Single molecule lifetime fluctuations reveal segmental dynamics in polymers.
ABSTRACT We present a single molecule fluorescence study that allows one to probe the nanoscale segmental dynamics in amorphous polymer matrices. By recording single molecular lifetime trajectories of embedded fluorophores, peculiar excursions towards longer lifetimes are observed. The asymmetric response is shown to reflect variations in the photonic mode density as a result of the local density fluctuations of the surrounding polymer. We determine the number of polymer segments involved in a local segmental rearrangement volume around the probe. A common decrease of the number of segments with temperature is found for both investigated polymers, poly(styrene) and poly(isobutylmethacrylate). Our novel approach will prove powerful for the understanding of the nanoscale rearrangements in functional polymers.
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ABSTRACT: Product releasing is an essential step of an enzymatic reaction, and a mechanistic understanding primarily depends on the active-site conformational changes and molecular interactions that involve in this step of an enzymatic reaction. Here we report our work on the enzymatic product releasing dynamics and mechanism of an enzyme, horseradish peroxidase (HRP), using combined single-molecule time-resolved fluorescence intensity, anisotropy, and lifetime measurements. Our results have shown a wide distribution of the multiple conformational states involving in active-site interacting with the product molecules during the product releasing. We have identified that there is a significant pathway that the product molecules are spilled out from the enzymatic active-site, driven by a squeezing effect from a tight active-site conformational state; although, the conventional pathway of releasing a product molecule from an open active-site conformational state is still a primary pathway. Our study provides a new insight of the enzymatic reaction dynamics and mechanism, and the information is uniquely obtainable from our combined single-molecule time-resolved single-molecule spectroscopic measurements and analyses.The Journal of Physical Chemistry B 07/2014; · 3.38 Impact Factor
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ABSTRACT: Rotational motion of fluorophores chemically attached to polystyrene chain-ends in ultra-thin films on solid substrates was studied by single-molecule fluorescence de-focus microscopy. The collective feature of the rotational motion was found and evidenced by the sharp change of the population of fluorophores undergoing rotational motion within a very narrow temperature range (named as the changing temperature, T c). The T c value was found to depend on film thickness and interfacial chemistry and the variation of the T c value is also dependent on the molecular weight of the polymer. The results demonstrate that the spatial confinement effect enhances the segmental mobility near the polymer chain-ends while the interfacial attraction restricts the segmental motion inside the thin film.Science China-Chemistry 03/2014; 57(3):389-396. · 1.52 Impact Factor
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ABSTRACT: A molecular rotor with a fluorescence lifetime depending on the local viscosity of its surroundings has been successfully used as a probe to monitor local viscosity changes during the bulk radical polymerization of methyl methacrylate.Polym. Chem. 03/2014; 5(8).
Single Molecule Lifetime Fluctuations Reveal Segmental Dynamics in Polymers
R. A. L.Valle ´e,1,2N. Tomczak,2L. Kuipers,1G.J.Vancso,2and N. F. van Hulst1,*
1Applied Optics Group, MESA?Research Institute, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands
2Materials Science and Technology of Polymers, MESA?Research Institute, University of Twente,
P.O. Box 217, 7500 AE Enschede, The Netherlands
(Received 6 March 2003; published 17 July 2003)
We present a single molecule fluorescence study that allows one to probe the nanoscale segmental
dynamics in amorphous polymer matrices. By recording single molecular lifetime trajectories of
embedded fluorophores, peculiar excursions towards longer lifetimes are observed. The asymmetric
response is shown to reflect variations in the photonic mode density as a result of the local density
fluctuations of the surrounding polymer. We determine the number of polymer segments involved in a
local segmental rearrangement volume around the probe. A common decrease of the number of
segments with temperature is found for both investigated polymers, poly(styrene) and poly(isobutyl-
methacrylate). Our novel approach will prove powerful for the understanding of the nanoscale
rearrangements in functional polymers.
DOI: 10.1103/PhysRevLett.91.038301 PACS numbers: 82.37.–j, 05.40.–a, 33.50.–j, 82.35.Np
Glasses are disordered materials usually obtained by
cooling a viscous liquid or a polymer melt fast enough to
avoid crystallization.Their static and dynamic properties
deviate largely from the simple Debye behavior. The
deviations are best interpreted in terms of dynamic het-
erogeneities of structure on the segmental (nanometer)
scale . Direct evidence for microscopic regions of
different relaxation time has been obtained by multidi-
mensional nuclear magnetic resonance , photobleach-
ing , excess light scattering near the glass transition
temperature (Tg) , dielectric hole burning , and,
recently, single molecule spectroscopy . However,
the key question concerning the temperature dependence
of the characteristic dimensions of the inhomogeneities
in glass forming liquids and amorphous solids is still
Single molecule detection has proven to be a unique
method to investigate the behavior of complex condensed
systems [7,8]. In contrast to ensemble methods, the single
molecule approach provides information on time trajec-
tories, distributions, and correlations of observables that
would otherwise be hidden. Individual members of a
heterogeneous population are examined, identified, and
sorted to quantitatively compare their subpopulations.
In the extreme case of cryogenic temperatures, it has
been shown  that most of the spectral trails of single
molecules obtained at around 1 K are consistent with the
standard two-level system model of glasses . At room
temperature, the broad spectra and complexity of the
system complicate the single molecule spectral analysis.
We present in this Letter a first study in the comple-
mentary time domain. The excited state lifetime of the
individual dye molecules is monitored in time. In a static
environment, the lifetime has a discrete value. Because of
the heterogeneity of the nanoenvironment, the lifetime
is different but constant for every molecule [11,12].
However, in a fluctuating environment, the lifetime will
vary and develop a certain distribution. We show that
lifetime fluctuations are due to variations of the radiative
density of states (RDOS) and consequently reflect the
local density fluctuations in the surroundingsof the single
molecule probe. We establish in a direct way the number
of segments involved in the rearrangement volume sur-
rounding the fluorophore, by connecting our observations
to the Simha-Somcynsky (SS) equation of state .
Interestingly, a common decrease of the number of seg-
mentswithtemperaturefordifferent polymersisfound, in
agreement with the predictions of the thermodynamic
Adam-Gibbs theory .
Dye-doped polymer films (70 and 200 nm) were pre-
pared by spin coating a solution of 1,1’ -dioctadecyl-
3, 3, 3’ , 3’- tetramethylindodicarbocyanine (DiD, 5 ?
10?10M, Molecular Probes) and polystyrene [PS,
89300 g=mol, polydispersity
Polymer Standard Service], or poly(isobutylmethacry-
late) (PIBMA, 67200 g=mol, PI ? 2:8, custom made
radical polymerization) in toluene onto a glass substrate.
Further annealing was performed in order to relax the
stresses induced by the deposition procedure. The choice
of the dyewas dictated by the following considerations: it
possesses a high fluorescence quantum yield (close to
unity), an absorption cross section of 7:5 ? 10?16cm2,
and is highly photostable when embedded in a polymer
matrix . PS (Tg? 100?C) and PIBMA (Tg? 56?C)
were chosen due to to their different glass transition
temperatures Tg.This allows us to probe polymer proper-
ties as a function of their relative distance to Tg, while
working at the same laboratory temperatures for both
polymers. Molecules in the sample were excited by 57 ps
pulses at a wavelength of 635 nm and repetition rate of
80MHz, generatedbya pspulseddiode laser (PicoQuant,
PDL 800-B, 100 ?W), at the focus of a confocal inverted
microscope (Zeiss). Fluorescence intensity and lifetime
of individual molecules were monitored in time, in
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VOLUME 91, NUMBER 3
2003 The American Physical Society038301-1
consecutive experiments, using a time-correlated single-
photon counting card (Becker & Hickl, SPC 500) .
Integrating over 200 ms time intervals, a lifetime accu-
racy of typically 0.1–0.3 ns was obtained.
Figure1 shows fluorescence lifetime trajectories of two
individual molecules emitting at approximately the same
intensity level. Both trajectories fluctuate in time, but in a
different way: while the first molecule (a) has a lifetime
restricted to small and rather symmetrical variations
around the mean value, the second molecule dwells occa-
sionally longer in the excited state, as is clear from the
excursions to longer lifetimes in the transient (b). The
corresponding single molecule fluorescence lifetime dis-
tributions built up from the transients further underline
the difference in behavior between the two molecules.
The characteristic shape varies from nearly symmetric
(c) to asymmetric (d).Why does the fluorescence lifetime
change? Three factors may potentially affect the fluores-
cence lifetime of dyes embedded in a dielectric medium:
quenching effects, changes in the conformation of the
fluorophore, and variations in the dielectric properties
surrounding the probe molecule.
First, quenching effects may be discarded since the
aperture of nonradiative decay channels in the matrix
would simultaneously induce a lowering of the measured
intensity and lifetime. This is not the case since intensity
and lifetime are not correlated in the plots of Fig. 1.
Furthermore, the quantum efficiency of the chosen fluo-
rophore (close to unity) excludes the possibility that the
large lifetime fluctuations result from fluctuations in the
number of decay channels. Moreover, the excursions in
the fluorescence lifetime trajectory [Fig. 1(b)] are always
towards higher values, which rules out any nonradiative
process as the cause of the fluctuations.
Second, the electronic properties of the conjugated
DiD molecule are dominated by the presence of delocal-
ized ? electrons. The lowest optical excitation corre-
sponds to a ? ! ?? transition and vice versa for the
emission.The molecule may thus be described as a simple
two-level system. In the electric dipole approximation
and in vacuum, the spontaneous emission rate ?0of the
molecule is given by the relation 
The radiative lifetime ?0is the inverse of ?0, where !0, d,
and ?0designate the transition frequency, the transition
dipole moment of the excited state of the fluorophore for
the ? ! ?? transition, and the dielectric constant of the
vacuum, respectively.This equation reveals that a change
in the transition frequency or the transition dipole mo-
ment of the dye due to the influence of the nanoenviron-
ment can be responsible for the observed fluctuations of
the lifetime. However, by quantum chemistry calcula-
tions, we showed  that low-cost energy motions of
the DiD molecule compatible with thermal agitation at
room temperature result in a fluctuation of at most 10% in
the fluorescence lifetime when compared to the rest struc-
ture.Therefore conformational changesdo not explainthe
large fluctuations observed.
in a 200 nm thick film of PIBMA at room temperature. Clearly, the second molecule exhibits excursions towards longer lifetime
values. (c),(d) For the two transients corresponding fluorescence lifetime distributions (bottom) and correlation between intensity
and lifetime (top) are shown.The shape of the distribution is more asymmetric for the second molecule.While the intensity remains
within a 10% fluctuating range, the lifetime shows changes up to 100%.
(a),(b) Transients of fluorescence intensity and fluorescence lifetime for two different individual DiD molecules embedded
PH YSICA L R EVI EW L ET T ERS
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VOLUME 91, NUMBER 3
As a result, we attribute the radiative lifetime fluctua-
tions observed to the local density fluctuations of the
surrounding polymer matrix. We quantified this effect
for 778 individual probe molecules embedded in different
polymer matrices, at various temperatures. For each
single molecule trajectory lasting at least 15 s, we were
able to build up a reliable distribution of fluorescence
lifetimes. Interestingly, the experimental lifetime distri-
bution is best fitted with a gamma distribution g?x?. This
distribution g?x? ? ???x???1e??x=???? is generally ac-
cepted for a lower bounded continuous variable fully
characterized by its first two moments xavand h?x2iav
or,equivalently,by itsshape (?)andscale (?)parameters,
gamma function. From the fitted gamma distributions,
we extract the value of the characteristic shape parameter
?. At each given temperature, collecting the shape pa-
rameters of 30 to 60 of these molecules, we constructed
the distribution of the shape parameter. Figure 2 shows
the shape distributions for three different temperatures
for DiD embedded in a 200 nm thick film of PIBMA.
Surprisingly, upon increasing temperature, the average
shape value as well as the width of the shape parameter
distribution decrease. This temperature dependence fur-
ther corroborates the conclusion that the density fluctua-
tion in the polymer is the main factor in the observed
How can density changes within the polymer affect the
fluorescence lifetime of an embedded fluorophore? The
probe molecule is placed in a nonoccupied space (cavity)
of a polymeric matrix.The energy it radiates thus depends
on the dielectric properties of the local surroundings.The
spontaneous emission rate ???? of the dye inside a homo-
geneous medium with dielectric constant ? is predicted to
dependence of the RDOS : ???? ?
the macroscopic electromagnetic field, while the dipole
couples to the local field at the position of the molecule.
Strictly the dipole-dipole interaction considering the sur-
rounding polymeric voids should be calculated ; in
?2, and ???? stands for the
p?0. However, this result was obtained by quantizing
practice, for complex systems, the concept of local field is
introduced. The microscopic local field differs from the
macroscopic field by a local field correction factor Lf
2? ? 1 ?2?
3??? ? 1?;
where here ? is the polarizability of the dye in a cavity of
volume ? . For substitutional or interstitial impuri-
ties such as dyes in an otherwise homogeneous medium,
Eq. (2) reduces to the empty-cavity (Lorentz) local field
polarizability thelocalfieldfluctuationsmight exceedthe
Lorentz factor. By including the local field factor the
spontaneous emission rate is given by ???? ? L2
Up to now, the polymer matrix has been considered as
a homogeneous medium with a dielectric constant ?.
However, polymer chain segments move in time in the
polymer matrix at room temperature. For the probe mole-
cule, the segmental rearrangements imply either creation
or annihilation of voids in its nanoenvironment, and thus
a change of its surrounding local dielectric constant.
Consequently, the system has to be considered as an
effective medium , consisting of polymer segments
and voids competing to occupy space. A local effective
dielectric constant ? modulated by the fraction h of holes
present in the medium is given by
2??1. For dye molecules with a substantial
? ? h?vac? ?1 ? h??pol;
where ?vac? 1 and ?pol? 2:5 designate the vacuum and
polymer (PS) dielectric constant, respectively.
Following usual statistical theory, the change of vari-
ables ? into h is accompanied by a corresponding change
of the probability density f??? into g?h? ? f???jd?
the effective medium considered here, this dependence is
smoothly linear: converting a gamma distribution of
holes, characterized by its first two moments hav? 10%
and h?x2iav? 0:4% to the corresponding distribution of
lifetimes, leads to a nonlinear deviation of only 5% in the
corresponding value of the shape parameter.
To relate our claim to a classical polymer theory, it is
interesting to note that the SS model  considers the
polymer as a lattice of sites that can accommodate the
chain segments of macromolecules. To account for mo-
lecular disorder, a temperature and volume dependent
fraction h of holes is introduced. Knowing the configura-
tional properties of the system, an equation of state has
been established , which permits the determination
of the fractional mean free volume havpresent in the
system. However, due to thermal fluctuations, the free
volume varies both in time and at every position. The
mean-squared deviation from the mean free volume can
be calculated once the number of polymer segments (Ns)
involved in a segmental rearrangement cell is known.
From the first two moments and by attributing a value
to Ns, a gamma distribution of free volume is built
[23,24]. Given a gamma distribution of free volume, the
embedded in a 200 nm thick film of PIBMA for three tem-
peratures. With increasing temperature, the distributions shift
towards lower shape values.
Distributions of shape parameter for DiD molecules
PH YSICA L R EVI EW L ET T ERS
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VOLUME 91, NUMBER 3
number of segments ?Ns? becomes a linear function of
the shape parameter ? multiplied by a temperature
and volume dependent factor, Ns? ?f?h;~ V V;~ T T?. The
shape distributions, as shown in Fig. 2, can therefore be
converted into corresponding distributions of the number
of segments Nsinvolved in a local segmental rearrange-
Figure 3 shows the peak positions of the Nsdistribu-
tions as a function of temperature for PIBMA, 70 and
200 nm thick films, and PS, 70 nm thick film. The
striking feature of this master plot is the appearance
of a general behavior, with the reduced temperature
?T ? Tg?=Tgas a common parameter. The observation
of the decrease of the number of segments when increas-
ing temperature is in agreement with the configurational
entropy model of Adam and Gibbs (AG), which predicts
that the length scale of the cooperatively rearranging
regions (CRR) decreases with increasing temperature.
Furthermore, the changes in width and position of the
distributions with temperature, shown in Fig. 2, clearly
reveal the existence of microheterogeneous domains of
different sizes and relaxation times, which is not consid-
ered in the AG theory, where the CRRs are assumed to be
equivalent. Admitting a distribution of sizes of the inde-
pendently relaxing CRRs, the theory thus does not give
any guidance as to what the distribution should be. Single
molecule spectroscopy and Fig. 2 provide such distribu-
tions and even show a reduction in the width of the
distributions with increasing temperature, related to the
appearance of a more homogeneous dynamics as the
temperature is increased.
Having opened a route towards direct microscopic in-
sight in the segmental dynamics of polymers, it will be
interesting to explore the significance of the Nsvalue
around Tgand the behavior towards higher temperatures.
We believe that our novel approach has large potential for
the understanding of the nanoscale dynamics of func-
tional polymers and biopolymers.
The authors are grateful to Jeroen Korterik and Frans
Segerink for technical support, Erik van Dijk for TCSPC
interfacing and helpful discussion of the results, and
Marı ´a Garcı ´a-Parajo ´ for introducing the first author to
the single molecule field. This research is supported by
the Council for Chemical Sciences of the Netherlands
Organisation for Scientific Research (NWO-CW).
*Electronic address: firstname.lastname@example.org
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a segmental rearrangement cell as a function of the reduced
temperature ?T-Tg?=Tgfor PS (squares, Tg? 100?C) and for
PIBMA (circles, Tg? 56?C). In the case of PIBMA, data for
both a 70 nm thick film (closed circles) and a 200 nm thick
film (open circles) are shown. At the glass transition tempera-
ture, typically six segments play a role in the segmental
Master plot of the number of segments Nsinvolved in
PH YSICA LR EVI EW L ET T ERS
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VOLUME 91, NUMBER 3