Pooria GillPhD of Nanobiotechnology

Assistant Professor at MAZUMSP.Gill@mazums.ac.ir

In The Name of Allah

Outline Motivation History How the AFM works

Two modes Contact Mode Non-Contact Mode Force Measurements Raster the Tip: Generating an Image Scanning Sample

OUr AFM Pictures

Examples The Good The Bad And the Ugly

Uses Topographical Analysis Thin Layer Depth RMS Roughness Calculations

Other types of Microscopy

Other Types of SPM Techniques Lateral Force Microscopy (LFM)

Frictional forces measured by twisting or “sideways” forces on cantilever. Magnetic Force Microscopy (MFM)

Magnetic tip detects magnetic fields/measures magnetic properties of the sample.

Electrostatic Force Microscopy (EFM) Electrically charged Pt tip detects electric fields/measures dielectric and

electrostatic properties of the sample Chemical Force Microscopy (CFM)

Chemically functionalized tip can interact with molecules on the surface –giving info on bond strengths, etc.

Near Field Scanning Optical Microscopy (NSOM) Optical technique in which a very small aperture is scanned very close to

sample Probe is a quartz fiber pulled to a sharp point and coated with aluminum to

give a sub-wavelength aperture (~100 nm)

Motivation Digitally image a topographical surface Determine the roughness of a surface sample or to

measure the thickness of a crystal growth layer Image non-conducting surfaces such as proteins and

DNA Study the dynamic behavior of living and fixed cells

History The Scanning Tunneling Microscope (STM) was

invented by G. Binnig and H. Rohrer, for which they were awarded the Nobel Prize in 1984

A few years later, the first Atomic Force Microscope (AFM) was developed by G. Binnig, Ch. Gerber, and C. Quate at Stanford University by gluing a tiny shard of diamond onto one end of a tiny strip of gold foil

Currently AFM is the most common form of scanning probe microscopy

Parts of AFM 1. Laser – deflected off cantilever 2. Mirror –reflects laser beam to

photodetector 3. Photodetector –dual element

photodiode that measures differences in light intensity and converts to voltage

4. Amplifier 5. Register 6. Sample 7. Probe –tip that scans sample

made of Si 8. Cantilever –moves as scanned

over sample and deflects laser beam

How the AFM Works The AFM brings a probe in

close proximity to the surface

The force is detected by the deflection of a spring, usually a cantilever (diving board)

Forces between the probe tip and the sample are sensed to control the distance between the the tip and the sample.

van der Waals force curve

Two ModesRepulsive (contact) At short probe-sample distances,

the forces are repulsiveAttractive Force (non-contact) At large probe-sample distances,

the forces are attractiveThe AFM cantelever can be used to

measure both attractive force mode and repulsive forces.

3 Modes of AFMContact ModeNon-Contact ModeTapping (Intermittent

contact) Mode

Contact Mode Measures repulsion between tip and sample Force of tip against sample remains constant Feedback regulation keeps cantilever deflection

constant Voltage required indicates height of sample Problems: excessive tracking forces applied by probe

to sample

Non-Contact Mode Measures attractive forces between tip and sample Tip doesn’t touch sample Van der Waals forces between tip and sample

detected Problems: Can’t use with samples in fluid Used to analyze semiconductors Doesn’t degrade or interfere with sample- better for

soft samples

Tapping (Intermittent-Contact) Mode

Tip vertically oscillates between contacting sample surface and lifting of at frequency of 50,000 to 500,000 cycles/sec.

Oscillation amplitude reduced as probe contacts surface due to loss of energy caused by tip contacting surface

Advantages: overcomes problems associated with friction, adhesion, electrostatic forces

More effective for larger scan sizes

Force Measurement The cantilever is designed with a

very low spring constant (easy to bend) so it is very sensitive to force.

The laser is focused to reflect off the cantilever and onto the sensor

The position of the beam in the sensor measures the deflection of the cantilever and in turn the force between the tip and the sample.

Raster the Tip: Generating an Image The tip passes back and forth in a

straight line across the sample (think old typewriter or CRT)

In the typical imaging mode, the tip-sample force is held constant by adjusting the vertical position of the tip (feedback).

A topographic image is built up by the computer by recording the vertical position as the tip is rastered across the sample.

Scan

ning

Tip

Rast

er M

otio

n

Top Image Courtesy of Nanodevices, Inc. (www.nanodevices.com)

Bottom Image Courtesy of Stefanie Roes(www.fz-borstel.de/biophysik/ de/methods/afm.html)

Constant-force scan vs.constant-height scanConstant-force Advantages:

Large vertical range Constant force (can be

optimized to the minimum)

Disadvantages: Requires feedback

control Slow response

Constant-height Advantages:

Simple structure (no feedback control)

Fast response Disadvantages:

Limited vertical range (cantilever bending and detector dynamic range)

Varied force

Scanning the Sample Tip brought within nanometers of

the sample (van der Waals)• Radius of tip limits the accuracy of

analysis/ resolution• Stiffer cantilevers protect against

sample damage because they deflect less in response to a small force • This means a more sensitive

detection scheme is needed• measure change in resonance

frequency and amplitude of oscillation

Image courtesy of (www.pacificnanotech.com)

General Applications Materials Investigated: Thin and thick film coatings,

ceramics, composites, glasses, synthetic and biological membranes, metals, polymers, and semiconductors.

Used to study phenomena of: Abrasion, adhesion, cleaning, corrosion, etching, friction, lubricating, plating, and polishing.

AFM can image surface of material in atomic resolution and also measure force at the nano-Newton scale.

What are the limitations of AFM?

AFM imaging is not ideally sharp

Advantages and Disadvantages of AFM

Easy sample preparation Accurate height

information Works in vacuum, air, and

liquids Living systems can be

studied

Limited vertical range Limited magnification

range Data not independent of

tip Tip or sample can be

damaged

A Better View

Now:

• Removed extreme points

• Digitally decreased the height of analysis

• Less than 1/3 as high as initial scan

•Lose resolution and data by clipping off extreme points

Thickness of a Thin Layerof Pd on Si WaferSi/Pd step

Step (where Pd coating ends)

Systematic error

Surface Roughness

Roughness typically measured as root mean squared (RMS)

Carbon Nanotube Tips• Well defined shape and composition.• High aspect ratio and small radius of curvature (“perfect” tip would be a delta

function tip).• Mechanically robust.• Chemical functionalization at tip.

DNACNT Tips

Images taken from Nanodevices, Inc. (www.nanodevices.com)and Wooley, et al., Nature Biotech. 18, 760

• STM can move atoms around on a surface.

SPM Lithography

Iron on Copper Iron on Copper

Eigler, et al. from IBM

• Dip Pen Lithography.SPM Lithography

Mirkin, et al. from Northwestern University

Million Cantilever Wafer

Millipede Memory

Cantilever Gas Sensors (Noses)

Cantilever Gas Sensors (Noses)

AFM Tips

80 – 320 µm

20 µm

35 µm

125 µm

Intermolecular interactions

Schematic of the force–extension characteristics of DNA: at 65 pN the molecule is overstretched to about 1.7 times its contour length, at 150 pN the double strand is separated into two single strands, one of which remains attached between tip and surface.

MFP

MFP is specially designed for force measurementpurpose

Adhesion Force ImagingHeight Adhesion

0102030

0 4 8 12

Hei

ght (

nm)

0.00.30.6

0 4 8 12

Ahes

ion

(V)

Albumin

Albumin

Polystyrene

Si

PS

pH 7

5 µm

Adhesion and Hardness Imaging

PLMA/PmMl6 blend on Si imaged in waterPLMA: poly (lauryl methacrylate)PmMl6: 2-methacryloyloxyethyl phosphorylcholine-co-lauryl methacrylate (1:6)

1 µm

Height Adhesion Stiffness

Simultaneous Height, Adhesion and Stiffness maps are obtainedwith “Pulsed-Force” AFM technique.

Conclusions How AFM works

Constant-height and constant-force scans (contact mode) Feedback control in constant-force mode Contact mode and tapping mode

Force measurements with AFM Force curves: contact part to measure hardness and adhesion to

measure intermolecular interactions Calibrations:

Detector sensitivity (nm/V) = Inverse of contact slope on a hard surface => Convert the measured T-B signal (V) to cantilever deflection (nm)

Spring constant (N/m) => Convert the cantilever deflection to force (N) [F=-kx]

Pooria GillPhD of Nanobiotechnology

Assistant Professor at MAZUMSP.Gill@mazums.ac.ir

In The Name of Allah

AFM : Contact Mode

Feedback Error:Deflection

Output:“Isoforce” Height

http://www.physik3.gwdg.de/~radmacher/publications/osteobla

AFM : Tapping Mode

Feedback Error:Amplitude

Output:“Isoamplitude” Height

Evaporated gold surface

Additional Feedback:Phase

(http://www.energosystems.ru/fgallery.htm)

SAMPLE REFERENCEgraphite Binnig, et al., Europhys. Lett. 3, 1281 (1987)

molybdenum sulfideboron nitride

Albrecht, et al., J. Vac. Sci. Tech. A 6 271 (1988)

goldsodium chloride (001)

lithium flouride

Manne, et al., Appl. Phys. Lett. 56 1758 (1990)Meyer, et al., Appl. Phys. Lett. 56 2100 (1990)

Meyer, et al., Z. Phys.B. 79 3 (1990)(1014) cleavage plane of a

calcite (CaCO3) crystalOhnesorge, et al., Science 260 1451 (1993)

Highly Oriented Pyrolytic Graphite (HOPG)

http://stm2.nrl.navy.mil/how-afm/how-afm.htmlhttp://www.physics.sfasu.edu/afm/afm.htm

LAYERED HARD CRYSTALLINE SOLID MATERIALS

AFM : First high resolution images

INTERVAL Au COATING :

homogeneous, smoother smaller

polydomain microstructure

Si chip

Si3N4cantilever

TOP VIEW

AFM: Tip Functionalization

ONE-TIME Au COATING :

heterogeneous, rougher larger

polydomain microstructure

1. Gold coatingPurpose:

Methods:

100 nm SIDE VIEW

100 nm SIDE VIEW

TOP VIEW

microfabricated Si3N4 probe tip

--

--

-

---

--

--

---

-

-+

+

+

+

++

synthetic polymers

polyelectrolytes

self-assembling monolayer

ligands

proteins

• Molecular Elasticity of Individual Polymer Chains• Protein Folding • DNA Interatomic Bonds • Receptor-Ligand Interactions• Covalent Bonds • Colloidal forces • Van der Waals forces • Hydration forces • Hydrophobic forces • Surface Adhesion • Nanoindentation• Electrostatic DLVO forces • Cell Adhesion• Steric Forces of Polymer Brushes

AFM: Tip Functionalization2. Chemical coating

Purpose:

Methods:

Applications:

antibodies

http://www.di.com/AppNotes/LatChem/LatChemMain.html

(c)

(d)nanotube with

individual ligand

(a) Benoit, M.; Gabriel, D.; Gerisch, G.; Gaub, H. E. Nature Cell. Bio 2000, 2 (6), 313.(b) Ong, Y-L.; Razatos, A.; Georgiou, G.; Sharma, M. K. Langmuir 1999, 15, 2719.

(c) J . Seog, Ortiz/ Grodzinsky Labs 2001(d) Wong S.S.; Joselevich E.; Woolley, A.T.; Cheung, C. L.; Lieber, C. M. Nature 1998, 394 (6688), 52.

(a) Single Cell Dictyostelium Discoideum

(b) E. Coli Bacteria

(c)colloidal particle

AFM: Tip Functionalization

IV. Chemical Force Microscopy (CFM) Frisbie, et al., 1994

Noncontact (NC) 1995

II. Friction or Lateral Force Microscopy (FFM/ LFM) Frisbie, et al., 1994

I. Normal Force Microscopy

III. Force / Volume Adhesion Microscopy Radmacher, et al., 1994

Contact DC and AC (Force Modulation Microscopy (FMM), Phase Imaging): Hansma, et al., 1991

Intermittant Contact/Tapping / Lift (AC): Hansma, et al., 1994

X=-OH,-CH3, -NH2

XX

XX

XXXXXXXXXXXXX

XXX

Surface Maps:Topography & Roughness, Electrostatic

Interactions, FrictionChemical, Adhesion , Hardness,

Elasticity /ViscoelasticityDynamic Processes :

Erosion, Degradation, Protein-DNA Interactions

AFM: Applications of modes

Timeline:

http://www.di.com/AppNotes/ForceVol/FV.array.html

PROBE TIP SHARPNESSSheng, et al. J. Microscopy 1999, 196, 1.CANTILEVER

THERMAL NOISELindsay Scanning Tunneling Microscopy

and Spectroscopy 1993, 335.Shao, et al. Ultramicroscopy 1996,

66, 141.

PIEZO AMPLIFIER, SENSOR AND CONTROL ELECTRONICS,

MECHANICAL PARAMETERSPhysik Instruments, Nanopositioning 1998

SPECIMEN DEFORMATION &

THERMAL FLUCTUATIONS

Hoh, et al. Biophys. J. 1998, 75, 1076.

ADHESION FORCEYang, et al. Ultramicroscopy 1993, 50, 157

(*http://cnst.rice.edu/pics.htmlLieber, et al., 2000)

AFM: Resolution factors/Artifact sources

Biological Applications: AFM Images of CellsContact mode image of human red blood cells - note cytoskeleton is

visible. blood obtained from Johathan Ashmore, Professor of Physiology University College, London. A false color table has been used here, as professorial blood is in fact blue. 15µm scan courtesy M. Miles and J.

Ashmore, University of Bristol, U.K.

Rat Embryo Fibroblast(*M. Stolz,C. Schoenenberger, M.E. Müller Institute,

Biozentrum, Basel Switzerland)

Height image of endothelial cells taking in fluid using Contact Mode AFM. 65 µm scan courtesy J. Struckmeier, S. Hohlbauch, P. Fowler, Digital Intruments/Veeco Metrology, Santa Barbara, USA.

Red Blood CellsShao, et al., : http://www.people.virginia.edu/~js6s/zsfig/random.html

Radmacher, et al., Cardiac Cellshttp://www.physik3.gwdg.de/~radmacher/

• rest cantilever on top of cell and monitor cantilever deflection up and down = beating of cell

• I. confluent layer of cells : beat regularly in terms of frequency and amplitude, enormous stability of pulsing, cell are synchronized and coupled together : diverse pulse shapes due to macroscopic moving centers of contraction and relaxation• II. individual cell : sequences of high mechanical activity alternate with times of quietness, irregular beating which often last for minutes, active sequences were irregular in frequency andamplitude• III. group of cells: “pulse mapping”

Biological Applications: Manipulation of Living Cells

http://www.people.virginia.edu/~js6s/zsfig/DNA.html

AFM image of short DNA fragment with RNA polymerase molecule bound to transcription

recognition site. 238nm scan size. Courtesy of Bustamante Lab, Chemistry Department, University

of Oregon, Eugene OR

Image of PtyrTlac supercoiled DNA. 750 nm scan courtesy C. Tolksdorf, Digital

Instruments/Veeco, Santa Barbara, USA, and R. Schneider and G. Muskhelishvili, Istitut für

Genetik und Mikrobiologie, Germany.

TappingMode image of nucleosomal DNA was the highlight of the "Practical Course on Atomic Force Microscopy in

Biology," held at the Biozentrum in Basel, Switzerland, July 1998. Image courtesy of Y. Lyubchenko.

The high resolution of the SPM is able to discern very subtle features such as these two linear dsDNA

molecules overlapping each other. 155nm scan. Image courtesy of W. Blaine Stine

Biological Applications: AFM Images of DNA

AFM: From Nano to MicroStructures

Human hair (C. Ortiz)

Eggshell

DIC (Differential Interference Contrast) image of human

lymphocyte metaphase chromosomes on microscopy

slide

dimensions 83 µm * 83 µm

DIC (Differential Interference Contrast) image of human lymphocyte metaphase chromosomes on microscopy slide

dimensions 83 µm * 83 µm

What does the future hold?

Atomic Force Microscopy;The Sample Preparation

Pooria GillPhD of Nanobiotechnology

Assistant Professor at MAZUMSP.Gill@mazums.ac.ir

In The Name of Allah

AFM sample preparation

19

Liquid AFM Images

41 45 48 56 60

70 nm t=0 min 20 2212

Effect of DNase I enzyme on G4-DNA (0.5:1) complex, the complex was immediately adsorbed onto mica and imaged until stable images were obtained, then the DNase I was introduced.

Nucleic Acids Research, 2003, Vol. 31, No. 14 4001-4005

Humidity affects the adhesion

AFM probeSalbutamol

Measurement of particle-particle interacti

onLactose1µm

Force (nN)

0

200

400

600

800

1000

1200

<10% 22% 44% 65%

‘Nanoscale’ contact‘Macroscale’ contact

Environmental AFM

Both temperature andhumidity can be controlledin this environmentalchamber.