4. Microscopy - static.uni-graz.at

Surface ScienceWS 2019/20, L. Grill and G. Simpson

4. Microscopy

From Ancient Greek: μικρός (mikrós, “small”) + σκοπέω (skopéō, “I look at”)

What is microscopy?

A microscope is able to produce a magnified image of a small object

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Radio Infrared Visible Ultraviolet γ- and x-rays

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4. Microscopy

A170125.124200.dat

A170125.124200.dat Ch: 1

Biasvoltage: 0.03610V

Current: 3.4E-08A

Temperature: 0.00000 [K]

0 1 2 3 40

1

2

3

4

-0.005062 nm

-0.003062 nm

-0.001062 nm

0.0009381 nm

0.002938 nm

0.004938 nm

Epithelial cells Red cells Bacteria

Mycoplasma Virus Proteins

Amino acidsAtoms


4.1 Optical Microscopy

History

Earliest form of microscope – Eyeglasses

Salvino D'Armato degli Armati of Florence (died 1317) ??

«Qui diace Salvino d'Armato degl' Armati di Fir., Inventor degl'occhiali. Dio gli perdoni la peccata. Anno D. MCCCXVII»

“Here lies Salvino, son of Armato degli Armati of Florence, inventor of eyeglasses. May God forgive his sins. A.D. 1317”

The first type of „microscope“

What is generally meant as a microscope today is the compound microscope

• A device which uses two or more lenses to produce a magnified image of an object

The unaided human eye can distinguish two points 0.2 mm apart


Credit for the first microscope is usually given to Zacharias Jansen, in Middleburg, Holland, around the year 1595. Since Zacharias was very young at that time, it's possible that his father Hans made the first one, but young Zach took over the production

A replication microscope was made but the exact details and specificationsremain unknown


History


Magnification


Lens equation:

1

𝑓=

1

𝑑𝑜+

1

𝑑𝑖

Magnification equation:

𝑀 =ℎ𝑖ℎ𝑜

= −𝑑𝑖𝑑𝑜

Gives image distance and image height if the object distance, object height, and focal length are known

Equation can be derived from simple geometric arguments


Compound microscope


• An object O is at distance a in front of objective

lens Lob

• Intermediate image O’ is projected

• O’ is further magnigifed by the eyepiece lens Ley

• Image O’’ is projected onto the retina

• The magnification is given by h‘/h

• Infinity space microscope has additional tube lens

• Creates a section into which accessories can be

added

• E.g. prisms, polarisers, retardation plates,

illuminators, etc

Compound microscope - two stage magnification• initial magnification with objective

• further magnification with eyepiece


Aberration


The failure of rays to converge at one focus because of a defect in a lens or mirror

Spherical aberration

Perfect lens

Non-perfect lens

• Peripheral rays and axial rays have different focal points

• Image appears blurred as a result

• Affects the resolution of the microscope

Refracted to a greater degree

Refracted only slightly

Monochromatic light

Can be reduced by limiting exposure oflight to the centre of the lens or usingspecial lens grinding techniques,


Aberration


The failure of rays to converge at one focus because of a defect in a lens or mirror

Chromatic aberration• The refractive index of materials varies with the

wavelength of light

• Known as dispersion:

• Image appears blurred as a result

• Affects the resolution of the microscope

white light

white lightdispersedlight

𝑣 =𝑐

𝑛

Blue light (low λ) refracted togreater extent than red (high λ)

Can be corrected by combiningmaterials of differing refractiveindex to bring different colours tofocus on same point


Image formation


Diffraction is the speading of light that occurs when a beam of light interacts with an object

Huygens Principle: every point which a luminous disturbance reaches becomes a source of a spherical wave. The sum of the secondary waves determines the form of the wave.

If we have two elementary waves interacting witheach other, the result is interference of light.

Construcive interference produces points ofmaximum intensity

Destructive interference produces points ofminimum intensity


Image formation


𝐼 𝛼 = 𝐼0𝑠𝑖𝑛

𝜋𝑎𝜆∙ sin 𝛼

𝜋𝑎𝜆∙ sin 𝛼

2

Intensity distribution given by:

tan𝛼 =𝑥

𝑑and sin 𝛼 =

𝑘𝜆

𝑎

An intensity minimum is given by:

tan𝛼 =𝑥

𝑑and sin 𝛼 =

(2𝑘+1)𝜆

2𝑎,

An intensity maximum is given by:

Of particular interest is the image of a point source of light. Images are madeup of myriad overlapping points of light.

The intensity distribution from single slit. Central maximum is 0th order, then 1st, 2nd, etc


Image formation


Theory of image formation 1873 Ernst Abbe

Diffraction is the reason we see contrast in opticalmicroscopy (if light simply passed through thesample – no contrast)

Objects under microscope act like complexdiffraction gratings

Interference occurs in two places: diffraction plane and image plane

In image plane is collection of Airy circles (nextslide) which collectively make the image

At back focal plane is Fourier transform of image

Usually di is large, but not in microscope

1

𝑓=

1

𝑑𝑜+

1

𝑑𝑖


Image formation


A point source of light consists of a central spot (Airy disk) surrounded by a series of diffraction rings

Central spot contains approx 84% of the light from thepoint source

Size of central disk is related to λ and the aperture angle ofthe lens

Radius of disk

Decreasing numerical aperture

𝑑 =1.22𝜆

2𝑁𝐴

Numerical aperture NA defineshow much light (brightness) andhow many diffraction orders arecaptured by the objective


Image formation


Rayleigh criterion for spatial resolution:

a) Profile of single diffraction pattern

b) Profile of two disks separated by theRayleigh limit. Maximum overlapsfirst minimum. „Barely resolved“.

c) Maximum overlaps with secondminimum. „Clearly resolved“

Obtaining an image whose resolution is limited by the size of the diffraction spot is said to bediffraction limited

Resolution limit with good optics and high NA is approximately 250 nm


Breaking the diffraction limit

4.2 Super Resolution Microscopy

As we have seen, the wave nature of light limits reolution of conventional microscopes toapproximately half of the wavelength of light used.

Many sub-diffraction limit techniques rely on flourescence methods

Eric Betzig Stefan W. Hell William E. Moerner

The Nobel Prize in Chemistry 2014 was awarded "for the development of super-resolved fluorescence microscopy".




As we have seen, the wave nature of light limits reolution of conventional microscopes toapproximately half of the wavelength of light used.

Many sub-diffraction limit techniques rely on flourescence methods

Jablonski Diagram

Shows energy levels occupied by an excited electronwithin a fluorescent molecule. (Chlorophyll a)

Absorption of a photon causes excitation fromground electronic state to higher excited state.

Collapse back to ground state can occur though:

Internal conversionFluorescencePhosphorescence

Blue photon

Red photon




Fluorophores

Fluorescein

Chlorophyll a

Green Fluorescent Protein (GFP)

Pulmonary artery epithelial cellsshowing photobleaching

Excitation Emission


STimulated Emission Depletion fluorescence microscopy (STED)


S0 – ground electronic stateS1 – 1st excited electronic stateL – Vibrational levels

The excitation is achieved by a laser focussed trough a pinhole andan objective lens

The sample contains a fluorophore

The laser light is scanned over the sample

The size of the laser spot is again limited by diffraction so, ifunmodified, the resolution will not be increased.




In order to reduce the area of each fluorescent point stimulated emission is employed

Point spread function (PSF):

Describes the profile of the excitationlight

J1 – first order Bessel functionν - 2πrNA/λexc

r – distance from focal pointNA - Numerical apertureλexc – excitation wavelength

In order to reduce the spatial extent of hexc(ν), thefluorescence is inhibited in the outer regions

For this, an additional beam of light is used – theSTED beam




Excitation photon promotes electron from singlet S1 groundstate to the some vibrational level in the first excited state S1

Excitation quickly followed by internal conversion to lowestexcited vibrational state (picoseconds).

The fluorescence occurs as the electron relaxes back to theground state

However:If an additional photon comes in with energy equal totransition from S1 to a vibrational state of S0 we getstimulated emission

Result is two photon with same properties (phase, polarisation, etc)

Importantly, the simulated emission photons have different energy (wavelength) to the fluorescence photons

This means they can be filtered out easily.

Excitation laser STED laser

Signal




The STED beam is donut shaped andsuperimposed on the excitation beam

The resulting PSF of the fluorescencesignal is reduced

The combined laser light is scanned over thesurface to reveal sub-diffraction-limit features




Octametic arrangement of peripheraltransmembrane GP210 protein aroundcore protein in a nuclear pore complexin immunolabeled Xenopus cells

Brings resolution to the order of50 nm

J. Biomed. Opt. 19(8), 080901


Single molecule imaging techniques


Another method to circumvent Abbe limit: single molecule emitters

Causing fluorescence from single, well-separated molecules allows individual molecules to be located

EYFP fluorescent protein anchored to ParA protein in bacterial cell


4.3 Electron microscopy

Ernst Ruska Gerd Binnig Heinrich Rohrer

The Nobel Prize in Physics 1986 was divided, one half awarded to Ernst Ruska "for his fundamental work in electron optics, and for the design of the first electron microscope", the other half jointly to Gerd Binnig and Heinrich Rohrer "for their design of the scanning tunneling microscope"

First prototype electron microscope from Ernst Ruska 1933



Why electrons?

Resolution is limited by the wavelength of the illuminating radiation

Electrons are particles with negative charge and mass 1/1836 that of a proton

Wave-particle dualism postulated by de Broglie (Nobel Prize 1929)

Electrons have wave nature and their wavelength is directly related to theirmomentum:

𝜆 =ℎ

𝑝=

ℎ

𝑚𝑣

λ - Wavelengthp - Momentumm - Massv – Velocityh – Plank constant

By controlling the energy and hence speed of the electrons we can choosethe wavelength

Electron microscopy can provide resolution down to 0.5 nm (atomic scale)



Electron generation

A low votage is applied tocathode filament (W or LaB6)

Through resistive heatingelectrons gain sufficientkinetic enery to escape fromits surface

Free electron in vacuum arepulled away from the hotsurface by placing the positive anode nearby

High voltage between anode and cathode accelerates electrons towards anode

Hole in anode allows a beam of electrons trough

Electrons continue unhindered through the vacuum

Wehnelt cylinder pre-focuses the electrons before acceleration towards to anode to increase intensity



Types of electron interaction

How can the electrons interact with the sample?




Elastic scattering

No energy is tranferred from the electronto the sample

Backscattered electrons are also elasticallyscattered

Due to electron being deflected in somedirection by interaction with the Coulomb potential of the atom




Inelastic scattering

Energy is transferred between the electronsand the sample

Information rich, many different signals:

X-RaysAuger electronsSecondary electronsPlasmonsPhonons



TEM



Electromagnetic lens

𝑭 = −𝑒 𝑬 + 𝒗 × 𝑩

𝑭 = 𝑒𝑣𝐵sin(𝒗, 𝑩)

E: electric field strengthB: magnetic field stregthe/v: charge/velocity of electrons

Magnetic lens is a copper coil inside iron pole pieces

Passing current through the wires creates a magnetic field

Field is strong at edges and weak in the centre

Electrons experience the Lorenz force F:

Electrons are deflected strongly/weakly at edges/centre

Incoming parallel beam of electrons is focussed

By adjusting the current in the copper wire the B fieldstrength can be adjusted

Focusing effect can therefore be controlled



Transmission electron microscopy (TEM)

A beam of electrons is produced in the electron gun

Focused through lenses

Transmitted through an ultra thin specimen

Magnified by further lenses

Detected by a phosphorescent screen or a CCD

Sample preparation is key.

Sample thickness vaires between 10 and 500 nm depending on the material

TEM grid

Microtomy

Solution dispersion

Focusedion beam (FIB)



Transmission electron microscopy (TEM)

Most common imaging technique is Bright Field

• Contrast is formed directly by diffraction and absorption of electronsby the sample (like optical microscopy)

• Simple 2D projection of the sample• Dark areas represent areas of higher electron scattering (denser or

higher atomic number)• Light areas are where electrons pass through the sample more easily• Similar to Lamber-Beer

Rat‘s liver 1 µm scale bar

Bright field anddark field image ofeutectic alloy(AlSi12(Mg))

Dark field imagehighlights Mg2Si precipitates

In Dark Field imaging, illuminationis tilted to select a specificdiffracted beam

Used to image a subset of thesample which satisifes the Bragg condition



High resolution TEM

Atomic resolution TEM image of a triple and a quadruple line at the interface between Σ3 boundaries and a Σ9 boundary in nanocrystalline palladium. H. Rösner and C. Kübel et al., Acta Mat., 2011, 59, 7380-7387

Very thin slice of crystal tilted so that lowindex direction is perpendicular to electronbeam

The electron wave interacts with theperiodic crystal structure.

Driffraction of electron beam due to Bragg condition

Interference of electron waves

Contrast mechanism difficult to interpret

2𝑑sin𝜃 = 𝑛𝜆



Scanning Electron Microscopy (SEM)



Scanning Electron Microscopy (SEM)

Electron beam is highly focused to a tiny spot and scanned pixel by pixel over a surface

At each pixel secondary electrons (SE) are generated

The SE are detected then amplified.

Signal is fed to a computer and image is displayed

SE (<50 eV) can only escape from a smallvolume (surface sensitve)

Backscattered electrons (BSE) have higherenergy and can therefore escape from deeper

"universal curve„Mean free path of SE



Imaging using SE

SE escape depth independent of PE energy

Intensity is increased with lower PE energy (i.e. higherPE energy causes more SE to be produced deeper andcannot escape)

Intensity of SE escaping from surface at each pixelrepresents contrast

Contrast a function of angle of PE and specimen(depth of field)

Escape depth aslo dependent on atomic number:10-100nm Carbon2-3nm Chromium1-2nm Platinum

Biological samples are coated with thin layer of Pt or Au

Electrical grounding (conducting samples) important to stop charging of sample



Imaging using SE



Imaging using BSE

Backscattered electrons can reveal differences in composition (heavy elements backscatter morestrongly than light elements)

BSE must be detected close to the objective lens

Red blood cell showing location of surface protein antibodiesaggregated with gold particles



Electron detection

SE collected by a grid with +200 – 500 V applied to it.

SE hit a scintillator which coverts electrons tophotons

Photons guided towards photomultiplier tube

Photoelectrons are amplified to produce a measurable current

Current is the signal


4.4 Scanning Tunnelling Microscopy

History

The Nobel Prize in Physics 1986

Gerd Binnig Heinrich Rohrer

IBM Zurich

STM is the first member in the larger ‘family’ of instruments known as Scanning Probe Microscopes

Use a sharp probe which is brought into close proximity with a surface.

Distance so small that a measurable tunnel current is present

Tip is raster scanned over the surface while measuring current/height.

Atomic scale features can be resolved



Overview of STM set-up

1 Metallic tip2 Piezoelectric scanner3 Current amplifier4 Feedback loop

The metallic tip is brought to within a few Ångströms of the conductingbiased surface using the coarse positioning system. Tunnel current isdetected and amplified. Feedback system maintains constant current byadjusting the height of the tip as it is scanned over the surface.


A particle with energy E encounters a potential energy barrier of height V

Classical case: A golf ball with kinetic energy reaches a hill. Not enough energy to overcome barrier. Ball rolls back down the hill (reflected)

Quantum case: Electron approaches potential barrier (vacuum). Barrier height exceeds electron energy. Electron is described by wavefunction. Wavefunction decay exponentially in the barrier.If barrier is thin enough probability density is non-zero at other side.


Tunnelling



Tunnelling

−ℏ2

2𝑚𝜵2 + 𝑉 𝒓 Ψ 𝒓 = 𝐸Ψ(𝒓)Time-independent Schrödinger Equation

Hamiltonian operator(total energy: kinetic+potential)

Wavefunction

Energy eigenvalues

Wavefunction:

Complex mathematical functionContains all measurable information about systemΨ*Ψ represents probability density (summed over all space = 1)For a free particle Ψ is a sine wave (SE exactly solvable)



Tunnelling

Particle reaches potential barrier

Wavefunction related toprobability of finding particle

Transmission probability isrelated to ratio of amplitude in c compared to a

A measure of the current

Probability decays exponentiallyin gap b

Local density of states (LDOS)

I depends exponentially on d!Also dependent on V and φ

𝐼 𝑑 = 𝐴𝑉𝑒−2𝜅𝑑 𝜅 =2𝑚𝜙

ℏ



Tunnelling

Metal: Electrons fill states upto the Fermi energy, EF. The work function Φ describesthe energy an electron at EF

needs in order to leave themetal

In the case of STM, the tip isbrought close to the sample. The wavefunction of theelectrons in the tip andsample overlap. Tunnellingdoes not occur at zero biasdue to absence of availablestates.

Applying a –ve bias tosample fills states in sample. Electrons in filled statestunnel thought the barrier toempty states of the tip. A net tunnel current ismeasured.

The measured current at the tip position is a measure of the local density of states (LDOS)



Tunnelling

The exponential relation leads to extreme sensitivity tochanges in the distance between the STM tip and the surface.

Back of envelope calculation:

Typical work function value for metal: 4 eVTypical atomic diameter: 3 Å

Change in d of this distance corresponds to factor1000 change in It.

High sensitivity to d means STM signal isdominated by the last atom of the tip.

𝐼 𝑑 = 𝐴𝑉𝑒−2𝜅𝑑 𝜅 =2𝑚𝜙

ℏ



Scanning

Signal measured is dominated by last atom of tip.

The tip is scanned over the surface to produce an STM image

In order to resolve atomic scale features of the surface it isimportant to be able to move the STM tip with subangstrom resolution.

Sub-Ångström control is achieved using piezoelectricelements.

Piezoelectric effect: reversible change in physicaldimensions of a meterial in response to an externallyapplied electric field.

Δ𝑙 = 𝑐𝑙

ℎ𝑈

lΔl – change in lengthc – piezoelectric coefficientl – lengthh – wall thiknessU – applied voltage

Typical resolution: 0.1 Å laterally; 0.01 Å vertically



Two main modes of scanning

Constant Current• A tunnel current is set• Tip moves over features of surface• Feedback loop responds quickly to

adjust the height of the tip• Tunnel current is maintained at

same value• Height of tip gives signal• Most common

Constant Height• Only x,y piezos used to scan• Z-position of tip is unchanged• Due to corrugation of surface,

tunnel gap changes• Leads to variation in current• Current gives signal• Can lead to tip crashes

https://www.youtube.com/watch?v=LDKnhfdBj3Y



Tip preparation

Electrochemical etching

Chemical reaction at the tip slowly depletes the metal of the tipTypically tungstenOften covered in layer of insulating oxide! Cleaning required



Tip preparation

Wire cutting

Much more simple, brutal methodWire (Pt80/Ir20) is stretched and cut with sharp tool

Leads to much poorer definition of apex

For most purposes irrelevant as the STM signal isdependent only on the last atom



Tip preparation

In situ tip forming is done toimprove the tip apex

STM tip is crashed into themetal surface

Slowly drawn out

In best case resulting in single atom apex

As tip is drawn out of surfacequantised level in theconductance can be seen

Aspect ratio of tip can beimportant for getting a sharp image.



Example images

Constant current STM image of Au(111) surfaceshowing step edge, herringbone reconstruction, and atomic resolution

Typical „multi-tip“ STM image

Si(111)-(7x7) reconstruction



Sample preparation - sputtering

Sputter yield – number of surfaceatoms ejected per incoming ion

On silicon

High energy ions impact a surface.

Series of collisions occurs in the top layers of atoms

Can lead to many processes(embedding, adsorption, disclocations, e- and M+ ejection)



Sample preparation - sputtering

Au(111) surface after sputtering at 400 K with 1 keV Ne+ ions

Aprroximately 10 layers removed



Sample preparation - Annealing

After sputtering where the top layers of the surface are removed, the surface is heated to inducethermal healing.

This induces mass transport on the surface (diffusion of atoms)

In order to acheive lowest energy large flat terraces are formed

Above: Ag(001) surface after sputtering at 180 K, 380 K, and 450 K (1 keV Ne+, 1200 s)



Sample preparation – Molecular deposition

Sublimation from filament heated crucible:

1. Electrical contacts for heating filament2. Thermocouple contacts3. CF flange for attachement to UHV system4. Molecules to be sublimed5. Glass tube with filament wrapped around it.

Low T

High T



Sample preparation – Molecular deposition

Electrospray from solvent

1. Molecules or nanoparticles dissolved/suspended in solution2. Solution injected from fine capillary3. High voltage applied to accellerate4. Uniform solvated droplets formed5. Differential pumping stages leads to evaporation of solvent6. Intact molecule/nanoparticles arrive at surface and adsorb Electrospray deposited unfolded CytC in

different charge states



Atomic/molecular manipulation

STM not only useful for imaging structures at the atomic scale. The STM tip can be used to maniulatesingle atoms/molecules




Van der Waals, chemical forces, or Pauli repulsion between tip and adsorbate.

Pulling (attractive),

Pushing (repulsive),

Sliding

https://www.youtube.com/watch?v=oSCX78-8-q0

Eigler, D. M. & Schweizer, E. K. Nature 344, 524–526 (1990)


https://www.youtube.com/watch?v=oSCX78-8-q0



Tunnelling electrons can inelastically interact with molecules to cause chemical reactions on surfaces

Cis-/trans- isomerisation of azobenzeneDissociation of oxygen molecules

Phys. Rev. Lett. 78, 4410-4413 (1997)




Bias between tip and sample means that a local electric field is present. Can be used to manipulatemolecules

Increasing d in the limit ofzero tunnel current

A161005.152354.dat

A161005.152354.dat Ch: 1


Current: 2.7E-13A


0 1 2 3 40

1

2

3

4

-0.1124 nm

-0.06237 nm

-0.01237 nm

0.03763 nm

0.08763 nm

Rotation of dipolar nanocar






A161005.152141.dat

A161005.152141.dat Ch: 1


Current: 2.7E-13A


0 1 2 3 40

1

2

3

4

-0.1124 nm

-0.06237 nm

-0.01237 nm

0.03763 nm

0.08763 nm






A161005.151944.dat

A161005.151944.dat Ch: 1


Current: 2.7E-13A


0 1 2 3 40

1

2

3

4

-0.1124 nm

-0.06237 nm

-0.01237 nm

0.03763 nm

0.08763 nm






A161005.151756.dat

A161005.151756.dat Ch: 1


Current: 2.7E-13A


0 1 2 3 40

1

2

3

4

-0.1124 nm

-0.06237 nm

-0.01237 nm

0.03763 nm

0.08763 nm






A161005.150850.dat

A161005.150850.dat Ch: 1


Current: 2.7E-13A


0 1 2 3 40

1

2

3

4

-0.1124 nm

-0.06237 nm

-0.01237 nm

0.03763 nm

0.08763 nm






A161005.150651.dat

A161005.150651.dat Ch: 1


Current: 2.7E-13A


0 1 2 3 40

1

2

3

4

-0.1124 nm

-0.06237 nm

-0.01237 nm

0.03763 nm

0.08763 nm



Scanning tunnelling spectroscopy

We know that the tunnel current in STM is a function of the local density of states of both thesample and the tip:

𝐼 ∝ න0

𝑒𝑉

𝜌𝑠 𝐸𝐹 − 𝑒𝑉 + 𝜖 𝜌𝑇 𝐸𝐹 + 𝜖 𝑑𝜖

𝑑𝐼

𝑑𝑉∝ 𝜌𝑠 𝐸𝐹 − 𝑒𝑉

surface tip

We are generally interested in the states of the surface only. However, above equation implies a convolution between surface and tip states. Further, tunelling transmission probability (not shownabove) depends in unknown manner on V.

Approximation: Assume that transmission probabiliy is constant; assume a smooth density ofstates of tip.

Integrated density of states

Density of states



Scanning tunnelling spectroscopySpectrum is acquired by ramping the biasand measuring the tunnel current

Feedback control is deactivated

Changes in the LDOS of the surface leadto features in I/V curve

More useful to inspect the differential conductance dI/dV and function of V

Corresponds to LDOS of sample (approximately – care must be takenupon interpretation)

Polarity of bias allows access tooccupied/unoccupied states

dI/dV can be obtained by numericaldifferentiation of I/V or by lock-in techniques.




Filled state (negative bias) andempty state (positive bias) images of Si(001)-(2x1) andmodel depicting shape of π andπ* orbitals

dI/dV spectra of the Si(111)-(7x7) surface.Differences observed when tunnel gap is changedShows that spectra affected by charge transportbetween surface and sample holder.

Physical review. B, Condensed matter 80(12)




STS can also be used to investigate theelectronic structure of molecules

Adsorption on NaCl bilayer decouples themolecule from the metallic surface

dI/dV spectrum then shows clearly theHOMO and LUMO

Resonant tunnelling at these resonancesallows one to image the MOs.

Science Vol. 317, Issue 5842, pp. 1203-1206




Further example of molecular orbital imaging – high temporal resolution.

Teraherz waveform excites only the HOMO and opens transient tunnel channel. Single electronsremoved from this molecular orbital and detected as shift in tunnel current.

Result is 100 fs snapshot of HOMO

Nature 539, 263 (2016)




Removal of electron from HOMO causes vertical vibration of molecule. Second time-delayed pulse canmeasure instantaneous height of molecule.

Nature 539, 263 (2016)




Also possible to access informationabout vibrational modes of adsorbedmolecules.

Inelastic tunnelling of electrons canexcite vibrational levels

Provides a small (~1%) increase in tunnelling current due to extra channel opening up

The second derivative shows peaks in the position corresponding to theenergy of the vibrations.

Requires high-stability and lowtemperature to ensure moleculesoccupy vibrational ground state


Science 280, 1732



Vibrational modes of single adsorbed ethene molecule on Cu(100)



Single molecule switching

Intramolecular hydrogen tautomerisation reaction observed in naphthalocyanine

Charge redistribution upon proton transfer leads to apparent rotation of LUMO

Changes are detected in tunnel current Science Vol. 317, Issue 5842, pp. 1203-1206



Single molecule chemistry

Tip-induced formation of biphenyl from iodobenzene on Cu(111)

Phys. Rev. Lett. 85, 2777



Single molecule chemistry

Thermally induced heirarchical growthof ordered 1D and 2D networks on Au(111)

Nature Chemistry 4, 215–220 (2012)


4.5 Atomic Force Microscopy

AFM

A sharp tip is mounted on the end of a cantilever.Depending on the mode of operation, the cantilever can be oscillated using the piezo driver.The force of attraction/repulsion between surface and tip modifies deflection of the cantilever or theoscillation frequency.This deflection is recorded by a photodetectorX,Y,Z piezos allow the tip to be scanned relative to the surface



Lennard Jones potential

Repulsive: Very small tip-sample distances

Exchange interactions due to overlapof electronic orbitals (Pauli repulsion)

Attractive:Instantaneous polarisation of atomsinduces polarisation of nearby atoms(van der Waals)

Other attractive forces can play large role (electrostatic – H-bonding, dipoleinteraction)

Results in attractive interaction



Contact Mode AFM

Replusive forces

Tip is in contact with the surface

The tip follows the topography of thesurface

As topography changes, the cantilever isdeflected

High resolution on hard surfaces (eg, metals)

Soft samples can be deformed due to high forces



Non-conact Mode AFM

Attractive forces

Tip is not in contact with the surface

The cantilever is oscillated above thesample

Two modes: frequency modulation, amplitude modulation

Frequeny modulation – feedback loopused to regulate amplitude and change in frequency of cantilever due to attractiveforces is measured

Amplitude modulation - Cantilever isdriven with a set frequency and theamplitude is measured.

Useful for biological samples



Tapping Mode AFM

Amplitude of oscillations is measured astip scans over the surface

Less destructive than contact mode

The tip only contacts the surface at a seriesof points over the surface

No dragging or tip-wear



Amplitude modulation

Sensor is excited at Fdrive, just off ofresonance frequency.

As tip scans over surface, resonancefrequecy changes depending on forcesbetween sample and tip.

Chance in amplitude of oscillation givesa measure of force.

Can be operated both with feedback control (tip height is adjusted in order to maintain A = Aset)Or in constant height mode.

Major disadvantage: in vacuum scan speed is very low due to use of high quality factor (Q) sensors and the long time for the oscillation to reach steady state after a change in Fres

-rather used in air or liquid



Frequency modulation

Damped oscillator:

The cantilever is driven with frequency ω

Energy exchange is maximised when drivingsignal has phase difference of 90°

Signal collected from deflection of cantileverand signal is adjusted to maintain oscillation ofcantilever at resonance



qPlus sensor Quartz tuning fork with immobilised upperprong.

Metal tip attached to lower prong

𝐹 = 𝑘𝑥

Hooke‘s lawF – forceK – spring constantX - displacement

Conventional cantilever has typical k = 10 N/m

To prevent „snap-to-contact“ restoring force must belarger than 100 nN

For cantilever – 10nm oscillation aplitude

k for qPlus is 1000- 10000 N/m – very stifftherefore very low aplitude oscillation



qPlus sensor



Kolibri sensor



Sub-molecular resolution

Pentacene on Cu(111) imaged both with STM and AFM with CO functionalised tip

AFM images using different tip modifications

A Ag tip;B CO tipC Cl tipD pentacene tip

CO tip gives tures image of molecular structure




The contrast depends stronglyon the tip-sample distance andhence the magnitude of theforces acting on the tip.

Featureless depression at high distance (attractive interaction) – van der Waals background

At very small tip-sample distances Pauli repulstionbecomes dominant

Areas of high electron density(i.e. chemical bonds) showhigher force than in the middleof carbon rings

Leads to bond imaging




Bond length and hence bond ordercan be qualitatively measured usingCO-tip NC-AFM



CO tip

2 CO molecules on Cu(111) surface

Picked up by retracting 0.3 Å and applying 2.5 V until sudden change in tunnel current is seen

Reimaging shows one CO has been picked up

Modified tip gives different contrast of remaining CO molecule


4. Microscopy - static.uni-graz.at

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