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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 

B 

A 

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. 

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2

What’s behind “cell mechanics” and why is it 

so important in biology 

Stiffness in Cell differentiation 

• Nature of the scaffold: 

(same stem cells) 

or 

1 kPa 0.1 kPa 0.01 kPa 

or 

or 

or 

different cell types 

• Tilt: 

(same stem cells and same substrate) 

or or or or 

0º 5º 10º 

different cell types 

• AFM and fluorescence microscopy can be used. 


3

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. 


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Principle of AFM 

Optical detection system 

Different feedbacks for different AFM modes 


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AFM Resolution 

Compared to other microscopy techniques 

0.1nm 1nm 10nm 100nm 1µm 10µm 100µm 

AFM 

light microscopy 

EM 

atoms 

molecules 

bacterioΦ chloroplasts 

bacteria 

cells

Combining AFM to Fluorescence 

2 techniques in 1 tool 

AFM 

Optics 

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Combining AFM to IOM 

Compatibility with various 

optical techniques 

AFM + … 

Sample courtesy 

INRA Nantes 

Sample 

courtesy Zeiss 

Sample 

courtesy 

NIH 

Sample courtesy 

SWMC Stanford 

Sample courtesy Nat. 

Sample courtesy Univ. of 

Univ. Singapore 

New South Wales 


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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. 

1) Import optical image 

into Nanoscope 

2) Target a location for 

the AFM scan 

3) Overlay optical and 

AFM images 


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Combining AFM to fluorescence 

Automatic Overlay (MIRO) 

Live Hela cells 


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Force Spectroscopy 

Get access to stiffness and adhesion 

2 

force (nN) 

1 

0 

-1 

0 

distance (nm) 

500 


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Contact theories in AFM 

Different models / samples 

Hertz DMT JKR 

Sneddon 

MD 

(Most adapted for 

biological samples) 

Standard for Peak Force QNM

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. 

Hela cells 

OS cells

FV/Fluo Applications in Biology 

CSK disrupting agents 

tubulin 

Effect of the 2 drugs: 

1) decay of fluorescence 

2) loss of tracking 

actin 

3) change in elasticity 

Nocodazole 

Latrunculin 

z 

z 

0 

x 

x 

Actin plays a much more important role in cell rigidity 

than tubulin. 

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

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 cm 2 .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 

80 

70 

60 

50 

40 

30 

20 

10 

0 

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. 


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Need for a new characterization 

technique 

Peak Force Tapping and Peak Force QNM 

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 


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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. 

5nm 

• 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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Preliminary test on a stiff sample 

FV/HMX/QNM comparison on a daphnia 

A 

A 

FV 

QNM 

A 

B 

C 

100 

µm 

Bright field image of Daphnia 

(20x) 

B 

• 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 

FV (32x32) 

QNM (128x128) 

• 90x90 µm 

• 5 min / image 

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Processed area 

QNM image + pfc image 

Bearing Analysis 

FV 

QNM 

36 curves Export + post-processing 

Export + post-processing 

144 curves 

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Line plot 

Indentation 

Modify force parameters 

Baseline correction 

Multiple line plots 

Boxcar filter 

Run History 

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) 

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FV/QNM accuracy in Biology 

Study on glioblastoma 

• Isogenic cell line U251 transfected with tumor suppressive factors. 

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

• 5-6 min per image. 

• 128x128 resolution. 

• Enough to observe contrast on all mechanical channels. 


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FV/QNM accuracy in Biology 

Study on glioblastoma 

Submitted to JJAP 

• 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,… 


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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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Background: Glyphosate 

Existing Data in Cytology and Main Challenges 

[H 2 O 2 ] marker 

• Glyphosate-treated samples show a 

higher number of necrotic and apoptotic 

cells, and a higher [H 2 O 2 ] 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. 

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PeakForce QNM: Much more information 

Probe changes in mechanical properties 

• 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. 

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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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PeakForce QNM: 

Easier imaging of Live Diatoms in Seawater 

5 µm 

• Large scale Catalyst images remarkably stable. 

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

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PeakForce QNM: Easier Imaging 

Mechanical Properties at High Resolution 

150 mV 

50 MPa 

0 0 

Peak force Error 

Young’s modulus 

20 nm 

0 

Deformation 

3d Height + YM skin 

• All PeakForce QNM channels are directly quantitative. 

• Unprecedented resolution from imaging to modulus. 

Pletikapic et al., J. 

Phycol 48 (2012) 174. 

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PeakForce QNM: Easier Imaging 

First AFM Investigation of the Frustule 

• 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. 

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QNM on Live Bacteria 

E. Coli K12 

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

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Correlating topography to Force curves 

HSDC files 

Export + post-processing 


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Correlating topography to Force curves 

HSDC files 

AFM tip 

fibril 

cell 

1 2 

Young’s modulus calculated 

over very specific areas 

Young’s modulus calculated 

over the entire cell 


39

Functionalized probes 

HSDC files - Detecting specific unbinding events 


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Combining QNM with functionalized 

probes - Pathogenesis of Malaria 

Erythrocyte (Red Blood Cell) Infection 

P. falciparum 

Anopheles mosquito 

Healthy RBC 

• 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. 

Knobs 

Infected RBC 

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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 http://cmr.asm.org/cgi/content-nw/full/13/3/439/F1. 

Adapted from the Plasmodium Genome Resource. 

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The Biological Question: 

Can we map the distribution of cytoadherent 

molecules to specific cell surface structures 

• 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. 

(CD36) 

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

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Molecular Recognition Imaging of IEs 

Colocalization of CD36 binding sites with knobs 

Topography Adhesion Overlay 

• 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). 

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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 


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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. 

1/22/2013 

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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/ 


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