Use of AFM for mechanical mapping of nanostructured surfaces Jan Přibyl Nanobio Core Facility CEITEC MU, Masaryk university, Brno pribyl@nanobio.cz Content ▪ Equipment – AFM, biosensors ▪ BioAFM – imaging, stiffness mapping, adhesion studies ▪ Samples, analysis ▪ Ongoing projects, cooperation, examples 2 AFM microscopy / spectroscopy 3 http://physics.mff.cuni.cz/kfpp/povrchy/method/afm imaging Stiffness mapping (nanoindentation) Affinity interaction AFM imaging Biosamples ▪ DNA – proteins – nanoparticles – liposomes – bacteria – single cells – cell clusters Working environment ▪ Lab atmosphere ▪ Liquid environment (drop/Petri dish) ▪ Elevated temperatures (37 oC) (AFM images: V. Hornakova, S. Solny, J. Pribyl. Photos by: nanophys.kth.se, anowerk.com) Nature Protocols 6, 1443–1452 (2011)www.bruker.com www.azonano.com Hybrid modes: QI, QNM → Topography&Mechanical properties 6 Max. loading force (SetPoint SP) Contact point (CP) Indentation process SP Height CP Height AFM tip AFM tip J. Vis. Exp. (76), e50497, doi:10.3791/50497 (2013). Force spectroscopy (nanoindentation studies) dECM → For tissue engineering ForceMapping as imaging method - High attraction forces - Soft material (macroscopic view) SP-height SP-height YM map YM map Samples by group of Gancarlo Forte, ICRC, Brno AFM operator: Guido Caluori AFM cantilever above the dECM (optical view) 28.8 kPa SP-height YM map 106.1 kPa Chromatin-protein interaction → For tissue engineering ForceMapping as imaging method - Collapsive material - Soft material (macroscopic view) Pesl M, Pribyl J, et al. 2016 Atomic force microscopy combined with human pluripotent stem cell derived cardiomyocytes for biomechanical sensing Biosensors and Bioelectronics 85 751–7 Pesl M, Pribyl J, et al. 2016 Phenotypic assays for analyses of pluripotent stem cell–derived cardiomyocytes J Mol Recognit n/a-n/a Pesl M, Acimovic I, Pribyl J, et al. 2014 Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells Heart Vessels 29 834–46 Primary CMCs EBs Water – YM 7.09 MPa Manitol YM 0.69 MPa by Marçal Gallemí Eva Benkova Lab & Jan Hejatko Lab Hypocotyl and root parts Plant samples under AFM spectroscopy investigation Fibroblasts thawing process model case to study IVF related thawing Together with I. Kratochvilova (Institute of Physics ASCR) Fibroblasts Feeder cells 60 min 25 % Rel YM Fibroblasts Feeder cells 120 min 25 % Rel YM Top view optical imgs SP height profile (12 hours) YM profile (12 hours) SP = 1.5 nN → deep indent. SP =0.4 nN → surface indent. Titanium nanotubes Osteoblast adhesion compatibility Aqueous phase Organic phase SEM – fixed cell TiNT stability by force spectrscopy Nanoindentation – living osteoblast 13 Improved Method for Surface Immobilization of DNA Molecules Used in AFM Single Molecule Imaging 1(3-Aminopropyl)silatrane (APS) •Chem. structure: K2O·Al2O3·SiO2 •Hydrophilic surface •Easy to be modified by chemical synthesis •pKa ~ 3, physiological pH → negative surface charge •Mica = silicate, hydrated SiO2 (~ Si-OH) NH2 NH2 NH2 NH2 NH2 HOOC HOOC HOOC HOOC HOOC HOPG HOPG HOPG DNA on graphite (HOPG) •Low roughness •High hydrophobicity SiO O O NH2 Si O NH2 SiO O O SH (3-Mercaptopropyl)trimethoxysilane MPTS(3-Aminopropyl)trimethoxysilane APTES 3-(Ethoxydimethylsilyl)propylamine APDMES Examples of alkylsiloxanes Silanization process SiO O O CH3 CH3 CH3 R OH Si R OO OH - CH3OH N(Et)3 OH OH OH OHOH OH OHOH OH OO O OO O R-propyltrimethoxysilane + cat. Si Si Si 16 • Especially with APTES during liquid silanization • Even vapors of water can cause this effect • Fixation for optical microscopy – expected factor • In contrary – in fixation for AFM – very disturbing • Solution: - silanization in vapours under vacuum (i.e. in desiccators) - monoalkoxysilanes – can not polymerize SiO O O R2 R1 R1 R1 SiO O O R2 R1 R1 R1 H O H Si OO R2 SiO O O R2 R1 R1 Si O O R2 + -R1OH Silanization process, practical complications Silanization Hydrophobization Si O OO NH2 Si O N O O NH2 Si O Si Si O OH NH2 Si OH Si OH or APTMS APS AP-mica + micaA B NH2 Si O N O O Si O O O NH2 APSAPTMS N OH OH OH + TEtOHA N O O O Si O O NH2 Si O O NH2 N OH OH SiO O NH2 Self-polymerization H2O Reactivity of aminosiloxanes 233.329 762.289 982.371 863.351 1083.412 316.382 643.251 1202.521 542.190 1303.578 1422.711 0 2 4 6 8 4x10 Intens.[a.u.] 1083.568 982.383 1202.548 863.352 1303.672 1422.803 762.276 1523.5520 1000 2000 3000 Intens.[a.u.] 200 400 600 800 1000 1200 1400 1600 1800 m/z APS purification •Reacts with many solid phases •Purification by solvent extraction and crystallization •Structure analysis by MS-ESi, MALDI-TOF and X-Ray Purified APS Crude APS •APS aquatic solution stable in water •One step, short time applicability •Low roughess of mica surfaces modified with purified APS APS use for DNA imaging Purified APS Crude APS Effect of substrate stiffness on mechanical and morphological properties of fibroblasts Extracellular matrix rigidity – plays a role: ▪ Locomotion ▪ Groth control ▪ Differenciation ▪ Phagocytosis ▪ Etc. O. Collin, et al. “Spatiotemporal dynamics of actin-rich adhesion microdomains: influence of substrate flexibility,” J. Cell. Sci., vol. 119, 1914–1925, 2006. Actin structure Cell height 21 Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating A.J. Engler, et al. “Embryonic cardiomyocytes beat best on a matrix with heartlike elasticity: scar-like rigidity inhibits beating” J Cell Sci. 2008, 121, 3794. Polyacrylamide polymer Acrylamide plus bisacrylamide crosslinking with different ratio 22 HA BSA EDAC (act.) HA HA BSA sklo sklo TEA APDMES Protein crosslinking In-situ covalent immobilization 2 types of gel: GHB = hyaluronan - BSA GHBG = hyaluronan – BSA - gelatin 23 PAA (polyacrylamide) gel – poorly adherent to the dish, instable and very sticky in aquatic solutions Practical aspects GHB and GHBG gels immobilized on microscopic slides 24 Surface roughness GHB gel Rms = 57.2 nm (aver. rough.) GHBG gel Rms = 39.7 nm (aver. rough.) Microscopic glass Rms (Sq): 228.8 pm 25 Surface stiffness Eaver 285.2 kPa 135.9 kPa Glass Eaver ~ 70 MPa RT 37oCGHB gel RT 37oCGHBZ gel Eaver 20.2 kPa 19.9 kPa Max. loading force (SetPoint SP) Contact point (CP) Indentation process SP Height CP Height AFM tip AFM tip J. Vis. Exp. (76), e50497, doi:10.3791/50497 (2013). Cell Stiffness by AFM nanoindentation GHB and GHBG gels immobilized on microscopic slides Mouse fibroblast cells Mouse fibroblast on glass 27 A) B) C) D) A) B) C) D) A) B) C) D) GHB 135.9 kPa GHBG 19.9 kPa Glass 70 MPa Cell morphology substrate stiffness Confocal microscopy ▪ DAPI nucleus staining ▪ Actin staining by Phalloidin 28 Glass 70 MPa GHBG 19.9 kPa GHB 135.9 kPa Effect of substrate stiffness on filopodia / lamellipodia structure 1.04 0.56 0.36 0 0.2 0.4 0.6 0.8 1 4.37 14.02 89.27 CP Height (µm) E (kPa) Substrate stiffness vs. cell stiffness and height 0 20 40 60 80 100 120 140 160 sklo HA gel bez želatiny HA gel s želatinou E (kPa) Glass GHB GHBG GHBG GHB Glass ▪ Mechanical properties similar to tissues ▪ Biocompatibility, non-toxic for cells ▪ Keeps adhesivity and mechanical properties (long term) ▪ Transparent – compatible with optical microscopies ▪ Adhesive for cells ▪ Outlook: application for cardiomyocytes (single cells, EBs) Gels based on crosslinked proteins and hyaluronan 5. Combination with other methods ▪ AFM+MEA (microelectrode array) → Mechanical&electrical prop. of CMCs ▪ AFM+electrochemistry (in-situ) → Combined study of electrochem. processes Samples Img & graph by Guido Caluori Nanoscale, 2009, 1, 40-49 asylumresearch.com Thank you for your attention! Acknowledgement Petr Skládal Guido Caluori Stěpán Solný Veronika Horňáková Biology Dept., Fac. of Medicine MU, Brno Vladimír Rotrekl Guido Caluori Šárka Jelínková Ivana Acimovic ICRC, FNUSA Brno Giancarlo Forte Giorgia Nardone Jorge Olivier De La Cruz IST Austria Marcal Gallemi Eva Benkova Institute of Physics Irena Kratochvilova Martin Golan 32 Projects Optical microscopy AFMConfocal microscopy Nanomechanical mapping Young’s modulus mapping ▪ Stiffness (Young’s modulus) mapping → stiffness = basic parameter of any material ▪ Elasticity-phenotype relation ship ▪ Mechanobiological characterization ▪ Driving of instrument properties (QNM, QI) Motivation Why to quantify elasticity of (living) objects? 34 Young’s modulus of materials www-materials.eng.cam.ac.uk/ Acta Biomater. 2007 Jul; 3(4): 413–438. Methods for YM measurement J. Vis. Exp. (76), e50497, doi:10.3791/50497 (2013). 35 Force distance curve analysis 22.16 kPa1.084 MPa 3D 36 Chromation-BAF complex on pLL-modified glass Similar structures found by Allen et al. (page 5) Smooth fibers with interspersed ellipsoids (ellipsoid size 60-90nm) Coiled nodular fibers (diameter 60-100nm, fiber width ~ 17nm, not corrected to tip geometry) ---- However, the complex is spread over the surface, not part of the complete chromatin piece 36 37 Chromatin only Tightly packed chains (linear / curled) + BAF Chromatin-BAF Granular structure (size of grains ~ 50-100nm) SUMMARY 38 Chromatin only Chains composed of ellipsoids Chromatin-BAF Granular structure, grains composed of fibers (?) (~17nm)SUMMARY + BAF 39 Biomechanical studies on cardiomyocytes ▪ Primary CMCs ▪ Embryonic bodies – iPS/HES cardiomyocytes ▪ Low noise ~ 10pN (~ 230 pm) ▪ Robust, low comp. requirements ▪ Possible combination with MEA ▪ Low throughput MCG = mechanocardiogramSetup scheme 40 Adrenergic reactivity Basic conditions Increased arrhythmic potential 70 uM metoprolol 1 mM caffeine Drug testing studies Mapping of force/beat rate Beta-adrenergic receptors diseases ▪ Duchenne muscular dystrophy ▪ CPVT In cooperation with V. Rotrekl, M. Pesl, Med Fac, MU 41 138 144 150 156 0 10 20 30 40 t (s) F (nN) -59 -58 -57 -56 -55 -54 -53 -52 -51 E (mV) d14 64 66 0 20 40 60 t (s) F (nN) -2 0 E (mV) Mechanical & Electrical properties of CMCs AFM & MEA/conductive tip MEA field EB cluster AFM cant. Exposed End insulated Conductive Tip In cooperation with V. Rotrekl, M. Pesl, Med Fac, MU Instrument interface: R. Raiteri, G. Caluori, Uni Genoa, Italy 42 Height Young’s mod. Use of AFM Force Mapping to study Integrin-Focal Adhesion (FA) hTER T AD M SC s hTER T AD M SC s PD G FP PD shTAZ FC hTER T AD M SC free 0 10 20 30 40 50 60 70 80 Youngmodulus[kPa] Masking G. Nardone et al., “YAP regulates cell mechanics by controlling focal adhesion assembly,” Nature Communications, vol. 8, p. 15321, May 2017. 43 3.60 kPa 1.92 kPa M C F7_C TR Lpl C F7_PD ZIM pl M C F7_PD LIM siR N A M LF7_C TR LsiR N A 0 2 4 6 8 10 12 Youngmodulus[kPa] Use of AFM Force Mapping to study cancer cells stiffness Two independent projects together with: ▪ Pavel Bouchal, Biochemistry Dept. MU ▪ Michal Masarik, Med Fac, MU 44 Hyaluronan Hyaluronan + MPO AFM measurements by Stepan Solny 158,8 2809,1 0 500 1000 1500 2000 2500 3000 3500 4000 YM(kPa) Hyaluronan - myeloperoxidase structure and mechanical properties https://wardroundstuff.com/tag/neutrophils/ 45 Gel 2 Gel 3 Gel 1 With Vladimir Vinarsky, Giorgia Nardone, Giancarlo Forte (ICRC, FNUSA, Brno) Flexible surfaces (gels) as support for single CMs PDMS based gels High density methacrylate Low density methacrylate Average YM = 27.4 kPa Average YM = 5.2 kPa 46 Equipment 1. BioAFM microscopes ▪ JPK NanoWizard3, ForceRobot300 mounted on the confocal fluor. mic. - Contact/Tapping imaging in liquid - QI, ForceMapping in liquid (elev. temp.) 46 Petri dish heater 47 ▪ NT-MDT Solver NEXT Ntegra Vita / Solaris - Contact/Tapping imaging - fully automated - education - in-situ elchem cell ▪ BrukerNano Dimension FastScan Bio - Contact/Tapping imaging in liquid - up to 1 image/sec - ScanAsyst - QNM/ForceMapping (Images by: Petr Skladal, nanowerk.com) 47 48 2. Microdeposition of liquids ▪ Scienion sciFlex Arrayers S1 and S3 - deposition and immobilization of biomolecules ▪ InnoScan 1100 - 2D fluorescence imaging (0.5 um resolution) 3. SPR biosensor ▪ Bionavis 220A - 4-channel SPR for real time kinetics of interaction 4. Supporting services ▪ Immobilization/conjugation of biomolecules ▪ ELISA (Biotek Synergy 2) ▪ QCM biosensors ▪ Electrochemistry (Autolab) (Images by: Petr Skladal, bionavis.com, trendbio.com.au, innopsys.com) 48 Equipment 49 Conclusions BioAFM allows: ▪ Visualize objects (biomolecules to cells) in under near physiological conditions ▪ Mapping of Young’s modulus of immobilized biosamples ▪ Time lapsed changes of mechanical properties Future outlook ▪ BioAFM instrumentation improvement – CO2 chamber, improved in-situ sterility, etc. ▪ Optical part improvement (objectives, cameras) → overlay imaging ▪ Tissue related experiments (i.e. heart valves)