Nanomechanical AFM measurements on biological samples

By Alex Berquand (Bruker Nano)

What’s behind “cell mechanics” and why is it so important in biology? Complexity of signal transduction in cells

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A B

C

• A, B and C have different stiffness and contain various molecules.

• Those molecules are associated to the inner components of the cell.

• They can regulate normal functions or lead to imbalance/disease.

• Identifying A, B and C and their components is of great importance.

• Nature of the scaffold: (same stem cells)

different cell types

• Tilt: (same stem cells and same substrate)

different cell types

• AFM and fluorescence microscopy can be used.

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What’s behind “cell mechanics” and why is it so important in biology? Stiffness in Cell differentiation

or or 1 kPa 0.1 kPa 0.01 kPa

or or

0º 5º 10º or or or or

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Concrete example Cancer: why is sensing differences in elasticity important?

• Cancer cells are usually softer than their counterparts, especially in the case of bladder (Lekka et al. 1999), prostate (Faria et al. 2008), breast (Cross et al. 2007), cartilage (Darling et al. 2007), blood (Rosenbluth et al. 2006) and ovarian (Sharma et al. 2011) tissues.

• Some organs are not permeable to antibiotics.

• Cancer surgery is often highly risky.

• Identifying differences in elasticity at an early stage is required. Possible by AFM on the corresponding cells lines.

- Lekka M. and Laidler P., Nat. Nanotechnol. 72 (2009) 72-73. - Faria E. C. et al., Analyst 133 (2008) 1498-500. - Cross S.E. et al. Nat. Nanotechnol. 2 (2007) 780-783. - Rosenbluth M.J. et al., Biophys. J. 90 (2006) 2994-3003. - Sharma S. et al., Nanomed. Nanotechnol. Biol. Med. (2011) under press.

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Usual tools to probe cell mechanics Major techniques

• Micropipette aspiration (HochMuth, 2000).

• Acoustic wave microscopy (Hildebrand 2001).

• Bio-imprinting (Dickert et al. 2002).

• Optical tweezers / optical traps (Dao et al. 2003).

• AFM (Force Spectroscopy) (Rotsch et al 2000).

- HochMuth R.M., J.Biomech 33 (2000) 15-22. - Hildebrand J.A. et al., PNAS 78 (1981) 1656-1660. - Dickert J.L. et al., Anal. Chem. 74 (2002) 1302-1306. - Dao M. et al., Mech. Phys. Sol. 51 (2003) 2259-2280. - Rotsch C. et al, Biophys J. 78 (2000) 520-535.

Principle of AFM Optical detection system

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Different feedbacks for different AFM modes

atoms

molecules bacterioΦ chloroplasts bacteria cells

0.1nm 1nm 10nm 100nm 1µm 10µm 100µm

AFM

light microscopy

EM

AFM Resolution Compared to other microscopy techniques

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Combining AFM to Fluorescence 2 techniques in 1 tool

AFM Optics

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AFM + …

Sample courtesy Zeiss

Sample courtesy NIH

Sample courtesy SWMC Stanford

Sample courtesy INRA Nantes

Sample courtesy Nat. Univ. Singapore

Sample courtesy Univ. of New South Wales

Combining AFM to IOM Compatibility with various optical techniques

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3) Overlay optical and AFM images

1) Import optical image into Nanoscope

2) Target a location for the AFM scan

Combining AFM to fluorescence Automatic Overlay (MIRO)

1) Capture 3 sample images at 3 different locations.

2) Capture 1 tip image.

3) Capture a background image and overlay it with an AFM image.

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Combining AFM to fluorescence Automatic Overlay (MIRO)

Live Hela cells

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distance (nm)

forc

e (n

N)

0

1

2

-1 500 0

Force Spectroscopy Get access to stiffness and adhesion

Hertz DMT JKR

MD

Sneddon

(Most adapted for biological samples)

Standard for Peak Force QNM

Contact theories in AFM Different models / samples

Hela cells OS cells

FV/Fluo Applications in Biology CSK disrupting agents

• Nocodazole can disrupt Tubulin CSK.

• Latrunculin can disrupt Actin CSK.

• Tested on 2 cell types in fluorescence, AFM imaging, AFM FV.

Effect of the 2 drugs:

1) decay of fluorescence

2) loss of tracking

3) change in elasticity

Actin plays a much more important role in cell rigidity than tubulin.

Too slow (low res.) + not enough information.

tubulin

actin

Nocodazole Latrunculin

0

z

x

z

x

FV/Fluo Applications in Biology CSK disrupting agents

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Popular AFM techniques Are they quantitative?

• Force measurements: Force Volume • Is it quantitative?

• What does it provide?

• Imaging: Tapping mode • Is it quantitative?

• What does it provide?

ϕ ϕ

Not Quantitative!

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FV to slow to probe biological processes? True for most of them

• Examples:

• Diffusion of proteins: 10-5-10-9 cm2.s-1.

• Polymerization of microtubules: sec-min.

• Protein translocation to nucleus (can be probed by fluorescence): 30 min to 1 h.

• Cell migration: µm/sec-µm/h.

• Cell division: 20 min to 3 days.

• Ideally: less than 10 min per AFM image. Impossible with FV or loss of resolution.

• Need for acceptable resolution to compare AFM and Fluorescence data (for instance: increasing number of STED publications).

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Resolution is important for biologists Example: STED

0

10

20

30

40

50

60

70

80

2007-2008 2009-2010 2011-2012

• Number of STED publications in Biology/Medicine research.

• AFM offers similar spatial resolution but is much slower.

• Need for higher resolution, higher force control and faster imaging to be complementary to fluorescence techniques.

Need for a new characterization technique Peak Force Tapping and Peak Force QNM

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PFT is based on ScanAsyst (fully Automated AFM)

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 1-2 kHz. 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.

The probe can be calibrated before the experiment so that all the channels are directly quantitative: PFQNM

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Needed range of Young’s moduli Example: Human Body

Overview: PeakForce QNM Basic Principle

• PeakForce Tapping is an oscillating technique that can be used to image a wide range of samples at a high resolution.

• The probe can easily be calibrated prior to the experiment. The technique is referred to as PeakForce QNM. In that case, all the recorded data are directly quantitative.

• Peak Force QNM can now be operated on live cells and can be used to test changes in morphology and any other property in response to various factors.

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5nm

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Preliminary test on a stiff sample FV/HMX/QNM comparison on a daphnia

100 µm Bright field image of Daphnia

(20x)

A

B

FV QNM A

B

C

A

• Daphnia scanned in FV, HMX and QNM modes (20 min per image).

• Young’s modulus calculated by using the 3 modes.

• QNM offers much higher resolution and information than FV. HMX returns intermediate results.

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Preliminary test on a stiff sample FV/HMX/QNM comparison on a daphnia

• QNM offers the best compromise in terms of resolution, ease-of-use and delivered information.

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Preliminary test on a soft sample. FV/QNM comparison on a cell

• 90x90 µm

• 5 min / image

FV (32x32) QNM (128x128)

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Preliminary test on a soft sample. FV/QNM comparison on a cell

FV QNM

144 curves

36 curves Export + post-processing

QNM image + pfc image

Processed area

Export + post-processing

Bearing Analysis

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Preliminary test on a soft sample. FV/QNM comparison on a cell

FV [Sneddon fit] = 10.9 MPa QNM (bearing Analysis) [Sneddon fit] = 21.72 MPa QNM (PFC+export) [Sneddon fit] = 18.42 MPa

• The 2 QNM results are very close.

• The FV result is different from the QNM results but the number of data point is also quite different + it’s hard to perform the analysis on exactly the same area.

• How about an analysis on higher scale (Bio experiment)?

Line plot Indentation

Multiple line plots

Modify force parameters Baseline correction

Boxcar filter Run History

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FV/QNM accuracy in Biology Study on glioblastoma

• Isogenic cell line U251 transfected with tumor suppressive factors.

• 5-6 min per image.

• 128x128 resolution.

• Enough to observe contrast on all mechanical channels.

PF error (z: 0-250 pN) Elasticity (z: 0-1.2 MPa) Deformation (z: 0-250 nm)

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FV/QNM accuracy in Biology Study on glioblastoma

• Same tendency (cancer cells softer than normal ones) but STD very different (number of data points: 589 824 for QNM / 9214 for FV; less than 10 min per image).

• Same remark for cell diffentiation, cell migration, cell remodeling,…

Submitted to JJAP

QNM study on live Hacats Effect of Glyphosate on Human Skin

• The Human skin is the first physiological barrier against physical and chemical aggressions.

• Glyphosate (N-(phosphonomethyl)Glycine) is a broad spectrum systemic herbicide used to kill weeds. It’s been extensively used in the late 2000’s.

• Its toxicity on lab animals has been clearly demonstrated but its effects on humans remain unclear.

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• Glyphosate-treated samples show a higher number of necrotic and apoptotic cells, and a higher [H2O2] than controls.

• Keratinocytes and HaCat cells are difficult to image by classical AFM modes. A more reliable technique is required.

• Need for a faster way to probe the oxidative stress. Changes in morphology and mechanical properties might be detectable by using PeakForce QNM.

Background: Glyphosate Existing Data in Cytology and Main Challenges

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[H2O2] marker

• Drug treatment induces a significant increase in Young’s modulus (x3) and a decrease in deformation (x2).

• Changes in mechanical properties can directly be correlated to changes in morphology.

PeakForce QNM: Much more information Probe changes in mechanical properties

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Journal of Structural Biology Publication January 2012

• Data was published in a paper entitled ‘Glyphosate-induced stiffening of HaCat keratinocytes, a Peak Force Tapping study on living cells’ in Journal of Structural Biology.

• The System: Bioscope Catalyst with PeakForce QNM and operated in fluid

• Authors are Celine Heu, Celine Elie and Laurence Nicod (FEMTO) and Alexandre Berquand (Bruker Nano GmbH)

Different Euk. cells: Diatoms Interest in Industry

• Diatoms are of interest in micro and nanoscale science for applications ranging from solar cells to optical systems.

• As a living vesicle, they also have potential for drug delivery.

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• Large scale Catalyst images remarkably stable.

• The Perfusing Stage Incubator accessory enables the active exchange of medium.

PeakForce QNM: Easier imaging of Live Diatoms in Seawater

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5 µm

150 mV

0 0

50 MPa

0

20 nm

Peak force Error Young’s modulus

Deformation 3d Height + YM skin

PeakForce QNM: Easier Imaging Mechanical Properties at High Resolution

• All PeakForce QNM channels are directly quantitative.

• Unprecedented resolution from imaging to modulus.

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Pletikapic et al., J. Phycol 48 (2012) 174.

• The frustule is a filtration wall allowing communication between the sea water and the inner part of the diatom.

• These are the first images of the frustle by AFM.

• Additionally, QNM channels provide quantitative mechanical properties, and show nice contrast between the pores, the rings & the rest of the frustule.

PeakForce QNM: Easier Imaging First AFM Investigation of the Frustule

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Bacteria with pili 3d-height (pili retracted) YM = 183 kPa

QNM on Live Bacteria E. Coli K12

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Correlating topography to Force curves HSDC files

Export + post-processing

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1 2

fibril

cell

AFM tip

Correlating topography to Force curves HSDC files

Young’s modulus calculated over very specific areas

Young’s modulus calculated over the entire cell

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Functionalized probes HSDC files - Detecting specific unbinding events

• Parasites enter bloodstream and infect RBCs.

• As parasites multiply, the RBCs breaks open and infects more RBCs.

• Infected RBCs are misshapen with knob-like structures on surface.

Combining QNM with functionalized probes - Pathogenesis of Malaria Erythrocyte (Red Blood Cell) Infection

P. falciparum

Anopheles mosquito Healthy RBC

Infected RBC

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Knobs

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Background: Cytoadherence Erythrocyte (Red Blood Cell) Infection

• P. falciparum infected erythrocytes (IE’s) exhibit cytoadherence (stick to blood vessel walls /endothelium).

• Prevents IE elimination by the spleen and causes vascular blockages.

• Interaction with endothelium surface receptors (eg. CD36).

Adapted from the Plasmodium Genome Resource. Adapted from http://cmr.asm.org/cgi/content-nw/full/13/3/439/F1.

• AFM probes were functionalized with endothelial surface receptor CD36.

• Used PeakForce QNM with functionalized probe to obtain 2D map of the distribution of CD36 molecular binding sites on IE.

The Biological Question: Can we map the distribution of cytoadherent molecules to specific cell surface structures?

(CD36)

Adapted from Li et al. (2011) PLoS ONE 6: 1-10.

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• CD36 binding sites (high adhesion) correlate to knob structures (circles).

• Adhesion is a result of specific interactions between CD36 and knobs and not due to crosstalk between data channels (arrow).

Molecular Recognition Imaging of IEs Colocalization of CD36 binding sites with knobs

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Topography Adhesion Overlay

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Challenge: Glycocalyx in force measurements Obstacle for access to the membrane

• Exists in bacteria and euk. Cells.

• Idea: use probes with various chemistries

Application Note #135 Quantitative imaging of living biological samples by Peak Force QNM Atomic Force Microscopy

• A comprehensive review of Peak Force QNM applications on soft biological samples

• Authors are Dr. Alex Berquand and Dr. Ben Ohler (Bruker Nano Inc.).

• + New App Note with latest developments and examples to be released soon.

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Contact information

Alexandre Berquand Life Science Applications Scientist Alexandre.Berquand@bruker-nano.com Tel: +49 174 333 94 62

www.bruker.com/en/products/surface-analysis/atomic-force-microscopy.html www.brukerafmprobes.com/ http://nanoscaleworld.bruker-axs.com/