Probing the dynamic responses of individual actin filaments under fluidic

mechanical stimulation via microfluidics

Chao-Min Cheng, Chung-Yao Yang, YongTae Kim, and Philip R. LeDuc

Citation: Appl. Phys. Lett. 102, 193704 (2013); doi: 10.1063/1.4806975

View online: http://dx.doi.org/10.1063/1.4806975
View Table of Contents: http://apl.aip.org/resource/1/APPLAB/v102/i19
Published by the American Institute of Physics.

Additional information on Appl. Phys. Lett.

Journal Homepage: http://apl.aip.org/
Journal Information: http://apl.aip.org/about/about_the_journal
Top downloads: http://apl.aip.org/features/most_downloaded
Information for Authors: http://apl.aip.org/authors

Downloaded 15 May 2013 to 140.114.125.14. This article is copyrighted as indicated in the abstract. Reuse of AIP content is subject to the terms at: http://apl.aip.org/about/rights_and_permissions
 APPLIED PHYSICS LETTERS 102, 193704 (2013)

Probing the dynamic responses of individual actin filaments under fluidic

mechanical stimulation via microfluidics
Chao-Min Cheng,1,a) Chung-Yao Yang,1 YongTae Kim,2 and Philip R. LeDuc2
1
Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu 30013, Taiwan
2
Departments of Mechanical Engineering, Biomedical Engineering, Computational Biology, and Biological
Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA
(Received 22 March 2013; accepted 22 April 2013; published online 15 May 2013)
Herein, we demonstrate an easy-to-handle approach that employs a combination of
microcurvilinear flow and fluorescence microscopy for probing the dynamic responses of
individual synthesized actin filaments. We observed morphological changes of single actin
filaments with different spatiotemporal responses when they were elongated with rotation or
underwent significant bending during fluidic shear stress, and found that they may initially increase
their curvature but then start releasing the external force immediately thereafter. Our approach
allowed us to visibly examine the dynamic responses of individual actin filaments under
simultaneous forces of rotation and elongation, as well as bending resulting from fluidic shear
C 2013 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4806975]
stress. V

This paper describes the development of an easy-to-use actin filaments, and microtubules) at the molecular
approach for probing the dynamic responses (i.e., elongation, level.17–21 For example, Kishino and Yanagida have demon-
rotation, and bending) of individual actin filaments. We stud- strated a method to catch and manipulate a single actin fila-
ied actin filaments, important structural proteins in many liv- ment (F-actin) using glass microneedles under conditions in
ing cells, using a unique microfluidic-based mechanical which an external force on a single filament can be applied
stimulation that employs a combination of microcurvilinear and measured.17 The tensile strength of a single filament—
flow via microfluidics and time-lapse epi-fluorescence mi- the force to break the bond between two actin monomers—
croscopy.1 Scientific understanding of structural and organiza- was measured directly through this method. Tsuda et al.
tional aspects in biologically relevant fields such as cell have also used optical tweezers and microneedles to deter-
biology or structural biology is enhanced through the develop- mine the torsional rigidity and the breaking force of single
ment of new experimental tools in chemical, physical, and en- actin filaments by measuring the rotational Brownian motion
gineering fields.1–5 These approaches, which can be grounded and tensile strength.19 This experiment indicated that an indi-
in technique development, have been used to study cytoskele- vidual actin filament exhibited comparable flexibility in the
tally relevant cellular behaviors such as cell migration and rotational and longitudinal directions but broke more easily
cell division as well as mechanics-related biological responses under a torsional load. These physically based approaches
by controlling specific mechanical stimulation (e.g., mechani- allow researchers to probe biologically relevant issues that
cal stretch, fluidic shear stress, or substrate rigidity) in living are not easily addressed thoroughly using conventional bio-
cells.6–11 The cellular responses under specific mechanical logically based methods or assays such as Western blotting,
stimulation, including cytoskeleton remodelling and stem cell immunofluorescence staining, or enzyme-linked immunosor-
differentiation, are highly tuned and relevant to the cell’s bent assay (ELISA). The main drawbacks of these physically
cytoskeletal structure, which is composed of actin filaments, based approaches include the following: (1) the challenges
microtubules, and intermediate filaments.12 The complicated and time required to build these techniques (e.g., it is chal-
dynamic responses of actin filaments and other cytoskeletal lenging to build optical tweezers with molecular-level reso-
elements facilitate cell division, motility, proliferation, and lution in a short period of time); and (2) difficulties with
determine cell shape.13 Among these cytoskeletal subunits, operating and maintaining the equipment (e.g., the require-
the actin cytoskeleton plays an important role in providing ments of high-cost equipment and well-trained specialists).
mechanical support for single cells and in delivering physio- The capacity to provide a technique-derived approach (i.e.,
logical functionality at the cellular level for a variety of func- chemical-/physical-/engineering-based approach) with the
tions including chemotaxis, phagocytosis, and mitosis.14–16 characteristics of robustness, ease-of-use, simplicity, and fru-
The actin cytoskeleton has distinct mechanical properties that gality would allow researchers to address biologically rele-
enable it to be part of the interconnected organizational struc- vant issues in novel and more direct ways. These approaches
ture both within and among living cells. Its elastic properties may also facilitate innovative research and fresh findings in
and interactions with the cell membrane are essential for a variety of research communities including biophysics,
understanding cellular morphology. polymer science, cell biology, and structural biology for
We, and others, have developed a variety of techniques which the use of other, more conventional, biologically
to probe the dynamic responses of biopolymers (e.g., DNA, based approaches may not.
To understand the structure and mechanics of actin fila-
a)
Author to whom correspondence should be addressed. Electronic mail: ments, we have taken a synthetically based approach to their
[email protected] biology. We have used this synthetic approach previously to

0003-6951/2013/102(19)/193704/5/$30.00 102, 193704-1 C 2013 AIP Publishing LLC

V

Downloaded 15 May 2013 to 140.114.125.14. This article is copyrighted as indicated in the abstract. Reuse of AIP content is subject to the terms at: http://apl.aip.org/about/rights_and_permissions
 193704-2 Cheng et al. Appl. Phys. Lett. 102, 193704 (2013)

create an artificial cell-like system with an artificial actin cy- provide a more comprehensive understanding of results and
toskeleton.22 This enabled us to obtain further insights into responses following imposition of the defined radial acceler-
cell and polymer behavior by mimicking the actin cytoskele- ation on multiple synthesized actin filaments in our micro-
ton in an artificial environment composed of giant liposomal curvilinear flow system while simultaneously controlling
vesicles and then using an atomic force microscope to exam- flow velocity, we have developed an approach that allowed
ine the resulting liposomes and force responses. In our us to characterize the flow profile in either the circular side
experiments, actin filaments appeared to influence the me- chamber or main channel by tracking and mapping out the
chanical properties (i.e., Young’s modulus) of this artificial flow path of 2-lm-diameter fluorescent beads (Spherotech
cell-like system so that the force response within this artifi- Inc., IL; No.: CFP-2052-2) with epi-fluorescence micros-
cial cell-like system was determined to be shared between copy. Fig. 1(e) displays the flow patterns in the circular side
cellular and polymer responses.22 Building off of our previ- chamber that were generated in our microfluidic device with
ous work, we have developed an easy-to-build, easy-to- flow rates of 50 and 75 ll/min. We found that the average
operate and robust microfluidic-based method to understand linear velocity of the pressure-driven flow in the circular side
the real-time dynamic responses of individual actin filaments chamber increased proportionately with the higher flow rate
undergoing elongation, strain, and curvature. Our approach from the syringe pump. Previously, we implemented a com-
leverages a combination of microcurvilinear flow through putational fluid dynamics (CFD) approach to determine the
microfluidics and time-lapse fluorescence microscopy to computational rotational velocities and radial accelerations
sequentially capture real-time images of single actin fila- when designing and fabricating our microfluidic device with
ments with mechanical stimulation in microfluidics. In this a circular side chamber. We did this to probe the real-time
study, we first fabricated a microfluidic device with a circu- dynamic responses of single DNA molecules in our previous
lar side chamber via soft lithography (Fig. 1(a); details study.24 Here, we have expanded upon this effort of examin-
described in the supplementary material23), in order to ac- ing actin filaments to generate greater understanding of the
complish three objectives. First, it allowed us to establish a effects of curvilinear flow on filaments in our microfluidic
microcurvilinear flow with flow controlled by our microflui- device. We were able to visualize and measure the real-time
dic device. Second, it enabled us to induce a high-radial dynamic responses of multiple actin filaments under curvi-
acceleration flow through our microfluidic device (maximum linear flow. Although we observed, recorded, and analyzed
fluid velocity of up to 0.22 m/s [z ¼ 1 lm] when the flow the real-time dynamic responses of phalloidin-labeled single
rate was 75 ll/min [as a radial acceleration of up to actin filaments via the same microfluidic-based system with
17 400 m/s2 and Reynolds number of about 0.6 as well]) with epi-fluorescence microscopy that we used in our previous
specific geometry that we designed and prepared. The high- DNA study, the scientific issues that we attempted to address
radial acceleration in the microcurvilinear flow system (with were quite distinct. For example, the dynamic responses
sharp streamlines) that we generated in our microfluidic de- between these two individual molecular systems of DNA
vice was controlled by either the dimensions of the circular and actin filaments under flow-stretching through using our
side chamber or the flow rate that we introduced, as shown microfluidic-based system address a longstanding scientific
in Fig. 1(b). Third, it allowed us to stimulate individual actin question that applies to several research communities includ-
filaments mechanically via fluidic shear stress induced ing skills and perspectives in polymer physics, biophysics,
through this microcurvilinear flow. Fig. 1(c) is an optical biomaterials, structural biology, and cell biology. This ques-
image of the microfluidic device that we used in this study. tion is grounded in the view of biopolymer research, where
The syringe pump (Harvard Apparatus, MA; No.: PHD researchers commonly view single DNA molecules as flexi-
22/2000), which was used to create and control the pressure- ble polymers. However, individual actin filaments are semi-
driven flow (e.g., the flow rate), was connected to our micro- flexible polymers (e.g., different persistence lengths for these
fluidic device through polyethylene tubing (Becton two biomolecules), indicating that their inherent properties
Dickinson, NJ; Intramedic tubing with an internal diameter would influence the dynamic responses (i.e., the final output
of 0.78 mm, No.: 427416). The synthesized actin filaments polymer behaviors) when under mechanical stimulation such
labelled with phalloidin (Molecular Probes, CA; No.: as in this study. Furthermore, in our previous work, we fixed
A12379) were imaged through an epi-fluorescence micro- one end of single DNA molecules to the bottom of our
scope with a 63 high numerical aperture (NA ¼ 1.4) oil microfluidic device (i.e., the glass coverslip surface) through
immersion objective, as shown in Fig. 1(d) (see more details a digoxigenin-antidigoxigenin linkage. Here, we were inter-
in supplementary material23). ested in free flowing biopolymer response. This approach,
We have used our microfluidic device with a circular then, was for a single-molecule-level polymer system with
side chamber to visualize, via epi-fluorescence microscopy, two free ends (versus one fixed end and one free end, in our
and measure the real-time dynamic responses of fluores- previous DNA study) under flow-stretching. We also did not
cently labelled k-phage DNA molecules including the elon- incorporate other biomolecules with our microfluidic-based
gation strain rate and the DNA curvatures at different system via any physical or chemical modification of the bot-
locations in the circular side chamber under different flow tom of our microfluidic device. We did not use specific
rates (i.e., different fluidic shear stresses). We found that biomolecules such as actin-binding proteins, myosin or
these DNA molecules displayed multimodal dynamic a-actinin, because we wanted to probe the real-time dynamic
responses—distinct conformations and adjustable curvatures responses of phalloidin-labeled actin filaments under flow-
(i.e., elongation and bending dynamics of single mole- stretching sans interactions with other biomolecules in our
cules)—within the microcurvilinear flow.24 In order to microfluidic device.

Downloaded 15 May 2013 to 140.114.125.14. This article is copyrighted as indicated in the abstract. Reuse of AIP content is subject to the terms at: http://apl.aip.org/about/rights_and_permissions
 193704-3 Cheng et al. Appl. Phys. Lett. 102, 193704 (2013)

FIG. 1. (a) Schematic of the pressure-driven

microfluidic-based approach designed to
observe, record, and analyze the real-time
dynamic responses of multiple actin filaments
under flow. (b) Schematic of the microfluidic-
based configuration designed and fabricated
to create the microcurvilinear flow in the cir-
cular side chamber. (c) An optical image of
our polydimethylsiloxane microfluidic device.
(d) A fluorescence image of multiple
phalloidin-labeled synthesized actin filaments
on a glass coverslip. (e) Two fluorescence
images of fluorescence beads moving from
the main channel into the circular side cham-
ber at flow rates of 50 ll/min and 75 ll/min.
The shutter was opened and a long duration
image was taken resulting in tracers indicated
the flow patterns of the beads. At flow rates
that were beyond this range (or higher than
100 ll/min), the curvilinear flow in our micro-
fluidic device was challenging to be fully
developed deep in the circular side chamber.

To establish device parameters, we began by character- of up to 6200 m/s2 and a Reynolds number of 0.9, it was
izing the curvilinear flow characteristics within our micro- challenging to visualize and record the real-time dynamic
fluidic device. We found that the initial flow response in the responses of fluorescently labeled actin filaments with two
microcurvilinear flow was different from that observed in the free ends at such a high flow rate. Thus, the flow rate
macroscale lid-driven cavity flow,25,26 as the combination of selected for use in this experiment was 50 ll/min, which was
a tight rotation radius (r < 50 lm) and a high rotational ve- generated by the syringe pump. Interestingly, at lower flow
locity (v  0.22 m/s) for the fluidic flow in the constricted rates (<50 ll/min), the curvilinear flow could be not fully
channel created a radial acceleration (v2/r) as high as developed deep into the circular side chamber. Our final
104 m/s2 (or 103 g) when the flow rate was 100 ll/min. We determination was our discovery that the thermally driven
then determined that the Reynolds numbers (Re) at the circu- fluctuations, which would influence the polymer behaviors
lar side chamber’s opening were 0.4 and 0.6 for the flow of synthesized actin filaments such as filamentous shapes,
rates of 50 ll/min and 75 ll/min, respectively, indicating that were minimal in the microcurvilinear flow system in our
the curvilinear flow in our microfluidic device was laminar; microfluidic device.24
this was important, as turbulence would likely induce other We observed, recorded, and analyzed the conformations
fluidic-based influences on synthesized actin filaments due to of individual actin filaments (Figs. 2 and 3) in the microcurvi-
the instability of turbulent flow. Although we were able to linear flow system under a pressure-driven flow in the circular
create a maximum fluid velocity of up to 0.27 m/s (z ¼ 1 lm) side chamber at different locations. An applied pressure gra-
when the flow rate was 100 ll/min with a radial acceleration dient from the syringe pump in this study, whether in the

FIG. 2. Real-time dynamic responses of multiple phalloidin-labeled synthesized actin filaments within the microcurvilinear flow of our microfluidic device
using epi-fluorescence microscopy. (a) Two actin filaments observed at different locations (marked as the locations (I) and (II)) in the circular side chamber
and (b, c) time-lapse snapshots of these two actin filaments under a pressure-driven flow at a flow rate of 50 ll/min at locations (I) and (II) in (a). (d, e) Time-
lapse fluorescence images of a single actin filament observed and recorded at position (III) in (d) under a pressure-driven flow at a flow rate of 50 ll/min.
There was no chemical or physical modulation of the bottom of our microfluidic device. (f) Schematic of the potential conformations (rotating and elongation)
of an individual actin filament under flow-stretching in the circular side chamber.

Downloaded 15 May 2013 to 140.114.125.14. This article is copyrighted as indicated in the abstract. Reuse of AIP content is subject to the terms at: http://apl.aip.org/about/rights_and_permissions
 193704-4 Cheng et al. Appl. Phys. Lett. 102, 193704 (2013)

TABLE I. Elongation strain rates (1/s) of single synthesized actin filaments Fig. 3(a) illustrates a single actin filament aligning with the
at different locations in the curvilinear flow system at various time points streamlines in the microcurvilinear flow system as deter-
through measuring the projected actin filament length via using ImageJ. The
“negative” values in this table mean that the projected actin filament length
mined experimentally and computationally. This response
at the current time point was shorter than at the previous time point. would induce a bending conformation of the semi-flexible
polymer under flow-stretching. We used our microfluidic-
Location (I) Location (II) Location (III) based approach to observe and record the curvature of a
single actin filament under flow-stretching. Due to the chal-
0 second to 1st second 0.80 0.40 2.75
1st second to 2nd second 0.56 0.57 0.10
lenges of capturing individual actin filaments in a single field
2nd second to 3rd second 0.29 0.27 0.15 of view in the circular side chamber in our microfluidic de-
Average elongation strain rate 0.33 0.60 0.83 vice, we developed an approach wherein we first turned off
(1/s; 0 second to 3rd second) the syringe pump, which had an initial flow rate of 50 ll/min
for capturing images. We then induced the biomolecules into
a curved conformation by allowing the biomolecules to ex-
rectangular main channel or circular side chamber, generated perience a relative low shear stress. We observed and
a parabolic fluid velocity profile that was at a maximum in recorded a single actin filament in the circular side chamber,
the microchannel center and zero at the walls. The fluidic as shown in Fig. 3(b). This actin filament continued to
shear stress distribution in the microchannel was, however, respond conformationally so that it was not only elongated
inversely related to the fluid velocity profile, indicating that but also underwent significant bending. It is worth noting
individual synthesized actin filaments in our microfluidic de-
vice underwent a non-uniform fluidic shear stress as shown in
Figs. 2(b), 2(c), and 2(e). These fluidic characteristics could
induce both the rotation and the elongation of these semi-
flexible polymers with two free ends at the different locations
in our microfluidic device.24 A schematic of how these two
polymer behaviors could occur based on rotation and elonga-
tion under flow-stretching is shown in Fig. 2(f). The differ-
ence in actin filament length could be due to out-of-plane
focusing issues, as the actin filament was not attached onto ei-
ther the bottom or the top of our microfluidic device in this
study and was allowed to freely move (Figs. 2(b), 2(c), and
2(e)) at the applied flow rate of 50 ll/min without chemically
or physically modulating the bottom of our microfluidic de-
vice. We also attempted to quantify the morphological
change of multiple actin filaments during the duration of the
flow (i.e., the projected length change versus time) by meas-
uring the projected actin filament length using ImageJ (public
software from National Institutes of Health; http://rsbweb.
nih.gov/ij/). This quantification is found in Table I, and indi-
cates the response of the semi-flexible polymers with two
free ends under flow-stretching. The elongation strain rates of
these individual synthesized actin filaments were slightly dif-
ferent when applying the same mechanical stimulation
(flow-stretching) in our microcurvilinear flow system, indi-
cating that each actin filament type has its own dynamic
responses. This is likely influenced by the length of the fila-
ment as well, as assembled actin filaments are known to have
a range of length distributions.12 This is supported by previ-
ous results wherein individual actin filaments have been
observed to undergo conformational elongation and rotating
at low shear stress.27 It is noted that this microfluidic-based
approach allowed us to simultaneously probe the real-time
dynamic responses of multiple actin filaments for each
experiment versus using one actin filament per experiment as
traditionally done in experiments using optical tweezers or
FIG. 3. (a) Schematic of the location and conformation of a single actin fila-
atomic force microscopy. Thus, direct comparisons, including ment in the microcurvilinear flow system of our microfluidic device. (b)
using averaging assumptions from a large number of polymer Time-lapse fluorescence images of the bending conformation of a phalloidin-
chain samples, could be employed as in previous actin fila- labeled synthesized actin filament in the microcurvilinear flow system under
high radial acceleration. The curvature of this individual actin filament
ment dynamics studies.17,28–30 noticeably increased at first, as this actin filament may start releasing the
Our microcurvilinear system can also be used to observe external force (attempted to reach a between internal energy and external
and record the bending of individual actin filaments (Fig. 3). mechanically relevant stimulation—fluidic shear stress, in this study).

Downloaded 15 May 2013 to 140.114.125.14. This article is copyrighted as indicated in the abstract. Reuse of AIP content is subject to the terms at: http://apl.aip.org/about/rights_and_permissions
 193704-5 Cheng et al. Appl. Phys. Lett. 102, 193704 (2013)

5
that we captured the time-lapse fluorescence images immedi- J. Doh and D. J. Irvine, Proc. Natl. Acad. Sci. U.S.A. 103, 5700 (2006).
6
ately after turning off the syringe pump (less one second), L. Dike, C. S. Chen, M. Mrksich, J. Tien, G. M. Whitesides, and D. E.
Ingber, In Vitro Cell. Dev. Biol.: Anim. 35, 441 (1999).
which indicated that this single actin filament was still under 7
N. Q. Balaban, U. S. Schwarz, D. Riveline, P. Goichberg, G. Tzur, I.
flow-stretching. But, when using this microcurvilinear flow Sabanay, D. Mahalu, S. Safran, A. Bershadsky, L. Addadi, and B. Geiger,
system, we had to control the flow rate to visualize a single Nat. Cell Biol. 3, 466 (2001).
8
J. L. Tan, J. Tien, D. M. Pirone, D. S. Gray, K. Bhadriraju, and C. S. Chen,
actin filament. The curvature of this individual actin filament
Proc. Natl. Acad. Sci. U.S.A. 100, 1484 (2003).
increased initially but afterwards this actin filament appeared 9
R. McBeath, D. M. Pirone, C. M. Nelson, K. Bhadriraju, and C. S. Chen,
to contract after the external shear stress began to decrease Dev. Cell 6, 483 (2004).
10
(Fig. 3(b) and the supplementary material23). 11
S.-Y. Chou, C.-M. Cheng, and P. R. LeDuc, Biomaterials 30, 3136 (2009).
Y.-W. Lin, C.-M. Cheng, P. R. LeDuc, and C.-C. Chen, PLoS ONE 4,
By using a microcurvilinear flow with epi-fluorescence e4293 (2009).
microscopy, we were able to observe, record, and analyze 12
R. Lenk, L. Ransom, Y. Kaufmann, and S. Penman, Cell 10, 67 (1977).
13
the real-time dynamic responses of individual semi-flexible D. E. Ingber, J. Cell Sci. 116, 1157 (2003).
14
polymers via a pressure-driven laminar flow approach capa- P. A. Janmey, Physiol. Rev. 78, 763 (1998).
15
M. Sato and T. Ohashi, Biorheology 42, 421 (2005).
ble of performing both high rotational velocity and high ra- 16
J. L. McGrath, Curr. Biol. 16, R326 (2006).
dial acceleration. This microfluidic-based approach enabled 17
A. Kishino and T. Yanagida, Nature 334, 74 (1988).
18
us to create and examine the influences of a microcurvilinear F. Gittes, B. Mickey, J. Nettleton, and J. Howard, J. Cell Biol. 120, 923
flow on individual molecules by simultaneously imaging (1993).
19
Y. Tsuda, H. Yasutake, A. Ishijima, and T. Yanagida, Proc. Natl. Acad.
them. This molecular level approach to non-equilibrium Sci. U.S.A. 93, 12937 (1996).
polymer physics demonstrates great potential for use in a va- 20
D. E. Dupuis, W. H. Guilford, J. Wu, and D. M. Warshaw, J. Muscle Res.
riety of research communities including fields of study Cell Motil. 18, 17 (1997).
21
examining entangled solutions of flexible polymers (DNA) W. C. Ruder, E. D. Pratt, S. Bakhru, S. Zappe, C.-M. Cheng, J. F. Antaki,
and P. R. LeDuc, Lab Chip 12, 1775 (2012).
and other polymer systems such as microtubules. 22
Y. Zhang, C.-M. Cheng, B. Cusick, and P. R. LeDuc, Langmuir 23, 8129
(2007).
23
We would like to thank the National Science Council of See supplementary material at http://dx.doi.org/10.1063/1.4806975 for ex-
perimental details and a video in real time.
Taiwan for financially supporting this research under Contract 24
C.-M. Cheng, Y. T. Kim, J.-M. Yang, S. H. Leuba, and P. R. LeDuc, Lab
No. NSC 101-2628-E-007-011-MY3, start-up funds, and Chip 9, 2339 (2009).
25
the Grant for Interactive Nano/MicroElectroMechanical P. N. Shankar and M. D. Deshpande, Annu. Rev. Fluid Mech. 32, 93
Components and Systems from National Tsing Hua (2000).
26
J. P. Shelby, D. S. W. Lim, J. S. Kuo, and D. T. Chiu, Nature 425, 38
University, Taiwan (to C.-M. Cheng). (2003).
27
P. LeDuc, C. Haber, G. Bao, and D. Wirtz, Nature 399, 564 (1999).
1 28
C. S. Chen, M. Mrksich, S. Huang, G. M. Whitesides, and D. E. Ingber, A. D. Mehta, M. Rief, J. A. Spudich, D. A. Smith, and R. M. Simmons,
Science 276, 1425 (1997). Science 283, 1689 (1999).
2 29
H. G. Craighead, S. W. Turner, R. C. Davis, C. James, and A. M. Perez, H. Clausen-Schaumann, M. Seitz, R. Krautbauer, and H. E. Gaub, Curr.
Biomed. Microdevices 1, 49 (1998). Opin. Chem. Biol. 4, 524 (2000).
3 30
S. Huang, C. S. Chen, and D. E. Ingber, Mol. Biol. Cell 9, 3179 (1998). T. Ikawa, F. Hoshino, O. Watanabe, Y. Li, P. Pincus, and C. R. Safinya,
4
P. S. Cremer and S. G. Boxer, J. Phys. Chem. B 103, 2554 (1999). Phys. Rev. Lett. 98, 018101 (2007).

Downloaded 15 May 2013 to 140.114.125.14. This article is copyrighted as indicated in the abstract. Reuse of AIP content is subject to the terms at: http://apl.aip.org/about/rights_and_permissions