Scanning Force Microscopy. i.e. Atomic Force Microscopy. Josef A. Käs Institute for Soft Matter Physics Physics Department. Atomic Force Microscopy

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1 Scanning Force Microscopy i.e. Atomic Force Microscopy Josef A. Käs Institute for Soft Matter Physics Physics Department Atomic Force Microscopy

2 Single Molecule Force Spectroscopy Zanjan 4 Hermann E. Gaub Applied Physics University Munich Ludwig Maximilians Universität

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4 Forced Ligand-Receptor Unbinding Moy, V. T.; Florin, E.-L.; Gaub, H. E. Science 1994, 266, Grandbois, M.; Beyer, M.; Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Science 1999, 283, p 1727 Keep in mind: 1k B T 3K = 25 mev = 4 pnnm =.6 kcal/mol = 2.5 kj/mol Thermal Energy Scale (k B T 3K ) 1 1 Myosin 1 1 stroke Molecular recognition

5 Probing a Single Metallo- Organic Bond the Ruthenium(II)-Terpyridine Complex O - PEG (76) - CH2CH2COOH N Functionalized AFM Tip N N Ru (II) G = 4 kt Metal Ion PEG 8 Terpyridine Counts SiOx 1 2 Force [pn] Probing Metalloorganic Bonds Force [pn] Force [pn] Extension [nm] 2 4 Extension [nm] Counts [%] Rupture Length [nm] 1

6 Unfolding Proteins Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science 1997, 276, Oesterhelt, F.; Oesterhelt, D.; Pfeiffer, M.; Engel, A.; Gaub, H. E.; Müller, D. J. Science 2, 288, The Giant Muscle Protein Titin I-Band A-Band I-Band Ig module Fn module unique insertion sequence PEVK region Z-Disc M-Line Titin Z-Disc mechanically active part I1 I15 I16 I19 I2 I41 Constructs I27 I3 Single Ig-Domain

7 Unfolding 4 and 8 Segment Long Recombinant Titin IgFragments 6 S Force Force (pn) 4 2 S 25 pn Extension (nm) Extension (nm) S Force 25 pn Extension (nm) Force (pn) Extension (nm) 2 pn 5 nm M. Rief, M. Gautel, F. Oesterhelt, J. M. Fernandez and H. E. Gaub, Science (1997),Vol 276, p 119- Unfolded Ig 8mer as a Worm Like Chain F(x)= kt p 1 1 x (1-x/L) 4 L ( ) 4 p= 3Å L(nm) Force (pn) Extension (nm)

8 5 Forced Unbinding of DNA Oligomer Duplexes PBS,T = 27 C, v = 1.2 µm/s 4 3 Force(pN) Extension(nm) 15 2 Bond rupture probability density mer DNA/DNA Force [pn] Bond rupture probability density mer DNA/DNA Force [pn] B-S and Melting Transition in l DNA Force [pn] Force [pn] Split Strand Extension [nm] WLC Model p=15 Å 4 Double Stranded DNA Single Stranded DNA Recombination of the Split Strands Reflects Sequence 1 2 Relative Extension 1 pn Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Nature Struct. Biol. 1999, 6,

9 Unzipping DNA Hairpins poly dgdc DNA poly dadt DNA G C G C G C G C δ d δ s F GC 2±3 pn F AT 9±3 pn 1 pn 25 pn Au Extension [nm] Extension [nm] Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Nat. Struct. Biol. 1999, 6, 346- Energy Landscape Changes under Tension G 2 < G 1 U U F G 2 = G 1 U 2 kon koff F = G 2 > G 1 kforced F > G 2 G 1 U 1 G xmin = x xmax F x x x2 x2 x ln (dfpull /dt) ² G - koff = λ e kb T - kforced = λ e ² G - F ² x kb T Fpull F = kb T ln ²x ²x koff kb T R.Merkel and E. Evans., Nature, 397, 5-53

10 Opportunities for Single Molecule Assays in Bio-Analytics -Mechanics provides orthogonal information -Extremely low amounts of analyte needed.. => Growing demand in Genomics & Proteomics Current bottle necks: Sensitivity Selectivity e.g. Quantitative SNP detection e.g. False positives High Throughput 1 6 Assays in parallel Improved Sensitivity by Smaller Sensors k T B Molecules as Sensors? Viani, Schäffer, Chand, Rief, Gaub, Hansma J. Appl. Phys. (1999), 4, p2258-

11 Absolute Versus Differential Force Measurements Scales measure absolute forces Balances compare unknown forces with a standard F apple = k d F apple? F standard Singe Molecule Differential Force Assay Detecting nucleic acid mismatches Discriminating energetically equivalent binding modes

12 Reference bond Single Molecule 1bit AD-Conversion Parallel Format Sample bond n I P bond Reference bond Single Molecule 1bit AD-Conversion Parallel Format Sample bond n I P bond Elastomerstamp Drainage DNA-chip

13 Cantilever Polymer Distance Distance Force Force Deflection sensor Analog signal 1 bit Force Force Deflection Position Chemically-Programmable Molecular AD Converter Spots with different reference bonds Reference bond Sample bond

14 DNA: a Programmable Force Sensor for Protein Chips -Kerstin Blank - Fillip Oesterhelt AFM-based Measurements of Cell Elasticity (-> R. Mahaffy et al., PRL, 2, R. Mahaffy et al., Biophys. J, 86, 1777 (24) )

15 AFM-based Measurements of Cell Elasticity Analyzing Viscoelastic AFM-Data f f The Hertz-model (sphere indenting an infinite elastic half plane): 4 3 1/ 2 3/ 2 f bead = KR δ with K =E/(1-ν 2 ) viscoelastic extension: ~ * i t δ δ δ e ω + with * osc ~ * = δ = δ + iδ => f bead R + 3 K 2 K R * 1 1 / 2 R δ 1 / 2 3 / 2 δ 1 / 2 * 2 * ( K ) / δ 3 K 1 δ δ 1 / 2 ~ ~ δ * => 2 * * fosc K1 = ~ = K' + ik" * 1/ 2 2δ ( Rδ )

16 Analyzing Viscoelastic AFM-Data Correcting for Substrate Effects (R. Mahaffy et al., Biophys. J, 86, 1777 (24) )

17 Correcting for Substrate Effects Tu (for non adhered parts) and Chen model (for adhered parts) correct for substrate effects and simultaneously determine the Poisson ratio. (R. Mahaffy et al., Biophys. J, 86, 1777 (24) ) Elasticity of Lamellipodia (S.Park et al., Biophys. J, submitted)

18 Elasticity of Lamellipodia BALB (n=1) SE SV-T2 (n=1) SE t-test (%) H-ras (n=1) SE t-test (%) Net path length (mm) l 3min Mean speed (mm/hr) v Directionality D Activity (%/min) a Average K (kpa) ± BALB 3T3 (n=37) SV-T2 (n=29) H-ras transformed (n=18) (S.Park et al., Biophys. J, submitted) Active Force Generation I Polymerization: 1. Thermal ratchet vs. polym.-stick-burst 2. Bead motility, nematode sperm motility (-> Formin) Molecular motors: 1. Knockouts (Spudich, Gerisch) vs. myosin II in the back of the lamellipodium (Small, Borisy) 2. Traction forces on soft substrates (Sheetz, etc) (-> myosin I actin-rail model, cell spreading) Retrograde flow: 1. Clutch hypothesis

19 Active Force Generation II (-> C. Brunner, M. Gögler, A. Ehrlicher, T. Jähnke, B. Kohlstrunk, Propulsive forces of fast moving cells, Nature, in preparation (24) ) Active Force Generation II lamellipodium cell body Deflection [nm] Translocation [µm] (-> C. Brunner, M. Gögler, A. Ehrlicher, T. Jähnke, B. Kohlstrunk, Propulsive forces of fast moving cells, Nature, in preparation (24) )

20 Active Force Generation III Active Force Generation III