Advanced biophysical methods for characterization of biosamples Atomic Force Microscopy of biosamples Jan PŘIBYL CEITEC MU, Core Facility NanoBio Masaryk university, Brno, Czech Republic E-mail: jan.pribyl@ceitec.muni.cz CEITEC at Masaryk University 2 Content ▪ AFM, BioAFM technology ▪ AFM operation modes: Imaging, Indentation, … ▪ Examples ▪ Core Facility NanoBiotechnology CEITEC at Masaryk University 3 Introduction Bio Atomic Force Microscopy (BioAFM) 4 by OverloardQ IMAGING Proteins, DNA, Nanoobjects ▪ Optical (fluorescence) microscopy ▪ Multielectrode Array (MEA) ▪ FluidFM AFFINITY INTERACTION NANOINDENTATION Stiffness mapping Combination with other techniques Jak funguje AFM CEITEC at Masaryk University 5 AFM (Atomic Force Microscope) ▪ Z rodiny tzv. Probe microscopies = skenující próbou ▪ Posun hrotu nad vzorkem = topografie povrchu ▪ Další vlastnosti vzorku (tuhost, adhezivita) z analýzy signálu AFM visualization of biomolecules and bioobjects Bare metal (gold) electrodesBacteria on glass slide SpermWhole cells (cell synapse)Individual biomolecules IgG Nanoparticles (gold nanoparticles BSA modified) DNA CEITEC at Masaryk University 7 AFM indentation ▪ AFM cantilever = nanosensor ▪ Force-distance curve (FDC) – describes interaction/deformation ▪ FDC = Young’s modulus, adhesivity, rigidity, etc. Imaging is not enough… Hyaluronan-myeloperoxidase complex myeloperoxidase Study by Stepan Solny Kashish Manchanda, et al. Arteriosclerosis, Thrombosis, and Vascular Biology. 2018;38:1859–1867 MPO (Myeloperoxidase) Reduces Endothelial Glycocalyx Thickness Dependent on Its Cationic Charge 9 Imaging hyaluronan myeloperoxidase + Molecular complex CEITEC at Masaryk University 10 FDC analysis Young’s modulus Experiment + FDC recording Tip (indenter) definition FDC fit CEITEC at Masaryk University 11 FDC automatic analysis Young’s modulus ▪ Force-Distance curves maps ▪ Typically, 32x32, 64x64 and more ▪ Automated FDC processing ▪ Results – stiffness maps, adhesivity maps, … 12 158,8 2809,1 0 500 1000 1500 2000 2500 3000 3500 4000 YM(kPa) FD curve recording and analysis Not too strong! Select the proper curve HA-MPO complex is 20x stiffer comparing to HA 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) Samples by group of Matthias Nowak, IST, Vienna Chromatin only Chains composed of ellipsoids Chromatin-BAF Granular structure, grains composed of fibers (?) (~17nm)SUMMARY + BAF CEITEC at Masaryk University 15 AFM as destructive sensor FDC, Rupture events ▪ PLB = PhosphoLipidic Bilayer (= cell membrane in-vitro) ▪ Force application = bilayer rupter (=rupture event) ▪ Z = PLB thickness ▪ F = PLB strength ▪ Difficult automatic analysis PLB = phospholipidic bilayer Hledání lokálních extrémů 16 Separation signal PLB thickness 3.7 nm Manual curve processing CEITEC at Masaryk University 18 Single-molecule force spectroscope (SMFS) Force dependency of biochemical reactions measured by single-molecule force-clamp spectroscopy Ionel Popa Pallav Kosuri Jorge Alegre-Cebollada Sergi Garcia-Manyes Julio M. Fernandez Nature Protocols June, 2013 http://biomechanicalregulation-lab.org/smfs CEITEC at Masaryk University 19 Protein immobilization ▪ Glass ▪ Gold: - Cysteine (SH-) tags - F ~ 1 nN → Ok for most of the unfolding forces ▪ Ni-NTA - F ~ 50 pN - OK for spectrin, ankyrin or C2 domains - NOT sufficient e.g., for Ig-domains - - - SiO2 P + + + + + + + + Adhesion force (= immobilization) Unfolding force Ionic interaction Adhesion force > Unfolding force CEITEC at Masaryk University 20 Probe (cantilever and tip) selection ▪ Gold-coated tip(few types on the market) ▪ Very soft (k < 0.01 N/m) ▪ Silicon Nitride material (typical for operations in liquid) ▪ Smaller is better (lower noise in liquid) CEITEC at Masaryk University 21 Probe (cantilever and tip) selection ▪ Olympus Biolever and Bruker MCLT-Au – most of publications ▪ Discontinued  CEITEC at Masaryk University 22 Probe (cantilever and tip) selection ▪ New cantilever types ▪ Ordered – to be tested JPK NanoWizard 3 Experimental settings ForceRobot measurement head Label-free, single molecule technique Measure forces between and within molecules. Characterize molecular and receptor-ligand interactions and protein (un)folding events. Target individual molecules. Experimental settings Modes of operation ▪ Force-clamp mode ▪ Protein folding/unfolding; Mechanisms of enzyme catalysis ▪ Force measurement with high speed of feedback gain – 0.1 to 5 milliseconds ▪ Typical settings: F = 50-500 pN, pulling speed 1 µm/s ▪ Length-clamp mode ▪ Difficult interpretation ▪ For multidomain proteins (native multi-domain proteins (such as titin, tenascin or spectrin) ▪ Typical settings: F = 50-500 pN, pulling speed 1 µm/s, no feedback driving Force-clamp mode “sawtooth” pattern Length-clamp mode “staircase” pattern Steps during polyprotein unfolding CEITEC at Masaryk University 25 Data processing JPK Data Processing software ▪ Predefined functions ▪ Batch processing of curves ▪ Data sorting Unfolding of artificial polyprotein GB1 Unfolding of individual titin domain Fitting of force-extension profile with worm-like fit Optical microscopy AFMConfocal microscopy Nanomechanical mapping of living cells 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? Young’s modulus (YM) 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). + Holographic Q-phase microscope Confocal vs. AFM Force Mapping on single cells A) B) C) D) A) B) C) D) Confocal microscopy ▪ DAPI nucleus staining ▪ Actin staining by Phalloidin NARDONE, Giorgia, Jorge Oliver-De La CRUZ, Jan VRBSKY, Cecilia MARTINI, Jan PRIBYL, et al., 2017. YAP regulates cell mechanics by controlling focal adhesion assembly. Nature Communications [online]. 8, ncomms15321. Cellular nanomechanics By means of AFM AFM mapping - correlation with fluorescence microscopy NARDONE, Giorgia, Jorge Oliver-De La CRUZ, Jan VRBSKY, Cecilia MARTINI, Jan PRIBYL, et al., 2017. YAP regulates cell mechanics by controlling focal adhesion assembly. Nature Communications [online]. 8, ncomms15321. GOLAN, Martin, et al. Front. Physiol., 29 June 2018 Timeline[h] 0 1 2 3 4 Evalutation - statistics Time-lapsed biomechanics Fibroblasts thawing process Time-lapsed monitoring of cell biomechanics SP height profile (12 hours) YM maps (12 hours) GOLAN, Martin, et al. IEEE transactions on nanobioscience 2018. 17(4), 485–497 SP = 1 nN, 100x100 m 2 NON IR IR 60 min 25% E rel *** NON IR IR 60 min 30% Erel SP = 0.4 nN, 10x10 m 2 *** ** High SP (2nN), 2.5um indent. Depth (35%) Low SP (0.35nN), 0.5um indent. Depth (6%) Cytoskeleton staining (Phalloidin/DAPI) stress fibers microfilaments Phenotypes in cells plated over night 0 50 100 150 NON IR IR NON IR IR b %ofcellswithphenotype 0 5 10 15 20 25 30 35 40 NON IR IR NON IR IR disrupted actin structure ring radial structure advanced radial structure linear stress fibers stress fibers with multiple microdomains cultured thawed a %ofcellswithphenotype Phenotypes in cells 25 min after plating cultured thawed Phenotypes in cells 25 min after plating GOLAN, Martin, et al. Front. Physiol., 29 June 2018 Timeline [h] 0 1 2 3 4 SiR live actin staining Černochová, P., Blahová, L., Medalová, J. et al. Cell type specific adhesion to surfaces functionalised by amine plasma polymers. Sci Rep 10, 9357 (2020). https://doi.org/10.1038/s41598-020-65889-y Cell-adhesion experiments www.jpk.com Relative adhesion of cells to plasma modified surfaces Other applications of AFM-based biomechanics Cell scratching = cell adhesion Pesl M, Pribyl J, et al. 2016 Biosensors and Bioelectronics 85 751–7 Pesl M, Pribyl J, et al. 2016 J Mol Recognit n/a-n/a Pesl M, Acimovic I, Pribyl J, et al. 2014 Heart Vessels 29 834–46 Cardiac cells biomechanics MCG = mechanocardiogramSetup scheme Other applications of AFM-based biomechanics Typical DFL vs. time curves recorded as a result of various drug treatment Base line (Thyroid medium) Isoproterenol (activator) Metoprolol (betablocker) Caffeine Pesl M, Acimovic I., Pribyl J., et. al. Heart Vessels 2013 Combination with other techniques CytoSurge Fluid FM module MultiElectrode Array Extracellular Cell Potential Cardiac cells and Neurons AFM + MEA = electromechanical (de)coupling NanoIndentation Single point indentation curves FluidFM – microfluidic force microscopy https://www.news-medical.net 2020/21 Innovations MEA – microelectrode array cellular electrophysiology Anal Bioanal Chem (2011) 399:2313–2329 CEITEC at Masaryk University 40 Raman imaging of amyloid deposits in snap-frozen Alzheimer’s disease human brain tissue Lochocki, B., Boon, B.D.C., Verheul, S.R. et al. Multimodal, label-free fluorescence and Raman imaging of amyloid deposits in snap-frozen Alzheimer’s disease human brain tissue. Commun Biol 4, 474 (2021). Raman microscopy On bio samples Calcification level and Collagen Fibers Arrangement in Bone Tissue + combination with AFM topography Tomasz Buchwald, et al. Spectroscopy 27(2):107-117 CEITEC at Masaryk University 41 Raman microscopy Chemical mapping + combination with AFM topography Nanoparticles loading study Fingerprinting Raman spectra by Marçal Gallemí Eva Benkova Lab & Jan Hejatko Lab Liver cirrhosis Correlation of Collagen fibers by polarized microscopy AFM nanoindentation AFM-based biomechanics On a tissue level Plant samples (hypocotyl) by Srikant Ojha Martin Gregor Lab CEITEC at Masaryk University 43 Core Facility NanoBiotechnology Standard operations of the CF • BioAFM microscopy – biomolecules, nano-objects • Stiffness mapping – cells, tissues • Combination of AFM with other techniqeus (BF/fluorescence microscopy) 2020/21 Innovations • FluidFM – microfluidic force microscopy • Raman chemical mapping • MEA – micro-electrode array cell electrophysiology 44 CEITEC at Masaryk University 45 Core Facility NanoBio CEITEC at Masaryk University 46 JPK NanoWizard 3 and 4 with extended scanning range + CytoSurge FluidFM module + Biosoft NanoIndenter BioAFM – living cells and tissues BioAFM – molecules, nanoobjects, molecular complexes Bruker Dimension Icon FastScan and MultiMode 8HR NTMDT Ntgra Vita Raman microscopy, SPR affinity biosensor, Upconverting particles UCNP reader Renishaw InVia Raman microscope Bionavis SPR biosensor device Labrox UPCON reader Bioelectrochemistry, Cellular electrophysiology Autolab Modular potentiostat MultiChannel MEA2100Lite CEITEC at Masaryk University 51 Technology and Expertise List of services FULL SERVICE / MEASUREMENT only / DATA PROCESSING only 1. Cells – mechanical properties 2. Cells - imaging 3. Biomolecules - imaging 4. Nano-objects imaging 5. Raman-AFM combined microscopy 6. Raman microscopy 7. Electrochemical measurements 8. Nanodeposition system 9. SPR biosensor 10. Scanning of upconversion luminescence 11. Multielectrode array recording of cellular potential CEITEC at Masaryk University 52 User Training ▪ 2019 – 2021: 6 workshops ▪ Over 200 participants ▪ Workshop content shared online – youtube, Data Storage Workshop title Date Main objectives No of participants Atomic Force Microscopy (AFM) for Bio Applications April 16-17, 2019 Combined characterization of biosamples by AFM, practical applications, hands-on session 20 Characterization of nanoparticles and proteins by Atomic Force Microscopy July 30-31, 2019 Characterization of nano-objects and proteins by AFM, practical applications, hands-on session 25 Spring Workshop on BioAFM Microscopy April 6-8, 2020 Theoretical background and new aspects of bio-AFM microscopy, sample preparation, hands-on session, social event Canceled Introduction to Raman microscope Renishaw inVia June 23rd, 2020 Introducing the Raman microscope Renishaw inVia, User samples characterization 12 (Bio) Atomic Force Microscopy (bioAFM), Basic Course October 1st, 2020 Basics of AFM, Sample preparation techniques, Data processing 80 Introduction to JPK NanoWizard 4 AFM microscope May 4th, 2021 Introduction to a new JPK NanoWizard AFM system combined with CytoSurge and NanoIndentor module 10 CEITEC at Masaryk University 53 User Survey CEITEC at Masaryk University 54 Technology offers for industrial partners Cooperation with industrial partners (http://industry.ceitec.cz/), Daniela Tršová manages this topic. Bio-AFM microscopy imaging and biomechanical studies AFM microscopy (structure and mechanical properties) of bio-samples (biomolecules, cells, tissues) under semiphysiological conditions (37 oC, liquid media). ▪ Raman microscopy of biosamples Raman mapping of biosamples (molecular complexes, cells, tissues) with high resolution (~ 500 nm) ▪ Drug testing on cardiac cells Biomechanical (bioAFM) and electrical field potential signal as a response to drug exposition. Human stem cells and/or primary animal cells can be used. ! Coordination with Vladimir Rotrekl RG – essential!!! ▪ Tuneable hydrogel system The new system of stable and Robust biocompatible hydrogel system with tunable mechanical properties. ! Coordination with Vladimir Rotrekl RG – essential!!! CEITEC at Masaryk University 55 Booking system Under development for last 2 years.. CEITEC at Masaryk University 56 Data Sharing ▪ Medium Storage of MU – complicated for external users ▪ OneDrive – limited space to 1 TB ▪ IT manager missing Sharing of ▪ Data ▪ Workshop content ▪ Software ▪ Guides CEITEC at Masaryk University 57 CF involvement in the Correlative Microscopy project Integration of our CF in the project correlative microscopy has been proposed in the Strategic Plan of the Central European Institute of Technology at Masaryk University for 2021-2028, section "At the frontiers of technology: Correlative approaches to connect dynamics and structure of living systems." Correlative AFM-STED microscopy highlight a new and essential aspect and generate a warning: Fluorescence techniques cannot characterize all the products derived from the in vitro aggregation of misfolded proteins. Therefore, the combination of microscopic techniques brings a better understanding of the physiological processes. Mechanical properties (AFM), calcium levels (fluorescent dyes), and cellular potential (MEA) – mechano-physical-electrical coupling "Mikroskopie" (= Microscopy) educational project and in the LLL remote training and propagation project started in the time of the corona crisis. CEITEC at Masaryk University 58 High-Resolution Correlative Microscopy: Bridging the Gap between Single Molecule Localization Microscopy and Atomic Force Microscopy P.D. Odermatt et al., Nano Lett. 2015, 15, 8, 4896–4904 F-Actin fibers study CEITEC at Masaryk University 59 Let's all the measurements end up with this... ▪ BioAFM microscopy - more than just imaging method ▪ Easy combination with other methods ▪ Sample range from molecules to tissue slices ▪ Mechanobiology - information related to the pathophysiology Conclusions ▪ Petr Skládal – FS MU ▪ J. Víteček, L. Kubala – IBP Brno ▪ M. Samwer, IMBA Wien ▪ G. Forte, G. Nardone – ICRC Brno ▪ M. Pesl, V. Rotrekl, S. Jelinkova, … - MU Brno ▪ I. Kratochvilova – IF CAS, Prague ▪ E. Benkova, M. Gallemi – IST Wien Acknowledgment Acknowledgment text - CIISB • Preferred version: „CIISB, Instruct-CZ Centre of Instruct-ERIC EU consortium, funded by MEYS CR infrastructure project LM2018127, is gratefully acknowledged for the financial support of the measurements at the CF Nanobiotechnology.“ • Short version: „We acknowledge CF Nanobiotechnology of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2018127).“​​​​​​​ CEITEC at Masaryk University 62 OP VVV CZ.02.1.01/0.0/0.0/18_046/0015974 (LM2018127) Thank you for your attention!