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Atomic Force Microscopy

Robyn Snow

2https://www.pinterest.com/lictorn/gary-larson-the-far-side

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Brief History of Microscopes

• 1590’s – Zacharias Jansen and father

• Several lenses in a tube– Enlarged objects only ~9X

http://www.history-of-the-microscope.org/history-of-the-microscope-who-invented-the-microscope.php

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Brief History of Microscopes• Anton van Leeuwenhoek (1632-1723)

– 1st to make a “real” microscope– 1st to see and describe bacteria, yeast plants…– Developed superior lenses

• 270X magnifying power

• Robert Hooke (1635-1703)– Verified van Leeuwenhoek’s work– Published Micrographia, 1665

• Observed pores in cork, called them “cells”

– Hooke’s Law IMAGE: http://www.nlm.nih.gov/exhibition/hooke/hookesbooks.html

Brief History of Microscopes• Nobel Prize in Physics, 1986

– Ernst Ruska• German physicist

– Fundamental work in electron optics– Designed 1st electron microscope

– Gerd Binnig , Heinrich Rohrer• IBM Zurich Research Lab

– Design of scanning tunneling microscope

5http://ernst.ruska.de/daten_e/library/documents/999.nobellecture/lecture.html

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General Overview of AFM

• Surface analysis technique– Surface topography– Elasticity– Friction– Magnetic forces– Electrostatic forces

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General Operation

– Small probe is scanned across surface – Data from interaction with surface is stored for

each point– Image is displayed as an intensity map, I(x,y) ; I =

parameter sensed by the probe• For AFM, the height of the surface is sensed• LFM, friction• MFM, magnetic fields

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Interaction force between probe and sample

Deflection of cantilever

Changes of the laser signal to the photosensitive position detector

Electric signal

Signal processing to generate image

Change of the surface properties along the scan line

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Atomic Force Microscopy

• Obtain image at or near atomic resolution

• Advantages:– Minimal to no surface prep– Non-destructive imaging– Atmospheric conditions– Sample not required to be conductive

• Polymers, ceramics, glass• Metals• Biological samples

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Surface Preparation-Depends on sample, generally:1. Clean substrate2. Sample must be adhered to the surface of

substrate– Mica, glass, gold– HOPG (highly ordered pyrolitic graphite)

3. Rigidly mount sample to the stage– No vibration

• If contamination layer present, use contact mode or UHV

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Resolution

Vertical Resolution:• Laser intensity noise• Photodiodes noise• Thermal noise of

cantilever• Vertical scanner resolution

<1Å(0.1nm)• Noise: electrical,

mechanical, acoustic

Lateral Resolution

• Lateral resolution ~30nm• Vertical resolution ~0.1nm• Limited to scan areas of 100μm

• Tip sharpness / shape• Scanner resolution in X,Y• Pixelization

– EX: 50μmX 50μm image– Samples/line @512 – Pixel size=0.098 μm =>cannot resolve features smaller than 98nm with 50μm scan size

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Gold Nanoparticles

http://www.afmworkshop.com/products-main/image-gallery.html#!nanotriangle_pm

http://www.afmworkshop.com/products-main/image-gallery.html#!gold_nanoparticles_100_nm_pm

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Diagram of Instrument

Probe

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Probe• Probe: Si or Si3N4

• Only part that contacts sample– Like the “eye” of the instrument– > shape is critical! -> Resolution depends on it

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Probe Tip• Conical Probe

• more preferable• higher resolution image

• Pyramidal Probe• can see distortion of image

• High Radius of Curvature

Artifact seen in image

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Fat-tip Effect: apparent width measured by large tip

x

w

Rtip

RSample

•When Rtip ~ ¼ Rsample, measured width = 2Rsample•Normal tip size, ~ 20 nm or larger.

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Si3N4 Tip Diamond Coated tip

SEM images of probes

Atomic Force Microscopy: Theory, Practice , ApplicationsPaul E. West, Ph.D.

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Conical Tip and Cantilever

Silicon Cantilever Length (µm): 225

Res. Frequency (kHz): 28Spring Constant (N/m): 0.1

Tip Radius ~8nmhttp://store.nanoscience.com/store/pc/viewPrd.asp?idproduct=2556&idcategory=4

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Cantilever and Probe

• Spring system/Force Sensor• Bends in presence of attractive/repulsive

forces• Cantilever deflection converted to force using:

– Hooke’s Law!

s : deflectionk : spring constant

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Hooke’s Law

IMAGE FROM : http://www.physics.usyd.edu.au/teach_res/jp/waves/hwaves1001.htm

Fe : Restorative force due to springF : force due to samples : springs displacement from equilibrium

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Ideal Spring System• Max deflection for given force

– Spring as soft as possible (small k)

• Minimize interference due to vibrations of building (~100Hz)– Stiff spring with high natural frequency()

Natural Frequency of spring(:

To achieve high frequency and max deflection:↓k and ↓m0

k = spring constantm0 = mass of spring

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Spring Constant, k

• For a rectangular cantilever:

𝑡

𝑙

𝑤

• E = Young’s modulus• Measure of stiffness • For Si, E = 1.3*1011N/m2

𝑘=𝐸𝑤𝑡3

4 𝑙3

• Typical values for k : 0.001 to 100 N/m• ~100microns in length , a few microns thick

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Piezoelectric Scanner• Piezoelectric material

– Typically (PZT) lead zirconate titanate (Pb(ZrTi)O3)

• Piezoelectric effect– Expand or contract in presence of potential

difference– Develop potential in response to mechanical pressure

• Allows for ability to precisely manipulate movement of sample or probe

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Piezoelectricity

http://www.ytca.com/lead_free_piezoelectric_ceramics

• Polarization in one direction occurs due to applied electric field– Change in length field strength

• ~0.1nm per applied Volt

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Light Lever Sensor• Force Sensor

– Detects changes in height/ deflection based on angle of reflected light

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Photodetector

A B

D C

• Photosensitive elements (photodiodes)• Photocurrent is produced upon

illumination for each quadrant

• The ratio between the photocurrent from each quadrant determines the relative position of the laser beam

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Probe-Surface Interaction• Interaction between

probe tip and surface =>Atomic Forces– Creates potential

energy(PE)• + PE : repulsive

forces , atoms very close

• - PE : attractive forces, van der waals, atoms further away

Attractive forces- takes minimal energy to bring atoms closer together at distance

Equilibrium : distance when potential energy minimized

Repulsive forces- very small r, takes a lot of energy to bring atoms closer together->repulsive forces dominant

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Lennard-Jones Potential (LJP)

• Empirical Model, describes potential energy (V) of interaction between outermost atom of tip and surface atoms

: well depth- measure of strength of attraction : distance at which V is zero (equilibrium)r : distance of separation

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n

http://chemwiki.ucdavis.edu/@api/deki/files/8914/Figure_B.jpg

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Van der Waals Forces𝑉 (𝑟 )=4∈[(𝜎𝑟 )

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−( 𝜎𝑟 )6](𝜎𝑟 )

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(𝜎𝑟 )6

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Modes of Imaging

Repulsive

Attractive

Tapping Mode

Non-Contact ModeContact Mode

http://virtual.itg.uiuc.edu/training/AFM_tutorial/

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Contact Mode• Tip is in very close contact with surface <0.5nm

-> Repulsive forces– Cantilever bends

s : displacement of cantilever ->height/Force measure– Force varies dramatically based on distance

between tip and sample• Two types of contact mode:

1. Constant Height2. Constant Force

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Constant Height Contact Mode

• Maintain constant height of sampler– Variations of deflection of lever are recorded as

topography• Deflection ↑ as height of sample ↑• Deflection ↓ as height of sample ↓

• Advantages– Higher scanning speeds– High resolution

Fig. 1 STM and AFM imaging of pentacene on Cu(111).

Leo Gross et al. Science 2009;325:1110-1114

Published by AAAS

A: Ball-and-stick model B: Constant current STM

C,D : Constant height AFM images

http://www.sciencemag.org/content/325/5944/1110.full

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Constant Force Contact Mode

• Maintain constant force between tip and sample – Regulate height of sample relative to the tip

• Feedback loop : photodetector and piezoelectric scanner

– Height of sample ↑ , Force ↑, lower height of sample to maintain constant force

• Slower scan speeds• Advantage:

– Can simultaneously measure other characteristics/forces

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Lateral Force Microscopy• In constant force mode:

– Scan perpendicular to longitudinal axis of cantilever– Measures surface friction – Friction-force map

• Four quadrant photodiode detector– Difference between

left and right segments friction

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Lateral Deflection

• Magnitude depends on:

– Frictional coefficient of the sample

– Topography of sample surface

– Cantilevers lateral spring constant

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Lateral Force Spring Constant• Lateral spring constant:

G : shear modulus-measure of elasticityr : length of tip

∆s

Force

𝒘

𝒕

𝒍

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Lateral Force Calibration

• Calibration of :– normal and lateral forces, F– Photodiode sensitivity, S

• Using reference sample• Normal Force• Lateral Force• lateral calibration factor

– Transforms lateral ΔV ->Friction Force (nN)

41http://www.parkafm.com/images/spmmodes/standard/Lateral-Force-Microscopy-(LFM).pdf

a) AFM image of topography b) LFM image

Human Hair Image : AFM vs LFM

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LFM Image

Topography LFM image

2μm x 2 μm of Nickel CD stamper matrixT.Göddenhenrich, S.Müller and C.Heiden, Rev. Sci. Instrum. 65, (1994) 2870

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Contact Mode: pros and cons

Advantages• High resolution >50nm• Fastest• No problem with

surface pollution– Can image in air or liquid

Disadvantages• High contact pressure

– Can damage/ not analyze soft samples

• Probe and sample experience lateral forces

• Lateral resolution limited by tip sharpness

• Lowers lifetime of tip

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Non-contact Mode• Lever(spring) oscillates close to its resonance

frequency from driving piezo– => Use z-piezo to vibrate the cantilever near its

resonant frequency

• Forces shift this oscillation

tip-sample distance of ~5-10nm

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Z-drive Piezo for Non-contact AFM

• Informs feedback loop of motion of tip/cantilever:• Frequency• Amplitude

• Allows for frequency modulation(FM-AFM) or amplitude modulation (AM-AFM/tapping mode)

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Frequency Modulation

• Excitation Amplitude constant• Tip-sample interaction-> frequency ↓

• Attractive forces

∆fNatural frequency, no interaction

Tip-sample interaction-> natural frequency shift

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Frequency Modulation• ∆f : info about tip-sample interaction• Feedback loop : adjusts tip-sample distance to

achieve constant Amplitude

48http://www.sciencemag.org/content/337/6100/1326.figures-only

Non-contact AFM of C60A : modelB to E : AFM showing Δf at differing tip heightsF : image used for measure of bond length• measured bond

lengths are Lh = 1.38Å

• Lp = 1.454 Å

STM image

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"C60a" by Original uploader was Mstroeck at en.wikipedia Later versions were uploaded by Bryn C at en.wikipedia. - Originally from en.wikipedia; description page is/was here.. Licensed under CC BY-SA 3.0 via Wikimedia Commons - http://commons.wikimedia.org/wiki/File:C60a.png#/media/File:C60a.png

“p”

“h”

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Amplitude Modulation/ Tapping Mode

• Cantilever excited to resonance frequency• Tip-sample distance ↓ , amplitude (A)↓• A reaches set point, below resonance A ->

height is measured• Feedback loop adjusts height to maintain set A

as sample is scanned

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Set-point

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Tapping mode pros and cons

Advantages• Reduced forces on

surface – Good for soft materials

• No friction forces– Can use sharper tips

• Can be in air or liquid• Improved lateral

resolution– ~5nm

Disadvantages• Slower than contact

mode– Up to20 minutes per

scan

• Tip is damaged after several scans

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Tapping mode image

http://www.veeco.com/pdfs/database_pdfs/B54_Rev_A1_Caliber_300.pdf

2.5μm scan sizeDNA deposited on mica

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High Resolution Image of muscovite mica in water

http://www.beilstein-journals.org/bjnano/single/articleFullText.htm?publicId=2190-4286-4-15

9.7ÅBlue : OxygenGreen : SiliconUnit Cell: 5.199Å

High-resolution dynamic atomic force microscopy in liquids with different feedback architecturesJohn Melcher, David Martínez-Martín, Miriam Jaafar, Julio Gómez-Herrero, Arvind Raman

Beilstein J. Nanotechnol. 2013, 4, 153–163.

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Force Modulation Microscopy(FMM)

• Tip in contact with the sample• z feedback loop maintains a constant

cantilever deflection• A periodic vertical oscillation signal is applied

to either the tip or the sample. • The amplitude varies according to the elastic

properties of the sample. • the system generates a force modulation

image --- a map of the sample's elastic properties

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FMM

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FMM vs AFM

Carbon fiber/polymer Composite Collected Simultaneously (5μm)

FMM gives more detailed information about the composition and distribution of the two components --- soft polymer (dark area) and hard carbon fiber.

FMM AFM

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2-phase block copolymer

AFM FMM

The softer, more compliant component of the polymer maps in black. 900nm scans. Veeco.

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Magnetic Force Microscopy (MFM)

• Tip is coated with a ferromagnetic film(Ni, Fe, Co)

• Scanned in non contact mode• Provides high resolution image of magnetic

patterns• Strength of local magnetic interaction

determines the vertical motion of the tip• Detectable magnetic field ~0.1 gauss (10

microteslas)

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Magnetic Force• Described as:

: magnetic permeability of free space (4π 10-7

WbA-1m-1)

: magnetic moment of the tip:strength of magnetic field from sample

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MFM Limitations

• Type of tip and magnetic coating affect image• Interaction of magnetic field of tip and sample

can alter each others field• Highly dependent on scan height• Inner and surface magnetic charges not able

to be deconstructed

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MFM Image

magnetic disk; 40 µm x 40 µm

http://www.afmworkshop.com/products-main/image-gallery.html#!

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MFM Image

http://commons.wikimedia.org/wiki/File:MFM_AFM_JANUSZ_REBIS_INFOCENTRE_PL_HDD_MAGNETIC_MEMORY_EVOLUTION.png#/media/File:MFM_AFM_JANUSZ_REBIS_INFOCENTRE_PL_HDD_MAGNETIC_MEMORY_EVOLUTION.png

3.2GB hard drive 30GB hard drive

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Electric Force Microscopy(EFM)

• Measures electric field gradient distribution on sample surface

• Tip : electrically conducting coating • applied Voltage between tip and sample• Deflection of cantilever charge density of

sample• Use photo-diode detector

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EFM

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Applications of EFM

• characterizing surface electrical properties• Interfacial charge transport and separation for

organic/electrode devices (conducting polymer, organic semiconductors, etc.)

• detecting defects of an integrated circuit (silicon surface)

• measuring the distribution of a particular material on a composite surface.

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Bio Applications

• Imaging• Ligand-receptor binding sites• Antibody-antigen binding sites• Proteins-folding/unfolding• Structural analysis – SMRFM• *need special surface preparation for bio samples

– Absorb sample onto a supported cationic bilayer (mica) surface and imaged with AFM in aqueous buffers

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DNA on multiple mica layers

2 μm X 2 μm

http://www.afmworkshop.com/products-main/image-gallery.html#!AFM_scan_DNA_mica_pm

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Red Blood Cells, 30μm X 30μm

http://www.afmworkshop.com/products-main/image-gallery.html#!07_img_blood_cells_01_big

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Single-Molecule Recognition Force Microscopy (SMRFM)

• Couple a “ligand” molecule to the tip– Thin PEG chain

• Ligand recognizes complementary receptor site in sample– Causes deflection of cantilever / change in

oscillation frequency => maps recognition sites

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SMRFM

http://www.jku.at/biophysics/content/e54633/e54706/e54710/#fig1

1. NH2 on tip reacts with NHS ester of PEG linker2. Protein attached to free end of PEG (amine-aldehyde linkage)

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How cell membranes respond to their environment

http://www.innovations-report.com/html/reports/life-sciences/report-47236.html

Simon Scheuring , Thomas Boudier , James N. Sturgis

AFM Image

Light harvesting Complex

Reaction centers

• Membrane organization in photosynthetic bacteria

• –Rsp. Photometricum• (exposed to strong

light)

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Questions

• THANK YOU!!