Journal of Modern Physics, 2013, 4, 27-42
Published Online November 2013 (
Open Access JMP
Single Molecule Applications of Quantum Dots
Thomas E. Rasmussen1, Liselotte Jauffred2, Jonathan Brewer1, Stefan Vogel1, Esben R. Torbensen1,
B. Christoffer Lagerholm1,3, Lene Oddershede2, Eva C. Arnspang1,4,5
1Departments of Physics, Chemistry, Pharmacy, Biochemistry and Molecular Biology,
University of Southern Denmark, Odense, Denmark
2Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark
3Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
4National Institutes of Health, Bethesda, USA
5Department of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center,
Aarhus University, Aarhus, Denmark
Received September 25, 2013; revised October 23, 2013; accepted November 22, 2013
Copyright © 2013 Thomas E. Rasmussen et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Fluorescent nanocrystals composed of semiconductor materials were first introduced for biological applications in the
late 1990s. The focus of this review is to give a brief survey of biological applications of quantum dots (QDs) at the
single QD sensitivity level. These are described as follows: 1) QD blinking and bleaching statistics, 2) the use of QDs in
high speed single particle tracking with a special focus on how to design the biofunctional coatings of QDs which en-
able specific targeting to single proteins or lipids of interest, 3) a hybrid lipid-DNA analogue binding QDs which allows
for tracking single lipids in lipid bilayers, 4) two-photon fluorescence correlation spectroscopy of QDs and 5) optical
trapping and excitation of single QDs. In all of these applications, the focus is on the single particle sensitivity level of
QDs. The high applicability of QDs in live cell imaging experiments held together with the prospects in localization
microscopy and single molecule manipulation experiments gave QDs a promising future in single molecule research.
Keywords: Quantum Dots; Single Particle Tracking; Fluorescence Correlation Spectroscopy; Optical Tweezers
1. Introduction
Fluorescent nanocrystals composed of semiconductor
materials are regularly referred to as quantum dots (QDs)
because of their optical properties. Quantum confinement
within the core material leads to the fluorescence emis-
sion wavelength being dependent on the core size and
material composition. These days QDs are regularly be-
ing used in a variety of biological applications both in
vitro and in vivo (References). Almost 15 years after
their initial application in biology [1], QDs have found
use in a multitude of different applications due to their
superior optical properties (brightness and resistance to
bleaching) as compared to conventional fluorophores. In
this review we present basic concepts of QDs with a fo-
cus on the optical properties that make QDs so special
and further give a survey of single QD applications in
biology. QDs have a higher brightness because of their
very high absorption excitation coefficients and high
quantum yield. QDs also display a higher resistance to-
wards photobleaching since their inorganic nature makes
them less prone to degradation resulting from photo-
induced bond cleavage. The resulting fluorescence life-
time (20 - 50 ns) [2] and high signal to noise ratio make
them ideal for time-resolved microscopy studies. Section
2 is an introduction to the material composition, quantum
physics and optical properties of QDs. In Section 3, there
are examples of two single particle applications of QDs:
QDs targeted to a protein of interest followed by high
speed single particle tracking (SPT) of the QD signal in
live cells, and QDs targeted to a DNA-lipid analogue and
SPT in lipid bilayers. In Section 4, there is an example of
the use of QDs as probe in two-photon fluorescence cor-
relation spectroscopy in which the size of the QD is
found. In Section 5 we present how the use of QDs as a
force-handle as well as a visualization probe using an
optical tweezers assay. In all the applications mentioned
in this review, the focus is on the use of QDs in single
particle detection experiments.
2. Quantum Dots—Fluorescent
Semiconductor Nanocrystals
Quantum dots (QDs) are inorganic fluorescent nanoscale
crystals with considerable enhanced optical properties in
terms of brightness, photostability, blinking and bleach-
ing compared to conventional organic and protein fluo-
rophores. QD core sizes are in the range 1 - 10 nm and
the individual nanocrystals contain from a few hundred
to several thousands of atoms each. The core of the QD
nanocrystals is typically composed of binary mixtures of
semiconductor materials (ZnS, CdS, CdSe, InP, CdTe,
PbS, PbTe). Early QDs were composed of only a naked
core but this led to inherent problems with fluorescence
output due to interaction of the exciton with the sur-
roundings. Increasing QD yield and efficiency was
achieved by adding a second shell layer semiconducting
material to the core resulting in a so-called core/shell QD
[3,4]. Core-shell QDs is the main type used today in bio-
logical applications.
QDs preserve some bulk properties from the material
they are made of, but because of their small size they
also retain new unique physical, chemical and electronic
properties due to quantum confinement effects. Probably
the most amazing feature of the confinement effects is
that the emission bands of QDs are dependent of the
composition and size. If semiconductors are exposed to
light excitation, the mobility of electrons in the material
increases. Characteristic for semiconductor materials is
that they have an unoccupied energy band called the
conduction band and an occupied energy band called the
valence band. When irradiated an electron from the va-
lence band can get excited and promoted to the conduc-
tion band. This result in the formation of a positive
charge called a hole and both the electron and the hole
are free to move around the bulk material kept together
by a Coulomb attraction. Together the electron and hole
constitute an electron-hole pair also known as an exciton,
the average distance between the electron and hole is
known as the Bohr radius of the exciton. Because the
excitons can be seen as particles in a box the following
quantum mechanical calculations can be made. The
Hamiltonian for a spherical QD of diameter R is given by
eh e
Hmm r
  
The first two terms are the kinetic energy operators of
the electron and the hole with masses me and mh respec-
tively. The last term denotes the potential energy interac-
tion of the electron and the hole that are in the positions
e and h from the center of the sphere. Due to the
charge on the electron (e) and the hole (+e) the Cou-
lombic attraction between the electron and the hole,
where ε is the permittivity of the material and
r r
the distance between the hole and the electron. Solving
the Schrödinger yields the following equation for the
approximation of the exciton energy [5].
11 1.8
Emm R
The equation states that the exciton energy decreases
with decreasing values of R and the term on the right will
decrease with small R-values and therefore the exciton
energy is largely of kinetic nature. Therefore the electri-
cal conductivity and creation of mobile charge carriers
depends on the size of the quantum dot. As a result the
fluorescence emission wavelength of the QD can be
tuned by size, such that the emission of smaller QDs will
be blue shifted while the emission of larger QDs will be
red shifted. In Figure 1 is a picture of a series of QDs
emitting at wavelengths from 490 nm to 650 nm.
2.1. Optical Properties of Quantum Dots
QDs distinguish themselves in several ways compared to
conventional organic and protein fluorophore molecules.
While the band gap of organic fluorescent dyes is defined
by the π-electron system of the molecule as well as elec-
tron donating and withdrawing groups, the QD band gap
is determined by the size of the nanocrystal even though
the material remains the same. Further, many of the most
commonly applied fluorophores are characterized by
relative narrow emission excitation and emission spectra.
QDs on the other hand have a much wider excitation
range while they exhibit very narrow and symmetrical
emission spectra separated by a distinct Stokes shift.
A lot of effort has been done in order to tune QD
emission to cover as large a wavelength range as possible.
Most commercial available QDs have emission spectra
Figure 1. Image showing fluorescence emission of CdSe/ZnS
with increasing size (490NC, 525NC, 565NC, 585NC, 605NC,
625NC & 650NC) and cadmium-free InGaP (700NC)
Quantum dots from eBioscience.
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situated in the visual part of the electromagnetic spec-
trum and include QD materials such as (CdS, CdSe,
CdTe). QDs emitting in the Ultra Violet range have
mainly been made of ZnS and ZnSe while emission in
the near infrared range has been accomplished with ma-
terials such as CdS/HgS/CdS, InP, InAs [6]. Mainly the
latter has received much attention in the development of
new QD types since the penetration of NIR light into
tissues is significantly higher than shorter wavelengths of
light, which are susceptible to significant scattering and
absorption by the tissue. Furthermore autofluorescence
from biological samples is less present in this part of the
spectrum resulting in a lower fluorescent background. In
Figure 2 is an overview of emission wavelengths of
commercially available QDs.
commercially available hybrid CdSe/CdTe QDs have
been shown to spend a majority of their time in a non-
fluorescent dark state [7-13]. The current consensus for
this observed QD intermittency is that non-charged QDs
are fluorescent, while charged QDs are not [8,14]. The
observed intermittency has further been shown to be par-
tially inhibited by small reducing agents such as β-mer-
captoethanol (BME), dithiothreitol (DTT) and mercap-
toethylamine (MEA) in mM concentrations [9,15,16].
Decreased blinking has also been accomplished by
growth of thick semiconductor shells around the QD
cores [17,18]. Non-blinking CdZnSe/ZnSe QDs have
also been reported, however, these QDs have a very
broad emission spectra with three characteristic emission
peaks making them non-useable for multiplexing appli-
cations [14].
QDs display one more characteristic optical feature.
QDs blink, periodically switching between an on and off
luminescent state. The cause of this behaviour is due to
trapping and untrapping of charges due to surface defects,
this enables the distinction of single QDs, which is es-
sential to perform single molecule measurements.
When this fluorescence blinking of QDs was first
observed, it was a surprise, since there was no known
quantum physical mechanism which could explain this
[19]. What was furthermore a surprise was that the off
times of QDs were distributed according to an inverse
power law rather than an exponential decay [20]. An ex-
ponential dependence of the probability P that a QD is off
for the time
would follow the equation (=
is the off time and
the slope of the linear
plot on a semilog scale. A power law dependence of the
probability P that a QD is off for the time
follow the equation
, where
is the slope of
2.2. Blinking of Quantum Dots
A major disadvantage of QDs is that the fluorescence
emission is intermittent, i.e., the QDs fluctuate between a
fluorescent emitting bright state and a non-emitting dark,
with the dark non-fluorescence emitting states lasting for
periods in the ms to second range timescale. For example,
Figure 2. An overview of emission wavelengths of quantum dots from different commercial sources.
the linear plot on a log-log scale. The power law de-
pendence of the blinking of QDs is unchanged for tem-
peratures from 10 K to room temperature, for core radii
from 15 Å to 27 Å, for different materials: CdSe, TeSe,
InP, for laser intensities from 100 W/cm2 to 20 kW/cm2
and pressure from atmospheric pressure to vacuum
[21-24]. Because QDs consist of two alloy compositions
and the bond length in the crystal lattice of the one alloy
is different from the bond length in the crystal lattice of
the other alloy there are imperfections in the crystal
structure. The quantum physical explanation of the
blinking of the QDs proposes that the intermittency is
caused by ionization of the QD. After photon excitation,
two electron-hole pairs could be excited simultaneously.
One pair could recombine by the Auger effect and emit
one hole or one electron outside the QD. This is also
called a trapped state as the emitted electron will be
trapped in the surrounding medium for some time. Dur-
ing this off-time the left behind electron hole in the QD
will generate a very fast non-radiative Auger channel for
any new excited electron-hole. The electron hole will
remain inside the QD during the off-period due to dif-
ferent barrier heights for electron and hole. The result is
that the radiative luminescence is quenched during the
off period, corresponding to the dark state of QDs
2.3. Bleaching of Quantum Dots
A major attraction of QDs is that they are more resistant
to photobleaching than is conventional fluorescent dye
and protein fluorophores. But QDs have also been shown
to photobleach under intense laser illumination [27,28]
and more recently even with Hg arc lamp illumination
[29]. However, contrary to fluorescent dyes and proteins
that display single step photobleaching from a fluores-
cent emitting state to a dark state, QDs have been shown
to photobleach by a gradual blue-shifting of their emis-
sion color eventually reaching a permanent dark state
[8,27,28]. This photobleaching of QDs is indicative of a
process in which the QD core is gradually shrinking as a
result of photooxidation of the core [8,27,28,30,31], a
hypothesis which is supported by the reported slowing of
blue shifting in a nitrogen atmosphere [28].
Photooxidation at the surface of QDs has shown to re-
sult in quenching of fluorescence emitted by CdSe QDs.
Formation of surface quenching states will cause a de-
crease in emitted fluorescence. The bleaching of QDs is
accompanied with a blueshift in the emission spectrum.
This bleaching process has been shown with laser powers
of 20 kW/cm2 and in both air and nitrogen atmospheres.
In nitrogen atmosphere the bleaching is slower but still
occurs [28]. It is possible to prevent the bleaching and
blueshifting of QDs by adding β-mercaptoethanol (BME)
[32] or mercaptoethylamine (MAE) [29]. Figure 3 de-
picts images of QD655s that were illuminated under
aqueous conditions for a few minutes with and without
BME added. Left and rightwimages were acquired about
7 minutes apart after continuous illumination with blue
filtered light. In the absence of BME, the QD655 emis-
sion color was observed to shift from an initial or-
ange/red hue to a yellow-green hue. In the presence of 25
μM BME, the QD655 emission color was observed to be
significantly stabilized to an orange/red hue for the dura-
tion of the experiment [32].
Bleaching of QDs happens at a slower rate than other
standard fluorophores. A direct comparison has been
made between Alexa488 and QDs. Alexa488 fades after
60 seconds of constant illumination whereas the QDs are
still emitting after 180 seconds. There are also many
examples of longer full intensity periods of QDs [33].
(a) (b)
(c) (d)
Figure 3. Fluorescence color switching of QDs emitting at
655 nm. QDs were non-specifically adsorbed to a glass cov-
erslip and imaged under aqueous conditions on a CoolS-
NAP-Procf color CD camera with 10 sec integration time.
Left and right images were acquired about 7 minutes apart
after continuous illumination with blue filtered light from a
100 W Hg arc lamp. (a, b) QD655 in the absence of
β-mercaptoethanol (BME), the QD emission color was ob-
served to shift from an initial orange/red hue to a yel-
low-green hue. (c, d) QD655 in the presence of 25 μM BME,
the QD emission color was observed to be significantly sta-
bilized to an orange/red hue for the duration of the experi-
ment (scale bar is 1 m) Reprinted with permission from
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2.4. Quantum Dot Coatings and Bioconjugation
In order for QDs to be used in single molecule applica-
tions certain surface modifications have to be done to the
bare core/shell QDs. Both core-shell and core only QDs
are coated with a thin layer of an organic ligand such as
trioctylphosphine oxide (TOPO) as a result of the organic
synthesis route that is used to make them [34,35]. While
a hydrophobic ligand such as TOPO stabilizes the QDs in
an organic solvent, these QDs need to be made more wa-
ter soluble for biological relevant applications [34-38].
Keeping the original ligands in place gives the brightest
QDs, and one way to do this is to use an amphiphilic
co-block polymer (e.g. an octylamine-modified poly-
acrylic acid) to coat the QDs [38]. The hydrophobic side
chains of these polymers interdigitate with the organic
ligands on the QDs, and the hydrophilic part constitute
the new surface of the now water soluble QDs, adding
another 1 - 2 nm to the diameter [39,40]. The hydrophilic
ends of these QDs have reactive groups that are available
for further bio-conjugation needed in order to direct the
QD binding towards a specific target. Bio-conjugation of
the QD will add to its final size and typical fully bio-
functional QDs have a size of ~20 nm.
Water stabilized QDs preferentially have reactive
chemical groups on the surface that are available for bio-
functionalization necessary in order to direct the QDs
towards a specific target for biological applications [36].
Often, the initial chemical groups on the surface are car-
boxylic groups, but these can be reacted with e.g. dia-
mine polyethylene glycols (PEG), resulting in amino
functionalized QDs. Covalent bio-functionalization of
carboxylic or amine QDs is easily achieved by chemical
cross-linkers, and various strategies exist depending on the
conjugation partners [40,41]. For example, the cross-linker
EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)
can be used to conjugate carboxylic groups on QDs to
amine groups on e.g. proteins or peptides [42,43]. An-
other bifunctional cross-linker SMCC (succinimidyl-4-
(N-maleimidomethyl)cyclohexane-1-carboxylate), which
has a maleimide reactive group and an NHS ester, can be
used to couple thiols on e.g. (mildly and selectively) re-
duced antibodies or antibody fragments to amines on QDs
[44]. QD conjugation of two amine groups has also been
done using Traut’s reagent and N-succinimidyl iodoace-
tate, a hetero bifunctional cross linker which couples thiols
and amines (SIA) [40]. Often, a linker such as PEG is
introduced between the QD and the bio-molecule to in-
crease steric freedom, and to minimize unspecific bind-
ing of the QDs [45,46]. Figure 4 illustrates several con-
jugation strategies between QDs and organic molecules.
3. Single QD Applications in Biology
The enhanced optical properties of QDs, in particular the
significant brightness and photostability, make these ma-
terials highly suitable for use in biological applica- tions
requiring even single molecule sensitivity.
3.1. Single Particle Tracking (SPT)
The foremost single molecule application where QDs
have been used is single particle tracking (SPT) [48]. In
SPT, single molecules of interests (MOIs) are sparsely
labeled with a luminescent or scattering probe and the
movement of the MOI:probe complexes is imaged by
time-lapse microscopy at repetition rates ranging from a
few to 50,000 Hz depending on the process investigated
and the signal of the probe [48]. In this way, trajectories
describing the motion of single MOI:probe conjugates
can be constructed with nanometer precision and milli-
second time resolution to provide details of the molecular
dynamic that ensures cellular structure and function.
More detail on technical aspects and data analysis in SPT
can be found in recent reviews (e.g. [48]).
The most common MOIs in SPT for biological appli-
cations are amphiphilic molecules such as lipids, lipid
anchored proteins, and transmembrane proteins. In this
case, the investigated motion of the MOIs is most fre-
quently restricted to lateral diffusion in two dimensions
within the plane of e.g. the plasma membrane or in a
model membrane. In this particular application more
leeway in the choice of probe is given because the limit-
ing factor in the lateral motion of the MOI:probe com-
plex is the viscosity of the membrane within which the
hydrophobic part of the MOI is residing. This is because
the viscosity of the membrane is 100 times that of vis-
cosity in the surrounding aqueous solution. For this rea-
son it is generally accepted that even probes that are
much larger than a particular MOI will only have a minor
impact of the motion of the MOI. In contrast, in the case
of an aqueous soluble MOI the motion will be severely
affected by large probes. In fact even fluorescent proteins,
e.g. green fluorescent protein which has a molecular
weight of about 28 kDa and a hydrodynamic radii of
about 3.4 nm will have a dramatic effect on the motion of
most biological MOIs.
Initially SPT experiments were done using interfere-
ence contrast video microscopy measuring the scatter
from micrometer-sized latex beads or 40 - 100 nm gold
nanoparticles [49]. Subsequently the technique has been
extended to track single fluorescent dyes and proteins
(sometimes called single molecule fluorescent tracking,
SMFT) [50]. By studying the motion of single molecules,
different modes of motion can be distinguished, and of-
ten the motion turns out to be very heterogeneous in a
way that cannot be described by ensemble measurements
which involve averaging over a large pool of indistin-
guishable molecules [51]. However, neither the latex
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R = small molecule, peptide,
protein, antibody, enzyme,
oligonucleotide, aptamer,
lipid, surface, etc.
(g) Histidine-Nickel-Carboxyl
(h) Maleimide Activation
(i) Active Esters
(f) Electrostatic
(e) Hydrophobic
(j) Streptavidin
(a) Thiol Coordination
(a) Imidazole/Histidine
(d) Functionalized
PEG Coatings
(c) Amine or
Carboxyl Coatings
Figure 4. An illustration of some selected surface chemistries and conjugation strategies that are applied to QDs. The grey
periphery around the QD represents a general coating. This coating can be associated with the surface of the QD via (e) hy-
drophobic interactions, or ligand coordination. Examples of the latter include: (a) monodentate or bidentatethiols, (b) imida-
zole, polyimidazole (e.g. polyhistidine), or dithiocarbamate (not shown) groups. The exterior of the coating mediates aqueous
solubility by the display of (c) amine or carboxyl groups, or (d) functionalized PEG. Common strategies for bioconjugation
include: (a) thiol modifications or (b) polyhistidine or metallothionein (not shown) tags that penetrate the coating and interact
with the surface of the QD; (f) electrostatic association with the coating; (g) nickel mediated assembly of polyhistidine to car-
boxyl coatings; (h) maleimide activation and coupling; (i) active ester formation and coupling; (j) biotin-labeling and strepta-
vidin-QD conjugates (not to scale) Reprinted with permission from [47].
beads or gold particles nor the fluorescent dyes or pro-
teins are ideal probes for SPT [52]. The former suffer
from being bulky, and the latter have limiting optical
properties, and none of them are ideal for multiplexing
studies. QDs on the other hand, are a great compromise
between those two categories of probes. They have a
moderate size, an extreme brightness, and an excellent
resistance to photobleaching, and are ideal for multi-
plexing studies even with simple setups [53]. The ease,
by which they are bio-functionalized, further makes it
possible to direct their binding towards almost any mo-
lecular target of interest, and makes them a preferred
choice for SPT (or single quantum dot tracking, SQT)
studies [40].
In a typical SPT experiment, the motion of sparsely
labeled single molecules is monitored by recording a
time-lapse image series [54,55]. Subsequently, the indi-
vidual main intensity peaks of the diffraction limited
point spread functions from the well separated probes are
detected and fitted computationally to a 2D Gaussian
distribution in order to localize the centroid positions
with a sub-diffraction limited spatial resolution of 10 - 40
nm. These centroid positions are then linked between
successive frames using advances linking algorithms to
build up single molecule trajectories [56-58]. In the case
of tracking QDs these algorithms take QD blinking into
account and are able to track molecules even if the QD is
“off” for some frames. The resulting trajectories are
typically analyzed by calculating the mean squared dis-
placement (MSD), or alternatively by calculating the
probability distributions of the squared displacements
[54,55]. For this analysis, QDs has the advantage that the
trajectories are long enough to allow for analysis of the
single trajectories. This is in contrast to tracking with
fluorescent dyes or proteins where the limited photosta-
bility of these probes requires that all trajectories are
pooled to give an analysis averaged in space and time
hindering the observation of transient and rare events. By
the shape of a MSD-vs-time plot it is possible to classify
the mode of motion. A freely moving molecule undergo-
ing Brownian motion will be a straight line in an MSD-
vs-time plot, according to MSD = 4 D t, with D being the
diffusion coefficient. Normally, however, plasma mem-
brane molecules in live cells experience hindrance in
their motion. This results in a MSD curve with a steep
slope at small times and a more moderately increasing
slope or a flat slope at longer times, indicating a time
dependent diffusion. The reasons for the confinement are
many, including interactions with membrane domains,
cytoskeleton barriers, molecular crowding, membrane
topology, and specific interactions with other membrane
molecules [59-62].
Since the first paper on tracking single QDs appeared
10 years ago [63], many have followed, and contributed
to the present understanding of structural, dynamical, and
functional aspects of the plasma membrane. Studies have
shown heterogeneous motion of individually labeled
plasma membrane proteins and lipids, and that e.g. actin
[64], the extracellular matrix [65], lipid microdomains
[66,67], and cholesterol all affect the movement of these
molecules. In neurobiology, single QD studies have been
used extensively to study the motion of specific receptors
in synapses under various cellular conditions and stimuli
(for review see [68]). For instance, it was shown how
GABAA receptors distribute asymmetrically across the
axon growth cone in a microtubule and calcium depend-
ent manner in response to a GABA gradient [69], and
how AMPA receptor mobility is functional in recovery
of synaptic activity [70]. Details of non-neural signal
transduction pathways have also been revealed. Lidke
and co-workers tracked EGF-conjugated QDs targeting
the EGF receptor (erbB1) that is often found dysregu-
lated in many cancers. Upon QD-ligand binding homo/
hetero dimerization and endosomal uptake was followed,
and further, a previously unknown mechanism of retro-
grade transport of the QD-EGF-EGFR complex from the
filopodia to the cell body was found [71,72]. Tracking of
membrane species using orthogonal multicolor QD la-
beling strategies have been conducted tracking the same
membrane species [64,73-76]. Recently, Clausen et al.
has extended this to the orthogonal and simultaneous
tracking of three different species, a lipid, a lipid-an-
chored protein, and a transmembrane protein (In press
PLOS ONE). Most QD tracking experiments are re-
corded at video rate (25 or 30 Hz), however, using a
camera with fast read-out, the extreme brightness of QDs
allows for imaging at up to 1750 Hz [77].
3.2. Single Particle Tracking of Hybrid
Lipid-DNA Analogues Using Quantum Dots
Labelling specificity is a major concern when performing
SPT experiments. Preferably the conjugation system used
to couple QDs to the target biomolecule should exhibit
high specificity and strong avidity towards the target and
as high degree of monovalency as possible. Another con-
sideration when designing a conjugation system for SPT
could be to make it as interchangeable as possible so the
same system could be designed to bind different sized
QDs to different targets enabling easy labelling for multi-
color SPT experiments. As a result Vogel et al. have
synthesized lipid-DNA analogues based on a polyaza
crown ether depicted in Figure 5 [78]. The membrane
anchors are linked to the nitrogens of the polyaza crown
ether building block and can be interchanged to encom-
pass either acyl chains or sterols such as cholesterol,
which in turn can be used to probe different environ-
ments in the plasma membrane or an artificial membrane
system. The lipid-DNA analogue is then coupled to an
oligomer of 17 bases at the 3’ end, which in turn can bind
to a complimentary strand that has a 5’-biotinylated oli-
gomer. Finally, streptavidin coated QDs can be bound to
the biotin end of the complimentary strand with high
specificity and avidity forming the SPT complex. The
inherent advantage of this conjugation system is the
specificity of the DNA complexation, since several dif-
ferent lipid-DNA analogues with different membrane-
anchor moieties could be labeled individually by using
unique DNA sequences that bind only to certain colored
3.3. Single Particle Tracking Studies in
Supported Lipid Bilayers
SPT studies were conducted to investigate the application
of previously mentioned lipid-DNA analogues in sup-
ported lipid bilayers made from a binary lipid mixture of
the phospholipids 1,2-Dipalmitoyl-sn-Glycero-3-phospho-
choline (DPPC) and 1,2-Dioleoyl-sn-Glycero-3-phospho-
choline (DOPC) in the ratio 1:1 supported on glass cov-
erslips. The supported lipid bilayers were made as de-
scribed in [79], briefly 24 mm in diameter glass cover-
slips were cleaned in a basic piranha solution composed
of Milli-Q water, ammonia and hydrogen peroxide in the
ratio 6.2:1:1 respectively and heated to 100˚C for two
hours. The processed cover slips were rinsed 3 times in
Milli-Q water and dried in the oven prior to use. The
phospholipids DPPC and DOPC (Avanti Polar Lipids)
were dissolved in a mixture of 90% chloroform and 10%
acetonitrile at a total lipid concentration of 10 mM. In
order to visualize the membn the microscope 0.5 ranes i
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Lipid/DNA analogue (SNV090)
Biotinylated olygomer
Stre p t av idi n
3 - T T T T G T G G A A G A A G T T G G T G - 5
A C A C C T T C T T C A A C C A C - 3
Figure 5. Schematic representation of the Streptavidin-coated 605 QD coupled to the lipid-DNA analogue by a biotinylated
complementary oligomer (not drawn to scale).
mol% of the fluorescent lipid dye NBD-PC (Avanti Polar
Lipids) was added to the lipid mixture. The supported
lipid bilayers were prepared on the glass cover slips by
spincoating using a Chemat technology spincoater KW-
4A. Spincoating is performed by adding 50 μl of the lipid
solution is placed in the middle of the coverslip and was
done using the following settings:
Spincoater settings
Steps Duration Speed
1 3 sec. 500 rpm
2 40 sec. 3000 rpm
Afterwards the spincoated coverslips are stored in a
vacuum desiccator for at least 24 hours to ensure the
solvent has evaporated. Depending on the lipid concen-
tration and of the applied volume of stock solution it is
possible to form several bilayer on top of each other.
Hydration of the dry spincoated bilayers is accomplished
by placing the spin-coated coverslip into a special heat-
ing stage composed of an aluminum holder and is fixed
in place by a plastic ring. The hydration of the multi-
laminar bilayer system enables one to wash of the top
layers by gentle pipetting of the revealing the bottom
bilayers on the coverslip. Heating of the setup is essential
since the phase transition of the lipid mixture is above
RT and it also aides in removing the top bilayers due to
the increased mobility of the lipids in the bilayers. The
hydration of the DOPC:DPPC 1:1 supported lipid bilay-
ers was performed in Dulbecco’s phosphate buffered
saline (PBS) (Sigma Aldrich) and the sample was heated
to 60˚C well above the phase transition of the mixture
and left to incubate for 30 minutes to ensure the mem-
brane is its fluid state. Gentle pipetting was used to re-
move excess lipid and to reveal the bottom bilayers on
the coverslip. Below the phase transition of the lipid
mixture DOPC should phase segregate to form ridged
flower-shaped domains in which the fluorescent dye
NBD-PC will not be present. Our microscopy studies
have shown that these flower-shaped domains do not
occur in the bottom lipid bilayer, possibly due to interact-
tions between the glass and the lipids as well as due to
the roughness of the glass surface. Therefore SPT studies
were only performed in bilayers that exhibited the
flower-like domains upon cooling below 40˚C - 36˚C.
Once a planar patch of membrane has been established
and characterized by fluorescent microscopy 100 μl of a
100 pM lipid-DNA analogue was added locally and in-
cubated for 30 minutes. The PBS was exchanged in order
to remove any non-incorporated membrane anchors. Af-
terwards 100 μl of a 100 pM biotin coupled complimen-
tary strand DNA solution was added and left to incubate
for 15 minutes. The PBS was exchanged in order to re-
move any unbound excess complimentary DNA, fol-
lowed by the addition of 10 μl of a 1 nM streptavidin
coated QD605 solution (Invitrogen), which was left to
incubate for 10 min. The unbound QDs were removed by
a final washing step.
SPT studies were done by first bleaching away the
signal from the NBD-PC in order to visualize the QDs
bound to the membrane surface, see Figure 6. Thereafter,
varying lengths (300 & 900 frames) were recorded and
from these the individual trajectories of the QDs were
extracted using ImageJ and a plugin called Spot tracker.
The mean squared displacements of these trajectories
were calculated using Wolfram Mathematica.
From the preliminary results obtained from the SPT
Figure 6. Epi-fluorescence images showing the membrane
(shown in green) labelled with 1 mol% NBD-PC and su-
perimposed tracks of QD605 coupled to the lipid-DNA ana-
logue imbedded in the membrane (shown in red) (scalebar
is 8 μm).The diffusion coefficient outside domains was 1.84
± 1.08 μm2/sec whereas the diffusion coefficient of mole-
cules in contact with domains was 1.22 ± 0.00 μm2/sec. The
diffusion coefficient inside domains was 0.42 ± 0.20 μm2/sec.
experiments three distinct diffusion patterns have been
observed: unhindered diffusion outside domains, hin-
dered diffusion at the border of the liquid and gel phase
domains and finally unhindered diffusion inside the gel
domains. It was unexpected to observe diffusion in the
supposedly immobile DPPC gel phase present in the
flower shaped domains—most likely this is due to the
phase not being completely equilibrated. As to be ex-
pected, the calculated 2D diffusion coefficients decrease
in relation to the amount of contact the SPT complex has
to the immobile phase. The most predominant diffusion
events were observed to be the unhindered diffusion in
the liquid phase. There were several observations of dif-
fusion along the edge of the flower domains. Finally,
some occasional events showed the SPT complex leaving
the rim of a domain at continuing the diffusion into the
fluid phase, whereas diffusion from inside a domain into
the fluid phase was not observed.
For biological membranes it has been reported that a
typical lateral diffusion values range between 1 - 4 μm2
sec1 and relating the values from this preliminary SPT
study the values seem comparable. As a control Fluores-
cence Recovery after Photobleaching (FRAP) was per-
formed on the NBD-PC labeled fluid phase of the sup-
ported bilayers. The measurements were performed on
both membrane systems with a confocal laser-scanning
microscope (CLSM) of the type (Zeiss LSM 510). Usu-
ally when doing FRAP only a spot is bleached in order to
determine the diffusion. However based on the work of
Braeckmans et al. line FRAP is now possible [80]. A
region of interest (ROI) was chosen to be 100*1 pixels,
which would be used for all measurements. Compared to
ordinary FRAP this improved technique is faster due to
the scanning motion and is able to determine the diffusion
constant and mobile fraction in more localized areas [80].
 
00 0
ec ne
Ft rnra nr
,,0 ,,FytFykFyt Fy 0 (4)
From the FRAP measurements the lateral diffusion
coefficient of the fluid phase was determined to be 2.9 ±
1.8 μm2·sec1, which is significantly higher than the 1.8
± 1.1 μm2·sec1 calculated from SPT measurement. This
is not surprising since the acquisition rate was slower in
the FRAP studies. When also keeping in mind that only
the mobile fraction is included in the FRAP studies, no
direct comparison between these numbers. Furthermore,
it cannot be excluded that the QDs studied by SPT are
bound to the membrane by more than only one lipid
DNA analogue. The quantum dots used for these ex-
periments from Invitrogen have approximately 37 strep-
tavidin binding sites on one quantum dot. Therefore
cross-linking of one quantum dot to several membrane
anchors can be a possibility. Although only preliminary
SPT data has been collected, the application of the
lipid-DNA analogue seems to be promising. Additional
data needs to be collected in order to evaluate the diffu-
sion coefficient values reported so far as well as if the
lipid-DNA analogue affects the physiochemical proper-
ties of the supported bilayer. One of the most interesting
applications of the anchor molecules is multi-color im-
aging, which can be performed with different sizes (and
hence colors) of quantum dots. If the anchors could be
designed to have a particular preference for a particular
lipid environment such as lipid domains these could then
be specifically labeled. If such a labeling probe could be
developed in vitro it would be possible to label domains
in cells and thus one would be able study the dynamics of
lipid rafts below the diffraction limit.
4. Quantum Dots as Probes for Two-Photon
Fluorescence Correlation Spectroscopy
Fluorescence correlation spectroscopy [FCS] is a very
useful technique to study the movement and interactions
of fluorescently labelled biomolecules and fluorophores.
Elson, Magde et al. [81] introduced FCS in 1972 as an
analogous technique to Dynamic Light Scattering and
Relaxation Kinetics, in an attempt to overcome inherent
limitations in both techniques [82]. In comparison with
optical scattering, fluorescence is much more sensitive
and selective enabling measurements with a low back-
ground intensity and detection sensitivity that spans from
nanomolar concentrations to the single molecule regime.
FCS can be performed on either a confocal microscope
or a two-photon fluorescence microscope [83], neverthe-
less in both experimental setups a laser is focused down
to a small focal volume in the order of a few femtoliters.
Open Access JMP
Open Access JMP
In the confocal setup this is achieved by the confocal
pinhole whereas the focal volume is inherent with a two-
photon setup since only a small volume has high enough
photon densities for two-photon excitation process to
occur [84]. In Figure 7 is a schematic representation of a
two-photon fluorescence correlation spectroscopy mi-
croscope setup.
The amplitude of the autocorrelation function is de-
and is inversely proportional to the aver-
age number of fluorescent species present in the focal
volume, and therefore can be used to determine the con-
centration of the fluorescent species. Consequently, the
amplitude will increase with low concentrations and de-
crease with high concentrations of the fluorescent species
respectively. The focal volume for a two-photon setup is defined as
(5) The decay rate of the autocorrelation function yields
information about the occupancy of the fluorescent spe-
cies in the focal volume. The function shows the prob-
ability of the fluorescent species being in the focal vol-
ume at 0t
and still remaining in the focal volume at a
later time point t
. Since large molecules diffuse
slower than smaller molecules the probability of finding
the same molecule in the focal volume at higher values
of t
will also increase—in turn autocorrelation
curves of large and small molecules will be shifted to the
right and left respectively.
where r0 is the beam waist in the radial direction and z0 is
the beam waist in the axial direction. In order to deter-
mine the size of the focal volume and the values of r0 and
z0 the setup is calibrated by measuring the diffusion of a
well-known fluorescent size standard such as a fluoro-
phore of a fluorescent polymeric bead. When fluorescent
or fluorescently labelled species diffuse randomly through
the focal volume due to Brownian motion the resulting
fluctuation fluorescence signal is recorded as a function
of time. These fluctuations can be analyzed by the use of
autocorrelation function
from which a diffusion
value can be attained.
The diffusion coefficient can be determined by relating
the diffusion time τD, which is the τ value corresponding
to the half value of the autocorrelation amplitude, to the
following relation [86].
  
GIt It
(6) 2
 
tIt It
 is the deviation from the
mean fluorescence intensity,
t. The auto-correla-
tion function for translational diffusion using two-photon
excitation can be calculated as [85].
Furthermore, assuming that the diffusion molecules
are spherical, the Stokes Einstein equation can be used to
determine the hydrodynamic radius, R, from the deter-
mined diffusion coefficient, D
 
GG rz
 
Figure 7. A schematic representation of a two-photon fluorescence correlation setup (a). Fluctuation measurements and the
esulting autocorrelation analysis (b). r
where kB is the Boltzmann constant, T is the absolute
temperature (kelvin), η is the viscosity of the medium
quantum dots from Invitrogen. The FCS
and R is the hydrodynamic radius of the diffusion mole-
cule [87].
In Figure 8 we present auto correlation curves and fits
of 605sAv
easurements were performed in 50 mM sodium borate
pH 8.2 with 1% (w:v) BSA at RT (293 K). The experi-
ments were performed on a custom built system which is
described in [88] and the data was fitted using the
Globalssoftware package developed at the Laboratory for
Fluorescence Dynamics at the University of California
Irvine. The excitation wavelength was 820 nm. It can be
seen from the figure that fitting the data to the simple
diffusion model (equation 8) does not fit the data satis-
factorily. Adding a term to equation 8 which takes the
power law nature [89] of the blinking of the QDs into
account is seen to greatly improve the fit to the data. The
auto-correlation function for translational diffusion and
blinking two-photon excitation takes the form
 
where B is the blinking amplitude and is the exponent.
The measured diffusion coefficient, of the QDs, found
from a global analysis of the data using the model in-
cluding the power law term was 19.7 ± 2 m2/sec. Using
Stokes Einstein’s equation, as in [76] we find the hydro-
dynamic radius to be 10.5 ± 1 nm.
1e-4 1e-3 1e-2 1e-1
Time [sec.]
Data 0.5 nM
Data 1 nM
0.5 nM
Fit 1 nM
Fit 0.5 nM
Figure 8. Auto correlation curves from FCS measurements
carried out on 605 sAv QDs from Invitrogen. The solid line
5. Optical Trapping of Quantum Dots
and mi-
are for 0.5 nM concentrations and the dotted lines are for 1
nM concentrations of QDs. The green lines represent the
data, the red lines are the best fit of a model for a single
freely diffusing species. The black lines are the best fit of a
model for a single freely diffusing molecule together with a
power law term to fit the blinking. It is clear from the figure
that a simple diffusion model does not completely describe
the data. Adding a term which takes the blinking of the
QDs into account, greatly improves the fit.
Optical tweezers are formed by tightly focusing a laser
beam. Most often, optical tweezers are implemented in a
microscope, equipped also with a quadrant photo-diode
or a CCD for position detection with nanometer
cro-second resolution. Using an optical trap, particles in
the nanometer to micrometer range can be manipulated
in 3D and corresponding values of forces (typically in the
pico-Newton range) and distances can be measured. An
inducible dipole in an optical field experiences a force in
the direction of the field gradient. This 3D restoring force
is harmonic in all three dimension and directed towards
the focus of the laser beam: F = κx, where κx is denoted
the trap stiffness in the x-direction and x is the position
of the particle with respect to the equilibrium position
within the trap, the center of the potential sketched in
Figure 9(a). From tracking the Brownian motion and
knowledge of the size of the trapped object and the vis-
cosity and temperature of the surrounding fluid, one can
deduce the force, F, acting on the trapped particle.
optical tweezers
Laser power (mW)
Intensity (S/N)
Figure 9. (a) Schematic of an optically trapped QD. The
particle performs thermal motions in the harmonic poten-
tial while the QD is simultaneously two-photon excited and
emits polarized red light. b) Emitted intensity (signal/noise)
versus laser power for QD agates of varying initial size. ggre
The dotted line has a slope of 2. The different aggregates
have different initial intensities; probably because of their
different sizes. Regardless of initial aggregate size, all traces
scales with a slope of 2; the characteristic of two-photon
absorption. Reprinted with permission from. Reprinted
with permission from [90]. Copyright (2012) American Che-
mical Society.
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QDs, with inducible dipole moments, can be optically
trapped by an infrared CW laser beam [91]. They serve
as excellent combined force handles and visualization
markers for investigations of individual cells, proteins,
and biological filaments. In general, a fluorescent mole-
cule can be two-photon excited if it simultaneously ab-
sorbs two photons, and if the energy difference between
the ground and excited state of the molecule corresponds
to the added value of the energies of the two photons.
QDs can be excited by a two-photon absorption process
[92,93], with a two-photon absorption cross section
which is large in comparison to other fluorophores used
in multiphoton microscopy [94-97]. Jauffred and Odder-
shede showed that QDs are readily excited through two-
photon excitation by a relatively weak CW infrared laser
beam, which can also be used to trap the QDs [98]. For a
two-photon absorption the emitted intensity scales as
P2, where P is the laser power, as shown in Figure 9(b).
One hallmark of optical trapping is that there exists a
linear relationship between κ and P. As shown in Figure
10(a), this is also true for optical trapping of two-photon
excited QDs, thus supporting that the nonlinear absor-
tion is only a weak perturbation that does not alter trap-
ping properties considerably. It is important to correctly
choose the QD such that it matches the given experi-
mental goals and conditions, e.g., available excitation
0.3 0.4 0.5 0.6 0.7 0.8
1.2 x 10-4
10 15 20 25 30
x 10-4
d (nm)
Figure 10. (a) Trapping strength versus laser power for
individual QDs. The error bars denote one SEM. The dot-
ted line is a linear fit to the data and it has a slope of 1.5 ×
203 pN/nm/W. Reprinted with permission from [90]. Copy
right (2012) American Chemical Society. (b) Trapping
strength dependence on diameters, d. All error bars denote
erred to the trapped parti-
n limit, will align with the laser’s po-
rod would rotate in an oscillatory man-
one SEM and the dotted line is the mean value of κ: κ = 1.6
× 104 pN/nm. Reprinted with permission from [99].
lasers and filters. The trapping strength for QDs with
different emission wavelengths λ (from 525 nm to 800
nm) and different physical sizes have similar trapping
capabilities, as seen in Figure 10(b). The typical trap-
ping strength measured for QDs is comparable to that
obtained from optical trapping of silver and gold nano-
particles of similar sizes [100-104]. See Reference [105]
for a review on optical tweezing and nanoparticles. In
summary, the QD can serve both as a handle for ma-
nipulation and controlled force transduction and at the
same time for visualization through two-photon absorp-
tion of the trapping laser light.
Optical trapping relies on light scattering, a linear
property. Within the recent years efforts were done also
to take advantage of the angular momentum of the laser
beam [106,107].
Angular momentum is transf
e by changing the direction of the laser polarization
vector. Most often optical tweezers are based on linearly
polarized light and an elongated trapped object, smaller
than the diffractio
rization vector, as shown in Figure 11(a). Objects lar-
ger than the diffraction limit, as for instance bacteria, will
align along the propagation direction of the trapping laser.
By changing the direction of the laser’s polarization vec-
tor, e.g., by using circularly polarized light, a trapped
elongated nano-particle can be forced to rotate in a con-
trolled fashion.
This was successfully done by Head et al. [108], who
used a focused laser bam to trap and two-photon excite
elongated quantum rods (with an aspect ratio of 10).
They used circularly polarized light and hence, the
trapped quantum
r. The quantum rod emitted light polarized along its
long axis and by analyzing emitted light using a polariza-
tion filter (as sketched in Figure 11(b)), they were able to
optical tweezers
180 0
240 270 300
(a) (b)
Figure 11.Schematic of an optically trapped quantum
rod that aligns along the laser polarization but still perform
significant 3D Brownian; (b) Schematic of the polarized
emission from an optically trapped semiconductor quantum
rod. The rod is
polarized ot shows
light emitted from the trapped quantum
along its long axis. The polar intensity pl
the light ideally measured by two orthogonally polarized
photomultipliers (x-PMT and y-PMT) as a function of the
polarization angle measured from the x-axis, this is indeed
consistent with experimental measurements [108].
Open Access JMP
detect that the trapped quantum rod was indeed rotating
with the same frequency as the applied polarization vec-
tor was changing with. The trapped particle could follow
field rotations of, at least, 320 Hz [108]. These results
pave the way for a system where the trapping and visu-
alization is further combined with the ability to measure
and transduce torque.
Altogether, with a single infrared tightly focused laser
beam one can optically trap, rotate, and perform two-
photon excitation of individual colloidal QDs. Hence,
individual quantum dots serve both as force and torque
tranducers as well as visualization markers.
article tracking, where their low
ake them favorable in comparison t
and for designed nano-scopic molecular electronics.
6. Conclusion
In this review we demonstrated the versatility of using
individual quantum dots as markers for individual mole-
cules and as force probes. QDs can be advously
applied for single p
bleaching rates mo
conventional fluorophores. Also, they give clear results
in FCS experiments, and can be individually optically
trapped, thus serving as force and torque transducing
handles. The great spectral properties of QDs include
high photostability, narrow emission spectra, blinking
and low bleaching rate. The photo-physical properties of
QDs make them ideal for multicolour experimental set-
ups, since several differently coloured QDs can be dis-
tinguished at the same time, while being illuminated by a
single light source. When performing single particle
tracking experiments, the blinking of QDs is a disadvan-
tage as certain visited positions remain undetected. How-
ever, blinking is also an advantage in localization mi-
croscopy, in particular in the novel super-resolution
techniques, as a subset of the QDs will emit in each im-
age frame. For super-resolution localization microscopy
QDs have a high potential in experiments involving posi-
tion localization microscopy of lipids and proteins in the
plasma membrane in live mammalian cells [7,109]. In
live-cell experiments as well as in vivo experiments in
animals, QDs have a strong potential as fluorescent probe
as they can be easily inserted into the cytoplasm and then
individually detected, conjugated and optically manipu-
lated [37,110-118]. In FCS experiments, the great advan-
tages of QDs are their well defined size and high fluores-
cence emission, which make QDs a trust-worthy refer-
ence or a good label of membrane proteins or lipids. The
possibility to excite QDs with two-photon excitation also
makes QDs applicable in skin permeability or drug up-
take receptor studies in which deep tissue microscopy is
necessary. Finally, as QDs can be individually manipu-
lated and visualized using a single laser beam, they serve
as the optimal handle and marker for the expanding ef-
forts in uncovering the action of individual molecules
7. Acknowledgements
We thank The Danish Council for Independent Research
for grant to EAC and funding from The Danish Research
Infrastructure to the Danish Molecular Biomedical Im-
aging Center and Dr. Mathias Clausen for reading the
manuscript before submission.
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