Surfaces, Interfaces and Colloids
Microscopy Techniques
José Paulo Farinha
Centro de Química-Física Molecular
Institute of Nanoscience and Nanotechnology
Instituto Superior Técnico
[email protected]
web.tecnico.ulisboa.pt/farinha
“Lucifer Yellow” (acrylic on canvas 130x70 cm) – detail
by Ana Tristany (www.anatristany.com)
Microscopy Techniques for Materials Characterization
Optical
Microscopy
Laser
Scanning
Microscopy
Scanning
Electron
Microscopy
(SEM)
Transmission
Electron
Microscopy
(TEM)
Scanning
Probe
Microscopy:
AFM, etc.
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Microscopy Techniques for Materials Characterization
Optical
Microscopy
Scanning
Electron
Microscopy
(SEM)
Laser
Scanning
Microscopy
Transmission
Electron
Microscopy
(TEM)
Scanning
Probe
Microscopy:
AFM, etc.
Optical Microscopy
The compound light microscope
"Microscope" (1625)
µικρόν (micron) "small“ +
σκοπεῖν (skopein) "to look at“
Giovanni Faber (1574–1629)
Galileo Galilei (1564 –1642)
First documented use of a
compound microscope (with
a convex and a concave lens)
“occhiolino”
(1609)
Academia dei Lincei
(1603 - )
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Magnification
1665 – “Micrographia”:
observation of thin slices
of cork. First use of the
term “Cell”
Illumination
Robert Hooke
(1635 –1703)
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What did Hooke see when he looked at cork?
Cork (Hooke, 1665)
A confocal microscope view of cork
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A compound microscope magnifies both
in the objective and the ocular
2f
imaginary
image
f
(magnifying glass)
object
Magnification
ocular
F
2f
objective
f
Illumination
sample
(Projector)
object
condensor
real image
F
light source
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A compound microscope magnifies both
in the objective and the ocular
ocular (magnifying glass)
Intermediate (real) image
(projector screen)
objective (projector)
Final (imaginary) image
Sample
light
Total magnification = Mobjective x Mocular
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Abbe’s law: resolving distance of an objective
“… minimum resolving distance is related to the wavelength of light
divided by the Numeric Aperture, which is proportional to the angle
of the light cone formed by a point on the object, to the objective”
Ernst Abbe and Carl Zeiss, 1877
d=
λ
2 NA
n - refractive index
NA - lens numerical aperture
NA = n sin θ
Carl Zeiss
(1816-1888)
Ernst Abbe
(1840-1905)
Friedrich Otto Schott
(1851-1935). Inventor
of borosilicate glass
Oil immersion systems
with NA = 1.4
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objective
θ
Resolving power
High NA Low NA
Ability of an objective to resolve
objects very close together
The resolving power is proportional to the
numerical aperture of the objective NA and
increases with:
– the angle θ the lens is capable of receiving light
– the refractive index n of the medium between
the object and the objective.
Depth of field
Optical thickness of the focused slice
Light
cone
d=
λ
2 NA
High NA
Low NA
NA = n sin θ
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Working distance
Objectives with higher NA (higher angle θ ) have shorter working
distance
NA = n sin θ
Refraction index n
air ≈ 1.00
water = 1.37
oil = 1.5
glass = 1.5
Magnification
Objective
NA < n
NA = 0.25
Magnification
Front lens
Objective
θ = 15º
n = 1.00 (air)
specimen
NA = 0.95
Front lens
θ = 74.7º
specimen
n = 1.00 (air)
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Refractive index matching
Refractive index mismatch
causes image distortion.
Use of oil-immersion or
water-immersion objectives.
Air
Coverslip
Water
Objective
Objective
n = 1.5
n = 1.5
n = 1.0
n = 1.5
n = 1.5
n = 1.3
Coverslip
Oil
n = 1.5
n = 1.5
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Fluorescence Microscopy
Stokes Shift
Absorption
Intensity
Emission
Emission
excitation
detection
Wavelength
Extinction Coefficient ε
Absorption
Efficiency of photon absorption
Fluorescence Quantum Yield φf
Integrated photon emission over the spectral band
For sub-saturation excitation, the fluorescence
intensity is proportional to the brightness : ε x φf
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Sir George Stokes
(1819–1903)
Due to the Stokes shift, the ilumination
and detected light are separated by a
dichroic mirror providing selective
imaging of fluorescent species.
Emission
Emission
Light
source
Excitation
DM
Absorption
Fluorescence microscopy is capable of imaging
the distribution of a single molecular fluorescent
species or label based solely on the properties
of fluorescence emission.
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Microscopy Techniques for Materials Characterization
Optical
Microscopy
Scanning
Electron
Microscopy
(SEM)
Laser
Scanning
Microscopy
Transmission
Electron
Microscopy
(TEM)
Scanning
Probe
Microscopy:
AFM, etc.
Laser Scaning Microscopy
Conventional
fluorescence microscope
full field detection
Laser scanning
microscope
point scan detection
full field illumination
point scan illumination
Lamp (Hg, Xe) + excitation filter
laser light source
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Laser Scanning
laser
y
y
x
z
x
point scan illumination
(fluorescence excitation)
point scan detection
(fluorescence emission)
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Confocal Microscope
Detector Signal
Detector
x
z
Discrimination of
out of focus light
y
pinhole
3D Imaging
Focal plan
z
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Multiphoton microscopy
hν*
hν*
Emission
hν
2-photon excitation
Absorbtion
hν
Emission
Absorbtion
single-photon excitation
Two photons at the same time and place?
Timescale for simultaneity ≈
s
(Heisenberg’s uncertainty principle)
10-16
hν
Maria Goeppert-Mayer
(1906 –1972)
Nobel Prize of Physics
(1963)
Photons with double wavelenght (> 750 nm)
Very high photon density
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1
1 
λ MP ≈  + 
 λ1 λ2 
λ
MP
−1
≈ 2 λSP
The required photon density for 2-photon excitation can only be
achieved in the focal plan during a laser pulse
laser pulse
focal plane
non-excited
dye molecule
2p-excited
dye molecule
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Excitation localized in very small volume (sub-femtoliter)
1-photon excitation
2-photon excitation
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Confocal microscope
Two-photon microscope
PMT
PMT
pinhole
No pinhole
needed
filter
Vis laser
excitation
emission
IR laser
excitation
emission
objective
z
y
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x
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MP excitation
- Reduced dye photobleaching and better z resolution.
- Reduced scattering of excitation and emission light.
Scattering ∝
- Wide excitation range.
1
λ4
- Uses red /NIR light: deeper penetration in biological tissue
- Reduced fluorescence emission...
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FLIM: fluorescence lifetime imaging microscopy
Image based on the difference in the decay rate of
fluorescent dyes instead of the emission intensity.
• Independent of local probe concentration
• Less affected by photobleaching and scattering
Excitation by pulsed laser and
detection in time domain.
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FLIM can distinguish
between probes with the
same emission spectra
(but different τ)
The fluorescence lifetime
of many dyes changes with
environmental conditions
(pH, polarity, temperature),
allowing their mapping.
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Confocal (single-photon) fluorescence microscopy
Multi-photon fluorescence microscoopy
FLIM - fluorescence lifetime imaging microscopy
AFM to couple to laser
scanning microscope
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Super-resolution Microscopy: Nanoscopy
Super-resolution Microscopy
Diffraction limit
Abbe’s law: resolving distance of an objective
“… minimum resolving distance is related to the wavelength of light divided by
the Numeric Aperture, which is proportional to the angle of the light cone
formed by a point on the object, to the objective”
d=
λ
2 NA
n: refractive index
NA: lens numerical aperture
NA = n sin θ
Spatial resolution using visible light is limited to ca. 0.3 µm
by the diffraction of visible light
By using fluorescence laser-scanning microscopy to detect
only a few molecules at a time, it is possible to reconstruct
the image with a resolution below the limit of diffraction
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Super-resolution Microscopy
PALM: photoactivated localization microscopy
Optical microscope resolution leads to blurry
images of small objects (ex.: cell mitochondria)
If specific proteins are tagged with fluorescent
molecules that can be activated one-at-a-time,
their positions can be determined with
nanometer resolution
All the data is assembled into a single image
Resolution comparable to an electron
microscope, but with the added benefit of
being specific to only the desired target
features.
Electron
microscopy
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Eric Betzig
William E. Moerner
Super-resolution Microscopy
PALM: photoactivated localization microscopy
Superposition of several thousand
images of individual molecules is
very time consuming
First PALM microscope, built by Eric Betzig
and Herald Hess (in Hess’ living room)
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Eric Betzig
William E. Moerner
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Super-resolution Microscopy
STED: stimulated emission depletion
http://4jif.eventos.chemistry.pt
Stefan Hell
Max Planck Institute for Biophysical Chemistry
“Super-resolution optical microscopy”
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Super-resolution Microscopy
STED: stimulated emission depletion
Einstein coefficients
Stimulated
absorption
Albert Einstein (1879-1955)
Nobel da Física em 1921
Spontaneous
emission
Stimulated
emission
Laser effect
Light Amplification by Stimulated
Emission of Radiation
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Super-resolution Microscopy
STED: stimulated emission depletion
STED uses a second laser to de-excite the all
fluorescent molecules by stimulated emission,
except those in a nanometer volume at the center
of the beam
Excitation laser
STED laser
Fluorescence
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Super-resolution Microscopy
STED: stimulated emission depletion
The wave front of the depletion beam is ring shaped,
featuring a dark spot of zero intensity in the center.
Fluorescence from the remaining excited dye
molecules is detected to obtain optical resolution in
the 10 nm range.
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Super-resolution Microscopy
STED: stimulated emission depletion
Example: Fluorescence image of
immuno-labeled amphibian pore
complexes (NPC)
STED reveals the NPC subunits
forming a octameric ring ~160
nm of 20 nm homodimers
around the 80 nm channel.
500 nm
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What is the best technique for my system?
Confocal fluorescence microscopy
- thin samples only (< 100 µm)
- multilabeling using different dyes
Multi-photon fluorescence microscoopy
- structures within thick samples
- reduced photobleaching but lower intensity
FLIM: fluorescence lifetime imaging microscopy
- local environment changes with only one probe
Super-resolution microscopy
- best optical resolution
- very limited choice of dyes and very expensive
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Microscopy Techniques for Materials Characterization
Optical
Microscopy
Laser
Scanning
Microscopy
Scanning
Electron
Microscopy
(SEM)
Transmission
Electron
Microscopy
(TEM)
Scanning
Probe
Microscopy:
AFM, etc.
Why use electrons?
Electrons behave as waves in
electron diffraction experiments
showing interference patterns
de Broglie relation
Louis de Broglie
Louis Victor Pierre Raymond
th
7 Duque de Broglie (1892-1987)
Nobel of Physics 1929
λ=
h
mv
Resolving distance
d=
λ
2 n sin θ
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Scanning Electron Microscopy (SEM)
An electron gun generates
electrons. A condenser focus the
beam in the smallest point
possible. Scanning coils deflect
the beam and make it raster the
sample surface.
Magnifications from ca. 50x to over 100 000x
Able to obtain local chemical information
JEOL 7100F FEG SEM @ IST
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SEM beam/specimen interactions (signal types)
Electron beam
Sample
SE - secondary electrons
(lower energy than BSE)
BSE - backscattered electrons
X-rays
SE are used for topographic contrast.
They have higher spatial resolution than BSE
because these electrons originate from a
smaller volume
BSE are sensitive to the atomic number due to
their high resolution.
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SEM - Topographic contrast (SE)
Where do the shades come from?
SE are low energy electrons and can
only escape the sample to reach the
detector if emitted from an interaction
volume near the surface.
Pollen
In “hills” there are more exposed surface, more
SE escape and the signal is higher, originating
bright regions in the image.
In “valleys” less interaction volume is exposed
and less SE reach the detector, originating
dark regions in the image.
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SEM - Backscattered electron imaging
BSE are high energy electrons, sensitive to composition (atomic number).
BSE imaging is used in conjunction with SE (topological) imaging.
Varistor
15 kV
M: 2000x
Topographic contrast (SE mode)
Atomic number contrast (BSE mode)
The bright regions in the SE image correspond to hills, whereas the
bright regions in the BSE image correspond to the presence of
heavier elements.
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SEM - Energy dispersive X-ray spectroscopy (EDS)
Electron
beam
Sample
X-rays are emitted from atoms when their
electrons make transitions between inner atomic
energy levels.
Each element has characteristic transition
energies
In EDS, the SEM image is built from X-rays with
specific energy, producing maps for each element
SE
BSE
SEM image, 2500x
Nickel
X-rays
Copper
Tin
Lead
Analysis
- Qualitative: element identification;
- Semi-quantitative, without standards;
- Quantitative, with standards of similar composition.
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SEM - Sample preparation for soft/biological materials
Biological structures and many soft materials collapse under vacuum...
1. Fixation: Stabilization of biological
material, usually by cross-linking with
aldehydes or OsO4.
E. coli
M: 10000x
2. Dehydration: Substitution of water with
solvent (ethanol, acetone).
3. Drying: critical point drying (CO2 at
31.1C and 1,073 psi, vented slowly) or
Hexametildisilizane (HMDS) drying (by
substitution
of
ethanol,
reducing
capillarity effects during drying).
4. Mounting: fixing on a sample holder.
5. Coating with conductive layer (Au,
carbon,...): Prevent charging effects that
hinder suitable image formation.
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Advantages & Disadvantages of SEM
Advantages
- high depth of field
- direct observation of the surface at high magnification
- wide range of magnifications (below 50 x to over 100 000x)
- local chemical and crystallographic information
Disadvantages of SEM
- Less resolution than TEM
- no internal detail
- conductive layer needed (non-conducting samples accumulate charge,
perturbing the beam and signal detection)
- high vacuum environment (damaging for biological and soft samples…)
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Microscopy Techniques for Materials Characterization
Optical
Microscopy
Laser
Scanning
Microscopy
Scanning
Electron
Microscopy
(SEM)
Transmission
Electron
Microscopy
(TEM)
Scanning
Probe
Microscopy:
AFM, etc.
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Transmission Electron Microscopy (TEM)
A TEM usually operates at 10-6 Torr
in the column and 10-7 Torr in the
electron gun chamber
Magnification up to 500 000x and
resolution down to 0.2 nm
Hitachi 8100 @ IST
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TEM contrast
The image contrast originates from:
• Diffraction: Crystalline materials
• Phase : High-resolution TEM (atomic resolution)
• Mass (amplitude) contrast: Soft and biological materials
TEM mass contrast for soft material
TEM image of cell
• Heavy atoms scatter more intensely (dark areas in the image).
• Fewer electrons are scattered at high electron accelerating voltages, since they
have less time to interact with atomic nuclei in the specimen.
However, high working voltages result in lower contrast
and damage to polymeric and biological samples
• Polymer and biological samples have low atomic number and similar electron
densities. Using staining agents that are selectively absorbed in one of the
phases increases imaging contrast (stained regions appear dark in the image).
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TEM sample preparation
The sample should be thin enough for electrons to penetrate without too large
energy loss, but still representative of the structure.
Important issues in biological samples:
• Preparation and preservation of fine structures
• Contrast generation
Microtome with cryo-chamber
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Advantages & Disadvantages of TEM
Advantages
- Highest special resolution (sub-Å already available)
- local chemical and crystallographic information at very high resolution
Disadvantages of TEM
- Extremely expensive (very high resolution at many M€…)
- Destructive technique (preparation and imaging)
- Complex and time-consuming sample preparation)
- high vacuum environment (damaging for soft/biological samples…)
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Microscopy Techniques for Materials Characterization
Optical
Microscopy
Laser
Scanning
Microscopy
Scanning
Electron
Microscopy
(SEM)
Transmission
Electron
Microscopy
(TEM)
Scanning
Probe
Microscopy:
AFM, etc.
Scanning Probe Microscopy
Scanning probe microscopy (SPM) images the surfaces using a physical
probe that scans the specimen. SPM was founded with the invention of
the scanning tunneling microscope (STM) in 1981.
Scanning tunneling microscopy
Gerd Binnig (1947)
(IBM Zürich)
Heinrich Rohrer (1933)
Nobel Prize of Physics,1986
Mica at atomic resolution
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STM measures the tunneling
current across the gap from a
tip to the sample.
Only for conductive samples
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Constant height
Constant current
I ~ Ve − cd
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Atomic force microscopy (AFM)
The information is gathered by
"feeling"
the
surface
with
a
mechanical probe (cantilever with
very sharp tip).
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Cantilever tip
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AFM can operate in contact or
dynamic (tapping, non-contact) mode
AFM imaging in water
can be used to study
biological samples
Hard samples (contact or taping)
Soft samples (tapping)
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AFM can give more information than
topology:
• Atomic Force Microscopy (AFM)
• Lateral Force Microscopy (LFM)
• Magnetic Force Microscopy (MFM)
• Electric Force Microscopy (EFM)
AFM of living fibroblast cell
• Nanolithografy, Nanoindentation,
Nanowear
• Other (ex: Force Modulation, Thermal
Scanning, etc.)
Nanoindentation
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