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Microrheology of complex fluids using optical tweezers: A comparison with

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Microrheology of complex fluids using optical tweezers: a comparison with macrorheological

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IOP PUBLISHING JOURNAL OF OPTICS A: PURE AND APPLIED OPTICS
J. Opt. A: Pure Appl. Opt. 11 (2009) 034016 (11pp) doi:10.1088/1464-4258/11/3/034016

Microrheology of complex fluids using

optical tweezers: a comparison with
macrorheological measurements
G Pesce1 , A C De Luca1 , G Rusciano1 , P A Netti2,3 , S Fusco2,3 and
A Sasso1
1
Dipartimento di Scienze Fisiche, Università di Napoli Federico II and CNISM (Consorzio
Interuniversitario per le Sienze Fisiche della Materia), Complesso Universitario Monte
Sant’Angelo, I-80126 Napoli, Italy
2
Dipartimento di Ingegneria dei Materiali e della Produzione, Università di Napoli ‘Federico
II’, Piazzale Tecchio 80, I-80125 Napoli, Italy
3
Fondazione IIT, Via Morego 30, I-16163 Genova, Italy

Received 1 July 2008, accepted for publication 3 September 2008

Published 20 January 2009
Online at stacks.iop.org/JOptA/11/034016

Abstract
The increasing interest in the mechanical properties of complex systems at mesoscopic scale
has recently fueled the development of new experimental techniques, collectively indicated as
microrheology. Unlike bulk-based approaches (macrorheology), these new techniques make use
of micrometric probes (usually microspheres) which explore the mechanical properties of the
surrounding medium.
In this paper we discuss the basic idea of microrheology and we will focus on one specific
technique based on optical tweezers (OT). The discussion starts from Newtonian fluids to tackle
the more general case of complex fluids, also showing results of these techniques on solutions
of a relevant biomolecule: hyaluronic acid (HA). In particular, we study the viscoelastic
properties of low molecular weight HA (155 kDa) at low ionic strength over an extended
frequency range (0.1–1000 Hz) and in a wide range of concentrations (0.01–20 mg ml−1 ),
which include both the dilute and semidilute regime. In the concentration range here explored
and within the test frequencies covered by our techniques, samples prevalently exhibit a viscous
behavior, the elastic contribution becoming significant at the highest concentrations. By
comparing OT outcomes to those obtained by a traditional rheometer, we found that they were
in good agreement in the overlapping frequency range of the two techniques, thus confirming
the reliability of the microrheological approach.

Keywords: optical tweezers, microrheology, biopolymers, hyaluronic acid, viscoelastic moduli

(Some figures in this article are in colour only in the electronic version)

1. Introduction a viscoelastic character. For this reason, the study of such

The study of complex fluids is an emerging area of
physics. Polymer solutions, colloids and many biomaterials
are commonly classified as complex fluids since they present
a mechanical behavior halfway between that of a solid (which
responds elastically to external stresses) and that of a liquid
(whose response has a viscous character). Viscoelastic
materials are ubiquitous in our daily life: common foods
and the cytoplasm of eukaryotic cells, as examples, present
materials has attracted the interest of both basic and applied
research.
Generally speaking, under shear stress, these materials
give rise to a viscoelastic rheological response, the energy
being partially stored (elasticity) and dissipated (viscosity).
Many equivalent representations exist for the viscoelastic
response of a complex medium; all of them can be expressed in
terms of the stress relaxation modulus G(t). Complex Fourier
transformation of G(t) can be used to obtain the frequency-

1464-4258/09/034016+11$30.00 1 © 2009 IOP Publishing Ltd Printed in the UK

J. Opt. A: Pure Appl. Opt. 11 (2009) 034016
dependent complex shear modulus G( f ) which consists of a
real part, G  ( f ), the elastic storage modulus, and an imaginary
part, G  ( f ), the viscous loss modulus. Since G  ( f ) and
G  ( f ) derive from the single function G(t), they are not
independent functions but are related by the Kramers–Kronig
relations.
For many years rheological investigations have been
performed by conventional rheometry [1, 2], which generally
provides the macroscopic response of the material (bulk)
over a restricted range of frequencies (up to few Hz).
Usually, a rheometer provides the in- and out-of-phase
responses of a material to an applied deformation or stress
which are intimately connected to the microstructure of
the fluid. However, obtaining structural information from
macrorheological data is an inverse problem which typically
does not present a unique solution. Moreover, many complex
materials are inhomogeneous on a small scale: they contain
extended molecular structures ranging from a few nanometers
to several microns. In complex media (for instance, the
cytoplasm of a cell) these heterogeneities play a crucial role
due to the complex structure of the medium itself.
In the recent past, increasing interest has been concerned
with the investigation of complex fluids at the mesoscopic
scale, which is an intermediate level between the molecular (re-
quiring molecular dynamics approaches) and the macroscopic
scale (well described by convention bulk rheometry). Efforts
in developing new techniques able to explore local properties
of soft materials have led to a new area normally indicated
as microrheology [3–5]. The basic idea of microrheology is
to employ microprobes to explore the viscoelastic response of
the medium where they are immersed. This local approach
offers, with respect to the conventional rheometers, the advan-
tage of investigating the heterogeneities of the medium. In ad-
dition, different sizes of the probe make it possible to explore
the network medium at different length scales [6]. Moreover,
microrheological techniques need small amounts of material
(a few microliters against milliliters requested by traditional
rheometry) and this becomes a crucial point when investigat-
ing biological samples or expensive materials. Finally, while
macrorheology is usually limited by the mechanical inertial of
the apparatus, microrheology is able to explore a much wider
frequency interval (up to tens or hundreds of kHz).
Nowadays, numerous microrheological techniques exist
such as particle tracking video-microrheology, atomic force
microscopy, diffusive wave spectroscopy, fluorescence recov-
ery after photobleaching, magnetic tweezers and optical tweez-
ers [7, 8].
All these techniques are usually separated into: (i) active
techniques, when a strain is recovered after an external applied
stress, and (ii) passive techniques, when the thermal motion of
microprobes is monitored.
The thermal motion can concern one particle (single-
particle tracking) or more particles (multiple-particle tracking).
From an experimental point of view thermal motion of
tracer particles is reconstructed with laser-particle tracking
(LPT) [4, 9] or with video-particle tracking (VPT) in the free
diffusion regime or in optical traps [10] (confined diffusion).
In particular, in LPT a laser beam shines on the probe bead
2
G Pesce et al
and a position sensor recovers the bead position. In VPT
standard image analysis is employed for monitoring the bead
centroid. If the tracer particle is spatially confined by an
optical trap, the Brownian motion amplitude is smaller than its
radius, therefore (i) only that selected volume is investigated
and (ii) the probe can be tracked for long times. However, even
though the viscoelastic moduli are inferred strictly locally, the
optical trap elasticity has to be properly taken into account.
A big advantage of LPT with respect to VPT is the different
frequency ranges that they can explore: VTP is usually limited
to about 30 frames s−1 (typical for standard video cameras),
while fast sampling, occurring with LPT, can cover up to a
five-decade range of frequencies. Therefore, LPT is typically
fast and sensitive enough to follow variations of the medium
under investigation.
The use of laser tweezers, first developed by Ashkin et al
[11], resulted in becoming a powerful tool for investigating
local viscosity [12]. In this kind of experiment, displacements
of optically trapped particles are often recorded by means of
quadrant photodiodes using back-focal-plane interferometry.
For absolute viscosity measurements such sensor positions
have to be calibrated.
Valentine et al [13] have measured the viscosity of
polymeric solutions through the displacement magnitude and
phase responses of a latex bead driven sinusoidally by optical
tweezers. In that experiment, the stiffness of the trap was
determined in a medium of known viscosity (water) and,
then, it was assumed to be the same for the determination
of polymeric solution viscosities. Pralle et al [14] measured
the diffusion coefficient of colloidal particles by analyzing the
autocorrelation function of the Brownian motion signal. In
that case, the trap stiffness was calculated using the Boltzmann
distribution of thermally driven position fluctuations [15] while
the position detector was calibrated by means of the so-
called attached-bead method. Following this method, the
position detector response is recorded while, a bead stuck
onto the coverslip surface is translated through the laser
focus by a known length within the linear response of the
detector. Although this method is currently employed in many
optical tweezer (OT) calibration procedures, it is affected by
significant bias caused by proximity surface effects and by
spherical aberrations, as recently demonstrated by us [16].
Recently, we have proposed alternative methods [17] which
combine an unbiased position detector calibration procedure
and frequency analysis of the Brownian motion of optically
confined polystyrene microspheres. More recently, Vermeulen
et al [18] have proposed a very interesting calibration method
which consists of very rapidly scanning the laser beam
across the solvent-immersed, trapped bead using acousto-
optic deflectors while recording the detector signals. It does
not require any knowledge of solvent viscosity and bead
diameter, and works in all types of samples (viscous or
viscoelastic samples). Bishop et al [19] have realized home-
made birefringent microspheres that were rotated by the optical
torque exerted by the trapping beam itself. The viscosity was
simply calculated from the damped rotator formula and, from
an experimental point of view, it was expressed in terms of the
optical power exerting the torque on the rotating particle and
J. Opt. A: Pure Appl. Opt. 11 (2009) 034016
its angular velocity. Although this technique does not need any
position detector calibration, a delicate calibration procedure to
estimate the real power at the sample is necessary. In addition,
the bead radius uncertainty due to the low reproducibility in
the fabrication process of birefringent beads was the main
contribution to the final precision of the measured viscosity.
Apart from simple fluids, use of OT has also been
successfully extended to studying complex fluids [20–22].
In this paper we compare viscoelastic properties of
hyaluronic acid prepared in water solutions obtained using OT
and traditional rheometry [23]. Hyaluronic acid (HA) [24] is a
natural linear polysaccharide (family of glycosaminoglycans)
which is present in the connective tissues of vertebrates,
the synovial fluid of joints and the vitreous humor of the
eye [25, 26]. The aim of this paper is to investigate the
rheological behavior of HA aqueous solutions at different
polymer concentrations ranging from 0.1 to 30 mg ml−1 . We
demonstrate that microscopic analysis of thermally driven
motion of a single particle confined in an optical trap can
provide the macroscopic linear viscoelasticity over an extended
frequency range. By extracting the particle time-dependent
mean square displacement from the equilibrium position of the
trapped bead, we obtain the elastic and viscous component
of the shear modulus G( f ), making use of the fluctuation–
dissipation principle and the generalized Stokes–Einstein
relation.
2. Theory
2.1. Macrorheology
Mechanical properties of viscoelastic media can be evaluated
by small-amplitude oscillatory shear experiments that allow
measurement of the unsteady response of the samples.
This approach has been successfully used to determine
the relationship between the structure and the mechanical
properties of materials [1].
In linear dynamic experiments the material is subjected to
a sinusoidal shear strain:
γ = γ0 sin(2π f t)
where γ0 is the shear strain amplitude and f is the oscillation
frequency. The mechanical response, expressed as shear stress
σ of viscoelastic materials, is intermediate between an ideal
pure elastic solid (in phase with the deformation) and an ideal
pure viscous fluid (90◦ out of phase with the deformation). In
general, σ can be expressed by the relation
σ = |G( f )| sin(2π f t + δ)
δ being the phase delay of the mechanical response with
respect to the imposed deformation or, equivalently:
σ = G  ( f )γ0 sin(2π f t) + G  ( f )γ0 cos(2π f t)
where G  ( f ) and G  ( f ), as given by the relations

G ( f ) = |G| cos δ
G  ( f ) = |G| sin δ
G Pesce et al
are the shear storage modulus and the shear loss G  ( f ) moduli,
respectively. The combined viscous and elastic behavior can be
expressed also in terms of the complex viscosity η defined as
G( f )
η( f ) = (5)
2π f
which is usually compared with the steady shear viscosity in
order to evaluate the effect of large deformations and shear
rates on the material structure.
2.2. Microrheology
A spherical particle of refractive index n 2 embedded in a fluid
of refractive index n 1 in the proximity of a focused laser beam
feels a potential U given by [27, 28]
 
−3V p n 1 n 22 − n 21
I0 e−r /w
2 2
U= (6)
c n 2 + 2n 1
2 2
where V p is the volume of the bead, c is the speed of light, I0
is the laser intensity and w is the value of the beam waist at the
focus. The force exerted on the particle as it moves from the
equilibrium position (r = 0) is calculated as
 
6V p n 1 n 22 − n 21
I0 e−r /w r r̂ .
2 2
F = −∇U = (7)
cw 2 n 2 + 2n 1
2 2
Therefore, for small displacement from the equilibrium
position, an optical trap exerts an elastic force Fi = ki ri
(i = x; y; z ), ki being the stiffness of the trap along a given
direction. Normally in an optical trap produced by a tightly
focused Gaussian beam the radial photonic forces are larger
than the axial one, i.e. k x ≈ k y > k z . Equations (6) and (7)
strictly hold for particles of linear sizes much smaller than the
laser wavelength. However, it is possible to demonstrate that
the optical potential is still harmonic, even for particle size
comparable with the laser wavelength [29].
A microsized bead embedded in a fluid undergoes a
continuous exchange of energy of the order of kB T (kB being
the Boltzmann constant and T the temperature) with the
(1)
surrounding medium. This interaction can be described by
random forces; for micrometric beads these are typically well
below the pN level, giving way to small deformation of the
mesh forming the network in which beads are embedded. In
turn, the medium response governs the particle’s movement,
which is the observable in microrheology. Unlike the free
Einstein diffusion, in an optical trap a bead moves following
a confined Brownian trajectory described by stochastic time-
(2) dependent displacements [x(t), y(t), z(t)]. The motion of a
spherical particle of radius a embedded in a Newtonian fluid is
described by the Langevin equation:
d2 x dx
(3) m = −kx − γ + F(t) (8)
dt 2 dt
where m is the mass of the particle, k is the trap stiffness and
γ = 6πηaε is the hydrodynamic factor. F(t) represents the
(4) random thermal force, which has an average value of zero and
a flat power spectrum, such that |F( f )|2 = 4γ kB T , kB being
3
J. Opt. A: Pure Appl. Opt. 11 (2009) 034016 G Pesce et al

the Boltzmann constant and T is the sample temperature. The
factor ε appearing in the expression of γ takes into account
viscosity correction due to the finite distance from the chamber
walls and it should be considered when particles approach the
glass surface [30].
For particles having a low Reynolds number the inertial
term in equation (8) is negligible. In this case, the solution of
equation (8) is given by
 2  kB T  k
 be caused by either local cytoplasmatic flow or sustained by
x (t) = 1 − e 3πηa t . (9)
k As mentioned above the mechanical response of a com-
At short times (t  3πηa/k ) equation (9) becomes
 2 
x (t) ≈ 2 Dt (10)
which is the well-known diffusion Einstein law, D = kB T /γ
being the diffusion coefficient. The steady-state solution of
equation (10) (t → ∞) is instead described by a plateau
∝ kB T /k which mainly depends on the trap stiffness k .
The Langevin equation can also be analyzed in the
frequency domain, i.e. in terms of the power spectral density
(PSD) of the stochastic signal x(t). Again, for a Newtonian
fluid the PSD is given by a Lorentzian curve [31]:
where the exponent α can be =1, <1 or >1. In equation (16),
the diffusion coefficient D and the exponent α are generally
dependent on the lag time. For a pure viscous fluid α = 1 and
we have again Einstein’s free diffusion. On the other hand, α <
1 indicates a sub-diffusion behavior (typical for a viscoelastic
medium) whereas α > 1 denotes a superdiffusive motion.
The terms v 2 t 2 take into account possible drift motion of the
fluid occurring at velocity v which, for biological samples, can
molecular motors [33, 34].
plex medium can also be described in terms of the frequency-
dependent complex shear modulus G( f ) = G  ( f ) + iG  ( f )
which represents the response (strain) to a given applied de-
formation (stress). This is the conventional approach based on
macrorheology. In order to find a connection between macro-
and microrheology we follow the procedure discussed in [35].
A bead embedded in a viscoelastic fluid experiences
stochastic force Fx produced by the interaction between the
beads and the fluid molecules. The Fourier transform x( f ) of
the displacement x is linearly related to the stochastic force by
the relation [35, 36]

kB T x( f ) = α ( f ) · Fx ( f ) (17)
Sx ( f ) =  (11)
γ π2 f c2 + f 2
where α( f ) = α  ( f ) + iα  ( f ) is the compliance. The
where fc denotes the corner frequency, which is related to the compliance α( f ) and the modulus G( f ) are connected
trap stiffness by the relation through the generalized Stokes–Einstein relation:

k = 2πγ f c . (12) 1
G(f)= . (18)
6π α a
At low frequencies (long times) the static PSD contains
information on the confinement in the optical trap: Equation (18) applies for an incompressible medium without
inertia (see [37] for a more detailed discussion on this
4γ k B T
S x (0 ) = . (13) point). Moreover, the validity of the continuum viscoelasticity
k2 requires that the bead radius a is larger than the mesh size of
At high frequencies (short time) the 1/ f 2 decay describes the the network. The knowledge of the chain length L in a dilute
free diffusive regime. regime and then of the correlation length when the HA chains
In principle, from the fit of the experimental curves start to overlap allows us also to deduce the average mesh size
of equations (9) or (11) it is possible to determine the ξ of the solution. The dependence of ξ upon the concentration
viscosity of the medium and the trap stiffness. Nevertheless, c is described by [38]
it is necessary to remind ourselves that, since the particle  −1/2
position is monitored through a voltage signal coming from a ξ ∝ L c/c∗ (19)
position detector (usually a quadrant photodiode), a calibration
procedure is required. Assuming a linear response of the where c∗ is the overlap concentration (≈0.06 mg ml−1 for HA
detector, this procedure consists in finding the position–voltage 155 kDa) which represents the border between the dilute and
calibration factor β such that semidilute regimes and it is determined as the point where the
concentration inside a single coil equals the bulk concentration.
x = βV. (14) So, from equation (19) at the overlap concentration the mesh
size is equal to the correlation length. In our study, at c∗ =
For a complex fluid the Langevin equation fails to describe the 0.06 mg ml−1 , L = ξ = 245 nm (as evaluated by Buhler and
stochastic motion of the trapped bead. In this case it can be Boué [39] using the small-angle neutron scattering technique).
useful to calculate the mean square displacement as a function The probes used in our investigation are 1.25 μm in diameter,
of the lag time τ : so they can be considered sufficiently larger than the mesh size.
 2    The fluctuation–dissipation theorem relates the dissipation

x (τ ) = [x(t + τ ) − x(t)]2 (15)
part α  to the power spectrum density (PSD) Sx ( f ) of the
which for a viscoelastic medium can be approximated as [32] trapped bead:
 2  2π f
α  ( f ) = Sx ( f ). (20)

x (τ ) = Dτ α + v 2 τ 2 (16) 2k B T

4
J. Opt. A: Pure Appl. Opt. 11 (2009) 034016
Figure 1. Essential scheme of the set-up for microrheology (a) and
macrorheology (b) measurements.
Once α  is determined, the real component α  can be obtained
by the Kramers–Kronig relation:
∞
 2 ζ α  (ζ )
α (f)= P dζ 2
π 0 ζ − f2
∞
∞ oscillation frequency has been varied from 0.1 to 1 Hz.
= cos(2π f t) dt α  ( f  ) sin(2π f  t) d f  . (21)
0 0
Finally, from equation (18), the elastic and viscous moduli,
G  ( f ) and G  ( f ), can be straightforwardly computed.
For a purely viscous fluid G = iG  ( f ) = −i2π f η,
leading to the well-known Stokes formula:
Fx = 6πηavx
where vx is the relative velocity of the bead with respect to the
fluid along the x axis.
3. Experimental apparatus
3.1. Laser tweezers’ set-up
In figure 1(a) is sketched the main parts of the apparatus:
a micrometric dielectric sphere is optically trapped and its
position monitored by means of a sensor position. The optical
tweezers system used in our experiment is described with
more detail in [17]. It consists in a Olympus IX70 inverted
microscope supplied with an infinitely corrected 60× water
immersion objective (UP-LAPO60XW3, NA = 1.2). A
semiconductor diode laser emitting about 80 mW at 830 nm
(Spectra Diode Labs, model SDL-5311-G1) was used as the
trapping laser. An anamorphic prism pair made the beam
spatial profile nearly circular while a telescope was used to
ensure a proper collimation and overfilling of the back aperture
of the objective. Typical laser power impinging on the sample
ranged from 1.7 to 15 mW.
The sample chamber consisted of a glass coverslip of
150 μm thickness over a microscope slide separated by a
100 μm Mylar spacer. Trapped particles were polystyrene
microspheres produced by Serva Electrophoresis (density:
G Pesce et al
1.06 g cm−3 , refractive index: 1.59) with a diameter of
(1.25 ± 0.01) μm diluted in distilled water. The sample cell
temperature was monitored using a NTC thermistor positioned
on the top surface of the microscope slide. The sample image
was projected by the focusing objective itself to the CCD port
of the microscope.
The laser beam after crossing the sample cell was collected
by the condenser lens whose back-focal plane was imaged
on the active surface of a quadrant photodiode (Centrovision
Silicon QD50-0-SD) with a bandwidth of about 250 kHz [40].
The voltage output signals coming from the QP were recorded
on a digital oscilloscope (Tektronix, model TDS5034B,
350 MHz bandwidth, 5 GS s−1 ) and then it was sent to a
personal computer for data analysis.
3.2. Conventional rheometer
The macrorheological mechanical properties were performed
by using a stress controlled rheometer (Gemini, Bohlin
Instruments) in a double gap and cone-plate geometry [41],
depending on the solution concentration (a scheme is shown
in figure 1(b)).
The instrument was preheated to the target temperature
(accuracy ±0.01 ◦ C) maintained throughout the tests. The
The tests were repeated at least four times. The
viscosity measurements were carried out by use of different
geometries (double gap and cone-plate), depending on sample
concentration, to investigate the broader shear rate possible.
The system was equipped with a special installation (humidity
chamber) to saturate the atmosphere with water to obviate
solvent evaporation.
(22)
4. Results
4.1. Rheology in Newtonian fluids: trap calibration
As previously described (see section 3), a quadrant photodiode
was used as the sensor. For small displacement from the
equilibrium position, for instance along the transverse x axis,
the QP output signal Vx is linearly dependent on the bead
position x = βVx , β (nm V−1 ) being the calibration factor.
In order to estimate the calibration factor and the trap
stiffness, we used the method of the power spectral density
(PSD). Therefore, we trapped a polystyrene bead in distilled
water and monitored the stochastic signal x(t) = βV (t). Then
we calculated the power spectral density:
kB T 1 A
SV ( f ) = = 2 . (23)
6πηaβ f + f c
2 2 2 f + f c2
From a fitting procedure of the voltage PSD, once the viscosity
of the water (9.5710−4 Pa s) and its temperature (T = 21.9 ◦ C)
are known, the calibration factor β and the trap stiffness k can
be easily estimated. We assumed this calibration factor in the
analysis of complex fluids (hyaluronic acid solutions in our
case).
In order to minimize the elastic term of the optical trap, the
laser power was reduced to achieve the minimum trap stiffness
5
J. Opt. A: Pure Appl. Opt. 11 (2009) 034016
Figure 2. Power spectral densities (PSDs) calculated at different
laser powers ( PLaser : 2.46, 4.20, 5.80 and 7.40 mW). In the inset, the
amplitude of the Lorentzian versus P 21 is plotted.
Laser
but still sufficient to trap 1 μm polystyrene beads. The PSDs
obtained at laser powers from 2.46 to 7.40 mW are reported in
figure 2. At high frequencies, according to equation (23):
kB T 1
SV ( f → ∞) =
γβ 2 f 2
i.e. all the PSDs collapse in a single straight line with slope −2.
At low frequencies, instead:
4γ k B T 1
S V (0 ) = ∝ 2
β 2k2 PLaser
so the PSD amplitude decreases as the inverse of the power
laser squared (see inset of figure 2).
We have tested the Kramers–Kronig procedure using
distilled water. Figures 3(a) and (b) show the behaviors
of G  and G  , respectively, obtained at the laser power of
2.46, 4.20, 5.80 and 7.40 mW. Although distilled water does
not exhibit any elastic response an elastic contribution was
observed (figure 3(a)), which is due to the elastic role of the
trap itself. The elastic contributions of the trap, calculated as
G trap = k/6πa , are highlighted by arrows in figure 3(a): as
can be seen, they match quite well with the G  calculations. On
the other side, for a Newtonian fluid G  is linearly dependent
on the frequency f , G  = 2πη f , and independent of the
laser power as shown in figure 3(b). We have checked that
the viscosity determined from the linear fit of the curves of
figure 3(b) is consistent (0.89 cP) with the expected water
viscosity (0.93 cP) at our working temperature (23.9 ◦ C).
4.2. Mean square displacements (MSD)
Similar information obtained in the frequency domain (PSD)
can be obtained in the time domain by monitoring the bead
position. First, in figure 4 we show typical stochastic
trajectories in the x – y plane obtained in pure water at different
laser powers. As can be seen, the higher the power the smaller
the region explored by the bead. Therefore, the trap stiffness
represents a sort of additional degree of freedom allowing us to
change the size of the explored volume.
G Pesce et al
(24)
(25)
Figure 3. Behaviors of the elastic (upper part) and viscous (lower
part) response of pure water solution. The non-zero elastic
contribution is due to the elastic effect of the optical trap.
4.3. Rheology of viscoelastic fluids
Viscoelastic properties of HA solutions were evaluated by
varying the HA concentration c from 0.01 and 20 mg ml−1 by
using both OT and a conventional rheometer in order to span a
concentration range including the overlap concentration (c∗ )
and the entanglement concentration (ce ). The entanglement
concentration (≈2.5 mg ml−1 ) represents the limit between
the unentangled and entangled regimes in which the polymer
chains start to overlap each other and to form a transient
network.
The stochastic signal of the bead position displays a
progressive transition from a simple fluid to a complex fluid.
In figure 5(a) are compared the stochastic signals measured
in pure water and that in a 0.1 mg ml−1 HA water solution,
while in figure 5(b) the respective PSDs are plotted. For pure
water the experimental curve is well fitted by a Lorentzian
curve (solution of the Langevin equation), whereas for the HA
mixture the fit quality is quite poor.
Furthermore, the elasticity effect can also be expressed in
terms of a power law of the Lorentzian decay. Increasing the
HA concentration, the PSDs’ frequency dependence changes
from f −2 , (purely viscous medium) to f −ν , ν being a
parameter tending asymptotically to 1.5 at the highest HA
concentrations [23].
6
J. Opt. A: Pure Appl. Opt. 11 (2009) 034016 G Pesce et al

200 200
P=7.40 mW P=5.80 mW
100 100

0 0
Y displacements (nm)

-100 -100

-200 -200
-200 0 200 -200 0 200

200 200
P=4.20 mW P=2.46 mW
100 100

0 0

-100 -100

-200 -200
-200 0 200 -200 0 200 Figure 6. Mean-square displacements (MSDs) of 1 μm spheres
X displacements (nm) measured at different laser HA concentrations.

4
x 10
2
Mean Square Displacement (nm2)

1.8 equation (13) fails to describe the PSD at high frequencies (see
1.6 figure 5), similarly the MSD of equation (10) is not able to
1.4 describe the experimental behavior of the MSD at short times
(fitting curves not shown in figure 6).
1.2
The moduli G  and G  were then evaluated from the
1 positions of a PS bead embedded in the solution. We recorded
0.8 stochastic signals x(t) with a sampling time dt = 20 μs and a
0.6 total acquisition time T = 4 s. From these data we computed
the PSDs, Sx ( f ). Finally, by equations (18), (20) and (22), we
0.4
calculated the moduli G  ( f ) and G  ( f ).
0.2
2 3 4 5 6 7 8 Figure 7 shows a typical spectra of G  ( f ) obtained at very
Power (mW) low concentrations, c = 0.01 mg ml−1 and c = 0.05 mg ml−1
( Plaser = 7.4 mW). For comparison, for each concentration,
Figure 4. Upper part: stochastic trajectories in distilled water of an G trap (pure water) is also reported. As can be noted, OT are able
optically trapped 1.25 μm bead in the x – y plane at different laser
powers. Lower part: plot of the maximum radius behavior as a to detect very small elastic moduli which are not measurable
function of the laser power (rmax = 2kκB T ∝ PLaser
1
). by conventional rheometry. However, at low frequencies,
G  ( f ) approaches the trap elastic contribution G trap . Hence,
to estimate the effective elastic contribution G eff of the sample,
the trap elasticity G trap should be subtracted from the measured
The MSDs calculated for different HA concentrations are elastic modulus G meas , i.e. G eff = G meas − G trap . This
plotted in figure 6. At high lag time all the traces reach subtraction procedure could not be trivial when G meas and G trap
the same plateau, representing the trap stiffness. As far as become comparable. Hence, weak traps are preferred because

Figure 5. Left part: stochastic signal in pure water (upper) and in a c = 0.1 mg ml−1 HA solution (lower). Right part: experimental PSDs
(points) and relative fitting curves (continuous lines).

7
J. Opt. A: Pure Appl. Opt. 11 (2009) 034016 G Pesce et al

3
10
HA 20 mg/ml
2
10

1
10

G', G'' [Pa]

0
10

-1
10

-2
10 G' Rheometer
-3
G'' Rheometer
10 G' OT
-4 G'' OT
10
-3 -2 -1 0 1 2 3
10 10 10 10 10 10 10
Freq [Hz]
Figure 7. Elastic modulus G  obtained in 0.01 and 0.05 mg ml−1 HA
solutions. For comparison, the elastic contribution of the optical trap Figure 9. G  and G  of a 20 mg ml−1 hyaluronic acid solution versus
measured in pure water samples is also reported (lower curve). frequency obtained with conventional rheometry (open symbols) and
optical tweezers (solid symbols).

(c = 20 mg ml−1 ) obtained using both OT and conventional

Figure 8. Shear loss moduli G  calculated at HA concentrations
between 0.05 and 20 mg ml−1 .
they are less perturbative, especially in the case of weak sample
elasticity.
Another technical problem concerning the evaluation of
G  and G  is the artifact produced at high frequencies
introduced by the Kramers–Kronig integral calculation.
Indeed, the finite sampling times of the stochastic signals
produce an artificial drop of both G  and G  curves. In
figure 7 the mathematical artifacts are around 200 Hz at c =
0.01 mg ml−1 and 400 Hz for c = 0.05 mg ml−1 (not visible in
the scale of figure 7), respectively. On the other hand, the lower
limit of the frequency range is limited by the total acquiring
time. These artifacts were properly taken into account and
eliminated in all the next figures by cutting the curves about
one decade before the cutoff. The shear loss moduli, instead,
are not affected by the optical trap effect and their evaluation is
straightforward. In figure 8 we reported the G  ( f ) curves for
all the HA concentrations here investigated.
Finally, we compared the results obtained from optical
tweezers experiments and from conventional rheometry.
Figure 9 shows the mechanical spectra of a HA solution
rheometry over a frequency range of 0.01 Hz–10 kHz. OT data
are presented after correction for the trap effects as described
above. The range of frequencies within the interval of 0.01
and 10 Hz was obtained by a conventional rheometer, while
the interval ranging from 0.25 to 1 kHz was explored by OT.
The data obtained with a conventional rheometer match fairly
well the OT data.
In particular, the agreement between macroscopic
and microscopic measurements obtained by a conventional
rheometer and optical tweezers is remarkable, especially for
high concentrations where the elastic contribution is quite
large. At low concentrations, instead, the agreement is not
good for the elastic shear modulus while it is still good for the
loss moduli. This is not surprising because it is well known that
a conventional rheometer is not reliable to measure low elastic
values.
Figure 10 shows the full-concentration dependence of the
shear storage and loss moduli versus frequency obtained with
OT: in the whole concentration range here investigated the
viscous modulus was always higher than the elastic modulus,
indicating a typical liquid-like viscoelastic response [1]. It is
worth noting that analysis of dilute solutions is rather difficult
to perform with a conventional rheometer due to the high
torque sensibility required and the relatively low absolute
values of the elastic modulus.
4.4. Role of probe–polymer interaction
In order to obtain reliable interpretations from microrheologi-
cal measurements a key role is played by the interactions be-
tween tracers and the networks they probe. In [42] Valentine
et al have discussed in detail this subject, especially for bio-
logical applications where the role of the probe–protein inter-
action is particularly significant. In our experiment, since both
HA and the polystyrene spheres are charged some electrostatic
interaction could be expected.

8
J. Opt. A: Pure Appl. Opt. 11 (2009) 034016 G Pesce et al

Figure 10. G  and G  versus the frequency at different HA concentrations ((A)–(F)). Solid and dotted lines are for G  and G  , respectively.
The shorter frequency range for G  is due to cutoff artifacts, as discussed in the text.

To assess the presence of any attractive or repulsive
interactions between the PS bead surfaces and HA molecules,
dynamic light scattering (DLS) (ALV 5000/E) measurements
were performed on polystyrene beads embedded in the
HA solution at different concentrations. The estimated
bead diffusion coefficient D was used to evaluate the ratio
Dη0 /D0 η, where η0 and η are the viscosity of the solvent
and of the HA solution, respectively, and D0 is the bead
diffusion coefficient in the solvent. Given the validity of the
Stokes–Einstein equation and in the absence of interactive
forces between the bead surfaces and HA molecules this ratio
is expected to be equal to 1. In our measurements we found
this ratio to be 0.98 ± 0.03 (see figure 11), indicating that no
significant interactions occur and therefore it is safe to assume
that interaction forces do not significantly affect the Brownian
motion of the polystyrene beads.

9
J. Opt. A: Pure Appl. Opt. 11 (2009) 034016 G Pesce et al

1,4 DLS PS sphere 1.245 μm

1,2
Dη /D η
0

1
0

0,8

0,6

Figure 13. Specific viscosity ηsp for rheometer (squares) and OT

-2 -1 0 1
10 10 10 10 (circles) as a function of hyaluronic acid concentration.
c [mg/ml]

Figure 11. Behavior of the ratio (D/η)/(D0 /η0 ) versus the

concentration obtained from dynamic light scattering (DLS)
measurements.
Figure 12. Behavior of η( f ) as a function of the hyaluronic acid
concentration obtained from optical tweezers.
4.5. Discussion
The frequency-dependent viscosity, calculated through the
relation G  ( f ) = 2π f η( f ), is plotted in figure 12 for different
HA concentrations. The well-known thinning effect is well
pronounced at high frequencies. Moreover, we also calculated
the specific viscosity ηsp = η−η η0
0
by using the viscosity values
η estimated by rheometer and OT. In the case of the rheometer,
the steady-state zero-shear viscosity η0 was extrapolated from
the flow curve in the limit of zero shear, while in the case of
OT η0 was extrapolated from the relation η( f ) = G  ( f )/2π f
in the limit f → 0 (see figure 12).
The specific viscosity values can be divided into three
different regions (see figure 13), separated by the two critical
concentrations, c∗ and ce . In these three regions we found:
(I)
ηsp ∼ c1.02(c < c∗ ), ηsp (II)
∼ c0.56(c∗ > c > ce ) and
(III) 1 .5
ηsp ∼ c (c > c ). e entanglements. Then the low molecular weight HA system
Linear polyelectrolytes, like HA in low salt concentration
solutions, show a weaker dependence of the viscosity
and viscoelastic parameters on the concentration compared
to a neutral polymer due to the chain resizing when
the concentration increases in the dilute and semidilute
regimes [43, 44]. According to polyelectrolyte scaling
(I)
theory [45], the dependence of specific viscosity is ηsp ∼ c for
c < c∗ , ηsp
(I)
∼ c0.5 for c∗ < c < ce and ηsp (III)
∼ c1.5 for c > ce .
So, our results, using both OT and rheometry techniques, are
in good agreement with theoretical predictions
For solutions at concentrations below ce (figures 10(A)–
(C)) G  α f 2 and G  α f . That is in agreement with the model
predictions for dilute solutions [46, 47].
In the semidilute entangled regime (c > ce ) the mechani-
cal spectra indicate that the polyelectrolyte macromolecules do
overlap but entanglements are readily untied due to the short
chain length between the molecules. The crossing between G 
and G  observed in entangled polymer solutions is due to the
persistence of mechanically active entanglements which occur
when the characteristic time of the imposed mechanical defor-
mation (∼1/ f ) is short compared to the characteristic disen-
tanglement time. In these conditions the entanglements act as
permanent crosslinks points and the solution exhibits an elas-
tic behavior. However, to observe this behavior it is necessary
that the molecules form a minimum number of entanglements,
n , which has been reported to be within 5–10 [47]. The num-
ber n could be approximately estimated from the ce and c∗ as
√
n = 4 ce /c∗ [48] which for our solutions is ∼2.5. This sug-
gests that in our case the low molecular weight HA molecules
do overlap with each other, as indicated by the change of the
ηsp slope above ce (figure 8), but most of the entanglements are
formed at the chain tail and therefore they might disentangle
readily.
The absence of G  and G  crossing was also reported
by Kobayashi et al [41] for 560 kDa HA solutions at
concentration up to 10 mg ml−1 , whereas higher molecular
weight HA promoted the formation of elastically active chain
holds a prevalent viscous behavior even at concentrations
greater than ce up to high frequency.

10
J. Opt. A: Pure Appl. Opt. 11 (2009) 034016
5. Conclusions
We show that the optical tweezers microrheological technique
can be used to measure the viscoelastic properties of
polymeric solutions. The use of OT enables us to extend
the range of frequencies investigated, usually not available
using traditional bulk measurements based on mechanical
rheometers. Particular care has to be taken in order to consider
properly the elastic contribution of the optical trap itself.
The viscoelastic properties of HA solutions measured with
OT agree with results obtained from classical rheometry in
the overlapping frequency range, validating the experimental
apparatus. Microrheological and rheological measurements
performed on samples show the same frequency dependence
for the elastic and viscous moduli. The dynamic mechanical
properties of the HA solution are in agreement with the
polyelectrolyte scaling theory. High frequency viscoelastic
behavior was investigated and no crossover between elastic and
viscous moduli was found, even at high concentrations.
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