Nanobiotechnology
Scanning Probe Microscopies
Jan Přibyl
CEITEC MU
Kamenice 5/A35, CZ-62500 Brno
pribyl@nanobio.cz
Microscopy techniques - resolution
Scanning Probe Microscope
basic scheme
signal
Reconstruction
of image
probe
Movement
element
Feedback
loop
Reconstruction
of image
surface
interaction
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SNOM (=NSOM)
Scanning NearField Optical Microscopy
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SNOM – basic principles
STM measures electric current, and AFM measures
forces, neither deals with light;
Light - crucial excitation source in both scientific research
and mother nature systems.and mother nature systems.
Scientific research fields: absorption, fluorescence,
photoinduced electron transfer, light-emitting devices,
photovoltaic cells.
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Why SNOM?
Light diffraction limit - conventional optical microscopy:
λ/2 ~ 250 nm ( Abbe diffraction limit)
Real cases - optical resolution ~ λ, 500 nm
NSOM offers higher resolution around 50 nm (or even < 30NSOM offers higher resolution around 50 nm (or even < 30
nm), depending on tip aperture size.
NSOM - simultaneous measurements of the:
- topography
- + optical properties (fluorescence)
direct correlation between surface nanofeatures and
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direct correlation between surface nanofeatures and
optical/electronic properties.
Useful for the studying:
inhomogeneous material surfaces (nanoparticles, polymer
blends, porous silicon, biological systems)
History of NSOM
1928 roots trace back – letters between Edward
Hutchinson Synge and Albert Einstein
Ideas started in mid-1980’s:Ideas started in mid-1980’s:
D.W. Pohl, W. Denk, and M. Lanz, Appl. Phys. Lett. 44,
651-3 (1984).
A. Lewis, M. Isaacson, A. Harootunian, and A. Murray,
Ultramicroscopy 13, 227 (1984);
Technology developed in 1990’s:
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Technology developed in 1990’s:
Eric Betzig, et al. Science, 262, 1422-1425 (1993).
Eric Betzig, et al. Nature, 2369, 40-42 (1994).
Prototype commercial available since 2000’s
Scheme of SNOM apparatus
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Major components of NSOM
Optical:
Light source (lasers: CW and pulsed), Fibers, Mirrors, Lenses,
Objectives (oil, large NA)
Photon detectors (Photon-Multiplier)
Probe (tip)
Mechanical:
Translation stage, Piezo scanner
Anti-vibration optical table
Electrical:
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Electrical:
Scanning drivers for piezo scanner
z distance control (feedback system)
Amplifiers, Signal processors
Software and Computer
SNOM probe
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What is Near-Field?
requires a nanometer sized aperture (much
smaller than the light wavelength).
A specimen is scanned very close to the aperture.A specimen is scanned very close to the aperture.
As long as the specimen remains within a
distance less than the aperture diameter, an
image with sub-wavelength resolution (aperture
size) can be generated.
There is a tradeoff between resolution and
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There is a tradeoff between resolution and
sensitivity (light intensity)
--- aperture size cannot be too small.
What is Near-Field?
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Near-field: For high spatial resolution, the probe must be
close to the sample
SNOM probe detail
Scheme
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NSOM
in operation Detail
Operation Modes
Transmission mode
Luminescence mode
Reflection mode
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Luminescence mode
Simoultaneously with Shear Force Microscopy (SFM)
Piezodriver via quartz tuning fork (change of oscillation amplitude is
monitored AFM-like imaging)
Key point is to use “AFM technology” to bring the light
very close to the surface (1-10 nm, distance<probe
diameter)
Real instrument example
Ntgra Vita AURA, Ntgra Vita SPECTRA (NTMDT, Zelenograd, Russia)
Bringing light close to the surface
Optical fiber
+ tuning fork
AFM transparent
probe
+ light via
objective
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TERS
Tip Enhanced Raman
Spectroscopy
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NSOM images
Tissues images
Living cells
Epi-illumination
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Living cells
Near-field illumination
Scanning Tunnelling Microscopy
STM
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STM - the first member of SPM family
Developed in 1982 by Gerd Binnig and Heinrich Rohrer
members of IBM in Zurich (Phys. Rev. Lett., 1982, vol 49,
p57)
1986 - Nobel prize in physics for their brilliant invention
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1982 - Triumph of Scanning
Probe Microscopy - image of
silicon surface 7x7
reconstruction.
Basic components of STM
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STM tip
STM tip - conductive (metals - Pt, W, Pt/Ir)
STM microscopy uses the very top (outermost) atom at the tip and the
nearest atom on sample
Tip is not necessarily very sharp in shape (different from AFM)
Tip preparation:
- Cutting with scissors
- Electrochemical etching
- Other techniques such as FIB (and combination)
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STM tip electrochemical etching
Surface tension helps to
create tip shape
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Or…
create tip shape
After etching the tipincluding
part of wire
remains in holder,
remaining part falls to
bottom
Tunneling current
Transfer of electrons without a contact of conductive is not
possible according to classical mechanics tunneling
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Tunneling of electrons… Fermi level
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Electron density of states - Fermi level
Electrons are happy sitting in either the tip or the sample
It takes energy to remove an electron into free space - vacuum around the tip as an
energy hill that the electron would need to climb in order to escape. The height of this
energy hill is called the work function, φ.
In order to bring an electron up and over the vacuum energy barrier from the tip into the
sample (or vice versa), we would need to supply a very large amount of energy.sample (or vice versa), we would need to supply a very large amount of energy.
Quantum mechanics tells us that the electron can tunnel right through the barrier.
Note: this only works for particles!
As long as both the tip and the sample are held at the same electrical potential, their
Fermi levels line up exactly. There are no empty states on either side available for
tunneling into! This is why we apply a bias voltage.
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A thin metal tip is brought in close proximity of the sample surface. At
a distance of only a few Å, the overlap of tip and sample electron
wavefunctions is large enough for an electron tunneling to occur.
When an electrical voltage V is applied between sample and tip, this
tunneling phenomenon results in a net electrical current, the
‘tunneling current’. This current depends on the tip-surface distance
d, on the voltage V, and on the height of the barrier Φ:
This (approximate) equation shows that the tunneling current obeys
Ohm’s law, i.e. the current I is proportional to the voltage V.
The current depends exponentially on the distance d.
For a typical value of the work function Φ of 4 eV for a metal, the
tunneling current reduces by a factor 10 for every 0.1 nm increase in
d. This means that over a typical atomic diameter of e.g. 0.3 nm, the
Φ - the work function (energy
barrier)
D - tip-sample distance
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d. This means that over a typical atomic diameter of e.g. 0.3 nm, the
tunneling current changes by a factor 1000! This is what makes the
STM so sensitive.
The tunneling current depends so strongly on the distance that it is
dominated by the contribution flowing between the last atom of the tip
and the nearest atom in the specimen
Single-atom imaging!
D - tip-sample distance
STM modes
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Factors affecting STM imaging
Corrugation - how much the electron density of surface
atoms varies in height above the surface.
Thermal drift – change of temperature cause extension ofThermal drift – change of temperature cause extension of
material
HOPG
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Graphite has a large corrugation, and is very planar, and thus is one of the
easiest materials to image with atomic resolution. (see next slide for example)
UHV-STM (UltraHigh Vacuum STM)
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UHV-STM examples
Si (111) 7x7, 40nm Clean Si(111)
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Si (111) 7x7, 40nm
empty states image,
room temperature, dark
spots represent missing
atoms or adsorbates
Ag-Si (111) 10nm
Clean Si(111)
reconstructed to
(7x7); STM image 50
x 50 nm2
Atomic Force Microscopy
AFM
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AFM microscope basic scheme
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AFM microscope block scheme
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Tip and cantileverTip and cantilever
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Cantilever and tip
tl. 1 mm
cantilever
hrot 50-100 um
Reflex layer (metal)
6 mm
2mm
Cantilever holder
hrot
100-200 um
50-100 um
5 um
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• Cantilever holder is quite universal
• Cantilever and tip – a variety of various types
Cantilevers
Material properties
– Stiffness Force Constant [N/m]
Force const.[N/m] 10-130 1-10 0.1-1.0 0.005-0.1
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Material cryst. silicon pol. silicon glass Si3N4
Res. f. [kHz] 200-500 100-200 15-100 1-20
Special applications – conductive, colloid, magnetic, tip less, …
Cantilever field
choose the one you like/need
Cantilever
characterization you may
find on box
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AFM probes (micro)fabrication is
quite complex
Tip properties
Shape – Curvature Radius R [nm]
Supersharp Standard Special app. Tip less
R
R 1 nm 10 nm 100 nm NA
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Cantilever fabrication
FIB (Focus Ion Beam) post-fabrication of AFM probes (tip)
44 Plateau Tip
Idealized force-distance curve describing a single approach-retract cycle of the AFM tip,
which is continuously repeated during surface scanning.
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Victor Shahin et al. J Cell Sci 2005;118:2881-2889
©2005 by The Company of Biologists Ltd
(DFL
deflection)
Torsion forces
(LF latheral
forces)
Cantilever bending – how to detect
Contact with surface
deflection)
Change of cantilever properties
(DFL/LF) is detected by laser
beam
DFL
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LF
Curvature radius (R) effect
SuperSharp tip
= real image
Standard tip
= R ~ 5-10nm
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Blunt tip
= affecting real shape
and size