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Quantitative imaging of living biological samples by Peak Force Tapping atomic force microscopy Alexandre Berquand, Bruker Nano, August 17 2011 Why force measurements are essential in biology? Mechanical properties of cells are determined


  1. Quantitative imaging of living biological samples by Peak Force Tapping atomic force microscopy Alexandre Berquand, Bruker Nano, August 17 2011

  2. Why force measurements are essential in biology? • Mechanical properties of cells are determined by the dynamic behavior of their cytoskeleton. • Alterations of the mechanical phenotype of the cell can lead to severe malfunctions or disease (cancer, malaria, neurodegeneration). • Cancer cells are known to be softer than their normal homologues. • AFM is the tool of choice to measure cells mechanical properties ex vivo and to correlate a change in mechanical properties with: • Drug treatment • Aging • Pathology 8/17/2011 BRUKER CONFIDENTIAL 2

  3. AFM under physiological conditions • Different types of perfusion systems to keep cells alive for a non-limited period of time: Perfusing Stage Incubator Regular fluid cell

  4. Tapping Mode and Phase imaging  depends on AFM parameters, surface and volume properties • The phase shift just reflects the energy dissipated but is a contribution of several factors and is not quantitative 8/17/2011 BRUKER CONFIDENTIAL 4

  5. Force Spectroscopy 2 Stiffness (Young’s force (nN) modulus) 1 0 Adhesion -1 500 0 distance (nm) Single force Force volume • Main drawbacks: slow, poor resolution and lack of information 8/17/2011 BRUKER CONFIDENTIAL 5

  6. Peak Force Tapping - principle • Works with most standard AFM probes in the standard AFM cantilever holders. • Z piezo is driven with sinusoidal waveform (not a triangle as in force-distance curves). • Z drive frequency is 2 kHz (Catalyst 1 kHz). That’s far below the cantilever’s resonance. • Z drive amplitude is fixed at typical value of 150 nm (300 nm peak-to-peak) • Vertical motion of probe produces force- distance plots as it taps on the sample. • Imaging feedback is based on the Peak Force of the force-distance curve. 8/17/2011 BRUKER CONFIDENTIAL 6

  7. Peak Force Tapping - features • SCANASYST: • uses automatic image optimization technology • simplifies and speeds up expert-level image acquisition • PEAKFORCE QNM: • generates quantitative maps of nanoscale material properties • does this simultaneously during imaging at consistently low force and high resolution • Data extraction: 8/17/2011 BRUKER CONFIDENTIAL 7

  8. PeakForce QNM - Calibration • Relative method • Calculate the defl. Sens. • Calculate the spring constant • Image a ref. sample and adjust the tip radius • Adjust the deformation • Absolute method • Calculate the defl. Sens. • Calculate the spring constant • Image a tip check sample and measure the tip radius

  9. PeakForce QNM - Modulus measurement Choose probe type according to range of expected modulus  Requirements:  Probe needs to deform sample (minimum: a few nm)  Probe needs to be deflected by sample (minimum a few nm)  8/17/2011 BRUKER CONFIDENTIAL

  10. Typical example: DNA • PeakForce QNM works in both air and liquid • Relevant and quantitative contrast on all the channels • Applications in liquids have not been as thoroughly explored: • DNA, most of polymers: OK • Cells? 2: Elasticity 3: Adhesion

  11. Any compromise between measurement of mechanical properties and resolution? Simon Scheuring , Physico-Chimie Institut Curie , ScanAsyst lever, 0.4 N/m ) Scale bar 10 nm Scheuring et al Eur Biophys J (2002) 8/17/2011 BRUKER CONFIDENTIAL 11

  12. Sea water samples: imaging of frustules 1 st time that such sample is imaged by AFM • • Very detailed contrast in Young’s modulus and deformation 8/17/2011 BRUKER CONFIDENTIAL 12

  13. Sea water samples: imaging of diatoms • First image of living diatoms with PFT and PFQNM. • YM of different parts: • Fibulae ~200 MPa • Silica stripes ~44 MPa • Core matrix ~21 MPa • … Under press (Journal of Phycology)

  14. Imaging of E. coli K12 • Strain very hard to image by AFM because they move very fast when under stress • b: 3d-height (10x10  m) image of a necklace of living k12 acquired in 20 min. • DMT modulus image of the same bacteria. Average Young’s modulus = 183 kPa 8/17/2011 BRUKER CONFIDENTIAL 14

  15. PFQNM study on human glioblastoma U251-MG cells (invasive) 1 st site-specific 2 nd site-specific recombination: recombination: Integration of expression vector Empty vector + GFP as which carries the gene of interest, integration site inside the GFP site Selection of cells having integrated the vector Test with TP53 and PTEN Possibly have ≠ mechanical properties

  16. PFQNM High Resolution images on glioblastoma - display 2 channels simultaneously 40x40 µm PF error image 3d-height + deformation skin 8/17/2011 BRUKER CONFIDENTIAL 16

  17. PFQNM Low Resolution images on glioblastoma - statistics Topography (z: 0-250 pN) Elasticity (z: 0-1.2 MPa) Adhesion (z: 0-800 pN) • 128x128 images (5 min per image): averaging on a high number of images • Highly quantitative • No damage of the sample Deformation (z: 0-250 nm)

  18. Results & Conclusion Young’s modulus (kPa) Elasticity (kPa) 140 120 100 80 60 40 20 0 TP53 and PTEN induced are Ctrl IND Ctrl non- tp53 non- tp53 IND pTEN non- pTEN IND IND IND IND significantly stiffer and less deformable than the other Deformation (nm) Deformation (nm) cell types 250 200 150 100 50 0 Ctrl IND Ctrl non-IND tp53 non- tp53 IND pTEN non- pTEN IND IND IND

  19. Imaging of living HaCaT and effect of Glyphosate Cell under stress: [Glyphosate] retracting & increase of YM by synthesizing stress factor 3 fibers Adhesion much Average dissipation = 1.3 keV = 2.10 -16 J higher between the cells than on the cells 8/17/2011 BRUKER CONFIDENTIAL 19

  20. MIRO: Overlay optical and AFM data in a few clicks 3) Overlay optical and AFM 1) Import optical image into 2) Target a location for the images Nanoscope AFM scan Hela HaCaT

  21. Combining MIRO and PFQNM • a: overlay of fluorescence (nucleus + actin) and AFM (PF error + YM) images. • b: PF error channel: 0-450 pN • c: YM channel: 0-4 MPa • d: deformation channel: 0-250 nm • Offers nice perspectives in biology: correlate fluorescence and AFM signals simultaneously in response to drug treatment 8/17/2011 BRUKER CONFIDENTIAL 21

  22. Typical samples and corresponding probes - Summary Calibration of Young’s Modulus by Gelatin or Agarose: ~1 to 100 kPa 8/17/2011 BRUKER CONFIDENTIAL 22

  23. Conclusions • Since its development, Peak Force Tapping and PeakForce QNM have greatly improved to extend the range on biological samples • Though it’s still not 100% quantitative for the softest samples, a very wide range of applications can be covered • We are still working on expanding the range… • Promising possibilities for recognition mapping with functionalized probes (still confidential) 8/17/2011 BRUKER CONFIDENTIAL 23

  24. New Application Note released… 8/17/2011 BRUKER CONFIDENTIAL 24

  25. Acknowledgements (sample providers) • Vesna Svetlicic, Tea Radic and Galja Pletikapic (Rudjer Boskovic Institute, Zagreb, Croatia) • Gregory Francius (LCPME, Nancy, France) • Andreas Holloschi, Leslie Ponce, Ina Schaeffer, Hella-Monika Kuhn, Petra Kioshis and Mathias Hafner (University of Applied Sciences, Mannheim, Germany) • Laurence Nicod, Celine Caille and Celine Heu (Institut FEMTO-ST, Besancon, France)

  26. Contact information • alexandre.berquand@bruker-nano.com +49 174 333 94 62 +49 621 842 10 66 • Contact email for Sales and Support ProductInfo@bruker-nano.com • Webinar Series www.bruker-axs.com/atomic-force-microscopy-webinar-series • PeakForce QNM www.bruker-axs.com/PeakForceQNM • ScanAsyst www.bruker-axs.com/ScanAsyst • BioScope Catalyst www.bruker-axs.com/bioscope-catalyst-atomic-force-microscope • Bruker NanoScale World Forum – Share, discuss, and learn about everything nano http://nanoscaleworld.bruker-axs.com/nanoscaleworld/

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