Krystyn J. Van Vlietkrystyn@mit.edu

3.052 Spring 2003March 4, 2003

HIGH-RESOLUTION IMAGING WITH FORCES(ATOMIC FORCE MICROSCOPY)

RAW DATA

Tip-Sample Separation Distance, D (nm)

Forc

e, F

(nN

)

adhesion

0

repulsiveregime

attractive regime

z-PiezoDeflection, z (nm)

Phot

odio

deSe

nsor

Out

put,

s (V)

CONVERTED DATA

jump-to-contact

substrate compression no interaction

0 0

kc

RAW DATA

Tip-Sample Separation Distance, D (nm)

Forc

e, F

(nN

)

adhesion

0

repulsiveregime

attractive regime

z-PiezoDeflection, z (nm)

Phot

odio

deSe

nsor

Out

put,

s (V)

CONVERTED DATA

jump-to-contact

substrate compression no interaction

0 0

kc

Review: Typical HRFS output on stiff substrate

RAW DATA

Tip-Sample Separation Distance, D (nm)

Forc

e, F

(nN

)adhesion

0

repulsiveregime

attractive regime

z-PiezoDeflection, z (nm)

Phot

odio

deSe

nsor

Out

put,

s (V)

CONVERTED DATA

jump-to-contact

substrate compression no interaction

0 0

kc

RAW DATA

Tip-Sample Separation Distance, D (nm)

Forc

e, F

(nN

)adhesion

0

repulsiveregime

attractive regime

z-PiezoDeflection, z (nm)

Phot

odio

deSe

nsor

Out

put,

s (V)

CONVERTED DATA

jump-to-contact

substrate compression no interaction

0 0

kc

Review: Typical HRFS output on stiff substrate

RAW DATA

Tip-Sample Separation Distance, D (nm)

Forc

e, F

(nN

)

adhesion

0

repulsiveregime

attractive regime

z-PiezoDeflection, z (nm)

Phot

odio

deSe

nsor

Out

put,

s (V)

CONVERTED DATA

jump-to-contact

substrate compression no interaction

0 0

kc

RAW DATA

Tip-Sample Separation Distance, D (nm)

Forc

e, F

(nN

)

adhesion

0

repulsiveregime

attractive regime

z-PiezoDeflection, z (nm)

Phot

odio

deSe

nsor

Out

put,

s (V)

CONVERTED DATA

jump-to-contact

substrate compression no interaction

0 0

kc

Review: Typical HRFS output on stiff substrate

Review: Experimental Aspects of Force Spectroscopy

Conversion of raw data in a high-resolution force spectroscopy experiment :• sensor output, s transducer displacement, force, F

• z-piezo deflection, z tip-sample separation distance, D

Typical force spectroscopy data for a weak cantilever on stiff substrate (ksample>> kcantilever) :APPROACH : (*sample and tip come together)• A: tip and sample out of contact, no interaction, cantilever undeflected, zero force (set F=0)• B/C: attractive interaction pulls tip down to surface and tip jumps to contact, cantilever exhibits mechanical instability• D: contact, constant compliance regime, no sample indentation, tip and sample move in unison (s/z=1)RETRACT :(*sample and tip move apart)• D: repulsive contact, constant compliance regime, tip deflected up • E: attractive force (adhesion) keep tip attached to surface, tip deflected down• F: tip pulls off from surface, cantilever instability • G: same as region A

s/m

D=z

RAW DATA

Tip-Sample Separation Distance, D (nm)

Forc

e, F

(nN

)

adhesion

0

repulsiveregime

attractive regime

z-PiezoDeflection, z (nm)

Phot

odio

deSe

nsor

Out

put,

s (V)

CONVERTED DATA

jump-to-contact

substrate compression no interaction

0 0

kc

RAW DATA

Tip-Sample Separation Distance, D (nm)

Forc

e, F

(nN

)

adhesion

0

repulsiveregime

attractive regime

z-PiezoDeflection, z (nm)

Phot

odio

deSe

nsor

Out

put,

s (V)

CONVERTED DATA

jump-to-contact

substrate compression no interaction

0 0

kc

AB/C

D

D

E F G

AB/C

DD

EF

G

*Note: For an adhesive interaction

F = k

Atomic Force Microscopy Imaging

• BASIC PRINCIPLES : piezo rasters or scans in x/y direction across sample surface cantilever deflects in response to a topographical feature

computer adjusts the z-piezo distance to keep the cantilever deflection constant and equal to the setpoint value

“feedback loop” : system continuously changes in response to anexperimental output (cantilever deflection)ERROR SIGNAL = actual signal- set point (*used to produce 2D topographical image in contact mode)

sample

sensor output, c, Fc

feedback loop • controls z-sample

position

position sensitivephotodetector

• measures deflection of cantilever

mirrorlaser diode A BC D

ERROR = actual signal - set point

c

xyz

10°-15°

cantilever• spring which deflects as probe tip

scans sample surface

computer • controls system

• performs data acquisition, display, and analysis

piezoelectricscanner

• positions sample(x, y, z) with Å accuracy

probe tip• senses surface

properties and causescantilever to deflect

sample

sensor output, c, Fc

feedback loop • controls z-sample

position

position sensitivephotodetector

• measures deflection of cantilever

mirrorlaser diode A BC D

ERROR = actual signal - set point

c

xyz

10°-15°

cantilever• spring which deflects as probe tip

scans sample surface

computer • controls system

• performs data acquisition, display, and analysis

piezoelectricscanner

• positions sample(x, y, z) with Å accuracy

probe tip• senses surface

properties and causescantilever to deflect

Atomic Force Microscopy:General components and functions

cantilever

AFM : Normal Force Spectroscopy Modes of Operation*AC=dynamic(tip is driven to oscillate), DC=static(no external oscillation on tip)

Contact (DC and AC) : Force Modulation

Non-Contact (AC)

Intermittent Contact : Tapping (AC)

AFM : Contact ModeFeedback Error:

DeflectionOutput:

“Isoforce” Height

http://www.physik3.gwdg.de/~radmacher/publications/osteoblasts.html

AFM : Tapping Mode

Feedback Error: Amplitude

Output: “Isoamplitude” Height

Evaporated gold surface

Additional Feedback: Phase

AFM : Normal Force Spectroscopy Modes of Operation

(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 acalcite (CaCO3) crystal

Ohnesorge, 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

Si3N4

cantilever

TOP VIEW

AFM: Tip Functionalization

ONE-TIME Au COATING : heterogeneous, rougher larger

polydomain microstructure

1. Gold coating Purpose: 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., 1991Intermittant Contact/Tapping / Lift (AC): Hansma, et al., 1994

X=-OH,-CH3, -NH2

XX

XX

XXXXXXXXXXXXX

XX

X

Surface Maps:Topography & Roughness, Electrostatic Interactions,

FrictionChemical, Adhesion , Hardness, Elasticity

/ViscoelasticityDynamic Processes :

Erosion, Degradation, Protein-DNA Interactions

AFM: Applications of modesTimeline:

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

voltageapplied

L

L+L

electrodes

connecting wires

d

+Y -X +X

D

D+D

polarization

x

yz

+Z

-Z

~

PROBE TIP SHARPNESS

Sheng, et al. J. Microscopy 1999, 196, 1.

CANTILEVER THERMAL NOISE

Lindsay Scanning Tunneling Microscopy and Spectroscopy 1993, 335.

Shao, et al. Ultramicroscopy 1996, 66, 141.

Distance, D (nm)

Forc

e, F

(nN

)

0

0

Fadhesion

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

=

t(max)

m

m

m

kt

cantilever

t(max)

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

AFM: Resolution factors/Artifact sources

AFM: Advantages as tool to assess biological responses

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/

cardiac cell

<0=0>0

restposition

• 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