AFM Atomic Force Microscopy (Atomic) Force Microscopy Scanning Probe Microscopy (SPM) Rasterkraftmikroskopie A cell biologist s hands-on view Binnig, Quate, and Gerber 1986
Cellular interactions with the extracellular matrix (ECM) Carlos P. Huang, et al., Lab on a Chip 2
Cellular interactions with the extracellular matrix (ECM) ECM Ligands Structure Mechanical properties 3
AFM a versatile tool in cell biology High-resolution imaging of living cells 4 25.09.2015
AFM a versatile tool in cell biology High-resolution imaging of living cells Single-cell force spectroscopy 5 25.09.2015
AFM a versatile tool in cell biology High-resolution imaging of living cells Single-cell force spectroscopy Elasticity measurements of cells and matrix 6 25.09.2015
Atomic force microscopy in cell biology Easy sample preparation: No staining No cutting No coating No fixation No drying Imaging under physiological conditions! 7
Atomic force microscopy imaging 8
Atomic force microscopy imaging Easy sample preparation: No staining No cutting No coating No fixation No drying y = slow x = Fast Imaging under physiological conditions! 9
10 0.1 mm
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Combined AFM and optical microscopy www.jpk.de Light microscopy AFM Temperature control BioCell
Cell-Matrix Adhesion 2D 3D cell-matrix cell-substrate
Formation of highly-ordered and parallel collagen I microfibrils on mica surfaces 400nm The matrices are ~ 3 nm high Stamov et al. UM 2015 Franz and Muller, MMB 2011 Cisneros et al. Small 2007 Friedrichs et al. JMB 2007 14
Collagen fibrils are highly ordered in the corneal stroma http://www.uni-mainz.de/fb/medizin/anatomie/workshop/em/emkollagen.html http://www.uni-mainz.de/fb/medizin/anatomie/workshop/em/emkollagen.html
Directional cell migration on nanostructured collagen matrices 100 nm AFM image of a nanostructured collagen matrix Corneal cells migrate directionally on the collagen matrix Ulbrich et al., Cells Tissues Organs 2011 Gruschwitz et al., Invest Ophthalmol Vis Sci 2010 16
Anisotropic deformation of the collagen matrix Early matrix deformation SEM Late matrix deformation Friedrichs et al. J Mol Biol 2007 17
Time-lapse AFM imaging of living cells Frame rate 4 min 18
Live cell timelapse AFM imaging of the cell periphery REF-52 (rat embryonic fibroblasts) Contact mode 10 x 50 µm 2-5 Hz ~2-4 min/frame
Matrix entanglement Deflection Height 3D reconstruction 20
Inverting adherent cells for visualizing ECM interactions at the basal cell side Gudzenko and Franz, Ultramicroscopy 2013 21
Inverting adherent cells for visualizing ECM interactions at the basal cell side Gudzenko and Franz, Ultramicroscopy 2013 22
Inverting cells and associated matrix proteins 23
Nanoscale matrix interactions at the basal cell side influence cell morphology 24
Proteoglycans - regulators of collagen fibrillogenesis Decorin -/- Decorin +/+ Danielson et al. JCB 1997 Kalamaiski et al. Matrix Biology 2010 25
Proteoglycans - regulators of collagen fibrillogenesis Decorin Collagen Weber et al JBS 1996 Kalamaiski et al. Matrix Biology 2010 26
Proteoglycans - regulators of collagen fibrillogenesis Core protein Core protein + GAG Kalamaiski et al. Matrix Biology 2010 Gautieri et al. Matrix Biology 2012 27
Glycosaminoglycan-dependent spacing of collagen fibrils Decorin w/o GAG chain 1:100 1:10 1:10 1:1 Decorin + GAG chain 1:200 1:20 1:10 1:1 Stamov et al. JSB 2013 28
29 25.09.2015 Prof. Max Mustermann - Präsentationstitel
Lumican bundles collagen I fibrils Stamov et al., JSB 2013 30
AFM-based single-cell force spectroscopy
AFM-based single-cell force spectroscopy (SCFS) I. cell-substrate adhesion II. cell-cell adhesion 32
AFM-based Single-Cell Force Spectroscopy cell-substrate adhesion - Maximal detachment force F max - Detachment work W - Single unbinding steps 33
The cellular integrin receptor repertoire matches the matrix composition of target tissues Tissue-specific ECM composition Tissue-specific integrin repertoire 34
The cellular integrin receptor repertoire matches the matrix composition of target tissues Pharmaworld 35
Producing bifunctional collagen/laminin adhesion substrates Dao et al., ECR 2012 Dao et al., JMR 2013 36
Studying differential adhesion using bifunctional adhesion substrates Dao et al., ECR 2012 Dao et al., JMR 2013 37 25.09.2015 Clemens Franz - FOR 1756 Retreat
Cell 1 Cell 2 Cell 3
Studying differential adhesion using bifunctional adhesion substrates Differential adhesion profiles of single cells Dao et al., ECR 2012 Dao et al., JMR 2013 39 25.09.2015 Clemens Franz - FOR 1756 Retreat
Differential spreading controlled by relative but not absolute differences in adhesion Dao et al., ECR 2012 Dao et al., JMR 2013 40
AFM-based (two) single-cell(s) force spectroscopy (SCFS) Gonnermann et al. IB 2015 41
SCFS cell-cell adhesion force spectroscopy - Maximal detachment force F max - Single unbinding steps Overall cell adhesion (F max ) Individual adhesion clusters (rupture force steps) Gonnermann et al. IB 2015
SCFS measuring cadherin receptors in their native environment artificial cadherin adhesion substrate chemically homogeneous defined molecular interactions
Covalent and density-controlled cadherin immobilization using the SNAP-tag O 6 -benzylguanine matrix thiol Engin et al., Langmuir, 2010 Fichtner et al., PLOS One 2014
Covalent and density-controlled cadherin immobilization using the SNAP-tag Extracellular cadherin domains 1-5 Engin et al., Langmuir, 2010 Fichtner et al., PLOS One 2014
Covalent and density-controlled cadherin immobilization using the SNAP-tag 1. Covalent 2. Quantitative 3. Orientated Optimal receptor spacing ~5-11 nm Engin et al., Langmuir, 2010 Fichtner et al., PLOS One 2014
Quantitating cell elasticity AFM indentation measurements 47
Cell nucleus deformation during tissue invasion 48
Lamins as regulators of nuclear stiffness Greiner et al. Biomaterials 2014
Circus Movement Membrane Blebbing top view side view Neural crest cells (Xenopus l.) 50
AFM force spectroscopy and an optical sideview setup
Testing Dynamic Membrane Blebbing 52
bleb event non bleb event bleb/non bleb transition 53 25.09.2015
Heart beat Force of a single cardiomyocyte? 54
Fabricating micro-structured cell culture substrates by direct laser writing (DLW) Nanoscribe 55
The Karlsruhe Wheel M. Wegener M. Bastmeyer Schloss und Stadt Karlsruhe Copper engraving Christian Thran, 1739 56
Flexible 3D-substrates produced by Direct Laser Writing Klein et al., Adv. Mater. 2010 Klein et al., Adv. Mater. 2011 57
Testing the stiffness of flexible cell culture substrates using AFM indentation measurements 0.8 µm Cellular contraction forces: ~ 40 to 60 nn 58
Thanks to Lu Dao Tetyana Gudzenko Anna Burcza Carina Gonnermann Dimitar Stamov Hella Baumann Anna Müller Bianca Schatz Ramona Ring Martin Bastmeyer Doris Wedlich Andreas Janshoff Martin Wegener Karine Anselme Stephane Brezillion Daniel Müller Pierre-Henri Puech Anna Taubenberger Jens Friedrichs David Cisneros Tomaso Zambelli FOR 1756 Dynamics of cell cell contacts 59