Micron 43 (2012) 1399–1407

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Increased imaging speed and force sensitivity for bio-applications with smallcantilevers using a conventional AFM setup
Michael Leitner a , Georg E. Fantner c , Ernest J. Fantner d , Katerina Ivanova d , Tzvetan Ivanov e ,Ivo Rangelow e , Andreas Ebner a , Martina Rangl a , Jilin Tang f , Peter Hinterdorfer a,b,∗aInstitute of Biophysics, Johannes Kepler University, A-4020 Linz, Austria
Center for Advanced Bioanalysis (CBL), A-4020 Linz, Austriac
École Polytechnique Fédéral de Lausanne, Laboratoire de bio- et nano-instrumentation, CH-1015 Lausanne, Switzerlandd
SCL-Sensortech, Tech Gate Vienna, Science and Technology Park, A-1220 Wien, AustriaeFachgebiet für Mikro- und nanoelektronische Systeme, Fakultät für Elektrotechnik und Informationstechnik, TU Ilmenau, D-98693 Ilmenau, GermanyfChinese Academy of Science, Chang Chun Institute of Applied Chemistry, 130021 Changchun, Chinab

a r t i c l e

i n f oArticle history:
Received 19 April 2012
Received in revised form 15 May 2012
Accepted 15 May 2012
Keywords:
Small cantileverHigh resolution imaging
Fast AFM imaging
Ultra-sensitive molecular recognition force
spectroscopya b s t r a c t
In this study, we demonstrate the increased performance in speed and sensitivity achieved by the use ofsmall AFM cantilevers on a standard AFM system. For this, small rectangular silicon oxynitride cantilevers
were utilized to arrive at faster atomic force microscopy (AFM) imaging times and more sensitive molecular recognition force spectroscopy (MRFS) experiments. The cantilevers we used had lengths between
13 and 46 ␮m, a width of about 11 ␮m, and a thickness between 150 and 600 nm. They were coatedwith chromium and gold on the backside for a better laser reﬂection. We characterized these small cantilevers through their frequency spectrum and with electron microscopy. Due to their small size and highresonance frequency we were able to increase the imaging speed by a factor of 10 without any loss inresolution for images from several ␮m scansize down to the nanometer scale. This was shown on bacterial surface layers (s-layer) with tapping mode under aqueous, near physiological conditions and on
nuclear membranes in contact mode in ambient environment. In addition, we showed that single molecular forces can be measured with an up to 5 times higher force sensitivity in comparison to conventional
cantilevers with similar spring constants.
© 2012 Elsevier Ltd. All rights reserved.

1. Introduction
Since the atomic force microscope was the ﬁrst time presented
by Binnig et al. (1986), it has evolved into a powerful, widely used
tool in nanoscience and nanotechnology. The applications span
the range from material science and biology to quality control
in semiconductor industry. Particularly important for biologicalsciences is that the AFM enables recording images under aqueous, near physiological conditions with nanometer (Kamruzzahan
et al., 2004; Kienberger et al., 2005, 2004) or even sub-nanometer(Muller et al., 1995) resolution. Chemical functionalization (Ebneret al., 2007a,b; Riener et al., 2003) of the AFM tip allows the measurement of pico-Newton forces (Hinterdorfer et al., 1996; Tanget al., 2007; Wielert-Badt et al., 2002), as well as simultaneously
topography and recognition imaging (Ebner et al., 2005; Leitneret al., 2011; Stroh et al., 2004; Tang et al., 2008b). Combination∗ Corresponding author at: Gruberstr. 40, A-4020 Linz, Austria.
Tel.: +43 732 2468 7631x7630; fax: +43 732 2468 822.
E-mail address: peter.hinterdorfer@jku.at (P. Hinterdorfer).
0968-4328/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.micron.2012.05.007

of AFM and optical (Madl et al., 2006) or ﬂuorescence microscopy(Duman et al., 2010) has also been realized. A main – if not themain – limitation for a much wider use of this technique, e.g.
industrial biotechnology, pharmacology, and medicine, is the longtime (several minutes) that is required to take a high resolution
AFM image. The foremost factors for this speed limitation in AFM
imaging are the scanner, the feedback loop, and the cantilever.
That it is possible to speed up the AFM has been demonstrated
by several research groups since the early 1990s (Ando et al., 2001,
2008; Barrett and Quate, 1991; Fantner et al., 2010; Hillner et al.,
1992; Hobbs et al., 2001; Humphris et al., 2005; Sulchek et al.,2000a; Viani et al., 2000). During this time a lot of progress has
been made in the ﬁeld, especially in the development of small andsensitive low noise cantilevers for imaging (Chand et al., 2000;
Hosaka et al., 2000, 1999; Viani et al., 1999a; Walters et al., 1996;
Yang et al., 2005), force spectroscopy (Hosaka et al., 2000; vander Mei et al., 2003; Viani et al., 1999a,b), and force mapping(Schaffer and Jiao, 2001), as well as in their detailed characterization (Schaffer and Hansma, 1998). Some groups also fabricatedspecial cantilevers with integrated actuators and/or sensors (Kimet al., 2003; Manalis et al., 1996; Pedrak et al., 2003; Rogers et al.,

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2003) for faster AFM imaging. With respect to scanners (Fukuma
et al., 2008; Kodera et al., 2005; Schitter et al., 2007), optimized
feedback (Ying et al., 2008), controlling (Ando, 2008; Kodera et al.,2006; Schitter et al., 2004; Schitter and Stemmer, 2003; Sulchek
et al., 2000b; Tien et al., 2004; Uchihashi et al., 2006), data acquisition (Fantner et al., 2005) or even complete high speed AFM
system (Fantner et al., 2006; Humphris et al., 2005), a lot of effort
has been undertaken, too. Nonetheless, most of these high speed
AFM components are limited to a certain number of researchlaboratories, because the developed instruments are highly
specialized.
Cantilevers which combine high resonance frequency, highbandwidth, and low spring constant have been developed here,
in which the cantilever design was optimized for several applications to achieve optimal performance. For example, molecular
recognition force spectroscopy requires a particular soft cantilever, whereas a cantilever for high speed imaging needs
a high resonance frequency. With cantilever lengths between
13 and 46 ␮m, widths of about 10 ␮m, and thicknesses of
150–600 nm, we were able to speed up a commercially available AFM by a factor of 10 in both ambient and aqueousconditions. We also detected diminished thermal force ﬂuctuations, when performing single molecular recognition force
spectroscopy.

2. Materials and methods
2.1. Tip preparation
All used chemicals were purchased from Sigma Aldrich and wereof highest quality.
AFM tip functionalization for molecular recognition force
microscopy: two functionalization methods, the ethanolamine.
HCL (Ebner et al., 2007a) and the gas phase silanization using
aminopropyltriethoxysilane (APTES) (Ebner et al., 2007a)
coating were tested. For ethanolamine-hydrochloride coating the small silicon oxynitride AFM cantilevers were cleaned
with ethanol (3 × 5 min) and chloroform (3 × 5 min) anddried in a gentle stream of nitrogen. Afterwards the tips
were incubated with 0.5 g/mL ethanolamine-hydrochloride
in DMSO in the presence of molecular sieve (3 Å) at room
temperature, to generate reactive amino groups on the tip
surface.
For APTES coating the tips were cleaned as described before.
APTES was freshly distilled under vacuum. For removal of air andmoisture a desiccator was ﬂooded with argon. Two small plastic
trays were placed in the desiccator. 30 ␮l of APTES and 10 ␮l triethylamine were pippeted into the two trays and the AFM tips wereplaced nearby. After 120 min of incubation in the closed desiccator,
APTES and triethylamine were removed and the desiccator again
ﬂooded with argon.
The Biotin-PEG-NHS crosslinker was covalently bound to thegenerated amino groups on the tips. For this, 1 mg of BiotinPEG-NHS crosslinker was dissolved in 0.5 mL chloroform and thecantilevers were placed in this solution. As a catalyst 10 ␮l triethylamine was added to start the reaction. After 2 h, the tips were
washed with chloroform (3 × 5 min) und dried with nitrogen. The
cantilevers were stored in PBS Buffer (150 mM NaCl, and 5 mM
NaH2 PO4 ) at 4 ◦ C and used within three days.
Sample preparation for molecular recognition force spectroscopy: avidin with a concentration of 0.1 mg/mL in PBS-Buffer
was incubated on freshly cleaved mica for 20 min. After the incubation step the mica was extensively cleaned by rinsing 50 times
with 1/10 PBS-Buffer (15 mM NaCl, 0.5 mM NaH2 PO4 ) and used
immediately.

2.2. Nucleopore complex preparation
The nuclear pore complex samples used were nuclear membranes prepared from Xenopus laevis oocytes. The oocytes wereextracted from X. laevis during operation. A single oocyte was put
into a BSA coated Petri dish with KHP (nuclear membrane puffer,
90 mM KCl, 10 mM NaCl, 2.2 mM MgCl2 , 1.1 mM EGTA, and 10 mM
HEPES, pH 7.4 adjusted with KOH). The oocyte was opened using
two needles so that the nucleus was spilled out. The nucleus wascleaned in KHP for 5 min to remove the cytosol. The cleaned nucleus
was transferred to a cell-TAK modiﬁed class slide in ultra-purewater. Afterwards the nucleus was opened with the two preparation needles to dispose the chromatin. The membrane was spanned
onto the cell-TAK modiﬁed position of the glass slide with the helpof the needles. The glass slide with the nucleus membrane was dried
and used within 5 days.
2.2.1. Class slide modiﬁcation using cell-TAK
The class slides were washed with 70% ethanol, rinsed withultra-pure water and dried in a gently steam of nitrogen. Cell-TAK
was diluted 1:6 with KHP Buffer. A 3 ␮l droplet of the diluted cellTAK was put on a glass slide and dried for 2 h. The modiﬁed glass
slide was washed with 70% ethanol, rinsed two times with ultrapure water and air-dried. The cell-TAK modiﬁed class slides wereused immediately.
2.3. Bacterial surface layer preparation
All bacterial surface layer measurements were performed on
the wildtype protein puriﬁed from Bacillus sphaericus CCM2177.
For this, 1 mg of protein was dissolved in 1 mL GHCl Buffer (5 M
GHCl, 50 mM Tris, pH adjusted to 7.2). The obtained solution was
dialyzed in 3 L of ultra-pure water for 3 h by changing the water
every hour. After dialysis the monomeric solution was centrifuged
for 10 min at 4 ◦ C to remove unsolved particles. The concentrationof the protein was adjusted to 0.1 mg/mL using ultra-pure water.
To start the recrystallization process 10 mM of CaCl2 were added.
A cleaned silicon chip (5 × 5 mm) was incubated to 1 mL recrystallization solution at room temperature. After 3 h of incubation thesilicon chips were gently rinsed with ultra-pure water and storedin physiological PBS-Buffer (1.8 mM KH2 PO4 , 10.1 mM Na2 HPO4 ,2.7 mM KCl, and 140 mM NaCl, pH adjusted to 7.2). 3 ␮L of NaN3
was added the next day to protect the samples from bacteria. The
samples were used within 5 days.
2.3.1. Cleaning silicon chips
5 mm × 5 mm sized silicon chips were cleaned with piranha(3:7 = H2 O2 :H2 SO4 ) for 30 min. Afterwards they were cleaned withultra-pure water (3 × 5 min) and air-dried. On the next day they
were immersed in ethanol (30 min), air-dried and immediately
used for incubation.
2.4. AFM imaging
All AFM imaging measurements were performed on a Pico Plus
setup (Pico SPM II 5500, Agilent, Chandler, USA), all the MRFS measurements were performed on a Pico SPM I setup (Agilent, Chandler,
USA). The molecular recognition force microscopy measurements
were performed in liquid with sweep duration of 1 s at a z-rangeof 300 nm. Bacterial surface layer measurements were performedin liquid environment using both tapping and contact mode. The
nucleopore complex measurements were performed using contact
mode in dry state. Tapping mode measurements were performed
with different acceleration frequencies using cantilevers with different sizes and characteristics.

M. Leitner et al. / Micron 43 (2012) 1399–1407

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Fig. 1. Electron microscope images of prototype cantilevers. (A and B) Typical images used for further characterization. The horn shaped and partly hollow tip from the front
side. The upper side of the silicon oxynitride cantilever is coated with chromium as adhesive agent and gold for laser reﬂection. The tip was visualized as cone with a heightof about 3 ␮m and a base radius of about 2 ␮m. The radius of the curvature is about 5–10 nm for the sharpest cantilevers. (C) The measured resonance frequency for the short
430 nm cantilever (Type C) is about 600 kHz. (D) The measured resonance frequency is about 280 kHz for the long 430 nm (Type D) cantilever. (E) Image of the 430 nm thickcantilevers (Type C and D).

3. Results
3.1. Characterization of small cantilevers
The used cantilevers were signiﬁcantly smaller than conventional cantilevers, but still large enough to be used with the opticaldetection system of a conventional AFM system (width of 11 ␮mfor all prototypes). A long 150 nm thick cantilever (Type B) witha low spring constant (about 21 pN/nm) was specially tailored forhigh sensitivity molecular recognition force spectroscopy (MRFS).
In contrast, long 430 nm thick cantilevers (Types C and D) and short
150 nm thick cantilevers (Type A) were designed for imaging ofsoft biological samples under aqueous and dry condition, both fortapping and contact mode. The latter represented a good compromise between high resonance frequency and low spring constant.
Prototypes E and F of 600 nm thickness were mainly used for characterization by electron microscopy. In particular type E turned
out too stiff for biological samples and its resonance frequency
exceeded the range for tapping mode imaging with our commercial
AFM.
First, the silicon oxynitride (Ohashi et al., 1997) prototypes werecharacterized using electron microscopy (EM) so as to determine
length, width, thickness, tip shape and radius (see Fig. 1A and B,
and table in Fig. 2). Using these dimensions, the spring constantand resonance frequencies were calculated (Fig. 2, for equations
see supporting information). Because of the reduced dimensionsof these cantilevers the mass of the tip and the different material constants of the reﬂex coating cannot be neglected. The shape(cone), the height, and the diameter at the base were extracted fromthe EM measurements. For coating a 10 nm ﬁlm of chromium asadhesive agent and a 20 nm thick gold layer for laser reﬂection wereused. The main results are summarized in the table of Fig. 2 and thecalculations are detailed in the supporting information. Calculated
resonance frequencies were compared with values measured using
the AFM frequency plot (Fig. 2, Type C and D) and they slightly disagree due to several assumptions made for the calculations. Moreprecisely, the tip was treated as point mass at the outer end of thecantilever and the cone shaped tip is partially hollow, so that theexact mass is difﬁcult to determine. In addition the thicknesses of
the chromium and gold layer can only be estimated from fabrication
parameters. Unfortunately, measuring the spring constant using
the more accurate thermal noise method was not possible withthe used equipment, because the cantilevers’ resonance frequency
exceeded the bandwidth of the A/D converter.
3.2. Molecular recognition force spectroscopy
AFM allows for the measurement of ultra-low inter- and intramolecular forces on the single molecule level. In molecular recognition force spectroscopy (MRFS) (Florin et al., 1994; Hinterdorfer
et al., 1996; Lee et al., 1994) a force is exerted on a receptor/ligand
complex and the dissociation process is followed over time. So
as to perform this type of measurement between a ligand on the
tip and a surface-bound receptor, a careful tip sensor design isrequired. Therefore the AFM tip had to be chemically modiﬁed(see Section 2) and, as a ﬁrst step, amino groups were generatedusing either APTES (Ebner et al., 2007a) or ethanolamine coating(Ebner et al., 2007a). Subsequently, a ﬂexible PEG-Biotin linker
was covalently bound to the amino groups on the tip. The suchlike biotin-functionalized AFM sensor was then moved towards the1402

M. Leitner et al. / Micron 43 (2012) 1399–1407

Fig. 2. The chart in Fig. 2 shows a summary of the results of the different characterized cantilever prototypes. The cantilever prototypes combine low spring constant witha high resonance frequency when compared to common cantilevers. The long 430 nm thick (Type D) and the short 150 nm thick (Type A) cantilevers were used for highspeed imaging at nanometer resolution in contact and tapping mode. The long 150 nm thick (Type B) cantilever was used for MRFS on avidin/biotin test system, showing
lower thermal ﬂuctuations then conventional cantilevers with similar spring constant. The long and short 600 nm (Type E and F) cantilever was only used for EM imaging
and characterization. The difference in the measured and calculated frequencies for Type C and D cantilever is mainly due to meanderings of the cantilever dimensions, thereﬂex coating, the tip mass and the shape of the tip.

sample containing the cognate receptors (avidin) and subsequently
retracted (Fig. 3). During contact time the biotin on the tip eventually bound to the avidin on the sample surface. Upon retraction the
cantilever was bended downwards and a force signal with a distinct
shape occurred. This force increased until the ligand receptor bondwas broken and the cantilever ﬂipped back into its resting position.
From the unbinding event, the binding force between tip tethered
ligand and surface bond receptor was determined. Importantly,
comparing our prototype cantilever with a typical commerciallyavailable force spectroscopy cantilever, the force noise was much
lower using the small prototype cantilever (Fig. 3).
For a clear discrimination between noise and unbinding, thethermal force ﬂuctuation of the cantilever needs to be signiﬁcantly lower than the measured forces. Thus the force–distancecycles recorded on the avidin/biotin system were analyzed regarding their thermal ﬂuctuation by extracting 50 nm intercepts
from the non-contact regime of the retraction curve. So as toevaluate the quantities of the prototype cantilevers, they werecompared to commercially available standard force spectroscopy
cantilevers (AUHW MSCT Type B-E, Bruker, USA; OMCL-TR400PSA
and BioLever, Olympus, Japan). The spring constant of typical MRFS
cantilevers is speciﬁed between 6 and 40 pN/nm. More precisely,
Type B cantilever showed a spring constant of about 21 pN/nm.
The BRUKER AUSHM MSCT B-D ranged from 10 to 30 pN/nm (B = 20,
C = 10, D = 30), whereas the Olympus BioLever was the softest available cantilever with a spring of 6 pN/nm. Finally, BRUKER AUHW
MSCT Type E, which is usually not used for MFRS, showed a typicalspring constant of 100 pN/nm and was compared to Type D cantilever. All measurements were performed using the same setup,
same scanner, and same photodiode to minimize instrumental
inﬂuences.
Evidently, the thermal force ﬂuctuations decreased with lower
spring constants (Fig. 4). Comparing commercial reference cantilevers to our prototypes (SCL) clearly indicated that smallcantilevers with similar spring constants have much lower force
ﬂuctuations (Figs. 4 and 5) under the same experimental conditions. In detail, the thermal ﬂuctuation for the Type B cantileverturned out to be 5–6 times lower than BRUKER cantilever B and D.
When we compared Type B cantilever to BRUKER C and Olympus
BioLever, the ﬂuctuation was still 2–3 times lower, even though thereference cantilevers showed a lower spring constant. Finally, thethermal force ﬂuctuation of Type D, with a value of about 21 pN, wascomparable BRUKER E, despite the considerably higher the spring
constant (535 pN/nm) (Figs. 4 and 5).
3.3. Imaging of biological samples under different conditions withhigher speed

Fig. 3. Fig. 3 shows a typical force distance cycle with a speciﬁc unbinding event
using a chemically modiﬁed cantilever prototype (Type B). Force distance cycles
were recorded on avidin adsorbed to freshly cleaved mica. The inset shows a typicalforce distance cycle recorded on the same system using a Bruker MSCT C (10 pN/nm)
cantilever. Obviously it can be seen that the force noise is much lower using the small
prototype cantilever.

The developed cantilever prototypes were utilized for imaging
of several biological samples under aqueous and ambient conditions in both tapping and contact mode. As test system for highresolution imaging in liquid bacterial surface layer proteins wereseparated from B. sphaericus and recrystallized on cleaned siliconchips. For imaging in dry state we used cell nuclear membranes
from X. laevi oocytes so as to visualize the embedded nuclear porecomplexes.
3.3.1. Tapping mode imaging
Bacterial surface layers comprise the outer surface layerof some bacteria (Sleytr and Messner, 1983). They consist ofM. Leitner et al. / Micron 43 (2012) 1399–1407

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Fig. 4. Fig. 4 shows force noise traces of different cantilevers which are common in molecular recognition force spectroscopy (MRFS). All curves have been extracted fromforce distance cycles, showing 50 nm ranges of the non-contact region of the retrace at the same loading rate (1 s sweep duration, 600 nm force distance cycles, 2000 data
points). (A and B) Force noise traces of the Type B and Type D prototype cantilevers. The 150 nm cantilever with a spring constant of 21 pN/nm showed the lowest thermal
force noise. BRUKER Cant. (B) (Fig. 4E, 20 pN/nm), (C) (Fig. 4F, 10 pN/nm) and (D) (Fig. 4G, 30 pN/nm) and the Olympus BioLever (Fig. 4C, 6 pN/nm) and TSP400 (Fig. 4D,
20 pN/nm) cantilever showed higher thermal force ﬂuctuations.

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M. Leitner et al. / Micron 43 (2012) 1399–1407

Fig. 5. The diagram in Fig. 5 summarizes the thermal force ﬂuctuation analysis for common MRFS cantilever and the prototype cantilevers. Type D cantilevershowed a thermal ﬂuctuation of about 22 pN which is comparable to Bruker cantilever E (∼28 pN). Type B cantilever showed a thermal ﬂuctuation of about
1.4 pN which is about 5 times lower when compared with Bruker cantilever B, and about 3 times lower when compared with Bruker cantilever C and Olympus
BioLever.

proteins which have the ability to self-assemble into monolayers
on various surfaces (e.g. silicon), resulting in well deﬁned, regular two-dimensional lattice structures. Depending on the bacterial
strain these layers from 5 to 20 nm thick oblique, square or hexagonal structures with a center-to-center spacing of 2.5–35 nm (Sleytr,
1997; Sleytr et al., 2007, 1994). Here B. sphaericus CCM2177 (Ilket al., 2002; Meyer and Neide, 1904) was used that forms a 2-D
square lattice (p-4 symmetry) with a spacing of about 14 nm anda thickness of 7 nm. As this layer is extremely ﬂat and displays
nm-sized features, it is perfectly suited to determine the lateral
resolution at different imaging speeds. The overview image in 6A
shows a silicon chip densely packed with bacterial surface layer
patches (for preparation see Section 2) recorded in contact mode ataqueous conditions using Type D cantilever. Image 6B was recorded
in tapping mode in the same environment. The typical square lattice structure with 14 nm (Tang et al., 2008a) can be clearly resolved
at larger magniﬁcations (Fig. 6B inset). The achieved resolution of
the recorded images was comparable to images recorded with conventional cantilevers at low speeds (5 ␮m/s) (Tang et al., 2008a).
The maximum tip velocity, where nm resolution could be observedwith Type D cantilever was about 55 ␮m/s. Therefore the usage ofthe small cantilever prototypes resulted in a speed increase by afactor of 10, reducing the total recording time of a 512 × 512 pixel
high resolution image to less than a minute.

4. Discussion

3.3.2. Contact mode imaging
Contact mode imaging in ambient conditions was performed on
nuclear membranes in which the nuclear pore complexes (NPC)

Speeding up AFM imaging often requires tedious electronic
developments and usage of special scanners. In this study weaimed at increasing the force sensitivity and the imaging speed of(Kaftan et al., 2002; Bednenko and Gerace, 2003; Stewart et al.,2001) are embedded. The NPCs are large protein assemblies consisting of about 30 different proteins, the nucleoporines (Nups),
each of which exists in several copies (Rout et al., 2000). In higher
eukaryotic cells an NPC assembles 120 of these proteins in total. It
reveals an eight-fold rational symmetry normal to the membrane
and an asymmetric assembly with respect to the horizontal planeof the membrane (Hinshaw et al., 1992).
The NPCs were chosen because they represent an example ofa rougher protein surface of several tens of nm in z scale. Due totheir stability they can also be prepared and imaged in ambientconditions. Fig. 7A shows a nuclear membrane in which the embedded nuclear pore complexes (NPC) (Kaftan et al., 2002) are clearlyvisible. The image was recorded using Type D cantilever in contact mode at ambient conditions. The maximum tip velocity forhigh resolution NPC imaging where the structures could be clearlyobserved was 33 ␮m/s (Fig. 7A). Fig. 7B shows NPC’s recorded witha conventional cantilever at 2.5 ␮m/s. Evidently, the lateral resolution is comparable. In contrast, using a conventional cantileverat high speed (25 ␮m/s) yields images with much less resolution(Fig. 7C).

Fig. 6. Bacterial surface layer from Bacillus Sphaericus were imaged with up to ten times higher speed compared to common cantilevers. (A) Overview image recorded incontact mode using Type D cantilever. Imaging speed was about 55 ␮m/s (∼10 lines/s). (B) Larger magniﬁcation image. The typical lattice structure with a constant of 14 nm
constant is clearly visible (see inset). Image recorded in tapping mode with nm resolution using the Type C Cantilever.

M. Leitner et al. / Micron 43 (2012) 1399–1407

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Fig. 7. (A) Nuclear pore complexes imaged with Type D cantilever using contact mode at ambient conditions. (B) The imaging speed of 12 lines per second was about ten times higher than in (A) using the same cantilever. (C) Conventional cantilever at a speed of 10 lines/s yielded images with much lessresolution.

a conventional AFM without any changes to the setup. Enhancements were accomplished by using small and sensitive cantilevers.
The force resolution for MRFS experiments was 3–5 times higher
than with usual cantilevers. Furthermore we achieved an increaseof the imaging speed by a factor of 10, while at the same timeretaining the tip sample interaction forces sufﬁciently low for
obtaining high resolution images soft biological samples in ﬂuid.
This reduced the time required for recording a 512 × 512 pixel
image to less than 1 min. It should be noted that this is stillquite far away from video AFM imaging reported by several othergroups (Ando et al., 2008; Anwar and Rousso, 2005; Fantner et al.,2006; Kobayashi et al., 2007; Miyagi et al., 2008; Picco et al.,2008, 2007), but nevertheless the achieved increase in scan speed
will allow the investigation of the dynamics of many biologicalprocesses.
The main challenge of using our prototype cantilevers in aconventional AFM is the small laser reﬂection area of the cantilever (∼11 ␮m) compared to the relatively large laser spotsof the AFM laser unit (∼30 ␮m). This makes the detected sum
signal up to 10 times lower compared to a conventional cantilever; improvements were achieved by the usage of an amplifying
photodiode. Another limitation was the line-rate speed limit of
the software used (13 scan lines/s). At much larger line rates
however, the resonances of the scanner become a limiting factor. Efforts for reducing these vibrations through input shaping
have yielded good results (Schitter et al., 2004; Schitter andStemmer, 2003, 2004). Optimization of imaging speed to video
rate requires signiﬁcant changes and improvements of the whole
AFM instrument (Ando et al., 2001). We believe, however, thatthe 10-fold increase in performance due to the use of thesmall cantilevers is sufﬁcient for many interesting biological processes and constitutes a signiﬁcant improvement for general AFM
users.
Acknowledgments
This work was supported by Fabican (project 819637) whichis part of the project cluster Austrian Science Foundation(project N00104-NAN), by FFG MNT.era.net (project 823980),
the Swiss National Science Foundation (205321 134786) and the
Schrödinger foundation. We thank Christian Rankl for help in data
evaluation.
Appendix A. Supplementary data
Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/
j.micron.2012.05.007.

Supporting Information Available: Detailed calculations forthe cantilever characterization
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