Quantitative imaging of living biological samples by Peak Force - - PowerPoint PPT Presentation

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?

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• 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

AFM under physiological conditions

• Different types of perfusion systems to keep cells alive for a non-limited

period of time: Regular fluid cell Perfusing Stage Incubator

Tapping Mode and Phase imaging

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• The phase shift just reflects the energy dissipated but is a contribution of

several factors and is not quantitative

 depends on AFM

parameters, surface and volume properties

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Force Spectroscopy

• Main drawbacks: slow, poor resolution and lack of information

distance (nm) force (nN)

1 2

• 1

500

Single force Force volume Stiffness (Young’s modulus) Adhesion

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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
• f 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.

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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:

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

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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)

2: Elasticity 3: Adhesion

• 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?

Typical example: DNA

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Simon Scheuring, Physico-Chimie Institut Curie ,

ScanAsyst lever, 0.4 N/m)

Scale bar 10 nm Scheuring et al Eur Biophys J (2002)

Any compromise between measurement of mechanical properties and resolution?

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Sea water samples: imaging of frustules

• 1st time that such sample is imaged by AFM
• Very detailed contrast in Young’s modulus and deformation

• 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)

Sea water samples: imaging of diatoms

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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

PFQNM study on human glioblastoma

U251-MG cells (invasive) 1st site-specific recombination: Empty vector + GFP as integration site Selection of cells having integrated the vector 2nd site-specific recombination: Integration of expression vector which carries the gene of interest, inside the GFP site

Test with TP53 and PTEN Possibly have ≠ mechanical properties

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PFQNM High Resolution images on glioblastoma - display 2 channels simultaneously

40x40 µm PF error image 3d-height + deformation skin

Topography (z: 0-250 pN) Elasticity (z: 0-1.2 MPa) Adhesion (z: 0-800 pN) Deformation (z: 0-250 nm)

• 128x128 images (5 min per image): averaging on a

high number of images

• Highly quantitative
• No damage of the sample

PFQNM Low Resolution images on glioblastoma - statistics

Elasticity (kPa) 20 40 60 80 100 120 140 Ctrl IND Ctrl non- IND tp53 non- IND tp53 IND pTEN non- IND pTEN IND Deformation (nm) 50 100 150 200 250 Ctrl IND Ctrl non-IND tp53 non- IND tp53 IND pTEN non- IND pTEN IND

Young’s modulus (kPa) Deformation (nm)

TP53 and PTEN induced are significantly stiffer and less deformable than the other cell types

Results & Conclusion

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Imaging of living HaCaT and effect

• f Glyphosate

Cell under stress: retracting & synthesizing stress fibers [Glyphosate] increase of YM by factor 3 Adhesion much higher between the cells than on the cells Average dissipation = 1.3 keV = 2.10-16 J

MIRO: Overlay optical and AFM data in a few clicks

3) Overlay optical and AFM images 1) Import optical image into Nanoscope 2) Target a location for the AFM scan

Hela HaCaT

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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

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Typical samples and corresponding probes - Summary

Calibration of Young’s Modulus by Gelatin or Agarose: ~1 to 100 kPa

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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)

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New Application Note released…

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)

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

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www.bruker-axs.com/atomic-force-microscopy-webinar-series

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www.bruker-axs.com/PeakForceQNM

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www.bruker-axs.com/ScanAsyst

• BioScope Catalyst

www.bruker-axs.com/bioscope-catalyst-atomic-force-microscope

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