51
51
-1
Wavenumber (cm )
1000
2000
2500
3000
Polyetherurethane
Intensity
Like 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.
1500
FTIR
Raman
The two techniques are
complementary, rather than
equivalent.
1000
1500
2000
2500
-1
Raman shift (cm )
3000
Raman spectroscopy
Raman Spectroscopy
46
52
52
Molecular and crystalline structure characterization
Raman is sensitive to the
atomic structure of the
material.
Raman intensity (arb. units)
Diamond
C Nanotube
Coal (Rock)-norm
Graphite
coal (wood)
1000
2000
3000
-1
Physics Reports, 409 (2005), 47
Raman Shift (cm )
Raman spectroscopy
Raman Spectroscopy
47
53
53
Chemical composition & component identification
Components distribution at micron & sub-micron scale
Distribution of ingredients in a pharmaceutical
tablet
The AAPS Journal 2004; 6 (4),
32
Renishaw, Inc.
Raman spectroscopy
Raman Spectroscopy
48
Molecular and crystalline structure characterisation
Presence of N vacancies
yields poor crystallinity
<C
Substitutional C fills N
vacancies improving the
crystallinity
>C
C incorporates interstitially
causing a degradation of
the crystal lattice
54
54
Raman spectroscopy
Raman Spectroscopy
49
Phase transition monitoring
Stress measurements
Mapping the Raman peak position of a
micro indentation in a silicon wafer.
Renishaw, Inc.
55
55
Raman spectroscopy
50
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
51
Raman spectroscopy
Raman Spectroscopy
57
57
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
K.S. Novoselov et al., Science 306, 666 (2004).
52
Raman spectroscopy
Raman Spectroscopy
58
58
Oriented CNT between two
electrodes
FS-MRL
Raman spectroscopy
Raman Spectroscopy
53
Primary Strengths:
•
•
•
•
•
Very little sample preparation.
Structural characterization.
Non destructive technique.
Chemical information.
Complementary to FTIR.
Super Optical Powers
59
59
Raman spectroscopy
Raman Spectroscopy
54
60
60
Primary Limitations:
•
•
•
Expensive apparatus (for high
spectral/spatial resolution and
sensitivity).
Weak signal, compared to fluorescence.
Limited spatial resolution.
Kryptonite
DC
Comics
Complementary techniques:
FTIR, EELS, Mass spectroscopy, EXAFS, XPS, AES, SIMS, XRD, SFG.
Raman spectroscopy
Raman Spectroscopy
55
61
Impurities
Virtual states
Excited
states
Excitation
Donnor
levels
Acceptor
levels
Vibrational states
Ground state
Luminescence
Raman scattering
Raman spectroscopy
Raman Spectroscopy
55
62
Impurities
Excited
states
Donnor
levels
Excitation
Virtual states
Acceptor
levels
Vibrational states
Ground state
Luminescence
Raman scattering
Luminescence
56
63
Lifetime: Phosphorescence, fluorescence
Mechanism: Photoluminescence, bioluminescence,
chemoluminescence, thermoluminescence,
piezoluminescence, etc.
Radim Schreiber
Trevor Morris
Photoluminescence
64
57
http://www.evidentcrimescene.com
Charles Hedgcock © University of Arizona, Tucson, AZ
http://kevincollinsphoto.smugmug.com
http://uclagettyprogram.wordpress.com
Photoluminescence
58
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.
Conduction
band
Valence band
direct band gap
indirect band gap
65
Photoluminescence
59
Excitations in materials
•Excitons
•Bound excitons
•Excitonic complexes
Peter Abbamonte et al., Proc. Nat. Acad. Sci. 105 (34)
Exciton describes the bound state of an electronhole pair due their mutual Coulomb attraction
Photoluminescence
60
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.
Davydov et al. Phys. Stat. Solidi (b) 230 (2002b), R4
67
Photoluminescence
68
61
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
Photoluminescence
Photoluminescence
69
62
GaAs
Conduction band
D
+
(D ,X)
FE
(Do,X)
(Ao,X)
A
Valence band
Photoluminescence
63
Using PL to determine the width and quality
of InGaAsN/GaAs quantum wells.
3nm
InGaAsN
QW
5nm
InGaAsN
QW
9nm
InGaAsN
QW
70
Photoluminescence
Photoluminescence
64
Strengths:
•
•
•
Very little to none sample
preparation.
Non destructive technique.
Very informative spectrum.
71
Photoluminescence
Photoluminescence
72
65
Limitations:
•
•
•
Often requires low temperature.
Data analysis may be complex.
Many materials luminescence
weakly.
Complementary techniques:
Ellipsometry, Modulation
spectroscopies,
Spectrophotometry, Raman.
Time-domain thermoreflectance
73
66
What is measured?
The reflectance variation with temperature.
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.
hn0
hn0
Time-domain thermoreflectance
74
67
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).
Time-domain thermoreflectance
68
The lowest thermal conductivity so far observed
for a fully dense solid. (48 mWm-1K-1)
75
Time-domain thermoreflectance
68
The lowest thermal conductivity so far observed
for a fully dense solid. (48 mWm-1K-1)
76
Time-domain thermoreflectance
69
77
Time-domain thermoreflectance
69
78
Time-domain thermoreflectance
70
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.
Science 315, 342 (2007)
79
Time-domain thermoreflectance
80
71
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.
Complementary techniques:
3w method, Raman spectroscopy, Nanoindentation.
Optical microscopy
72
Optical microscopy
73
"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).
Optical microscopy
74
Optical microscopy
75
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
Optical microscopy
76
Phase contrast
Bright field
Dark field
Polarizing
Optical microscopy
77
Difference interference contrast
Optical microscopy
78
Phase contrast
Bright field
Fibers in bright field and dark field
Dark field
Polarizing
Optical microscopy
79
Fluorescence
Optical microscopy
80
Contrast-Enhancing Techniques for Optical Microscopy
Specimen
Type
Imaging
Technique
Transmitted Light
Transparent Specimens
Phase Objects
Bacteria, Spermatozoa,
Cells in Glass Containers,
Protozoa, Mites, Fibers, etc.
Light Scattering Objects
Diatoms, Fibers, Hairs,
Fresh Water Microorganisms,
Radiolarians, etc.
Light Refracting Specimens
Colloidal Suspensions
powders and minerals
Liquids
Amplitude Specimens
Stained Tissue
Naturally Colored Specimens
Hair and Fibers
Insects and Marine Algae
Fluorescent Specimens
Cells in Tissue Culture
Fluorochrome-Stained Sections
Smears and Spreads
Birefringent Specimens
Mineral Thin Sections
Liquid Crystals
Melted and Recrystallized Chemicals
Hairs and Fibers
Bones and Feathers
Phase Contrast
Differential Interference Contrast (DIC)
Hoffman Modulation Contrast
Oblique Illumination
Rheinberg Illumination
Darkfield Illumination
Phase Contrast and DIC
Phase Contrast
Dispersion Staining
DIC
Brightfield Illumination
Fluorescence Illumination
Polarized Illumination
http://micro.magnet.fsu.edu
Optical microscopy
81
Contrast-Enhancing Techniques for Optical Microscopy
Reflected Light
Specular (Reflecting) Surface
Thin Films, Mirrors
Polished Metallurgical Samples
Integrated Circuits
Diffuse (Non-Reflecting) Surface
Thin and Thick Films
Rocks and Minerals
Hairs, Fibers, and Bone
Insects
Amplitude Surface Features
Dyed Fibers
Diffuse Metallic Specimens
Composite Materials
Polymers
Birefringent Specimens
Mineral Thin Sections
Hairs and Fibers
Bones and Feathers
Single Crystals
Oriented Films
Fluorescent Specimens
Mounted Cells
Fluorochrome-Stained Sections
Smears and Spreads
Brightfield Illumination
Phase Contrast, DIC
Darkfield Illumination
Brightfield Illumination
Phase Contrast, DIC
Darkfield Illumination
Brightfield Illumination
Darkfield Illumination
Polarized Illumination
Fluorescence Illumination
http://micro.magnet.fsu.edu
Optical microscopy
Near-field Scanning
Optical microscopy
82
Resolution
Based on the Airy discs formation Abbé
derived a theoretical limit to an optical
instrument resolution.
Arch. Microskop. Anat. 9, 413 (1873)
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  0.61l
NA
Rayleigh criterion (light emitting particle)
𝜆
𝜆
𝑑≈
≈
𝑁𝐴𝑐𝑜𝑙 + 𝑁𝐴𝑜𝑏𝑗 2 𝑁𝐴
Abbé criterion (illuminated particle)
91
Optical microscopy
83
0.61 𝜆
𝑑≈
𝑛 sin 𝜇
𝜆
𝑑≈
2 𝑛 sin 𝜇
Rayleigh criterion (light emitting particle)
Abbé criterion (illuminated particle)
Confocal microscopy
84
• 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.
Confocal microscopy
85
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%.
Confocal microscopy
86
Confocal microscopy
87
Anna-Katerina Hadjantonakis; Virginia E Papaioannou
BMC Biotechnology 2004, 4:33
Confocal microscopy
88
Strengths:
Limitations:
– Optical sectioning (0.5 m).
– three-dimensional images.
– Improved contrast (200:1).
– Better resolution (1.5x).
– Field of view defined by the
scanning range.
– 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).
Beyond confocal microscopy
89
Beyond confocal microscopy
90
Nature Reviews Genetics 4, 613 (2003)
Biophysical J. 75, 2015 (1998)
(courtesy of Brad Amos MRC, Cambridge)
Beyond confocal microscopy
91
Beyond confocal microscopy
92
Beyond confocal microscopy
93
•
•
•
•
•
•
•
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.
Beyond confocal microscopy
94
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.
Near-field
scanning
optical
microscopy
(NSOM)
Near-field
Scanning
Optical
microscopy
95
10
4
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
(NSOM)
Near-field
Scanning
Optical
microscopy
96
Instrumentation:
10
5
Near-field scanning optical microscopy (NSOM)
97
10
6
Strengths:
–
–
–
–
Powerful, very attractive.
No sample preparation.
Non destructive technique.
Sub diffraction limit
resolution (50 nm).
CBS Broadcasting Inc.
Near-field scanning optical microscopy (NSOM)
98
10
7
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.
Complementary techniques:
AFM, SEM,TEM, Confocal microscopy.
It is Important to use complementary techniques!
99
Thank you!
10
8
100
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