See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/257310458

Combination of fluorescence microscopy and

nanomotion detection to characterize bacteria

Article in Journal of Molecular Recognition · November 2013

DOI: 10.1002/jmr.2306 · Source: PubMed

CITATIONS READS

9 154

6 authors, including:

Carine Benadiba Giovanni Longo

École Polytechnique Fédérale de Lausanne Istituto di Struttura della Materia - CNR
30 PUBLICATIONS 235 CITATIONS 64 PUBLICATIONS 579 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Thorough understanding the human erythrocytes' aging by investigating the ultra-structural,

nanomechanical and biochemical properties of the cells View project

Human erythrocyte’s ageing in healthy and pathological subjects, studied by DSC and AFM
techniques View project

All content following this page was uploaded by Giovanni Longo on 01 July 2017.

The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document
and are linked to publications on ResearchGate, letting you access and read them immediately.
 Special Issue Article
Received: 30 June 2013, Revised: 23 July 2013, Accepted: 29 July 2013, Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/jmr.2306

Combination of fluorescence microscopy and

nanomotion detection to characterize bacteria†
S. Aghayeea, C. Benadibaa, J. Notza, S. Kasasa,b, G. Dietlera and G. Longoa,c*

Antibiotic-resistant pathogens are a major health concern in everyday clinical practice. Because their detection by
conventional microbial techniques requires minimally 24 h, some of us have recently introduced a nanomechanical
sensor, which can reveal motion at the nanoscale. By monitoring the fluctuations of the sensor, this technique can
evidence the presence of bacteria and their susceptibility to antibiotics in less than 1 h. Their amplitude correlates
to the metabolism of the bacteria and is a powerful tool to characterize these microorganisms at low densities. This
technique is new and calls for an effort to optimize its protocol and determine its limits. Indeed, many questions
remain unanswered, such as the detection limits or the correlation between the bacterial distribution on the sensor
and the detection’s output. In this work, we couple fluorescence microscopy to the nanomotion investigation to
determine the optimal experimental protocols and to highlight the effect of the different bacterial distributions
on the sensor. Copyright © 2013 John Wiley & Sons, Ltd.

Keywords: Nanomechanical sensors; Fluorescence microscopy; Bacteria; Nanomotion detector; metabolism; AFM

INTRODUCTION environment (Park et al., 2010) and the combination of a cantilever

Inappropriate use of antibiotics has caused an increasing number of
multi-drug-resistant bacterial strains, making the resulting diseases
more difficult to diagnose or treat with the common first-line
medical therapies (Alanis, 2005). An early detection of the responsi-
ble microorganism and possible susceptibility to specific antibiotics
will allow quick initiation of an appropriate treatment, causing a
decrease in mortality risk and hospitalization costs, which can put
a heavy burden on the economy of society and individuals.
To reduce the time needed to characterize bacterial growth and
proliferation, new technological solutions have been introduced.
Among them, nanomechanical sensors stand out as one of the
most promising techniques (Huber et al., 2013, Shekhawat and
Dravid, 2013). A very sensitive oscillator (typically an atomic force
microscope cantilever [AFM]) is coupled with a displacement
detector to produce extremely powerful and versatile sensors
(Boisen et al., 2011, Tamayo et al., 2013, Waggoner and Craighead,
2007) capable of characterizing biological systems with unprece-
dented detail and time resolution (Alvarez and Lechuga, 2010,
Calleja et al., 2012, Djuric and Jokic, 2007, Hansen and Thundat,
2005, Lang et al., 1999, McKendry et al., 2002). These devices are
often employed to investigate the effects of environmental stimuli
on bacteria. Some solutions rely on a high-resolution determina-
tion of the mechanical properties of one (Wali et al., 2010) or
several parallel sensors (Reed et al., 2006) to determine the
presence of added mass on the lever’s surface. Other works
consider coating of the sensor’s surface with a thin nutritive layer
(typically agarose) in humid atmosphere to support the local
growth of bacteria (Detzel et al., 2006, Gfeller et al., 2005).
Cantilevers containing microfluidic components have been
conceived to monitor the presence and growth of cells and
bacteria without modifying the mechanical properties of the
sensor (Barton et al., 2010). Recently, new, more complex solutions
have been proposed. Among them, particularly elegant are
590
sensor and a high-resolution laser scanning to characterize in real
time the vibration modes (Tamayo et al., 2012).
We have recently introduced a versatile and rapid diagnostic tool
(Kasas et al., 2011) capable of a rapid and quantitative determina-
tion of bacterial resistances. This technique (nanomotion detector)
uses an AFM (Binnig et al., 1986) cantilever as nanomechanical
sensor (Gimzewski et al., 1994, Huber et al., 2008, Lang and Gerber,
2008). By monitoring the nanoscale oscillations of the cantilever
that arise when viable specimens are attached to its surface, this
technique is able to investigate the bacterial metabolism when
the specimens are exposed to different environmental stimuli,
including bactericidal drugs (Longo et al., 2013).
These fluctuations could be caused by very diverse meta-
bolically related phenomena, such as the bacterial activity
within the membrane. Therefore, it allows determining the
viability and reaction of the microorganisms to the stimuli
long before their replication. This technique is of great
* Correspondence to: G. Longo, Laboratoire de Physique de la Matière Vivante,
Ecole Polytechnique Fédérale de Lausanne, EPFL SB IPSB LPMV BSP 414
(Cubotron UNIL), Rte de la Sorge CH-1015 Lausanne, Switzerland.
E-mail: giovanni.longo@epfl.ch
†
This article is published in Journal of Molecular Recognition as part of the vir-
tual Special Issue ‘AFM BioMed Shanghai 2013, edited by Jun Hu, SINAP, China
and Pierre Parot and Jean-Luc Pellequer, CEA, France’.
a S. Aghayee, C. Benadiba, J. Notz, S. Kasas, G. Dietler, G. Longo
Laboratoire de Physique de la Matière Vivante, Ecole Polytechnique Fédérale
de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
b S. Kasas
Département des Neurosciences Fondamentales, Université de Lausanne,
CH-1015 Lausanne, Switzerland
c G. Longo
Istituto di Struttura della Materia, Consiglio Nazionale delle Ricerche, 00133

micromechanical pedestals used as sensitive oscillators in liquid Rome, Italy

J. Mol. Recognit. 2013; 26: 590–595 Copyright © 2013 John Wiley & Sons, Ltd.
NANOMOTION DETECTION AND FLUORESCENCE MICROSCOPY TO STUDY BACTERIA
relevance because it can characterize the bacterial resistance
to antibiotics in less than 1 h, instead of the several hours or
days needed using conventional microbiological techniques
(Horvat, 2010, Pfyffer et al., 1997).
However, many questions have remained unanswered to date,
and further investigations are still required, such as the optimal im-
mobilization protocol for the bacteria or the correlation between
the amplitude of the measured fluctuations and the number of bac-
teria attached to the cantilever and their distribution on its surface.
In this work, we intend to shed some light on this problem. We
have employed fluorescent optical microscopy to provide a
reliable measure of the amount of germs under investigation,
and we have compared these results with the resulting
fluctuations of the cantilever sensor.
MATERIALS AND METHODS
Description of the setup
To perform the measurements, we used a JPK NanoWizard III AFM
(JPK, Berlin, Germany) coupled with an Axiovert X optical
microscope with an HXP 120C fluorescent lamp (Zeiss Microscopy,
DE, USA). All the fluorescence images were collected using 553 nm
excitation wavelength and 585 nm emission fluorescence through
a standard 40× objective and using a ProgRes MFCool digital
camera (Jenoptik, DE, USA). The in-program camera-control soft-
ware was used to collect the images in an automated way. The
AFM was equipped with a custom liquid cell (Kasas et al., 2013)
to reduce the amount of liquid medium needed to perform the
measurements and to allow a noiseless medium transition during
the experiments.
Materials
Sigma-Aldrich (St Louis, MO, USA) supplied all chemicals,
phosphate buffered saline (PBS, pH 7.4), lysogeny broth (LB),
glutaraldehyde, and ampicillin, all with analytical grade.
Preparation of the bacteria
In this work, we used colonies of Escherichia coli (E. coli) DH5-α
strain, transformed by a construct of pRSET-B vector. The
tdTomato gene was inserted inside the pRSET-B backbone,
pRSET-B-tdTomato (the construct was obtained by the group
of Professor Tsien) (Shaner et al., 2004). Bacteria transformed
with this plasmid express a red fluorescent protein as well as
ampicillin resistance. Only those colonies of E. coli that are
successfully transformed by this plasmid can survive in the
presence of ampicillin. This kind of transformation is increas-
ingly used in the study of several unicellular organisms
(Graewe et al., 2009), including fungi (Kaufmann, 2009) and
bacteria (Lakins et al., 2009).
To insert the vector into E. coli, certain steps were taken
according to the laboratory protocol. A frozen stock of DH5-α E. coli
at 80°C was put into ice. A volume of 50 μl of bacteria was added
to a ligation solution and incubated on ice for 30 min. The tube was
then placed at 42°C for 45 s to cause a heat shock. The bacteria
were then incubated on ice for 2 min. At this stage, the bacterial
membrane was ready to accept the plasmid. We added 250 μl of
LB solution to the tube containing the germ, which was incubated
at 37°C for 1 h. The bacteria solution was inoculated on LB-agar
plates containing a 50 μg/ml ampicillin. The agar plates were
incubated at 37°C overnight, to allow the growth of the
J. Mol. Recognit. 2013; 26: 590–595 Copyright © 2013 John Wiley & Sons, Ltd.
transformed bacteria. After this period, a colony was collected from
the agar plates and incubated overnight at 37°C in 10 ml of LB with
50 μg/ml concentration of ampicillin. A stock of bacteria solution
was stored in 80°C for future use.
In preparation for the experiments, a bacterial colony was
collected from the frozen stocks of transformed E. coli strain
with pRSET-B-tdTomato plasmid and incubated overnight at
37°C in 10 ml LB solution with 50 μg/ml concentration of
ampicillin. After incubation, 4 ml of the bacteria was washed
in PBS. Before each rinse, they were sedimented by centrifuga-
tion at 6000 rpm for 10 min, and were finally re-suspended in
1 ml PBS. The sensors for each experiment were prepared
using a small volume (namely 50 μl) of the germs in PBS.
Preparation of the cantilevers
Silicon nitride triangular cantilevers (DNP-10, Bruker, Santa
Barbara, CA, USA) with a nominal length of 205 μm and spring
constant of 0.06 N/m were used as sensors. To bind the
bacteria, the surface of the cantilever was functionalized with
0.5% glutaraldehyde for 7 min after which it was rinsed three
times using ultrapure water (Meyer et al., 2010). The cantilever
was dried using nitrogen and then exposed to a 50 μl PBS
solution of the prepared bacteria solution.
Once the microorganisms were firmly adsorbed to the sensor’s
surface, it was inserted in the analysis chamber, flushed with PBS,
and imaged using fluorescence microscopy to determine the
number and distribution of the bacteria.
The fluorescent microscopy images of the sensor suggested
that the number of attached bacteria, their viability, and their
stability over time strongly depended on the sensor’s prepara-
tion protocol. To find the optimal conditions, we tested several
exposure times for glutaraldehyde functionalization and bacteria
adsorption, determining for each procedure the number and the
stability of the attached bacteria.
The result of this screening indicated that the best combina-
tion involves functionalizing the cantilever for 7 min using a
0.5% glutaraldehyde aqueous solution followed by 40 min
incubation with the bacteria.
To highlight the different outcomes following the various
preparation protocols, we show in Figure 1(a)–(c), the effect of
exposing the cantilevers to the solution containing the bacteria
for three different time lengths.
Setup of the nanomotion experiment
The setup of a nanomotion experiment is sketched in Figure 1
(d) and described in detail in the study of Barton et al., 2010.
Briefly, the bacteria-loaded cantilever was placed into an
analysis chamber and flushed with PBS. Its fluctuations were
recorded using an AFM’s laser-detection transduction unit.
External electronics was used to collect the fluctuations of the
cantilever sensor. All acquisitions were performed using a
10 kHz rate in PBS. In all cases, we performed minimally
30-min measurements, which were analyzed by calculating
the average variance of the amplitudes.
Before the start of each experiment, the JPK data acquisition
software was used to calibrate the cantilever deflection in order
to convert the microscope’s output in nanometers.
The thermal fluctuations of each cantilever recorded before
the attachment of the bacteria were chosen as baseline for the
591
successive experiment.
wileyonlinelibrary.com/journal/jmr
 S. AGHAYEE ET AL.

a b

c d

Figure 1. The optimization of the setup. (a), (b), and (c): fluorescent microscopy images of the treated cantilevers with a. 20 min, b. 30 min, and c.
40 min of incubation with the bacteria. All of the cantilevers were functionalized by a 6-min exposure to glutaraldehyde. (d): the core setup of the
experiment with laser (L), cantilever (C), and detector (D).

The fluorescence analyses RESULTS AND DISCUSSION

The AFM microscope used for these measurements is
coupled to an optical microscope with fluorescence imaging
capabilities. This setup was used to monitor the position
and viability of the attached bacteria throughout each
nanomotion experiment. During the time span of a complete
experiment (around 1 h), the microscope’s camera was used
to collect fluorescence images of the cantilever every 20 s,
allowing a quantitative investigation of the number and
distribution of the attached germs.
At first, we compared the number of bacteria that were
attached to the cantilever’s surface over several preparations
using the very same optimized protocol described previously.
In our previous work, we had estimated a total number of
almost 1000 bacteria attached to the lever, but we had no
independent way to determine their viability or their contri-
bution to the overall increase in the amplitude of the
fluctuations (Longo et al., 2013). Most of the cantilever

Figure 2. Determination of number and distribution of the bacteria on the sensor. Three cantilevers prepared using the optimized protocol exhibit a
different distribution of the microorganisms on their surface. To determine the importance of the germs located in different areas of the sensor, we
592

divided its surface in four segments of ~50 μm in length. The number of bacteria for each section is summarized in Table 1.

wileyonlinelibrary.com/journal/jmr Copyright © 2013 John Wiley & Sons, Ltd. J. Mol. Recognit. 2013; 26: 590–595
NANOMOTION DETECTION AND FLUORESCENCE MICROSCOPY TO STUDY BACTERIA

Table 1. The bacteria present on the different sections of preparations resulted in a roughly constant number of living
the three cantilevers shown in Figure 4 (fluorescent) bacteria adsorbed to the sensor’s surface. Inter-
estingly, such a small number of attached bacteria (~110 bac-
Section A Section B Section C Section D teria are attached on the three cantilevers depicted in
Cantilever 1 8 14 40 50 Figure 2) were sufficient to produce a measurable fluctuation
Cantilever 2 10 24 40 40 of the sensor.
Cantilever 3 40 50 15 7 Each preparation led to a different distribution of the germs
on the surface. In some cases, most of the adsorbed bacteria
The three cantilevers show a similar overall number of attached were concentrated on the arms of the cantilever, whereas in
bacteria. Only the bacteria on the cantilever surface are taken other cases, the bacteria were more abundant near the apical
into consideration. area. As expected, this difference in the distribution had a large
effect on the resulting outcome of the nanomotion experiments
(Ramos et al., 2008). To better define these differences, we
divided the cantilever in four areas ~50 μm in length (shown as
8
red rectangles in Figure 2) and determined the number of
4
bacteria present in each area (Table 1).
While performing the fluorescence images, we monitored the
0 living bacteria through the fluctuations induced on the cantile-
ver. An example of the outcome of such nanomotion experiment
-4 is shown in the top panels of Figure 3. The analyses indicated
that, if the number and the viability of the attached bacteria
-8 were constant, the induced movements were stable throughout
a period of more than 2 h.
2 Next, we exploited fluorescent proteins to estimate the
stability of the attached bacteria. If the preparation or the
1 attachment procedures cause the death of the microorganisms,
the fluorescence should rapidly fade, because of photobleaching
0
phenomena (Shaner et al., 2008, Straight, 2007). The images
shown in the lower panel of Figure 3 indicate that the microor-
ganisms were very well attached and viable for the entire time
scale of an experiment.
Then, we investigated the impact of the bacteria’s distribution
on the deflection amplitude. We collected simultaneously
fluorescence images and nanomotion data from several cantile-
vers, calculating, for each case, the number of bacteria and the
variance of the resulting fluctuations. By comparing these data,
Figure 3. Stability of the system. Nanomotion measurements and
we were able to determine that the variance depends mainly
corresponding fluorescent images collected from a sensor throughout
a 2-h period. The fluorescent images show that the bacteria are firmly
on the number of viable bacteria present on the apical region
attached to the surface. No microorganism appears to have moved or of the cantilever (section A): the amplitude of the fluctuations
detached. The corresponding deflection information indicates that if grows with this subset of the attached microorganisms. Consid-
the distribution and viability of the bacteria is constant, so is the ering the overall number of viable germs for each case does
amplitude of the sensor’s movement. not deliver a similar correlation (Figure 4).

Figure 4. Effect of the number of bacteria on the induced fluctuations of the cantilever. A comparison between the number of bacteria attached
to the apical region of the cantilever and the resulting variance of the fluctuations of the cantilever. The correspondence between the bacteria
attached to the section A of the sensor and the variance of the fluctuations are accurate. Such relation does not hold if all the bacteria bound
593

to the surface of the sensor are taken into consideration.

J. Mol. Recognit. 2013; 26: 590–595 Copyright © 2013 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jmr

Figure 5. Effect on fluorescence and bacterial movements of the
exposure to ampicillin. Top panel: fluctuations of the cantilever produced
by the presence of E. coli before and after the exposure to ampicillin. The
effect of the drug is a rapid decrease of the fluctuations, indicating the
death of the germs. Bottom panel: fluorescence images of the cantilever
showing the effect of the antibiotic on the viability of the E. coli. Minutes
after the exposure to the drug, the fluorescence dimmed. The normalized
fluorescence of the apical region dropped more than three-fold in
10 min, because of photobleaching.
In fact, the maximum variance of the measured fluctuations
(3.4 nm2) is much smaller than the variance values measured in
our previous work (5–20 nm2), where the number of adsorbed
bacteria was 10-times higher (Longo et al., 2013). Yet, these
measurements ensure that as few as 100 microorganisms can
produce a measurable signal, highlighting the extreme sensitivity
of the technique. The main advantage, in this case, is that we are
able to define the subset of bacteria that contribute most to these
fluctuations, indicating that the effective sensitivity limit of the
technique is in the order of few tens of microorganisms.
Finally, to highlight further the direct correlation between the
viability of the bacteria and the amplitude of the fluctuation of
the sensor, we monitored the fluorescence produced by the bacte-
ria and the induced nanometric movements of the cantilever when
exposed to a bactericidal dose of antibiotics (top panels in Figure 5).
We chose a concentration that is more than twice the Minimum
REFERENCES
Ai H-W, Hazelwood KL, Davidson MW, Campbell RE. 2008. Fluorescent
protein FRET pairs for ratiometric imaging of dual biosensors.
Nat Meth. 5: 401–403.
Alanis AJ. 2005. Resistance to antibiotics: are we in the post-antibiotic
era? Arch. Med. Res. 36: 697–705.
Alvarez M, Lechuga LM. 2010. Microcantilever-based platforms as
biosensing tools. Analyst 135: 827–836.
Barton RA, Ilic B, Verbridge SS, Cipriany BR, Parpia JM, Craighead HG.
2010. Fabrication of a nanomechanical mass sensor containing a
nanofluidic channel. Nano Lett. 10: 2058–2063.
Berg CM, Anderson SV. 1976. Penicillin-induced lysis of Escherichia-
coli in medium that does not support sustained growth.
594
Antimicrob. Agents Chemother. 9: 713–715.
wileyonlinelibrary.com/journal/jmr Copyright © 2013 John Wiley & Sons, Ltd.
S. AGHAYEE ET AL.
Inhibitory Concentration (MIC) for these modified bacteria (Singer
et al., 2010) in order to ensure a rapid death of the germs. Namely,
we injected in the analysis chamber a solution containing 0.5 mg/ml
of ampicillin. This antibiotic is a competitive inhibitor of
transpeptidase, actively inhibiting the cell wall synthesis (Kaldalu
et al., 2004). This ultimately produces lysis of the cell wall, resulting
in the death of the microorganisms (Berg and Anderson, 1976,
Boman and Eriksson, 1963). The dying bacteria ceased producing
the fluorescent proteins, and the ones already present underwent
a rapid photobleaching (lower panel in Figure 5). This produced,
in less than 10 min, a more than three-fold reduction of the emitted
light. Indeed, this photobleaching time scale is compatible with the
results obtained by other groups under similar illumination condi-
tions (Ai et al., 2008, Shaner et al., 2005). At the same time, the bac-
teria stopped their metabolically related movements, causing the
reduction of the sensor’s fluctuations.
CONCLUSIONS
In this study, we demonstrated that fluorescently labeled bacteria
can be used to assess the viability of microorganisms attached to a
nanomechanical sensor. We employed fluorescent optical micros-
copy to determine the exact number and distribution of the germs
attached to the sensor as well as their stability and viability
throughout an entire experiment.
The use of fluorescent bacteria was extremely important to
determine the optimal conditions to be used in the preparation
of a nanomotion experiment. Furthermore, by coupling fluores-
cent imaging and fluctuation analysis, we were able to determine
that the bacteria present in the apical section of the cantilever play
the most important role in defining the sensor’s fluctuation
amplitude.
The sensitivity of the technique indicates that even a very small
quantity of bacteria can deliver a measurable signal. This opens the
way to the use of this diagnostic tool to study infections in cases
where only a very small volume of bacteria is available.
All these information will be of utmost importance in optimizing
the nanomotion to obtain a fast determination of the susceptibility
of bacteria towards antibiotics. Moreover, these results will allow a
better understanding of the origin of the recorded fluctuations and
of their correlation with the bacterial metabolism. Finally, these
results will be the base for the development of an independent
and automated diagnostic technique.
ACKNOWLEDGEMENT
This work has been supported by the FN-CR 32I3-130676 grant.
Binnig G, Quate CF, Gerber C. 1986. Atomic force microscope. Phys.
Rev. Lett. 56: 930–933.
Boisen A, Dohn S, Keller SS, Schmid S, Tenje M. 2011. Cantilever-like
micromechanical sensors. Rep. Prog. Phys. 74: 036101.
Boman HG, Eriksson KG. 1963. Penicillin induced lysis in Escherichia
coli. J. Gen. Microbiol. 31: 339–352.
Calleja M, Kosaka PM, San Paulo, A, Tamayo, J. 2012. Challenges for
nanomechanical sensors in biological detection. Nanoscale 4:
4925–4938.
Detzel AJ, Campbell GA, Mutharasan R. 2006. Rapid assessment of
escherichia coli by growth rate on piezoelectric-excited
millimeter-sized cantilever (PEMC) sensors. Sens. Actuators B
117: 58–64.
J. Mol. Recognit. 2013; 26: 590–595
NANOMOTION DETECTION AND FLUORESCENCE MICROSCOPY TO STUDY BACTERIA
Djuric ZG, Jokic IM. 2007. Thermomechanical noise of nanooscillators
with time-dependent mass. Microelectronic Eng. 84: 1639–1642.
Gfeller KY, Nugaeva N, Hegner M. 2005. Rapid biosensor for detection of
antibiotic-selective growth of Escherichia coli. Appl. Environ. Microbiol.
71: 2626–2631.
Gimzewski JK, Gerber C, Meyer E, Schlittler RR. 1994. Observation of a
chemical-reaction using a micromechanical sensor. Chem. Phys. Lett.
217: 589–594.
Graewe S, Retzlaff S, Struck N, Janse CJ, Heussler VT. 2009. Going live: a
comparative analysis of the suitability of the RFP derivatives RedStar,
mCherry and tdTomato for intravital and in vitro live imaging of
plasmodium parasites. Biotechnol. J. 4: 895–902.
Hansen KM, Thundat T. 2005. Microcantilever biosensors. Methods 37: 57–64.
Horvat RT. 2010. Review of antibiogram preparation and susceptibility
testing systems. Hosp. Pharm. 45: S6-S9.
Huber F, Lang HP, Backmann N, Rimoldi D, Gerber C. 2013. Direct
detection of a BRAF mutation in total RNA from melanoma cells
using cantilever arrays. Nat. Nanotechnol. 8: 125–129.
Huber F, Lang HP, Gerber C. 2008. BIOSENSORS new leverage against
superbugs. Nat. Nanotechnol. 3: 645–646.
Kaldalu N, Mei R, Lewis K. 2004. Killing by ampicillin and ofloxacin induces
overlapping changes in Escherichia coli transcription profile.
Antimicrob. agents chemothe. 48: 890–896.
Kasas S, Longo G, Alonso-Sarduy L, Dietler G. 2011. Nanoscale motion
detector. Patent n. PCT/IB2011054553, Switzerland.
Kasas S, Radotic K, Longo G, Saha B, Alonso-Sarduy L, Dietler G, Roduit C.
2013. A universal fluid cell for the imaging of biological specimens in
the atomic force microscope. Microsc. Res. Tech. 76: 357–363.
Kaufmann A. 2009. A plasmid collection for PCR-based gene targeting in
the filamentous ascomycete Ashbya gossypii. Fungal Genet. Biol. 46:
595–603.
Lakins MA, Marrison JL, O’Toole PJ, Van Der Woude MW. 2009. Exploiting
advances in imaging technology to study biofilms by applying
multiphoton laser scanning microscopy as an imaging and
manipulation tool. J. Microsc. 235: 128–137.
Lang HP, Baller MK, Berger R, Gerber C, Gimzewski JK, Battiston FM, Fornaro
P, Ramseyer JP, Meyer E, Guntherodt HJ: 1999. An artificial nose based
on a micromechanical cantilever array. Anal. Chim. Acta 393: 59–65.
Lang HP, Gerber C. 2008. Microcantilever sensors. STM and AFM Studies
on (Bio)molecular Systems: Unravelling the Nanoworld, Samori P (ed).
Springer: Berlin Heidelberg, Vol. 285; 1–27.
Longo G, Alonso-Sarduy L, Rio LM, Bizzini A, Trampuz A, Dietler G, Kasas S.
2013. Rapid detection of bacterial resistance to antibiotics using AFM
cantilevers as nano-mechanical sensors. Nat. Nanotechnol. 8: 522–526.
McKendry R, Zhang J, Arntz Y, Strunz T, Hegner M, Lang HP, Baller
MK, Certa U, Meyer E, Güntherodt H-J, Gerber C. 2002. Multiple
label-free biodetection and quantitative DNA-binding assays on
a nanomechanical cantilever array. Proc. Natl. Acad. Sci. 99:
9783–9788.
J. Mol. Recognit. 2013; 26: 590–595 Copyright © 2013 John Wiley & Sons, Ltd.
View publication stats
Meyer RL, Zhou X, Tang L, Arpanaei A, Kingshott P, Besenbacher F. 2010.
Immobilisation of living bacteria for AFM imaging under physiologi-
cal conditions. Ultramicroscopy 110: 1349–1357.
Park K, Millet LJ, Kim N, Li H, Jin X, Popescu G, Aluru NR, Hsia KJ, Bashir R.
2010. Measurement of adherent cell mass and growth. Proc. Natl.
Acad. Sci. 107: 20691–20696.
Pfyffer GE, Welscher HM, Kissling P, Cieslak C, Casal MJ, Gutierrez J,
RuschGerdes S. 1997. Comparison of the mycobacteria growth indi-
cator tube (MGIT) with radiometric and solid culture for recovery of
acid-fast bacilli. J. Clin. Microbiol. 35: 364–368.
Ramos D, Tamayo J, Mertens J, Calleja M, Villanueva LG, Zaballos A. 2008.
Detection of bacteria based on the thermomechanical noise of a
nanomechanical resonator: origin of the response and detection
limits. Nanotechnology 19.
Reed J, Wilkinson P, Schmit J, Klug W, Gimzewski JK. 2006. Observation of
nanoscale dynamics in cantilever sensor arrays. Nanotechnology 17:
3873–3879.
Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien
RY. 2004. Improved monomeric red, orange and yellow fluorescent
proteins derived from Discosoma sp. red fluorescent protein. Nat.
biotechnol.. 22: 1567–1572.
Shaner NC, Lin MZ, McKeown MR, Steinbach PA, Hazelwood KL,
Davidson MW, Tsien RY. 2008. Improving the photostability of
bright monomeric orange and red fluorescent proteins. Nat.
Methods 5: 545–551.
Shaner NC, Steinbach PA, Tsien RY. 2005. A guide to choosing fluorescent
proteins. Nat Meth. 2: 905–909.
Shekhawat GS, Dravid VP. 2013. Nanomechanical sensors: bent on
detecting cancer. Nat. Nanotechnol. 8: 77–78.
Singer JT, Phennicie RT, Sullivan MJ, Porter LA, Shaffer VJ, Kim CH. 2010.
Broad-host-range plasmids for red fluorescent protein labeling of
gram-negative bacteria for use in the zebrafish model system. Appl.
Environ. Microbiol. 76: 3467–3474.
Straight AF. 2007. Fluorescent protein applications in microscopy. Methods
in Cell Biology: Digital microscopy, Sluder G, Wolf DE (eds). Academic
Press: San Diego (USA), 3rd Edition, Vol. 81; 93–113.
Tamayo J, Kosaka PM, Ruz JJ, San Paulo A, Calleja M. 2013. Biosensors
based on nanomechanical systems. Chem. Soc. Rev. 42: 1287–1311.
Tamayo J, Pini V, Kosaka P, Martinez NF, Ahumada O, Calleja M. 2012.
Imaging the surface stress and vibration modes of a microcantilever
by laser beam deflection microscopy. Nanotechnology 23.
Waggoner PS, Craighead HG. 2007. Micro- and nanomechanical sensors
for environmental, chemical, and biological detection. Lab Chip 7:
1238–1255.
Wali RP, Wilkinson PR, Eaimkhong SP, Hernando-Garcia J, Luis Sanchez-Rojas
J, Ababneh A, Gimzewski JK. 2010. Fourier transform mechanical
spectroscopy of micro-fabricated electromechanical resonators: a novel,
information-rich pulse method for sensor applications. Sensor. Actuator
B-Chem. 147: 508–516.
595
wileyonlinelibrary.com/journal/jmr