J Fluoresc
DOI 10.1007/s10895-016-1814-3
Two-Photon Fluorescence Tracking of Colloidal Clusters
Debjit Roy 1 & Dipankar Mondal 1 & Debabrata Goswami 1
Received: 13 December 2015 / Accepted: 26 April 2016
# Springer Science+Business Media New York 2016
Abstract In situ dynamics of colloidal cluster formation from
nanoparticles is yet to be addressed. Using two-photon fluo-
rescence (TPF) that has been amply used for single particle
tracking, we demonstrate in situ measurement of effective
three-dimensional optical trap stiffness of nanoparticles and
their aggregates without using any position sensitive detector.
Optical trap stiffness is an essential measure of the strength of
an optical trap. TPF is a zero-background detection scheme
and has excellent signal-to-noise-ratio, which can be easily
extended to study the formation of colloidal cluster of
nanospheres in the optical trapping regime. TPF tracking can
successfully distinguish colloidal cluster from its monomer.
Keywords Optical tweezers . Fluorescence . Trapping
Introduction
Single beam gradient optical trap is commonly known as op-
tical tweezer [1, 2]. It uses the radiation pressure of a tightly
focused laser beam to generate force of ~pN [2, 3] ranges that
can spatially trap the colloidal particles [4–6]. Over the years,
it has gained a huge popularity in the fields of physics, chem-
istry, biology, nanotechnology etc. [2–9] due to the wide range
of forces generated by it with nanometer spatial resolution [10,
11] for the non-invasive single molecule manipulation tech-
niques in solution. Without going into the details of the force
field calculation, it can be stated that if a TEM00 profile laser
* Debabrata Goswami
dgoswami@iitk.ac.in
1
Department of Chemistry, Indian Institute of Technology Kanpur,
Uttar Pradesh 208016, India
beam is used for the tweezing action, a three dimensional
harmonic potential well is generated and the trapped objects
follows Hookean law for the small displacements from the
trap center [12]. The strength of an optical tweezer is usually
expressed by the term trap stiffness which is eventually the
force constant of the Hooke’s law [3]. The most common way
of measuring the trap stiffness is by the back focal plane
scattered light detection [13–15]. This basic principle of this
detection scheme is that the scattered light from the trapped
object interferes with the trapping laser beam to give rise to a
spatial interference pattern [16]. With change in the position of
the trapped object, the interference pattern gets changed. By
recording this interference pattern by position sensitive detec-
tors like quadrant photo diodes [13], high speed CCD cameras
[17] etc., the trap stiffness is measured. In general, a continu-
ous wave (CW) laser is used for the trapping events and some-
times a second very low power detection beam is used to track
the trapped particle’s position. Over the last few decades, the
usage of the femtosecond laser pulses has also been used for
the optical trapping experiments [18–20] as it gives both spa-
tial and temporal control. Another advantage of using a fem-
tosecond laser is that in addition to the scattering, it can gen-
erate multi-photon processes [21] like two-photon fluores-
cence (TPF) [20, 22] that can be used for background free
imaging of the trapping process. TPF detection has better sig-
nal to noise ratio than conventional scattered light detection
schemes as it has zero background.
Since the effect of thousands of pulses are averaged during
measurement, the trapping efficiency of a high repetition rate
(HRR) femtosecond laser is same as that of a CW laser twee-
zer [19, 23]. On the same token, the time averaged trapping
force generated using a HRR femtosecond laser and that of a
CW laser are the same. Here we are going to present the appli-
cation of TPF generated from the fluorophore coated trapped
nanospheres to measure the trap stiffness of a femtosecond
 J Fluoresc

optical tweezer. To the best of our knowledge, this is the first
attempt to measure trap stiffness by using nonlinear processes,
such as, TPF. Additionally, TPF tracking method distinguishes
the trapped nanosphere monomer from its dimer.
Theory
The optical force acting on a trapped colloidal particle can be
written as [3]: Fopt = − κq, where κ is called the trap stiffness
and x is the distance of the trapped particle from trap center.
The Langevin equation for an optically trapped spherical col-
loidal particle with radius r can be written as [24]:
⋅⋅ ⋅
m q ðt Þ þ γ q ðt Þ þ κqðt Þ ¼ ζ ðt Þ ð1Þ
where, m is the mass of the trapped particle; γ is the hydrody-
namics drag coefficient of the spherical particle in a fluid
having dynamic viscosity η and is given by: γ = 6πrη, and
ζ(t) is the random Brownian force acting on the trapped parti-
cle by the fluid. For a fluid with a low Reynolds number, the
first term of the Eq. (1) can be neglected and it transforms to
[12]:
⋅ (1–2αq2). Thus, we can have,
γ q ðt Þ þ κqðt Þ ¼ ζ ðt Þ ð2Þ
The Fourier analysis of Eq. (2) gives rise to the power
spectrum of the trapped object. The time averaged power
spectrum is given by:


1 D
Pq ¼ 2 ð3Þ
2π ω20 þ ω2
where, D = kBT/γ, is called Einstein-Stokes diffusion coeffi-
cient with Boltzmann constant kB and T is the absolute tem-
perature of the sample, ω is the frequency and ω0 = κ/2πγ, is
the corner frequency (roll off frequency) where the value of
Pq becomes 3 dB of its initial value. In a dense colloidal
suspension, multiple particle trapping can also take place
[25, 26]. For the trapping of two identical colloidal particles,
the time averaged power spectrum becomes:


1 D
Pq;2 Particle ¼ ð4Þ
π2 ω20 þ ω2
If the trapped colloidal particles are fluorescent, then
the fluorescence signal coming from the trapped ob-
jects can be used to track the motion of the trapped
particle. Additionally, if the trapped fluorescent colloi-
dal particle undergoes TPF at the trapping wavelength,
then the situation is similar to the two photon fluores-
cence single particle tracking [27–30] process where
the position sensitive detectors are not essentials as
the total TPF signals are themselves position sensitive.
The intensity profile of a TEM 00 profile Gaussian
shaped femtosecond laser pulsed at a given time can be
written as:
I ðqÞ ¼ I 0 e−αq
2
ð5Þ
where, I0 is the amplitude of the laser pulse and α is a constant.
The TPF intensity of the trapped particle at position q from the
trap center at time t is given by:
F ðq; t Þ∝I 2 ðq; t Þ ¼ a:e−2αq ðtÞ
2
ð6Þ
where, a is the proportionality constant. For the small
displacement (q ≪ 1) value of F(q,t) is proportional to
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 F ðq; t Þ−a 
 
 2αa  ¼ qðt Þ ð7Þ
Thus the term in the left hand side of Eq. 7 would
directly give position of the trapped nanosphere from
the trap center while its Fourier analysis would directly
produce power spectra of the trapped particles according
to Eqs. 5 and 6. Using this procedure, the trap stiffness of
singly and doubly trapped fluorophore coated polystyrene
nanospheres were measured.

Thus the time averaged power spectra for both the one and
two trapped colloidal particles are Lorentzian with same cor-
ner frequency. In fact, from Eqs. 3 to 4, it can be seen that the
time averaged power spectra of doubly trapped nanosphere is
just the double of the power spectra of the singly trapped
nanosphere. From the interference pattern, using position sen-
sitive detectors, the value of q(t) is obtained which eventually
gives rise to the power spectra. Fig. 1 Schematic of the experimental set–up
J Fluoresc

CW trap (UPlanSApo, 100XO, 1.4 NA, Olympus Inc., Japan) was used
0.12 ML trap
Linear fit of CW for trapping so that the gradient force in the laser propagation
Critical Velocity (mm/s)

Linear fit of ML
0.10
0.08
0.06
0.04
0.02
5 10 15 20
Power (mW)
Fig. 2 Plot of critical velocity, vc, versus power for the 250 nm radius
polystyrene nanospheres under both CW and mode–locked laser
Experimental Techniques
The experimental set-up is based on a home-built inverted
microscope reported earlier [20, 22, 31]. A schematic of the
setup is shown in Fig. 1. ATi:sapphire oscillator that can work
both in CW and Mode-locked condition (MIRA 900-F
pumped by Verdi5, Coherent Inc., Santa Clara, California)
was used for trapping. The pulse width of the laser was
~180 fs with 76 MHz repetition rate and centered at 780 nm
wavelength. The TEM00 mode laser beam was first expanded
and then collimated to slightly overfill the back aperture of the
objective to obtain nearly the diffraction limited focal spot
size. A high numerical aperture microscopic objective
axis overcomes the scattering force. The dichroic mirror be-
fore the objective reflects the 780 nm light and transmits the
TPF and the bright field image. The real time video of the
trapping was captured using a CCD camera (350 K pixel, e-
Marks Inc.). The TPF signal coming from trapped
fluorophore-coated nanospheres was collected using a photo
multiplier tube (PMT) (1P28, Hamamatsu, Japan) connected
to a digital oscilloscope (waverunner 6100 A, LeCroy Corp.,
Chestnut Ridge, New York). The power was measured at the
25 30 35 40
sample stage using power meter (PM30 sensor connected to
FieldMate laser power meter, Coherent Inc.). The sample
stage was attached to a linear motorized stage (UE1724SR
driven by ESP300, Newport) connected to a tunable velocity
controller (ESP300, Newport Inc.) for force measurement
using the viscous drag method. To measure the trap stiffness
along x and y axes, another 532 nm auxiliary laser (DJ532–40,
Thorlabs Inc.) was used. The forward scattered light, collected
by condenser lens (UPlanApo 60X, Olympus Inc.) was ana-
lyzed by the quadrant photodiode (2901, Newport Inc.) con-
nected to the digital oscilloscope. The sample used for trap-
ping was fluorophore coated 0.50 (±0.016) μm diameter poly-
styrene nanospheres (F-8887, Molecular probes). The λmax for
single photon absorption and emission of the fluorophore is at
580 nm and 605 nm respectively. The fluorophore molecules
have two-photon absorption at 780 nm. The original solution
was 100 times diluted by Phosphate Buffered Saline solution
to prepare the final solution to be used for tweezing.

Fig. 3 Plot of TPF signal with Data @ 15 mW 0.026 Data @ 18 mW

0.019
Step Fit Step Fit
time for trapping of 250 nm radius 0.017
0.012
dye coated polystyrene
nanosphere suspended in water at 0.006 0.009
various trapping laser power 0.000 0.000

0 2 4 6 8 0 5 10 15 20
0.072 Data @ 21 mW 0.075 Data @ 24 mW
Step Fit Step Fit
0.048 0.050

0.024 0.025
TPF Signal (Volts)

0.000 0.000
0 2 4 6 8 0 2 4 6 8 10 12
0.126 Data @ 27 mW 0.126 Data @ 30 mW
0.084 Step Fit 0.084
Step Fit
0.042 0.042

0.000 0.000
-1 0 1 2 3 4 5 6 7 8 0 2 4 6 8 10
0.159 Data @ 33 mW 0.189 Data @ 36 mW
Step Fit Step Fit
0.106 0.126

0.053 0.063

0.000 0.000
0 1 2 3 4 5 0.0 0.5 1.0 1.5 2.0 2.5

Time (sec)
 J Fluoresc

-0.8 2.8
Single Microsphere Trap Trap Stiffness
Linear Fit of Trap Stiffness
Double Microsphere Trap 2.6
-1.0 Linear Fit of Single Micrpsphere Trap
Linear Fit of Double Micrpsphere Trap 2.4

Trap Stiffness (pN/µm)

2.2
log (TPF Intensity)

-1.2
2.0

-1.4 1.8

1.6
-1.6
1.4

-1.8
-2.0
1.20 1.25 1.30 1.35 1.40 1.45 1.50
log (Power)
Fig. 4 Log-log plot for the voltage rise for the single and double
nanosphere trapping (after background subtraction) vs. power. Linear
fits with slope = 2 confirm TPF signal
Polystyrene micro beads with diameter greater than 700 nm
were not used for the trap stiffness measurements to avoid
decay in TPF due to FRET [31]. All data were collected at
20 kHz sampling rate and plotting and fitting were done using
Origin 8.5 (Origin Inc.) software. The entire experiment was
performed at 298 (±1) K.
Results and Discussions
To check if the CW and pulsed lasers works similarly for the
trapping of 250 nm radius polystyrene nanospheres, the total
average force acting on the trapped nanospheres were mea-
sured using the viscous drag method. In viscous drag method,
the sample stage is moved with a fixed velocity to measure the
critical velocity, vc, beyond which the trapped particle gets
untrapped as the Brownian forces acting on the trapped parti-
cle becomes larger than the optical force. Thus at velocity vc,
the optical force and the Brownian force becomes equal.
1.2
1.0
15 20 25 30 35
Power (mW)
1.55
Fig. 6 Trap stiffness (κ) versus power for singly trapped 250 nm radius
polystyrene nanosphere
Using the Stokes’ law for a continuous fluid with low
Reynolds number, the Fopt is given as: Fopt = 6πrηvc
The optical force acting on the trapped nanosphere can
also be written as [2, 32]:
ns P
F opt ¼ Q ð8Þ
c
where, ns is the refractive index of the fluid, P is the incident
trapping laser power, c is the velocity of light in vacuum and Q
is a dimensionless quantity which is called as the trapping
efficiency. Thus the critical velocity of the trapped object
would linearly vary with the incident laser power and the
slope of the vc versus P plot would give the trapping efficien-
cy. The plot of vc versus P for the 250 nm radius polystyrene
nanospheres under both CW and mode–locked laser is given
in Fig. 2 which tells that the trapping efficiency of the CW and
mode–locked lasers for trapping of 250 nm radius polystyrene
nanospheres are similar and thus the Langevin equation stated
earlier (Eq. 1) can be safely used in this situation.

100
1000

10

100
Power(V /Hz)

1
Power (V /Hz)

15 mW
2
2

18 mW 0.1
10
21 mW 15 mW
18 mW
24 mW 0.01 21 mW
27 mW 24 mW
1
30 mW 27 mW
1E-3 30 mW
33 mW 33mW
36 mW 36 mW
0.1 1E-4
10 100 1000 1 10 100 1000
Frequency (Hz) Frequency (Hz)
Fig. 5 Power spectrum for singly trapped 250 nm radius nanosphere Fig. 7 Power spectrum at different laser powers when there is no particle
with Lorentzian fit at various trapping power trapped (this is the background data)
J Fluoresc

Single Nanosphere
1000 Double Nanosphere
100
Linear fit of Single Nanosphere
Linear fit of Double Nanosphere
90

Corner Frequency (Hz)

100
Power (V /Hz)

80
21 mW
2

10
24 mW 70
27 mW
30 mW 60

1 33 mW
36 mW 50

10 100 1000 40
Frequency (Hz) 15 20 25 30 35

Fig. 8 Power spectrum for two trapped 250 nm radius nanospheres with Power (mW)
Lorentzian fit at various trapping powers Fig. 10 Plot of corner frequency versus laser power for single as well as
two simultaneously trapped 250 nm radius nanospheres

There is a sharp increase in the TPF signal whenever a
fluorophore coated nanosphere is trapped [22, 24]. When a
second identical nanosphere is trapped, the TPF signal (after
background subtraction) shows nearly a two-fold increase.
The plot for TPF versus time for 250 nm radius fluorophore
coated nanospheres, which are trapped singly and subsequent-
ly doubly, at different laser power are shown in Fig. 3. The
voltage rise for the single and double nanosphere trapping
(after background subtraction) versus power plot in the log–
log scale (Fig. 4) shows a slope of ~2, indicating that the
collected fluorescence signal occurs due to two–photon ab-
sorption (TPA). As the probability of TPA is very small (typ-
ically 10−6), it does not affect the trapping force and so we can
easily apply Eq. (1) for the trapped nanospheres. From Eq. (7),
the term [(|(F(q,t) − a)/2αa)|)1/2], is taken as the position, q(t),
in Eq. (1) to generate the power spectra for these optically
trapped 250 nm radius fluorophore coated polystyrene nano-
spheres. Power spectrum for singly trapped 250 nm radius
nanosphere with their Lorentzian fit at different powers is
given in Fig. 5. It can be seen that with increase in the trapping
laser power, the corner frequency is increased linearly. The
plot of κ versus trapping power (Fig. 6) shows a linear rela-
tionship confirming the practicality of using TPF as position
marker for the tweezed object [33, 34]. The power spectrum
when no particle is trapped (dark spectrum) at different trap-
ping laser powers is shown in Fig. 7. Figure 7 shows that all
values are ~102 to 104 times smaller than our calibration spec-
tra, indicating that they can only contribute very little to the
spectra. Also as the spikes are narrow with small amplitude
and are few in number, they are not statistically relevant dur-
ing the data fitting. Thus white noise has very little contribu-
tion to the power spectra (Figs. 7 and 8).
It should be noted that, measurement of trap stiffness using
conventional techniques like using quadrant photodiode or
CCD camera, the motion of the trapped object in the x-y plane
is measured. But, in reality, the trapped object explores the
whole trapping potential well landscape, which indeed is three
dimensional in nature. Moreover, the trapping potential is not
only elongated but also shallow along axial axis compared to
radial axis which, in turn, implies that the trapped object

4.0 Table 1 Value of Einstein-Stokes diffusion coefficient (D) obtained

from the fitted power spectra of the singly as well as doubly trapped
3.5 linear fit of 250 nm radius nanospheres at different powers
linear fit of
D (×106) (arb. unit) from D (×106) (arb. unit) from
Trap Stiffness

Power
3.0
(mW) single nanosphere trapping double nanosphere trapping
2.5
15 21.0 (± 0.7) –
2.0 18 21.6 (± 0.4) –
21 22.0 (± 1.5) 23.0 (±0.8)
1.5 24 20.9 (± 0.7) 22.0 (±1.5)
27 21.6 (±1. 4) 21. 8 (±1.6)
15 20 25 30
Power (mW) 30 19.2 (±1.4) 21.7 (±1. 9)
Fig. 9 Trap stiffness along x-axis (κx) and along y-axis (κy) plotted 33 22.3 (±3.1) 23.1 (±2.2)
against trapping laser power. It is important to note that κx and κy at 36 19.8 (±4.0) 21.8 (±3.5)
any power is greater than the effective three dimensional trap stiffness

fluctuates more along axial axis than along radial axis. These
facts lead us to conclude that TPF signal fluctuation has a
major contribution from the movement of the trapped object
along axial direction along with the radial position fluctuation.
This is further confirmed from the fact that trap stiffness along
x and y axes (Fig. 9) are greater than the measured three
dimensional trap stiffness, which consists of trap stiffness
from all three axes. Thus measuring trap stiffness using TPF
tracking method provides us effective three dimensional trap
stiffness that is actually felt by the trapped object. However,
since TPF signal fluctuates around its non-zero mean value,
exact position of the trapped object cannot be found by solely
using this technique. This, in fact, has restricted us from mea-
suring the individual contribution of trap stiffness along x, y
and z axis.
From Fig. 3, it can also be seen that at laser powers
≥21 mW, trapping of two 0.5 μm nanosphere occur in our
experiments. For such doubly trapped nanospheres at different
trapping laser powers, Fig. 8 shows the power spectrum with
their respective Lorentzian fits according to Eq. 3. Although
from Eqs. 3 to 4, it is expected that the corner frequencies for
singly and doubly trapped nanospheres should have been the
same for the same trapping laser power, we find that the corner
frequency for the doubly trapped nanosphere is always slight-
ly lower than that of the single nanosphere (Fig. 10). This can
be explained from the fact that in the presence of one particle,
the other particle’s time averaged position cannot be at the
minima of the potential well. Also the force field felt by each
of these trapped nanosphere are different from the force
felt by the singly trapped nanosphere. Thus the doubly
trapped nanosphere system effectively experiences dif-
ferent force field compared to that of the singly trapped
nanosphere. Alternatively, we can argue that the optical gra-
dient force is responsible for the formation of a dumbbell
shaped optically bound cluster for doubly trapped 0.5 μm
polystyrene nanospheres. This would have a different corner
frequency as compared to that of the single 0.5 μm polysty-
rene nanosphere and in our situation, the corner frequency of
this dumbbell shaped optically bound cluster is lower than the
single nanosphere (Fig. 10). Thus the corner frequency of the
trapped monomers and dimers of 0.5 μm diameter nano-
spheres determined from the TPF tracking method can easily
differentiate them. Table 1 shows that the value of diffusion
coefficient (D) for the trapped nanosphere from singly and
doubly trapped nanospheres are consistently higher.
Conclusions
We have demonstrated an effective three-dimensional optical
trap stiffness measurement technique by using single particle
tracking of two photon fluorescence signal from an optically
trapped colloidal fluorescent nanoparticles without using any
J Fluoresc
position sensitive detector. This technique allows probing the
early time formation of dimers of 250 nm radius nanospheres
and enabled its differentiation from its monomer.
Acknowledgments DG thanks the support from the Wellcome Trust
Senior Research Fellowship (UK) and Swarnajayanti Fellowship from
DST, India for funding. DR and DM thank CSIR and UGC, India respec-
tively for graduate fellowship. We thank Mrs. S. Goswami for language
check.
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