General Introduction: AFM Principles and Operation For Bio€¦ · General Introduction: AFM...

Simon [email protected]

http://perso.curie.fr/Simon.Scheuring/

Monday, 29 August, 2011

General Introduction:

AFM Principles and Operation For Bio

Simon Scheuring

Email: [email protected]: http://perso.curie.fr/Simon.Scheuring/

COST AFM-BioMed Summerschool

Monday, August 29, 2011

mailto:[email protected]









Atomic Force Microscopy (AFM)

Invention of the STM and the AFM

Atomic force microscope.Phys. Rev. Lett. 1986 Mar 3;56(9):930-933Binnig G, Quate CF, Gerber C.

Scanning tunneling microscopy - from birth to adolescenceNobel lecture, December 8, 1986Binnig G. and Rohrer H.












Invention of the STM and the AFM

Perhaps we were fortunate in having common tra ining in superconductivity, a field which radiates beauty and elegance. For scanning tunneling microscopy, we brought along some experience in tunneling and angstroms, but none in microscopy or surface science. This probably gave us the courage and light-heartedness to start something which should “not have worked in principle” as we were so often told.Nobel lecture, December 8, 1986 by GERD BINNIG AND HEINRICH ROHRER











STM (Scanning tunneling microscope)AFM (atomic force microscope)SNOM (Scanning nearfield optical microscope)

probe

surface

Different types of ‚Scanning Probe Microscopy (SPM)ʻ for different applications













AFM tip

Atomic force microscope

Lever length: ~ µmTip radius: ~ nm

Phonograph

Phonograph tipMounting: ~ mmTip radius: ~ µm











Pierre Curie Left: Piezo

1878-1882 Pierre et son frère Jacques mènent une série de travaux sur la piézo-électricité.

Découverte de la piézo-électricitéSon premier travail a trait à la détermination des longueurs d'onde calorifiques. Puis, avec son frère, alors préparateur de Friedel, au laboratoire de minéralogie de la Sorbonne, il étudie les cristaux. Tous deux découvrent alors le phénomène de piézo-électricité qui consiste en une polarisation électrique produite par la compression ou la dilatation des cristaux dépourvus de centre de symétrie. A partir de cette constatation, ils conçurent un quartz piézo-électrique qui servit à mesurer des courants électriques de faible intensité. Peu après, ils élaborèrent un électromètre à quadrants, appelé par la suite électromètre Curie.

Pierre and Jacques Curie discover the piezo electricity













The X-, Y- and Z- piezo tube












The tip and the protein

AFM-Tip / Protein

=

Tour-Eiffel / Basket-Ball

AFM-Tip~ 3 µm = 3 * 10-6 m

Protein~ 3 nm = 3 * 10-9 m

Basket-Ball~ 3 dm = 3 * 10-1 m

Tour-Eiffel~ 3 dm = 3 * 102 m

~ 1000











High-Resolution AFM Imaging

The force measured by the cantilever / The spring constant (k) of a cantilever

F: Forcek: spring constantz: z-displacement

E: Youngʻs modulusw: cantilever widtht: cantilever thicknessl: cantilever thickness

kB: Boltzmann constantT: temperaturex2: cantilever thickness

frequenzy (kHz)no

ise p

ower

(dB)

Geometrical consideration

Thermal noise analysis











The Reality

Thatʼs what the Tip Does

Thatʼs what the Piezo Drives

The contouring mechanism













The principle of the AFM











The Reality

Thatʼs what blunt tip ʻseesʼ

Thatʼs what sharp tip ʻseesʼ

“The sharper the tip - The better the image”

The tip contours the surface and itself













Tip - Sample : force-distance curve

distance

defle

ctio

n / f

orce











Force-distance curve


solid surface

approaching curves

repulsion

indentationdistance

defle

ctio

n / f

orce











Force-distance curve


retracting curves

solid surface

adhesion

indentationdistance

defle

ctio

n / f

orce











Force-distance curve - real case


approaching curves

repulsion / indentation

retracting curve

indentation / adhesion

distance

defle

ctio

n / f

orce











AFM Force Spectroscopy

Cantilever deflection correction











Piezo P1 to P2: ∆z = 10 nm

1 2













Piezo P1 to P2: ∆z = 10 nm

1 2

Cantilever P1 to P2: ∆d = 3 nm

1 2













Protein: ∆s = 7 nmPiezo P1 to P2: ∆z = 10 nm

1 2

Cantilever P1 to P2: ∆d = 3 nm

1 2













Surface

tip

membrane


Ions are important











Surfacemembrane

tip

Counter-ion concentration

Co-ion concentration

Debye-length

distance

conc

entra

tion


Adsorption and imaging - Rationales











Surfacemembrane

tip



Debye-length

Debye-length

distance

conc

entra

tion













SurfaceMembrane

Tip



Debye-length

distance

conc

entra

tion

distance

concentration

SurfaceMembrane

Tip














Tip - Sample : Repulsive forces

Daniel J Müller, Dimitrios Fotiadis, Simon Scheuring, Shirley A Müller & Andreas Engel*Electrostatically balanced subnanometer imaging of biological specimens by atomic force microscopyBiophys J, 1999, 76 (2): 1101-1111











Contact mode - Deflection / Force

(deflection when in contact)

Imaging modes


Oscillating mode - Deflection / Force - Amplitude - Phase

(Amplitude decreases and phase changes when in contact)











Dimensions in life sciences - what technique is appropriate

plant cells animal cells bacteria viruses ribosomes proteins molecules atoms

light microscopyelectron microscopy (EM)

atomic force microscopy (AFM)NMRX-ray

1cm 1mm 100µm 10µm 1µm 100nm 10nm 1nm 1Å

Techniques In Structural Biology











The signal-to-noise ratio (SNR) vs. The number of molecules to analyze

101 102 103 104 105 106 107 108 109 1010 1011 1012 1013 1014 1015 1016 1017 10181

>10mg>10mg>10mg>1mg>10µg >100µg>10µg

SolubilizationPurification

Crystallization

LabellingSolubilizationPurification


Crystallization


-


-

---


Crystallization

3D (~2Å)3D (~3.5Å)3D (~6Å) 3D (~1Å)2D (~10Å)3D (~10Å)3D (~20Å)Topo (~10Å)

AFM - Techniques In Structural Biology

AFMmembranes

NMRmolecules

X-ray3D-crystal

EM (cryo)molecules

EM (stain)molecules

EM (cryo)2D-crystal











100 % 90 % 80 % 70 %

Schiele AFM

50 % 40 % 30 %60 %

TEMstained

TEM cryoholey C-film

20 % 10 % 0 %

TEM cryo C-film

AFM - The Signal-To-Noise Ratio (SNR)

<1 %

AFM

(X-ray)(NMR)











2µm

100Å

Membranes on mica - The color code












The AFM false color table: Sunset at Los Lobos (California) - Paul Hansma












AFM - In Membrane Protein Research

The nativeness of the membrane protein sample

3D-crystal

AFM

2D crystal

Densely packed reconstitution

Loosely packed reconstition

Native membrane ex cellula

Native membrane in cellula








Simon Scheuring* & James SturgisChromatic adaptation of photosynthetic membranes

Science, 2005, 309 (5733): 484-487


The best Image

10nm

Native membrane











Never Ever Give Up!








Simon Scheuring, Henning Stahlberg, Mohamed Chami, Christine Houssin, Jean-Louis Rigaud & Andreas Engel*Charting and unzipping the surface-layer of Corynebacterium glutamicum with the atomic force microscope

Mol Microbiol, 2002, 44 (3): 675-684


S-Layer Comparison

20nm

Corynebacterium glutamicum Deinococcus radiodurans

20nm

Raw data Raw data









Mol Microbiol, 2002, 44 (3): 675-684


S-Layer Comparison

20nm


20nm

Raw data Raw dataAverage Average









Mol Microbiol, 2002, 44 (3): 675-684


S-Layer Comparison

20nm


20nm

Raw data Raw dataAverage Average

Model Model









Mol Microbiol, 2002, 44 (3): 675-684


300pN

20nm

300pN

20nm

S-Layer Comparison

Corynebacterium glutamicum

Deinococcus radiodurans

Müller et al. (1999)









Mol Microbiol, 2002, 44 (3): 675-684


300pN

20nm

300pN

20nm

S-Layer Comparison




1

11









Mol Microbiol, 2002, 44 (3): 675-684


300pN

20nm

300pN

20nm

S-Layer Comparison




1

11

2

22









Mol Microbiol, 2002, 44 (3): 675-684


300pN

20nm

300pN

20nm

S-Layer Comparison




1

11

2

22

33

3









Mol Microbiol, 2002, 44 (3): 675-684


300pN

20nm

300pN

20nm

S-Layer Comparison




1

11

2

22

33

3

4

44









Mol Microbiol, 2002, 44 (3): 675-684


300pN

20nm

300pN

20nm

S-Layer Comparison




1

11

2

22

33

3

5

55

4

44









Mol Microbiol, 2002, 44 (3): 675-684


300pN

20nm

300pN

20nm

S-Layer Comparison




1

11

2

22

33

3

6

66

5

55

4

44









Mol Microbiol, 2002, 44 (3): 675-684


300pN

20nm

300pN

20nm

S-Layer Comparison




1

11

2

22

33

3

6

66

5

55

4

44

1

11









Mol Microbiol, 2002, 44 (3): 675-684


300pN

20nm

300pN

20nm

S-Layer Comparison




1

11

2

22

33

3

6

66

5

55

4

44

1

11

2

22









Mol Microbiol, 2002, 44 (3): 675-684


300pN

20nm

300pN

20nm

S-Layer Comparison




1

11

2

22

33

3

6

66

5

55

4

44

1

11

2

22

3

33











Thank you!

Simon Scheuring

Email: [email protected]: http://perso.curie.fr/Simon.Scheuring/

COST AFM-BioMed Summerschool

Images and Movieswww: http://perso.curie.fr/Simon.Scheuring/

„The sharper the tip-

The better the image“














General Introduction: AFM Principles and Operation For Bio€¦ · General Introduction: AFM...

Documents

Transcript of General Introduction: AFM Principles and Operation For Bio€¦ · General Introduction: AFM...