AFM. Atomic Force Microscopy. (Atomic) Force Microscopy. Scanning Probe Microscopy (SPM) Rasterkraftmikroskopie. A cell biologist s hands-on view

Transcription

1 AFM Atomic Force Microscopy (Atomic) Force Microscopy Scanning Probe Microscopy (SPM) Rasterkraftmikroskopie A cell biologist s hands-on view Binnig, Quate, and Gerber 1986

2 Cellular interactions with the extracellular matrix (ECM) Carlos P. Huang, et al., Lab on a Chip 2

3 Cellular interactions with the extracellular matrix (ECM) ECM Ligands Structure Mechanical properties 3

4 AFM a versatile tool in cell biology High-resolution imaging of living cells

5 AFM a versatile tool in cell biology High-resolution imaging of living cells Single-cell force spectroscopy

6 AFM a versatile tool in cell biology High-resolution imaging of living cells Single-cell force spectroscopy Elasticity measurements of cells and matrix

7 Atomic force microscopy in cell biology Easy sample preparation: No staining No cutting No coating No fixation No drying Imaging under physiological conditions! 7

8 Atomic force microscopy imaging 8

9 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 mm

11 11

12 Combined AFM and optical microscopy Light microscopy AFM Temperature control BioCell

13 Cell-Matrix Adhesion 2D 3D cell-matrix cell-substrate

14 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

15 Collagen fibrils are highly ordered in the corneal stroma

16 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

17 Anisotropic deformation of the collagen matrix Early matrix deformation SEM Late matrix deformation Friedrichs et al. J Mol Biol

18 Time-lapse AFM imaging of living cells Frame rate 4 min 18

19 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

20 Matrix entanglement Deflection Height 3D reconstruction 20

21 Inverting adherent cells for visualizing ECM interactions at the basal cell side Gudzenko and Franz, Ultramicroscopy

22 Inverting adherent cells for visualizing ECM interactions at the basal cell side Gudzenko and Franz, Ultramicroscopy

23 Inverting cells and associated matrix proteins 23

24 Nanoscale matrix interactions at the basal cell side influence cell morphology 24

25 Proteoglycans - regulators of collagen fibrillogenesis Decorin -/- Decorin +/+ Danielson et al. JCB 1997 Kalamaiski et al. Matrix Biology

26 Proteoglycans - regulators of collagen fibrillogenesis Decorin Collagen Weber et al JBS 1996 Kalamaiski et al. Matrix Biology

27 Proteoglycans - regulators of collagen fibrillogenesis Core protein Core protein + GAG Kalamaiski et al. Matrix Biology 2010 Gautieri et al. Matrix Biology

28 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

29 Prof. Max Mustermann - Präsentationstitel

30 Lumican bundles collagen I fibrils Stamov et al., JSB

31 AFM-based single-cell force spectroscopy

32 AFM-based single-cell force spectroscopy (SCFS) I. cell-substrate adhesion II. cell-cell adhesion 32

33 AFM-based Single-Cell Force Spectroscopy cell-substrate adhesion - Maximal detachment force F max - Detachment work W - Single unbinding steps 33

34 The cellular integrin receptor repertoire matches the matrix composition of target tissues Tissue-specific ECM composition Tissue-specific integrin repertoire 34

35 The cellular integrin receptor repertoire matches the matrix composition of target tissues Pharmaworld 35

36 Producing bifunctional collagen/laminin adhesion substrates Dao et al., ECR 2012 Dao et al., JMR

37 Studying differential adhesion using bifunctional adhesion substrates Dao et al., ECR 2012 Dao et al., JMR Clemens Franz - FOR 1756 Retreat

38 Cell 1 Cell 2 Cell 3

39 Studying differential adhesion using bifunctional adhesion substrates Differential adhesion profiles of single cells Dao et al., ECR 2012 Dao et al., JMR Clemens Franz - FOR 1756 Retreat

40 Differential spreading controlled by relative but not absolute differences in adhesion Dao et al., ECR 2012 Dao et al., JMR

41 AFM-based (two) single-cell(s) force spectroscopy (SCFS) Gonnermann et al. IB

42 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

43 SCFS measuring cadherin receptors in their native environment artificial cadherin adhesion substrate chemically homogeneous defined molecular interactions

44 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

45 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

46 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

47 Quantitating cell elasticity AFM indentation measurements 47

48 Cell nucleus deformation during tissue invasion 48

49 Lamins as regulators of nuclear stiffness Greiner et al. Biomaterials 2014

50 Circus Movement Membrane Blebbing top view side view Neural crest cells (Xenopus l.) 50

51 AFM force spectroscopy and an optical sideview setup

52 Testing Dynamic Membrane Blebbing 52

53 bleb event non bleb event bleb/non bleb transition

54 Heart beat Force of a single cardiomyocyte? 54

55 Fabricating micro-structured cell culture substrates by direct laser writing (DLW) Nanoscribe 55

56 The Karlsruhe Wheel M. Wegener M. Bastmeyer Schloss und Stadt Karlsruhe Copper engraving Christian Thran,

57 Flexible 3D-substrates produced by Direct Laser Writing Klein et al., Adv. Mater Klein et al., Adv. Mater

58 Testing the stiffness of flexible cell culture substrates using AFM indentation measurements 0.8 µm Cellular contraction forces: ~ 40 to 60 nn 58

59 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