CHO cell

Cell under Mitosis
Calcium waves in
DRG Neurites

Application Note #135
Quantitative Imaging of Living Biological
Samples by PeakForce QNM Atomic ForceMicroscopy
It is now well established that measuring the mechanicalproperties of living cells ex vivo can be a good indicatorof the health of the organism from which they wereextracted. Atomic force microscopy (AFM) is a powerfulinvestigation and diagnostic tool, especially in force mode.
Nevertheless, force spectroscopy suffers from several
limitations, including the speed of acquisition, a relative lackof resolution, and the fact that it doesn’t directly provide
quantitative information. In order to fill this gap, Bruker has
developed a new imaging technique, PeakForce QNM®,
which provides much more informative data at a highresolution and with remarkable ease-of-use. Up to now,
this very promising technique has been tested successfully
on a wide range of samples but not on living organisms.
This application note gives an overview of the biologicalsamples that can be imaged using this technique and theinformation that can be extracted from the atomic force
microscopy data.

HaCat cluster

can be quantified by AFM and used to estimate the effectof drug treatment or different types of pathologies, as wellas such natural processes as aging.5-7 For instance, it is
becoming more and more evident that cancer cells and theirnormal homologues express clearly different mechanicalproperties.8-11 Moreover, AFM is often used to correlate
elastic behavior and cell migration or division.12-15 The vast
majority of these studies are based on TappingMode,
single-force curves, or force-volume measurements.

AFM and Cell Mechanics

TappingMode offers the advantage of applying negligible
nominal, friction and shear forces, and phase imaging
reflects the energy dissipated between the tip and thesample during each tap on the surface. Nevertheless, thenature of the phase signal remains always unclear since
it is a mixed contribution of several surface and volume
parameters, including adhesion forces, contact area,elasticity, viscosity and dissipation. Even if working at
specific ratio setpoints allows more sensitivity to surface orvolume properties, a quantitative extraction is impossible.

Since its development,1 AFM has proven itself to be atool of choice to image super soft biological samples,
especially with the emergence of TappingMode™ 2 andforce spectroscopy3 and the fact that it is one of the
few microscopy techniques that allows observation ofcells under near-physiological conditions.4 It is now well
documented that the mechanical properties of living cells

Force volume is another powerful technique based onforce measurements achieved on a matrix of points definedby the user. Stiffness (and by extrapolation the sample’s
Young’s modulus) and the adhesion between the tip andthe sample can be extracted from each force curve. If the
tip is functionalized with a molecule of interest, specificunbinding events can also be detected on the retraction

curve. Although it is very commonly used in the AFM world
to probe cell mechanics, force volume suffers from two
main drawbacks. First, acquiring high-resolution data ishighly time consuming. This is why in the majority of force
volume studies, the stiffness and adhesion images have amuch lower resolution than the height channel. Secondly,
the elastic properties cannot be directly quantified. The data
have to be post-processed by using external software sothat the Young’s modulus can be extracted.
To overcome these bottlenecks, Bruker has developed
PeakForce QNM. This revolutionary new mode is based on
Bruker’s new Peak Force Tapping™ technology, with which
the probe is oscillated at a low frequency while force curves
are generated each time the AFM tip taps on the sample’s
surface. Many parameters can be extracted quantitatively
in real time from each of these force curves, which makes
PeakForce QNM the most powerful AFM technique fornanoscale material property characterization available today.

Easy, High-Resolution Quantification of Sample
Mechanical Properties
PeakForce QNM enables direct extraction of quantitative
nanomechanical information from biological samples
without sample damage. It’s based on Peak ForceTapping technology, during which the probe is oscillated
in a similar fashion as it is in TappingMode, but at far
below the resonance frequency (1 or 2 kHz depending on
the tool). Each time the tip and the sample are brought
together, a force curve is captured. However, where thefeedback loop maintains the tapping amplitude constant in
TappingMode, Peak Force Tapping controls the maximum
peak force on the probe. These forces can be controlled at
levels much lower than contact mode and even lower thanC

70

By eliminating the time variable, one can plot the force
versus the tip-sample distance (figure 1b), from which muchinformation can be obtained. As in regular force mode, themaximum adhesion force between the tip and the sample
can be extracted as the step height between the baseline and the pull-off point. The peak force is defined as
the vertical distance between the base line and the turnaway point. The deformation corresponds to the horizontal
distance between the contact point and the turn-away
point. By extrapolating the retraction curve close to thecontact area, the software determines the Young’s modulus
using a DMT16 fit. This mechanics model assumes that thecontact principle remains the same as in the Hertzian model

70

APPROACH

WITHDRAWL

60

50

50

40

40

30
20
10
0

E

A

20

dissip.

10

150

200

250

300 350

Time (µs)

adh.

–20

D
100

def.

30

–10

–20
50

peak F.

0

B

–10

a

Figure 1 illustrates the different force fields experienced by
the probe during an approach-retract cycle, as well as all theinformation that can be extracted from the generated force
curves. When the probe approaches the sample (figure 1a),it’s pulled down toward the surface by attractive forces,
which are mainly capillary, Van der Waals and electrostaticforces. At point B, those negative forces become higher
than the cantilever’s stiffness, which causes the tip to pullto the surface and then start indenting into the sample
until the Z-position of the modulation reaches its maximum(point C). This position represents the maximum peakforce value, which is used for the feedback control. After
this point, the probe starts withdrawing until it reaches thepull-off point (the maximum adhesion point, which also
corresponds to the minimum force). Then the tip continues
retracting and reaches back to its original position (E) where(as in A) no more force field affects its motion.

Force (nN)

Force (nN)60

TappingMode, allowing operation on even the most delicate
biological samples.

400

450

b

100

200

300

YM
400

500

600

Z (nm)

Figure 1: Working principle of PeakForce QNM. While the probe is oscillated, a force curve is recorded for each pixel of the image. To
distinguish between the different portions of the tip trajectory, this example was recorded by using a TAP150A probe, which is typically used
to image rather stiff and poorly compliant samples. On biological samples, the typical peak force can be up to a thousand times lower.

2

but considers additional attractive interactions focused
inside an annulus located outside of the contact area (figure2a). In that case, and considering the contact between asphere and an elastic half-space, the force is related to thedeformation by:

F = 43 E* R 1/2 d 3/2and

α = Rdwhere E* represents the reduced Young’s modulus, R the
tip radius and d the deformation depth.
Eventually, the energy dissipated by the tip and the sample
during each tap on the surface is obtained by integrating thearea between the approach and the retraction curves.

Direct Quantification of AFM Signals on
Biological Samples
When the probe is calibrated prior to the experiment, all thesignals mentioned above will be directly quantitative. Thiscalibration can be done as follows:
1) Engage on a stiff part of the sample (like glass) andrecord a force curve from which the deflection sensitivity
can be calculated.
2) Withdraw and calculate the spring constant using“Thermal Tune”.
3) Record a topography image of the Tipcheck sample to geta value of the tip radius R.
4) Having entered the estimated R value, adjust the
deformation on a sample of choice. The sample to bescanned should have similar mechanical properties as
the biological sample that will be investigated during
the experiment. For non-biological samples, a list ofreference samples, as well as a list of suitable AFM
probes, can be found in Bruker Application Note #128.17
For purely biological samples, the tip can be calibrated oncommercial gelatin gels, if the Young’s modulus of thesample of choice is around 100 kPa. For softer samples,
home-made calibration samples based on gelatin18 or
agarose19 can enable measurements down to 1 kPa.
As the exploration of the mechanical properties of living
cells is a key requirement today, and also a real technicalchallenge, the present document will review a list of
typical applications that can be addressed using Bruker’s
PeakForce QNM technique, from the simplest to the mostcomplex examples. Although this newly released technique
has been successfully tested on polymers,20 this applicationnote marks the first time that it has been applied to living
and super soft samples.
In most of the existing publications, the Hertz theory is used
to estimate the contact mechanics because it’s by far thesimplest since it considers that there are no adhesion forces
between the tip and the sample. This is not true, although
they can be greatly reduced in liquid. The DMT assumption
is based on a Hertzian geometry but considers that theadhesion forces exist and are located outside the contact
area and tends to underestimate it. It is mainly applicable topoorly compliant samples, having low adhesive properties
and low contact radius.
On most of the tested samples, a Sneddon fit21 was used
to extract the Young’s modulus by capturing a HSDC (High
Speed Data Capture) file on a scan line at a very highresolution (a few thousand of force curves). By comparing
the force and height profiles, the non-desired parts (force
curves captured on a portion of the sample that is not ofinterest, such as glass) can be excluded manually. The
remaining force curves can be exported as a single file,post-processed by an external program,22 and the average
Young’s modulus can be calculated by considering different
contact theories, such as the Sneddon model.
This mechanical theory considers the contact between
an elastic half-space deformed by a rigid conical indenter(figure 2b), determining that the load is proportional to
the square of the penetration depth. It is not applicable toceramics or polymers but is commonly considered as mostadapted for biological samples. The indentation depth andthe tip radius are related by:

Figure 2: Contact mechanics in AFM. In a, the DMT fit is basedon a Hertzian assumption but states that the adhesion forces arefocused outside the contact area. This is well adapted to highdensity polymers and poorly deformable samples. In b, the Sneddon
fit considers the tip as an infinite conical indenter, which is well
adapted to soft (biological) and deformable samples.

On such samples, a wide range of AFM probes have beentested and the recommendation is given in figure 3. The
softest existing probes on the market are OBL-B, whichhave a nominal spring constant of 0.006 N/m and are thus
appropriate to investigate super soft living cells, suchas neurons.

3

OBL B: 0.006 N/m*Tested probes: 0.01, 0.03, 0.07 and 0.1 N/mcorresponding
probesScanAsyst Fluid: 0.7 N/mScanAsyst Fluid
Prototype probes*
Prototype probes*
OBL B
Silicified organisms (diatoms)

Typical samples

sporesbacteria
Euk. cells
understressOsteoblasts
90% of euk. cells
Neuronal cells

1

10

100

1000

kPa
10000

Figure 3: Range of compliance of various biological samples and corresponding AFM probes recommended for Peak Force Tapping.
Depending on their type, eukaryotic cells can exhibit very different mechanical properties. Neurons can be extremely soft (down to 1kPa)
whereas bone cell can be as robust as bacteria. In order to properly probe the cell properties, selecting the right spring constant and thus
sensitivity is mandatory.

Imaging Biological Samples with PeakForce QNMMarine biological samples are often composed of a mixtureof soft and rigid components. As an example, diatoms,
unicellular algae that appeared 180 million years ago, havea cell wall called frustule that is made of pure silica coatedwith organic molecules, such as pectin and other types ofpolysaccharides.23 Those barriers are riddled with holes thatact as filters and pores, enabling communication between
the inner part of the diatom and the external environment.
A sample of water taken from the Adriatic Sea was put ona glass slide and investigated by PeakForce QNM. Other
than very relevant observations on living diatoms,24 somecell wall remnants were also found in the suspension.
Figure 4 gives an example how those structures look.
The 3D-topography profile reveals a characteristic wafflelike structure with pores 100 nm in size and an averageheight of 20 nm. The adhesion channel shows a marked
contrast between the bottom of the pores (about 50 pN
in average) and the rest of the cell wall (less than 20 pN).
However, the most informative channels are the elasticity
and the deformation data. On both channels the three4

portions of the frustule are distinguished, each exhibiting
clearly different mechanical properties: the center of thepore (average Young’s modulus of ~300 kPa and averagedeformation of ~7 nm), the ring around the pore (~75 kPa
and ~25 nm) and the core part of the cell wall, which seemto have intermediate mechanical properties (~200 kPa
and ~10 nm). Those differences might be explained by thepresence of components of different nature. As expected,
the deformation is low for stiffer portions of the sample andvice-versa.
Additional experiments were carried out on Escherichia coli
K12 bacteria. E. coli are gram-negative, aerobic/anaerobic
and non-sporulating bacteria of about 2 μm in lengthand 500 nm in width and height. They are rather robust
bacteria that can live on a wide variety of substrates. Some
non-pathogenic stains are of high interest because they
can be used as therapeutic, so-called probiotic, agents totreat various diseases.25,26 Unlike most of E. coli species,
K12 strains are able to multiply in the intestine and areparticularly resistant to antibodies. One of their othercharacteristics is that they possess pili (see figure 5a) that

Figure 4: Imaging of phytoplankton cell wall with a BioScope Catalyst AFM. Top left: electron microscopy image of a diatom, sample courtesyof Dennis Kunkel, Astrographics. Most of the PeakForce QNM channels provide a remarkable contrast and high-resolution features.

typically retract under depletion conditions or any stressing
environment. Until now, imaging those bacteria alive with
the AFM, in any mode, has been a considerable challenge
and a historically elusive result.

dishes.28 The inlets and outlets for gas and liquids, as well
as a heating plate enable introduction of the appropriate
serum, gas and temperature to bacteria or eukaryotic cells
for their growth and maintenance.

Figure 5 shows high-resolution images of such living
bacteria, easily obtained in less than one hour. As can beseen on the 3D-representation of the height channel (figure5b), the pili are no longer visible, which can be explained by
the fact that extracting from their suspension medium andspreading them on a dish induces a stress that causes thosepili to retract. Prior to the experiment, the AFM probe wascalibrated on a gelatin sample and thus the elastic modulus
could be calculated. Figure 5c shows the DMT modulus
channel. By using a Sneddon fit, the average Young’s
modulus was determined to be 183 kPa, which perfectly
matches previous observations. 27

In another set of experiments, PeakForce QNM was used
to investigate glioblastoma cells. Glioblastoma are by far
the most common and malignant form of brain cancer. The
most common factors underlying the cause of the disease
are exposure to cytomegalovirus,29 smoking30 and electricfields.31 Moreover, this affection is not easy to cure, due
to the fact that most of therapeutic agents cannot crossthe hemato-encephalic barrier and because conventional
therapy is very likely to damage the brain. For thesereasons, most existing treatments have a more palliative
than healing effect. This is why there is an increasing
interest in trying to achieve better insight in how the tumor
is mediated.

Monitoring Cell Dynamics in Real TimeAll living cells are dynamic, changing shape due torearrangement of their cytoskeleton scaffold and spreading
and migrating on the cell culture substrate. These processes
and the mechanical changes that accompany them canbe monitored with PeakForce QNM. Moreover, cells canbe kept alive for long periods of time by using Bruker’s
Perfusing Stage Incubator (PSI) accessory, which consistsof an enclosed incubation chamber that fits with cell culture

Though the signaling pathways are particularly complex,
it is well known that the capacity of a cell to be invasive isdirectly connected to its intrinsic mechanical properties.
Living glioblastoma cells have been imaged by PeakForceQNM on the BioScope Catalyst™ and maintained alive
for the duration of the experiment by the use of the PSI.
This technology allows the user to apply a very gentle
to moderate force on the sample, depending on the5Figure 5: E. Coli K12 bacteria imaged by PeakForce QNM on a BioScope Catalyst AFM. In a, the structure of the strain is drawn. In b, an AFM
10x10μm 3D-height representation of a cluster of bacteria is shown. In c, Young’s modulus channel (z-scale: 0-4GPa) is depicted. This is thefirst time that such bacteria has been imaged alive by AFM.

information needed. When applying a very light force on
the sample, the topmost features of the cell (glycocalyx,
protrusions) can be probed. On the other hand, applying aslightly higher force is required to sense the organelles andthe cytoskeleton located underneath the plasma membrane.

Probing the real mechanical properties of the sample also
requires indentation of the sample (and thus flex in the
cantilever) by at least a hundred nanometers. Figure 6ashows a typical high-resolution image obtained on living
glioblastoma while applying a moderate force (~300 pN).

Figure 6: Images of living glioblastoma cells by PeakForce QNM and the BioScope Catalyst AFM. In a, 40x40μm height image recorded at
a moderate force shows both topmost and internal structures. In b, 15x15μm 3D overlay of topography and deformation channels is shown.
Bruker’s BioScope Catalyst with Perfusing Stage Incubator offers the best balance of living cell imaging for long-term experiments.
6

At such a scanning force, both topmost and more innerfeatures (most likely the cell cystoekeleton) can be detected.
Figure 6b is a 3D representation of the cell topography witha “deformation skin.”This technology and setup can also be used to monitorchanges in cell properties in response to external stimuli.
Keratinocytes are the major components of the outermost
layer of the human skin. They establish tight junctions withother types of cells, such as neurons, and represent themain barrier against UV radiations for the human body.
Studying such cells by AFM helps researchers understandthe process of skin cancer or other impairments. It has also
been shown that an excess of keratin produced by thosecells can undergo programmed cell death, though the fullmechanism is still unknown.32

HaCat is an immortal cell line of human keratinocytes thatis widely investigated in cytology and also represents agood candidate to explore the potential of PeakForce QNM.
The cells were exposed to an oxidative agent capable ofinducing a stress. In response to this chemical aggression,
the cells tend to transform and synthesize so-called actin
stress fibers. A typical medium-resolution image is shownin figure 7. On the peak force error channel, structuralchanges, such as the morphology of the cells themselves
are observed. These tend to adopt a cocoon-like structure.
Stress fibrils are also clearly visible around the center ofthe cell. The changes in mechanical properties are also
obvious, the Young’s modulus increases and deformation
decreases in response to the drug treatment. The adhesion
channel also provides some interesting information, though

Figure 7: 75x75μm BioScope Catalyst and PeakForce QNM image of living HaCat cells under oxidative stress. The cells react by rapidly
synthesizing stress fibrils to establish contacts with adjacent cells. Such dynamic processes can also be tracked by using this technique.

7the adhesion contrast between the cell and the glass israther homogeneous (which could be expected as nonspecific adhesion forces tend to be more uniform in liquid
environment). There is a dramatic decrease in the averageadhesion between neighbor cells. This might indicate
the presence of cellular adhesion molecules (CAMs).
Such an assumption could be verified by the use of AFM
probes functionalized with specific antibodies directedagainst different types of CAMs. In PeakForce QNM, aforce curve is made for each pixel of the image, thus theresolution is the same on all the channels. This example
illustrates how easy and fast (384x384 pixel resolution
images can be captured in 6 to 9 minutes) it is to directly
and in a quantitative manner probe changes in topography
and mechanical properties of living cells in response to
drug treatments.

Figure 8: Overlay of fluorescence and AFM images of living HUVEC cells created with MIRO on a BioScope Catalyst. The main benefit of
MIRO is to enable the display of optical and AFM information simultaneously. When operating with functionalized probes, a “Point & Shoot”
option can also be used to accurately trigger force measurement at desired locations without losing the ligand.

8

Overlaying AFM and Optical Channels

References

Another of the current key challenges for biologicalapplications is to be able to get optical and AFM information
simultaneously. Bruker’s exclusive Microscope Image
Registration and Overlay (MIRO™) feature can be used toeasily import optical/fluorescence images into NanoScope®
software and overlay them with AFM images. After a shortcalibration, the user can select the location to make theAFM scan. Thus the sample automatically can be moved tothe desired position and the AFM image can be captured
pixel by pixel, and fully integrated into the optical image.

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Figure 8 shows an overlay achieved on living endothelial
cells. The fluorescence image (double-staining DAPI for
nucleus and α-phalloidin for actin filaments) is set as the
background and overlapped with an AFM image madeof a mix of two channels: peak force error and Young’s
modulus. The transparency was set at 50% so that a direct
correlation can be made between the different parts ofthe cells (visible by AFM topography and fluorescence)and their corresponding mechanical properties (Young’s
modulus AFM channel). In b, c and d the individual peakforce error, Young’s modulus and deformation AFM images
are represented. It can clearly be seen in the elasticity anddeformation channels that on the edges of cells where thethickness is too low, the influence of the scaffold (glass) on
the mechanical properties of the sample is non negligible,
whereas on the core part of the cells, the average Young’s
modulus is much more reliable (45.3 kPa). For a matter ofclarity, only three AFM channels are shown here, but eight
different signals can be displayed simultaneously.

Conclusion
The applications shown above demonstrate that Peak
Force Tapping is by far the most powerful and quantitative
high-resolution AFM technique available today to probe
quantitative chemical and mechanical properties of living
biological samples with an acquisition speed comparable
to TappingMode. The number of different mechanicalproperties that can be characterized exceeds that of othercommonly used AFM modes. Its potential paves the way
for many exciting new applications in the field of biology,
especially in cancer research and cardiovascular diseases.

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9

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AuthorAlexandre Berquand, Bruker Nano Surfaces(alexandre.berquand@bruker-nano.com)

Acknowledgements
We thank the following persons for having provided high-quality samples:
Vesna Svetlicic and Galja Pletikapic (Rudjer Boskovic Institute, Zagreb, Croatia)
Gregory Francius (LCPME, Nancy, France)
Andreas Holloschi, Leslie Ponce, Petra Kioshis and Mathias Hafner (Universityof Applied Sciences, Mannheim, Germany)
Laurence Nicod and Celine Heu (Institut FEMTO-ST, Besancon, France)

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©2011 Bruker Corporation. All rights reserved. Peak Force Tapping, TappingMode, ScanAsyst, PeakForce QNM, BioScope Catalyst,

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Bruker Nano Surfaces Divison
Santa Barbara, CA · USA+1.805.967.1400/800.873.9750
productinfo@bruker-nano.com

www.bruker-axs.com

MIRO, and NanoScope are trademarks or registered trademarks of Bruker Corporation. AN135, Rev. A0

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