BioAFM spectroscopy for mapping of Young s modulus of living cells Jan Přibyl pribyl@nanobio.cz
Optical microscopy AFM Confocal microscopy Young modulus
Content Introduction (theory) Hook s law, Young s modulus Force-distance curves Nanomechanical mapping Force-distance curves Young modulus evaluation Experimental part Instruments available Practical application Young s modulus mapping of living cells Hyaluronane-myeloperoxidase interaction PDMS gels 3
4 Introduction
Young s modulus of materials 5 http://www-materials.eng.cam.ac.uk/
Young s Modulus - an extension to Hooke s Law Hook s law The amount of force applied is proportional to the amount of displacement (length of stretch or compression). F - applied force k spring constant x - amount of displacement 6
Similarly to Hooke s Law the stretching of a spring is proportional to the applied force F = -k x σ = Ε ε stress F A Young s Modulus (modulus of elasticity) strain L L E stress strain E stress strain F A L L E ( L L Li f i) A F 7
Stress vs. strain graphs The Young modulus 2 large strain for little stress _ material is flexible, easy to stretch little strain for large stress _ material is stiff, hard to stretch 0 0 strain 0 0 strain Young modulus is large for a stiff material slope of The Young modulus is large for a stiff material (large stress, small strain). Graph is steep. graph is steep Property The Young modulus of is a the property of the material not - the independent specimen. Units of the Young of modulus weight and MN m shape 2 or MPa; for stiff materials GN m 2 or GPa. Same as units of stress, because strain is a ratio of two lengths, e.g. extension is 1% of length Units Pa 8 e.g. polymer e.g. diamond, steel
Methods for YM measurement Olympus 38DL PLUS Measure the longitudinal and shear wave sound velocity of the test piece using the appropriate transducers and instrument setup. 9 Reference: Battelle PNNL MST Handbook, U.S. Department of Energy, Pacific Northwest Laboratory
Cell Young s modulus - methods 10 Acta Biomater. 2007 Jul; 3(4): 413 438.
The microfluidic optical stretcher Stretching forces, F (major and minor axes) Optical deformability, ODt, Optically induced surface forces - trapping and stretching of cells Cells in a microfluidic channel trapped (A) and deformed (B) Divergent laser beams Distribution of surface forces monitored by laser beam (Gaussian int.) 11 Biophys J. 2005 May; 88(5): 3689 3698.
Mechanical Properties of Living Cells Using Atomic Force Microscopy 12 J. Vis. Exp. (76), e50497, doi:10.3791/50497 (2013).
QI mode Hertzian fit (A) Typical force distance curves for hard (green) and soft (blue) materials. (B) Adhesion on a hard surface. (C) Molecule molecule and cell surface detachment process with three unbinding events. 13 Phys. Chem. Chem. Phys., 2015, 17, 2950-2959
QI-imaging examples 14 JPK supporting info
Proud (ma) TiO2 NT biosensing 1,5 1,0 0,5 0,0-0,5-1,0-1,5-2,0 PBS 1,7 mm H 2 O 2 3,3 mm H 2 O 2 5,0 mm H 2 O 2 6,7 mm H 2 O 2 8,3 mm H 2 O 2-2,5-2,0-1,5-1,0-0,5 0,0 0,5 1,0 1,5 2,0 2,5 Napětí (V) 15
Quantitative NanoMechanics (QNM) attractive forces (capillary, VdW, elstat) negative forces > cantilever stiffness indentation withdrawing Peak force feed back control PeakForce QNM = quantitative nanomechanical information (biological samples without damaging) Based on Peak Force Tapping technology - probe is oscillated (~TappingMode), res. freq 1-8 khz (=sampling rate) depending on the tool). Difference: Tapping Mode const. amplitude, Peak Force Tapping maximum peak force on the probe (much lower comparing to contact mode biological samples) 16
PeakForce QNM on Bacteria (A) PeakForce QNM (250Hz) Sneddon modulus (B) PeakForce curves (C) Force volume Sneddon modulus image of the same bacteria collected at a ramp rate of 2Hz. (Standard DNP-A probe in water with 300nm modulation amplitude, Scan size 5µm.) 17 BrukerNano supporting info
18 Nanomechanical mapping
Young s modulus determination by AFM Force-distance curves are measured by lifting the cantilever above the surface Sphere-type of the probe If the cantilever is moved in the x-y axis, the Force Maps are thus produced. Young s modulus values, are obtained by the curve post-processing 19
Hertzian fit Measured curves were fitted to following function: where F is force, E is Young modulus, α face angle, δ tip-sample separation, ν Poisson ratio: Tip-sample separation = correction of measured curve (height) for cantilever bending 20
Parabolic tip shape Four sided pyramid Spherical tip 21
22 Examples of Force-distance curves and Force Maps evaluation
Force-distance curves Height Slope Adhesion Height Adhesion Young modulus With Giancarlo Forte, ICRC 23
Experimental part Instruments available 24
JPK NanoWizard3 AFM microscope JPK NanoWizard Scanning by probe standard AFM head (area up to 115x115um) ForceRobot AFM head for fully automated AFM spectroscopy Incl. inverted optical microscope Olympus IX80 25
Young s modulus mapping Quantification of kinetics, affinity and molecular mechanisms of biological interactions Binding studies such as receptor/ligand or antibody/antigene Analysis of protein (un)folding and function Mechanical studies on soft materials Force Robot head extension 26
Bruker Dimension Icon/FastScan Scanning by probe (area up to 115x115um) Very fast imaging speed (up to 4Hz, Incl. inverted optical microscope Olympus IX80 DNA on mica With H. Kolarova, H. Zapletalova, UPOL Lipozomes on graphite electrode 27 With J. Vacek, UPOL
Bruker Dimension Icon/FastScan In liquid (Petri dish) Glass slides holder Elevated temperatures Closed liquid chamber 28 http://www.nanophys.kth.se
Practical applications 29 29
Single human immortalized adipose tissuederived mesenchymal stem cells (htert AD- MSCs) on micropatterned substrates Together with group of Giancarlo Forte, ICRC Experimental notes AFM Microscope: JPK NanoWizard 3 AFM Probes: AppNano Hydra 6R-200N (low stress SiN, tip silicon, rectangular shape, k~0.083 N/m, no coating on cantilever) Batch processing of curves - Hertz fit - parameters: Tip shape: quadratic pyramid Pyramid angle: half-angle to face 35deg Poisson ratio: 0.5 X-channel Tip-Sample separation Y-channel: vertical dfl in extension movement 30
2015.07.09-12.17.03 shtaz Height Young modulus 2015.05.05-14.40.56 shyap 31
2015.02.19-12.34.49 ADMSC Height Young modulus 2015.07.09-14.11.16 shtaz 32
2015.07.09-16.40.01 shtaz Height Young modulus corrected 2015.11.19-17.54.21 ADMSC 33
2015.11.19-16.58.44.504 ADMSC Height Young modulus corrected 34
Evaluation of Young s modulus maps? 25,380 kpa 26,707 kpa 35
Masking of cell surrounding area 15.4 kpa 20.3 kpa 36 Gwyddion software
q [kpa -1 ] 5,8903 kpa 6,6263 kpa 5,5307 kpa 6,0019 kpa 25,380 kpa 26,707 kpa 1.2 Y = A + B * X 0.8 A B 9.24347E-7-2.08031E-12 0.4 0.0 37 0 5 10 15 20 25 z [MPa] Automatic edge detection inclination finds the cell border precisely, however does not keep the values of Young modulus (Z-scale)
Young modulus [kpa] htert ADMSCs on petri dish shtaz free cells htert ADMSCs on PADO1 GFP cells on petri dish 80 70 60 50 40 30 20 10 0 38 htert ADMSCs htert ADMSCs PD GFP PD shtaz FC htert ADMSC free
Cancer cell stiffness Together with group of Pavel Bouchal, Biochemistry Dept Experimental notes AFM Microscope: JPK NanoWizard 3 AFM Probes: AppNano Hydra 6R-200N (low stress SiN, tip silicon, rectangular shape, k~0.083 N/m, no coating on cantilever) Batch processing of curves - Hertz fit - parameters: Tip shape: quadratic pyramid Pyramid angle: half-angle to face 35deg Poisson ratio: 0.5 X-channel Tip-Sample separation Y-channel: vertical dfl in extension movement 39
Cell: MCF7_CTRLpl Position 1 Optical images 4x 40 10x
Cells CF7_PDZIMpl Position 1 Optical image 4x 10.099 kpa 3.822 kpa 41
Cells MCF7_PDLIMsiRNA Position 3 Optical image 4x 1.897 kpa 1.684 kpa 42
Cells MCF7_PDLIMsiRNA Position 1 Optical image 4x 3.60 kpa 1.92 kpa 43
Position 1 Cell: CTRLsiRNA Optical images (4x) Position 3 Position 2 44
0.9170 kpa 0.7281 kpa results-mlf7_ctrlsirna_pos1a-data- 2015.09.04-08.46.572015.09.04-08.46.57.png 6.25 kpa 2.72 kpa results-mlf7_ctrlsirna_pos1b-data- 2015.09.04-08.28.032015.09.04-08.28.03.png 1.7595 kpa results-mlf7_ctrlsirna_pos2a-data- 2015.09.04-10.14.172015.09.04-10.14.17.png 1.7261 kpa 0.9628 kpa 0.5083 kpa results-mlf7_ctrlsirna_pos2b-data- 2015.09.04-09.43.032015.09.04-09.43.03.png 0,337 kpa 2.00 kpa 1.25 kpa results-mlf7_ctrlsirna_pos3a-data-2015.09.04-11.14.002015.09.04-11.14.00.png 0,305 kpa 45 2.52 kpa 1.28 kpa results-mlf7_ctrlsirna_pos3b-data-2015.09.04-10.47.192015.09.04-10.47.19.png
Young modulus [kpa] 0.9717 kpa (1.0192 kpa) results-mcf7_ctrlpl_pos1b-data- 2015.09.03-21.15.572015.09.03-21.15.57.png 0,337 kpa 0.6537 kpa (0.4415 kpa) 12 10 0,305 kpa 8 6 4 2 0 46 MCF7_CTRLpl CF7_PDZIMpl MCF7_PDLIMsiRNA MLF7_CTRLsiRNA
47 Effect of myeloperoxidase activity on hyaluronan layer elasticity
YM [MPa] Young modulus dependency on SetPiont value With Jan Vitecek & Lukas Kubala, IBP SetPoint [nn] is maximal value of force applied to cantilever Higher values of force can cause penetration of layer with sharp tip of AFM probe 3000 Surface Glass Glass-AP-Hyaluronan 2000 1000 0 48 0.0 0.5 1.0 1.5 2.0 2.5 SP [nn]
YM (kpa) 1400 Myeloperoxidase AFM setting: - AFM Hydra-50N-2R (AppNano) probe -Z-height (lift height) = 450 nm - SetPoint = 400 pn - Force-Maps 36x36 points - Time for a single force map: 7 minutes -All experiments at 37 o C, PBS buffer ph=7.4 (0.22um filtered. 1200 1000 AFM adjustment 800? 600 400 200 49 0 13:12 14:24 15:36 16:48 18:00 19:12 20:24 21:36 Time stamp
PDMS gels for single CMs With Vladimir Vinarsky, ICRC Gel 1 Gel 2 Gel 3 50
Summary Nanomechanical mapping Force-distance curves: Young modulus evaluation maps of Young s modulus QI imaging mode QNM imaging mode ScanAssyst Instruments available JPK NanoWizard 3 QI, YM maps ForceRobot head long term measurements Bruker FastScan QNM, ScanAssyst, high resolution, user friendly Samples Living cells (Petri dishes, glass slides, etc.) Biomolecules (single molecular imaging) (Bio)polymers Do not hesitate to contact me> pribyl@nanobio.cz 51