Ultramicroscopy 114 (2012) 56–61

Contents lists available at SciVerse ScienceDirect

Ultramicroscopy

0304-39

doi:10.1

n Corr

E-m

journal homepage: www.elsevier.com/locate/ultramic

Minimizing tip–sample forces in jumping mode atomic force microscopyin liquid

A. Ortega-Esteban a, I. Horcas b, M. Hernando-Perez a, P. Ares b, A.J. Perez-Berna c,C. San Martın c, J.L. Carrascosa c, P.J. de Pablo a, J. Gomez-Herrero a,n

a Departamento de Fısica de la Materia Condensada, C-3, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spainb Nanotec Electronica S.L., Centro Empresarial Euronova 3, Ronda de Poniente 12, 28760 Tres Cantos, Madrid, Spainc Centro Nacional de Biotecnologıa (CNB-CSIC), Darwin 3, 28049 Madrid, Spain

a r t i c l e i n f o

Article history:

Received 1 July 2011

Received in revised form

20 November 2011

Accepted 9 January 2012Available online 20 January 2012

Keywords:

Atomic force microscopy

Scanning in liquids

Virus

91/$ - see front matter & 2012 Elsevier B.V. A

016/j.ultramic.2012.01.007

esponding author.

ail address: julio.gomez@uam.es (J. Gomez-H

a b s t r a c t

Control and minimization of tip–sample interaction forces are imperative tasks to maximize the

performance of atomic force microscopy. In particular, when imaging soft biological matter in liquids,

the cantilever dragging force prevents identification of the tip–sample mechanical contact, resulting

in deleterious interaction with the specimen. In this work we present an improved jumping mode

procedure that allows detecting the tip–sample contact with high accuracy, thus minimizing the

scanning forces (�100 pN) during the approach cycles. To illustrate this method we report images of

human adenovirus and T7 bacteriophage particles which are prone to uncontrolled modifications when

using conventional jumping mode.

& 2012 Elsevier B.V. All rights reserved.

1. Introduction

Atomic force microscopy (AFM) [1] operates in a number ofenvironments including ambient air, ultra high vacuum and liquids[2,3]. This technique enables to study individual biological mole-cules and it allows not only to see but also to touch the materialunder study. In an AFM, a sharp stylus of tens of nanometers indiameter attached to the end of a cantilever is approached to thesurface. As a consequence a force appears between the tip andsurface causing the cantilever to bend. By controlling this bendingwith a feedback algorithm, it is possible to obtain a topographic mapby scanning the surface. To study biological samples, such as cells,membranes, proteins, viruses, etc. it is convenient to work in liquidmilieu in order to preserve the functionality of the biomolecules.Imaging of soft biological specimens in liquid is a daunting taskwhose methods extremely depend on the kind of sample. Forinstance, contact mode, where the tip sweeps the surface withoutdetaching, is adequate for scanning two dimensional protein crys-tals, such as purple membrane [4] which can stand the high lateralforces exerted by the tip [5]. Conversely, individual weakly adsorbedbiomolecules, such as small viruses, are prone to undesired mod-ifications by lateral forces since they are not held and surrounded bya neighborhood. Although dynamic mode, where the tip oscillates atthe resonance frequency, can image individual biomolecules, [6] it is

ll rights reserved.

errero).

technically complicated in liquid (for instance the well known forestof peaks characteristic of liquids cells) [7] and its interpretation isstill under debate. On the other hand, Jumping Mode (JM) [8,9] is anelegant method that works by performing a quick force vs. distancecurve (FZ) at each point of the scanned area, moving the tip laterallyat the farthest tip–sample distance minimizing lateral forces [10]. Inorder to reduce piezoelectric resonances, the FZ is performed using asinusoidal voltage wave that is applied to the scanning piezoelectric.From an operational point of view JM can be seen as a mid-waytechnique between contact [1] and dynamic modes [6,11]. Using JMit is possible to obtain, in addition to the topography image, relevantinformation about other magnitudes such as adhesion [12,13] andstiffness of the sample resolved at the nanometer scale. JM isparticularly suitable for scanning in liquids, where the low adhesionforces allow using small Z displacements at each point [10]. Inaddition, the high damping (low Q factor) exhibited by the canti-levers when scanning in liquids makes JM comparable, if not better,than classic dynamic modes such as amplitude modulation [14–16].In addition, theoretical interpretation of the topography imagestaken in JM is simpler than in the case of dynamic modes. Whenusing JM in conventional operation the feedback parameter is thecantilever deflection, as in the case of contact mode [17]. Animportant additional advantage with respect to contact mode isthat the zero force level can be reset at each image point to the valueat the maximum tip–sample distance, thus avoiding the typicalnormal force drift observed in contact mode experiments [18].However, there are residual forces yet in JM that can be veryharmful when scanning samples which present a steep up hill, such

A. Ortega-Esteban et al. / Ultramicroscopy 114 (2012) 56–61 57

as viruses. Therefore in here we introduce some new developmentsin JM which improve its performance to conveniently identify thetip–sample contact at each image point and minimize the maximumforces applied during scanning. We have termed this new mode asjumping mode plus (JMþ).

2. Working mechanism

Jumping mode operation encompasses the following steps(see Fig. 1a): at time t1 the cantilever tip is at position x1, z1

moving to the right. The sequence describes a conventionaljumping cycle on the s type region (i). (1) The tip is lifted. (2)The tip is moved parallel to the sample surface. (3) The tipapproaches the sample the same distance as is lifted in (1). (4)The feedback is switched on, adjusting the tip height to accom-plish the set point force and acquiring the topography data. (5)The tip is detached from the surface and a new cycle begins. (ii)Tip–sample lateral (lower) and vertical (medium) positions as afunction of the time. The relevant cycle points are shown for thesake of clarity. The upper chart portraits the cantilever deflection(FN). At position 3 a clear peak is produced as a consequence ofthe Z piezo approach.

One undesirable characteristic of this procedure is that whenthe tip moves uphill its vertical excursion is overestimated andthe sample is over-indented, resulting in an additional force thatcan damage delicate samples.

The remedy for this problem is quite simple; instead of movingthe tip a previously set distance z for the forward and backwardcycles, it is better to check for the cantilever deflection as it isapproached to the surface, and stop it if the deflection is greaterthan the set point (see Fig. 1b). This option had already beenimplemented in JM operation, but was seldom used when workingin liquid because its functionality was limited by the viscousdragging of water on the cantilever; as the cantilever approachesthe surface the dragging force produces a spurious deflection thathides the tip–sample contact point. If the dragging deflection equals

a b

(i)

(ii)

1 2

3

4

5

t

Z

t1, x1 ti, xi

Z (t)

X (t)

FN (t)

1 3 2 5 4

Fig. 1. Diagrams showing a cantilever scanning a sample with two flat surfaces spanne

force correction).

the feedback set point force, then the z piezo retracts before tip–sample contact. This problem required performing JM force curvesslowly enough to reduce the dragging as much as needed decreasingthe imaging speed below acceptable values. In order to surpass thissubject we should consider the Reynolds number that is given by

Re¼ rvD=Z

where r is the density of the liquid (�1000 kg m�3), v the relativevelocity between the fluid and the cantilever (1–10 mm/s in jumpingmode), D is a characteristic size for the cantilever (0.1 mm) and Zthe dynamic viscosity of the liquid (�10�3 Pa s). With these figuresthe characteristic Re for our problem is much smaller than 1 indicat-ing that the dragging force that a cantilever undergoes duringmotion can be approximated by the Stokes’ law Ff¼�bv where b

is a constant that depends on both the viscosity of the liquid and thegeometry of the cantilever, and v is the velocity of the cantilever.

Fig. 1(b) shows JMþ (with dragging force correction) opera-tion. The cartoon shows the same time sequence as Fig. 1(a) butnow for the new version of jumping, the main difference is atpoint 3 where the tip is moved towards the sample until thenormal force exceeds the set point. As a consequence thecantilever deflection exhibits a smooth variation as the tipcontacts the surface as shown in (ii).

Fig. 2(a) shows a typical FZ taken in air ambient conditions, wherethe viscous dragging forces are insignificant. As the tip approaches thesurface the cantilever deflection remains zero (from 1 to 2), until thetip–sample gradient becomes larger than the cantilever stiffness.At this point instability arises, the tip contacts the surface and thecantilever is positively deflected (3). In the retraction branch anegative deflection of the cantilever originated by the tip–surfaceadhesion is observed (4). When the stiffness of the cantilever over-comes the adhesion force, the tip is released and the cantileverrecovers the free equilibrium position. Obviously, when the tip is notin contact with the sample, the cantilever deflection is zero and thebackward and forward paths in the FZ plot coincide. This makes thedetection of the tip–sample contact event quite straightforward asshown in Fig. 2(b). Fig. 2(c) shows a FZ taken in liquids where the

1 2

3

4

5

(i)

(ii)

t

Z

t1, x1 ti, xi

Z (t)

X (t)

FN (t)

1 3 2 5 4

d by a s type region. (a) Standard Jumping. (b) Jumping mode plus (with dragging

-270

0.0

0.5

1.0

1.5

time (ms)

Forc

e (n

N)

-2.0

-1.5

-1.0

z p

iezo

(V)

-400

0

1

2

time (ms)

Forc

e (n

N)

Force

-2.0

-1.5

-1.0

z p

iezo

(V)

z piezo

scan

Virus

-200 0 200

-260 -250 -240

Fig 3. (a) Representation of the z piezo (black) and normal force (red) along one line on

uncontrolled peak forces in the positive slope. (c) Piezo and force curves obtained when

any relevant peaks in the force. (For interpretation of the references to color in this fig

a

10 nm

10 n

m

25nm

25 n

m

c

e

1 2 3

4

1 2

Set point

d

b

fSet point

Set point

Fig. 2. (a) Typical FZ curve in air. (b) Cartoon showing the set point level (thin

horizontal line) and the feedback point (filled circle). (c) FZ taken in liquids where

the dragging force effect is clearly observed. (d) Cartoon showing the set point

level (thin horizontal line) and the feedback point (filled circle) for a FZ in liquid.

The empty circle marks the Z turning point if the dragging force is not removed.

(e) FZ curve in liquids after applying the new algorithm filters in the normal force.

(f) Cartoon showing the set point level (thin horizontal line) and the feedback

point (filled circle) for a FZ in liquid after applying the new algorithm filters. Since

the dragging force effect is removed, there is no Z turning point.

A. Ortega-Esteban et al. / Ultramicroscopy 114 (2012) 56–6158

effect of the dragging force is clearly observed. As the tip movestowards the surface, the cantilever suffers an additional bendingbecause of the hydrodynamic friction effect (from 1 to 2). Since, asmentioned above, the piezoelectric follows a sinusoidal displacement,the velocity v has a maximum at the midpoint of this displacementand so is the dragging force (Fig. 2(c)). Then the force drops untilreaching the contact point when it starts growing again (2). If thedragging force equals or exceeds the set point, the stop condition isfulfilled before reaching the surface, thus hindering the determinationof the contact point (Fig. 2(d)). We have developed an algorithm thatassumes a viscous resistance under a laminar flow expression for thedragging force, and included it in the well-known WSxM software[18]. The procedure starts by acquiring some FZ curves, filtering thehigh frequency noise and fitting the data to the theoretical expressioncalculating b. Then a simple algorithm filters the normal force in realtime during data acquisition by subtracting the previously calculateddragging force from the raw normal force signal (Fig. 2(c)), and usesthis filtered normal force to stop the tip when a secure set force (aninput parameter) is reached. Fig. 2(e) shows a typical FZ curve inliquid environment where the normal force has been corrected usingthis method. The force applied using this high efficient digital filterwill be almost as low as the noise level of the normal force.

3. Experimental results

JM has been applied for imaging individual viruses [19–23]with extraordinary success. Small viruses such as parvovirus

245-0.2

0.0

0.2

0.4

0.6

0.8

1.0

Forc

e (n

N)

time (ms)

3.5

4.0

z p

iezo

(V)

200

0

1

2

Forc

e (n

N)

time (ms)

3.5

4.0

z p

iezo

(V)

scan

Virus

400 600

250 255 260

a virus with the conventional JM procedure. (b) Zoom in the enclosed region shows

the new JMþ procedure is applied. (d) Zoom in the enclosed region does not show

ure legend, the reader is referred to the web version of this article.)

A. Ortega-Esteban et al. / Ultramicroscopy 114 (2012) 56–61 59

(25 nm in diameter), can be repeatedly scanned tens of timeswithout showing further modification. However, bigger virusespresent higher uphill slopes, and they are prone to be modified bythe conventional version of JM after recording a few images. It isimportant to recall that while eukaryotic viruses disassembleinside the host to release the DNA, bacteriophages translocatetheir DNA from outside the host leaving an intact protein shell.Since phages do not need disassembly on their infection pathway,it is likely that they would be more stable than eukaryotic virusesagainst external aggressions such as the mechanical stress

Fig. 4. (a) 250 nm�330 nm AFM topography image of two HAdV particles acquired w

figure (a). At the horizontal solid line the new algorithm is switched off, reverting to c

imaging of the same area with JMþ procedure demonstrates total destruction of the s

Fig. 5. (a) 500 nm�500 nm AFM topography image acquired applying the new algor

(circled particle in Fig. 5a). (c) Switching back to JMþ confirms that the particle has b

induced by the AFM tip during scanning. Thus, we have chosenhuman adenovirus (HAdV) and T7 bacteriophage as test speci-mens to show the advantages of our new JM methodology.

Measurements have been carried out using a Cervantes FullModeAFM (Nanotec Electronica S.L., Madrid, Spain, www.nanotec.es) andstandard silicon nitride Olympus cantilevers RC800PSA (Olympus,Tokyo, Japan) with a nominal spring constant of 0.05 N/m.

HAdV has a 95 nm diameter icosahedral, non enveloped capsidenclosing a double stranded DNA genome [24]. Fig. 3(a) and (c)shows the evolution of the force as the virus particle is scanned with

ith JMþ . (b) Time evolution (downwards) of the topography at the dashed line of

onventional JM. Notice the sudden change in the scanning profile. (c) Subsequent

canned virus while the other particle remains unmodified. Set point 150 pN.

ithms of bacteriophage T7. (b) When JMþ is switched off a particle is detached

een completely removed. Set point 115 pN.

A. Ortega-Esteban et al. / Ultramicroscopy 114 (2012) 56–6160

the conventional and the new JM version, respectively. The forcepeaks [25] of about 1 nN observed when the tip scans the positiveslope (Fig. 3(b)) of the virus are completely absent when the new JMis used (Fig. 3(d)). We have observed that under the application ofthe new JM procedure, HAdV particles can be scanned for a longtime without significant damage. Fig. 4(a) is an AFM topographyimage acquired using the new JM procedure. Two virus particlesthat are structurally intact can be observed after imaging with thenew JM operation. When we switch from the new to the standardJM (green line shown in Fig. 4(b)) the result is the complete andimmediate destruction of the scanned particle. Setting back to thenew JM, we confirm the total destruction of one virus particle, whileits neighbor where no standard JM scan was performed remainsunaltered, thus validating the method (Fig. 4(c)).

Bacteriophage T7 belongs to the Podoviridae family, and it hasan icosahedral capsid around 51 nm in diameter, with a triangu-lation number T¼7, and a non-contractile tail (reviewed in [26]).The shell is made of 415 copies of the gp10 protein [27] thatencloses a double stranded DNA 40 kb in size. The T7 head alsocontains the dodecameric connector (gp8) and a singular core(built by proteins gp14, 15 and 16), which is attached to the5-fold vertex by the connector [28]. This same vertex is where thetail also assembles in the mature virion. Fig. 5(a) portraits a fewT7 particles adsorbed on the surface presenting different symme-tries. We note that the same image is obtained reproducibly forhours. In Fig. 5(b) we switched to conventional JM observing asudden detachment of one particle (see the dashed circle inFig. 5(a)). When switching back to the new JM we confirm thedetachment of one T7 particle. We conclude that while weaklyanchored T7 viruses tend to be detached using the conventionalversion of JM, JMþ can obtain reproducible topographies even ofslightly attached viruses. The rationale behind this observationhas profound biological implications. While eukaryotic viruses,such as adenovirus, disassemble inside the host to release theDNA, bacteriophages (T7 phage) translocate their DNA fromoutside the host leaving an intact protein shell. Since phages donot need disassembly on their infection pathway, it is likely thatT7 would be more stable than adenovirus for external aggressionssuch as the mechanical stress induced by the AFM tip duringscanning. Therefore adenovirus particles are readily destroyedwhen using the conventional JM (Fig. 4). Conversely, conventionalJM do not destroy T7 particles, but remove, probably to the liquidmedium, those which are not strongly anchored to the surface.

The main goal of designing and improving JM is to obtaintopographies of delicate samples by controlling the normal forceand removing the lateral force as much as possible. Since thecontact slope and the snap off point of the FZ curves performedwhile applying this new algorithm do not change, adhesion andstiffness mapping are not modified respect to the standard JM.

4. Conclusions

In summary, we present a new version of jumping modeoperation (JMþ) that removes the dragging force, detecting accu-rately the tip–sample contact. We have tested the advantages of thenew implementation by showing improved preservation upon JMscanning of the large HAdV particles and T7 bacteriophages.

Acknowledgments

The authors thank the economical support through ProjectsMAT2010-20843-C02-01, CSD2010_00024, Comunidad de MadridS2009/MAT-1467. P.J. de Pablo acknowledges projects MAT2008-02533/NAN and PIB2010US-00233. Work in the C. San Martın lab

supported by grants BFU2007-60228 and BFU2010-16382 fromthe Spanish Ministry of Science. A. Ortega-Esteban is a recipient ofa FPU fellowship from the Spanish Ministry of Education.

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