Measuring Boltzmanns constant through holographic video

microscopy of a single colloidal sphere

Bhaskar Jyoti Krishnatreya
Department of Physics and Center for Soft Matter Research, New York University, New York 10003
Arielle Colen-Landy
Packer Collegiate Institute, 170 Joralemon Street, Brooklyn, New York 11201
Paige Hasebe
Department of Physics, NYU Abu Dhabi, United Arab Emirates
Breanna A. Bell, Jasmine R. Jones, and Anderson Sunda-Meya
Department of Physics, Xavier University of Louisiana, New Orleans, Louisiana 70125
David G. Grier
Department of Physics and Center for Soft Matter Research, New York University, New York 10003
(Received 21 June 2013; accepted 15 October 2013)
The trajectory of a colloidal sphere diffusing in water records a history of the random forces exerted
on the sphere by thermally driven uctuations in the suspending uid. The trajectory therefore can be
used to characterize the spectrum of thermal uctuations and thus to obtain an estimate for
Boltzmanns constant. We demonstrate how to use holographic video microscopy to track a colloidal
spheres three-dimensional motions with nanometer precision while simultaneously measuring its
radius to within a few nanometers. The combination of tracking and characterization data reliably
yields Boltzmanns constant to within two percent and also provides the basis for many other
useful and interesting measurements in statistical physics, physical chemistry, and materials science.
VC
2014 American Association of Physics Teachers.
[http://dx.doi.org/10.1119/1.4827275]
I. INTRODUCTION
Early in his annus mirabilis in 1905, Albert Einstein pub-
lished an explanation for the constant random motion that
small objects undergo when suspended in uids.
1
He pro-
posed that these visible motions are caused by invisibly
small uid molecules colliding at random with the objects
surface. The statistical properties of the objects position and
velocity uctuations therefore should reect those of the
underlying thermal bath of molecules. Specically, Einstein
predicted that the particles mean-squared displacement
would be proportional to the thermal energy scale k
B
T, where
T is the uids absolute temperature and k
B
is Boltzmanns
constant. These predictions were borne out by Jean Perrins
direct microscopic observations,
2
rst published in 1912,
which yielded a value for k
B
that was consistent with values
that previously had been obtained from tests of the ideal gas
law and from measurements of black-body radiation. These
observations constituted the rst experimental demonstration
of the physical reality of atoms and molecules and earned
Perrin the Nobel Prize for Physics in 1926.
Perrin carried out his experiments entirely manually, meas-
uring the positions of micrometer-scale colloidal spheres
through the eyepiece of an optical microscope and recording
the results on graph paper. This process has since been auto-
mated with the introduction of digital video microscopy
3
and
the introduction of automated numerical methods
4
to identify
individual spheres in digitized video images and to link their
positions in consecutive video frames into time-resolved tra-
jectories for analysis. Quantitative video microscopy of colloi-
dal spheres has been used not only to reproduce Perrins
experiment
38
but also to measure colloidal particles interac-
tions with each other,
911
to probe the statistical physics of
particles moving through structured force elds,
1216
and as
the basis for particle-tracking microrheology,
17
which is used
to characterize the viscoelastic properties of complex uids
and biological materials.
Conventional video microscopy is most useful for meas-
uring colloidal particles in-plane positions and provides lim-
ited information about their motion along the microscopes
optical axis.
4,18
The axial range in conventional microscopy is
limited to a few micrometers by the microscopes depth of
focus, and axial tracking requires either calibration of each
particles changing appearance
4,18
or substantially modifying
the microscope itself.
19,20
The size and shape of individual
colloidal particles are difcult to measure from conventional
images because diffraction fringes obscure their outlines.
This article describes how holographic video microscopy
can be used to track micrometer-scale colloidal spheres with
nanometer precision in all three dimensions while simultane-
ously measuring each spheres radius and refractive index
with part-per-thousand precision. Implemented with low-
cost equipment, holographic video microscopy yields such a
wealth of high-resolution information that a ve-minute
video of a single diffusing sphere can yield a measurement
of Boltzmanns constant to a few parts per thousand. The
same information can also be used to weigh an individual
colloidal sphere, to measure its porosity, and to perform self-
consistent experimental tests of the uctuation-dissipation
theorem.
II. COLLOIDAL SAMPLES
The experiment consists of tracking a single micrometer-
scale colloidal sphere as it moves unhindered through quies-
cent water at a xed temperature. The particles used for this
study are chemically synthesized by emulsion polymerization,
which ensures that they are spherical to within a percent
23 Am. J. Phys. 82 (1), January 2014 http://aapt.org/ajp VC
2014 American Association of Physics Teachers 23
and have well characterized and consistent chemical and
optical properties.
21
Before the sample can be mounted in a
microscope, it must be sealed in a suitable container. Our
container consists of the small rectangular volume made by
gluing the edges of a glass microscope cover slip (Corning
Life Sciences, 22 mm 22 mm, #1.5) to the face of a stand-
ard glass microscope slide (Fisher Scientic, Fisherbrand
Economy Plain). Even this simple apparatus requires some
care in its preparation.
Small colloidal spheres tend to stick to surfaces irreversibly
because of van der Waals attraction.
21
This fate can be
avoided if the spheres and the walls of their container all carry
a high enough electrostatic charge to prevent deposition.
Many commercially available brands of colloidal spheres are
synthesized with large numbers of ionizable surface groups
specically for this purpose. The surface groups dissociate in
water, leaving behind bound charges. Our samples consist of
polystyrene sulfonate spheres (Duke Scientic, catalog
4016A, nominal diameter 1.587 60.018 lm) that develop
large negative surface charges. Clean glass surfaces similarly
develop negative surface charges in contact with water,
22
which will tend to repel the negatively charged particles.
As delivered, microscope slides and cover slips often are
coated with lubricants to prevent them from sticking to each
other. These lubricants tend to suppress the surface charge
and thus tend to allow colloidal particles to stick. We there-
fore thoroughly clean the glass surfaces before assembling
our samples.
First, we use a soap solution and a soft bristled brush to
clean the slide and cover slip, and then rinse them clean with
deionized water. To prevent water-borne residue from con-
taminating the surfaces, we dry them with a stream of nitro-
gen gas, taking care to blow excess liquid off the corners.
Then we place the slide and cover slip in an oven at 70

C
for 10 min until it is thoroughly dry. To minimize contamina-
tion, the glass pieces rest on their edges during drying. Once
the glass is dry, we rinse it rst with acetone to remove
remaining organic residue, then isopropanol, and nally
ethanol to facilitate drying. Once the pieces are dried under
streaming nitrogen, they are clean enough to assemble a
sample that will remain viable for a day or two.
Creating samples that are stable over longer periods
requires surfaces with higher and more uniform surface
charge. We achieve this by further cleaning the glass pieces
in a 100 W oxygen plasma etcher (SPI Plasma Prep II) for
30 min. This process removes any remaining organic residue
and oxygenates the silica surface so it develops a higher sur-
face charge when it comes into contact with water. The
entire cleaning process including plasma etching takes two
hours.
As shown in Fig. 1, the sample container is assembled by
placing the cover slip over the microscope slide with thin
glass spacers to set the separation. We use two slivers of
glass cut from a clean cover to set the spacing at roughly
120 lm. The spacers are aligned with the edge of the cover
slip and then glued in place with optical grade adhesive
(Norland Industries, UV-cured adhesive No. 68). The two
remaining edges of the cover slip are left open to form a
channel into which the colloidal dispersion is introduced.
The goal in preparing the colloidal sample is to have no
more than one or two particles in a typical eld of view.
Substantially fewer particles would make it difcult to nd a
particle to study, while substantially more would interfere
with particle tracking. Assuming a eld of view of 50 lm
50 lm120 lm and a single-particle volume of 2 lm
3
, we
prepare samples with a volume fraction of roughly 10
5
by
diluting the stock solution of spheres from the manufacturer
with deionized water. A little more than 80 ll of the diluted
suspension lls the glass channel. We then seal the ends of
the channel with more optical adhesive, taking care to avoid
bubbles, and cure the glue thoroughly under an ultraviolet
light. This last step requires an additional half hour.
III. HOLOGRAPHIC VIDEO MICROSCOPY
A. Hardware
A holographic microscope suitable for tracking and char-
acterizing micrometer-scale colloidal spheres can be created
from a conventional optical microscope by replacing the illu-
minator with a collimated laser source.
2325
Our custom-
made implementation is shown schematically in Fig. 1. The
collimated light from a ber-coupled laser passes through
the sample and is collected by an objective lens before being
relayed by a tube lens to a video camera.
Laser light scattered by the sample interferes with the rest
of the illumination in the microscopes focal plane. The cam-
era records the intensity of the interference pattern. Each
snapshot in the resulting video stream is a hologram of the
sample, which encodes comprehensive information on the
samples position, size, and optical properties. A sequence of
holographic snapshots constitutes a time-resolved record of
the samples behavior and characteristics.
An ideal laser illuminator provides a circular, linearly
polarized beam in a single Gaussian mode with no longitu-
dinal or transverse mode hopping. Many low-cost diode
lasers have these characteristics. The beam should be colli-
mated to within 1 mrad and should be aligned with the
microscopes optical axis to within 10 mrad.
26
The beams
diameter should be large enough to fully illuminate the eld
of view with roughly uniform intensity, and the beam
should be bright enough to use the cameras full dynamic
range without saturation. In our implementation, the 3-mm-
diameter beam from a 25-mW diode laser (Coherent Cube,
ber coupled, operating at a vacuum wavelength of
k = 447 nm) provides more than enough light. Stable oper-
ation is important for this application; some laser pointers,
Fig. 1. (Color online) Basic components of a holographic video microscope.
The microscope slide containing the sample is mounted on a three-axis posi-
tioner that is not shown for clarity.
24 Am. J. Phys., Vol. 82, No. 1, January 2014 Krishnatreya et al. 24
for example, suffer from an instability known as mode hop-
ping that renders them unsuitable.
The magnication and axial range of the holographic
microscope are determined by the combination of the objec-
tive lens, the tube lens, and the video camera. We use a
Nikon 100oil-immersion objective with a numerical aper-
ture of 1.4, which also provides a good basis for optical trap-
ping applications.
27
Any oil-immersion objective lens with
well-corrected geometric aberrations should perform well for
holographic microscopy. Long working distance is not nec-
essary because the lens gathers light scattered along the
entire length of the illuminating laser beam. Short depth of
focus is desirable because it reduces blurring. This consider-
ation favors lenses with higher numerical apertures.
When combined with a 200-mm tube lens, a 100objective
yields a magnication of 135nm/pixel on the half-inch detector
of a 640480-pixel video camera. We conrmed this gure
by measuring the spacing of a grid calibration standard. This
overall magnication factor is the only instrumental calibration
constant required for the measurement.
The magnication establishes the resolution with which
interference fringes can be measured. These fringes become
more closely spaced as an object approaches the focal plane.
The magnication thus establishes the lower limit of the ac-
cessible axial range. Our system can reliably measure the
position and properties of colloidal spheres that are more
than 5 lm above the focal plane.
The interference pattern spreads out and loses contrast as
the particle moves away from the focal plane. The limited
eld of view and the sensitivity of the camera together estab-
lish the upper limit of the axial range. We are able to track
and characterize micrometer-diameter colloidal spheres up
to 100 lm above the focal plane. This represents a ten-fold
improvement over conventional microscopy, whose axial
range is limited by the microscopes depth of focus.
The video camera that records the holograms ultimately deter-
mines the quality of the recorded data and the time resolution
with which colloidal particles can be tracked and characterized.
Remarkably, even a relatively low-cost surveillance camera suf-
ces for high-precision holographic tracking and characteriza-
tion, both because commodity video sensors have evolved to
high standards of performance, and also because the hologram
of a sphere can subtend a large number of pixels. Our instrument
uses an NEC TI-324A grayscale camera, whose analog video
output is recorded with a consumer-grade digital video recorder
(Panasonic EH50S) as an uncompressed digital video stream
with a nominal dynamic range for intensity of 8 bits per pixel.
B. Lorenz-Mie analysis
The intensity I(r) recorded at position r in the image plane
results from the interference of the illuminating wave with
light scattered by the particle. We describe the system in
Cartesian coordinates r =(x, y, z), with the origin located at
the center of the microscopes focal plane. The ^ x- and ^ y-
coordinates lie in the focal plane and ^z is directed along the
optical axis. We model the electric eld of the illumination
as a monochromatic plane wave polarized in the ^ x-direction
and propagating in the ^z-direction (downward), so that
E
0
(r; t) = u(r) e
ikz
e
ixt
^ x: (1)
The wavenumber k of the light is related to its frequency x
through the dispersion relation k = n
m
x=c, where c is the
speed of light in vacuum and n
m
is the refractive index of the
medium. Most lasers are characterized by the vacuum wave-
length of the light they emit, rather than the frequency; these
are related by k = 2pc=x. Ideally, the amplitude prole of
the beam u(r) would be a smoothly varying function such as
a Gaussian. But in practice, as we shall see, light scattered
by dust and other imperfections in the optical train can make
u(r) quite complicated.
A small particle centered at r
p
scatters a small amount of
the incident light from r
p
to position r in the imaging plane,
as given by
E
s
(r; t) = E
0
(r
p
; t) f
s
(k(r r
p
)): (2)
The scattering function f
s
(kr) describes how light of wave-
number k that is initially polarized in the ^ x direction is scat-
tered by a particle with specied properties located at the
origin of the coordinate system. In Eq. (2), r r
p
is the dis-
placement from the particles center to the point of
observation.
Calculating the scattering function is notoriously difcult,
even for the simplest cases. Scattering of a plane wave by an
isotropic homogeneous dielectric sphere is described by the
Lorenz-Mie theory
28,29
and is parameterized by the radius of
the sphere a
p
and its complex refractive index n
p
.
The intensity of the interference pattern due to a sphere of
radius a
p
and refractive index n
p
located at position r
p
rela-
tive to the center of the microscopes focal plane is
I(r) = jE
0
(r; t) E
s
(r; t)j
2
: (3)
A recording of this intensity distribution is a hologram of the
sphere and contains information about the spheres position
r
p
, its radius a
p
, and its refractive index n
p
. Because a
spheres scattering pattern is largely symmetric about the
axis passing through its center, such a hologram should
resemble concentric rings on a featureless background. In
practice, the expected ringlike pattern usually is obscured by
a rugged landscape of interference fringes due to imperfec-
tions in the illumination. These characteristics are apparent
in the measured hologram of a colloidal polystyrene sphere,
as shown in Fig. 2(a).
The inuence of these distracting intensity variations can
be minimized by factoring illumination artifacts out of the
observed interference pattern so that
I(r) = I
0
(r)

^ x
u(r
p
)
u(r)
e
ikz
p
f
s
(k(r r
p
))

2
: (4)
Here, I
0
(r) = ju(r)j
2
is the intensity distribution of the
illumination in the absence of the scatterer. Figure 2(b)
shows this background intensity distribution for the holo-
gram in Fig. 2(a). Dividing I(r) by I
0
(r) yields a normalized
hologram
24,25
b(r) =
I(r)
I
0
(r)
; (5)
that is comparatively free of illumination artifacts, as shown
in Fig. 2(c).
If we assume that the amplitude and phase of the illumina-
tion vary little over the size of the recorded scattering pat-
tern, we can approximate u(r
p
)/u(r) by a complex-valued
parameter a to obtain
25,30,31
25 Am. J. Phys., Vol. 82, No. 1, January 2014 Krishnatreya et al. 25
b(r) ~ b(rjfr
p
; a
p
; n
p
; ag) = j^ x a e
ikz
p
f
s
(k(r r
p
))j
2
:
(6)
Here, the Lorenz-Mie model for a spheres normalized holo-
gram b(rjfr
p
; a
p
; n
p
; ag) is an exceedingly complicated func-
tion of the particles three-dimensional position r
p
, radius a
p
,
and refractive index n
p
. It also depends on the adjustable pa-
rameter a that accounts for variations in illumination at the
position of the particle. Fortunately, open-source software is
available
32
for calculating b(rjfr
p
; a
p
; n
p
; ag). Normalized
holograms therefore can be t pixel by pixel to Eq. (6) to
locate and characterize each colloidal sphere in each holo-
graphic snapshot.
Fitting b(r) to b(rjfr
p
; a
p
; n
p
; ag) can be performed with
standard numerical packages for nonlinear least-squares t-
ting. Our implementation uses an open-source Levenberg-
Marquardt tting routine.
33
Assuming that Eq. (6) is the cor-
rect model for the data and that the reduced chi-squared sta-
tistic for the t is of order unity, the uncertainties in the t
parameters can be interpreted as estimates for the associated
measurement errors. Using this interpretation, ts to Eq. (6)
routinely yield the in-plane position of a micrometer-
diameter sphere to within one or two nanometers, the axial
position to within ve nanometers, the radius to about a
nanometer, and the refractive index to within a part per
thousand.
25,30,34
Obtaining meaningful estimates for the measurement
errors in the position, size, and refractive index requires
good estimates for the error in the value of b(r) at each pixel
in the normalized hologram. Each video snapshot suffers
from additive noise arising from the recording process,
which is likely to be inuenced by normalization with the
background image. We estimate an overall value for the
noise in each image using the median-absolute deviation
(MAD) estimator.
35
For the normalized hologram in Fig.
2(c) this value is Db = 0:009 or roughly 1% of the average
valuea reasonable gure for images digitized with 8 bits
per pixel.
In addition to random noise, the normalized hologram also
suffers from uncorrected background variations that tend to
break the circular symmetry of a spheres hologram and
therefore reduce the quality of the ts. To estimate this con-
tribution to the measurement error, we azimuthally average a
spheres image about the center of the scattering pattern to
obtain the radial intensity prole b(r), and then compute the
variance Db(r) = jb(r) b(r)j of the intensity at each radius
r. These contributions might be added in quadrature. Instead,
we estimate the single-pixel measurement error as
max(Db(r); Db), treating Db as a simple noise oor. This
choice has no substantial inuence on the results of the ts.
The range of radii r over which normalized images are t
to Eq. (6) is then limited by the range over which the signal
peeks out over the noise and uncorrected background varia-
tions. A convenient estimate for consistent particle tracking
and characterization is to identify the maxima and minima in
the radial prole b(r) and to set the range to the radius of a
particular extremum. The images in Fig. 2 were cropped to
the radius of the twentieth extremum. With these considera-
tions, the chi-squared parameter for a typical t is of order 1,
suggesting that computed uncertainties in the adjustable pa-
rameters are reasonable estimates for the measurement errors
in those parameters.
Figure 2(d) shows the result of tting the normalized
hologram to b(rjfr
p
; a
p
; n
p
; ag) using n
m
=1.3397, which
is the accepted value
36
for the refractive index of water at
k = 447 nm and T =24.0

C. The only other calibration

constant for the measurement is the overall magnication
of the optical train, which is found to be 0.135 lm/pixel
using a standard reticule. With these two calibration con-
stants, the t yields the particles in-plane position to within
0.8 nm in the plane and 5.7 nm axially.
The t values for the radius and refractive index are
a
p
=805 61 nm and n
p
=1.5730 60.0006. The former is
substantially larger than the nominal 780-nm radius specied
by the manufacturer. The latter is signicantly smaller than
the refractive index of bulk polystyrene at this temperature
and wavelength, which is 1.592. Chemically synthesized
polystyrene spheres are not fully dense, however. The dis-
crepancy presumably results from an effective porosity of 1
or 2 percent assuming the pores to be full of water.
37
C. Background estimation
The background intensity I
0
(r) used to normalize each
hologram can be obtained by taking a snapshot of an empty
eld of view. Moving the microscopes stage is not an effec-
tive way to nd a particle-free area, however, because the
background can change from place to place within a sample.
Instead, moving the particle out of the eld of view with op-
tical tweezers or another non-contact manipulation technique
can be effective.
If optical tweezers are not available, the background inten-
sity can be estimated by recording a sequence of images as
the particle moves freely across the eld of view. The meas-
ured intensity deviates from the background intensity only in
the vicinity of the particles scattering pattern. These inten-
sity variations are either brighter or darker than the local
background. The background at each pixel therefore can be
estimated as the median value of the intensities measured at
each pixel as the particle moves across the eld of view.
Fig. 2. Video holography of a colloidal polystyrene sphere in water. (a)
Measured hologram intensity I(r) of a colloidal polystyrene sphere in water.
(b) Background intensity I
0
(r), estimated from the running median of 10,000
video frames. (c) Normalized hologram b(r) obtained by dividing the holo-
gram in (a) by the background in (b). (d) Fit of (c) to the prediction of
Lorenz-Mie theory using Eq. (6).
26 Am. J. Phys., Vol. 82, No. 1, January 2014 Krishnatreya et al. 26
A sufciently long video sequence of a particle diffusing
across the eld of view therefore can be used rst to estimate
the background intensity and then to analyze the particles
diffusion. The background in Fig. 2(b) was estimated as the
median of 900 consecutive images obtained over 30 s, begin-
ning with the image in Fig. 2(a).
D. Video holography
The sequence of snapshots in a holographic video stream
can be analyzed individually to obtain time-resolved infor-
mation on a particles position and characteristics. The time
interval between snapshots is determined by the video cam-
eras shutter interval t
0
, which is 1/29.97 s for cameras con-
forming to the National Television Systems Committee
(NTSC) standard used in the United States.
Most consumer-grade video cameras do not acquire all of
the pixels in a frame simultaneously. Rather they rst record
the odd-numbered scan lines and then the even, the time
between these elds acquisition being half the shutter
period. Such images are said to be interlaced. Because a par-
ticle may have moved substantially during the interval
between recording the even and odd elds, the two elds of
each interlaced image should be analyzed separately. The
images in Fig. 2 show the even eld of an interlaced holo-
gram, which was interpolated back to full resolution for
presentation.
The loss of half the spatial resolution in one direction is
compensated by the doubling of the measurements temporal
resolution. If results from the two elds are to be combined
into a single time sequence, care must be taken to ensure that
the two data streams are interleaved in the correct order, and
that the geometric offset between the two elds is accounted
for correctly.
In addition to providing time-resolved particle-tracking
data, holographic video microscopy also yields time series
for the particles radius a
p
and refractive index n
p
. These
quantities are useful for monitoring the samples stability,
particularly the stability of the temperature. Ideally, a
p
and
n
p
should not vary over the course of a measurement by
more than the uncertainties in their values. Changes in tem-
perature, however, cause changes in the relative refractive
index of the medium and the sphere and therefore inuence
extracted values for a
p
, n
p
, and the axial position z
p
.
The 10,000 data points in Fig. 3 were obtained from the
even and odd elds of 5000 consecutive video snapshots of
the same sphere shown in Fig. 2. Variations from the mean
values for a
p
and n
p
are signicantly larger than the single-t
uncertainty estimates. Much of this variability is likely to be
due to uncorrected illumination artifacts and slow mechani-
cal drifts in the optical train, including ow in the immersion
oil. In addition to these variations, the overall mean values
drift slightly over time, particularly near the end of the mea-
surement. This trend suggests that more condence should
be attached to the rst 100 s of this data set.
IV. ANALYZING A SPHERES SEDIMENTATION
Figure 4(a) shows the three-dimensional trajectory r
p
(t) of
the same colloidal sphere. The plot is colored by time, and
the data from Fig. 3 contributed the rst of 10,000 points.
In-plane distances in Fig. 4(a) are measured relative to one
corner of the eld of view. The axial distance is measured
relative to the height of the focal plane. Based on the
Fig. 3. (Color online) Time-resolved measurements of the radius a
p
and re-
fractive index n
p
of a single diffusing colloidal sphere. Dashed lines indicate
the mean values and standard deviations from the mean.
Fig. 4. (Color online) Free diffusion of a sedimenting colloidal sphere. (a) Three-dimensional trajectory measured over 160 s. The points are colored to indicate
the passage of time, and the in-plane components of the motion are projected onto the xy-plane. (b) Axial component of the trajectory showing steady sedimen-
tation together with a linear least-squares t for the sedimentation velocity. (c) Components of the mean-squared displacement of the trajectory in (a).
27 Am. J. Phys., Vol. 82, No. 1, January 2014 Krishnatreya et al. 27
particles motion at very long times, we estimate the axial
position of the lower glass surface of the sample chamber to
be roughly z
0
=5 lm on this scale. Because hydrodynamic
coupling can substantially inuence a particles diffusion
when it approaches a solid surface,
3840
we restrict our anal-
ysis to z
p
(t) > 15 lm. Being able to rule out hydrodynamic
coupling as a source of systematic error is one of the note-
worthy strengths of using holographic video microscopy to
measure Boltzmanns constant.
Although the in-plane diffusion shown in projection in
Fig. 4(a) appears to be random, there is a clear downward
trend in the axial position plotted in Fig. 4(b). This trend
shows that the sphere is slightly more dense than the sur-
rounding water and therefore sediments under gravity.
Fitting z
p
(t) to a linear trend yields a mean axial velocity of
v
z
=75.27 6 0.01 nm/s. A sphere of radius a
p
and mass den-
sity q
p
should achieve a terminal velocity of
v
z
=
2
9
a
2
p
(q
p
q
m
)g
g
(7)
in a medium of mass density q
m
and dynamic viscosity g. From
this, we can estimate the particles density. Taking the viscosity
of the water to be g = 0:912 mPa s at T=24.08C and its den-
sity to be q
m
= 997:3 kg=m
3
, and using g=9.80363m/s
3
for
the gravitational eld strength in New York City, we obtain for
the density of the sphere q
p
= 1; 045:7 kg=m
3
. When compared
with the density of bulk polystyrene, q
PS
= 1; 047 kg=m
3
, the
value for q
p
estimated from sedimentation is consistent with a
porosity of 1 or 2%, assuming the pores to be lled with
water.
37
This agrees well with the estimate for the spheres po-
rosity obtained from optical data alone in Sec. III B.
V. ANALYZING A SPHERES DIFFUSION
A small particle immersed in a uid responds both to
deterministic forces, such as the force of gravity, and to ran-
dom thermal forces resulting from collisions with the uids
molecules. The former inuence causes the particle to drift
with a constant velocity v, while the latter renders a diffusing
particles trajectory r
p
(t) unpredictable and is a dening
characteristic of Brownian motion. For clarity, we will drop
the subscript p in the following analysis of the particles
trajectory.
Even though a Brownian particles position cannot be
anticipated, a statistical ensemble of such trajectories never-
theless has predictable properties. In the absence of drift due
to external forces, the ensemble average of the particles dis-
placements over a time interval Dt is expected to vanish.
This is not surprising because the random forces themselves
vanish on average in accord with the second law of thermo-
dynamics. More remarkably, Einstein
1
and Smoluchowski
41
predicted that a diffusing particles mean-squared displace-
ment should increase linearly with time. For a particle that
diffuses as it drifts, the Einstein-Smoluchowski relation
predicts
Dr
2
(Dt) = hjr(t Dt) r(t)j
2
i = 2dDDt (vDt)
2
; (8)
where d is the dimensionality of the trajectory and D is a
constant called the diffusion coefcient. The angle brackets
in Eq. (8) indicate either an average over different but equiv-
alent trajectories or else an average over statistically inde-
pendent starting times t in a single trajectory. In the limit
where D=Dt is much smaller than v
2
, Eq. (8) reduces to the
familiar result for motion with constant velocity, r(t Dt)
= r(t) vDt.
By considering the statistics of molecular motion in the
suspending uid, Einstein showed that the macroscopic par-
ticles diffusion coefcient is proportional to the thermal
energy scale of the microscopic molecules:
D = c
1
k
B
T: (9)
The constant of proportionality is the viscous drag coef-
cient c that characterizes the force F = cv required to move
the particle through the uid at speed v. The drag coefcient
therefore establishes the power P = cv
2
that is dissipated in
moving the particle through the uid. In this sense, Eq. (9)
relates microscopic uctuations to macroscopic dissipation
and so is an example of a uctuation-dissipation theorem.
For the particular case of a spherical particle of radius a
p
moving through an unbounded Newtonian uid of dynamic
viscosity g, Sir George Stokes calculated the viscous drag
coefcient to be c = 6pga
p
. The associated Stokes-Einstein
diffusion coefcient
D =
k
B
T
6pga
p
; (10)
together with the Einstein-Smoluchowski equation in Eq.
(8), provides a basis for estimating Boltzmanns constant
from tracking data.
Holographic particle tracking offers the substantial benet
over conventional video microscopy that it yields a precise
estimate for the radius a
p
of the particular sphere being
tracked, including an estimate for the uncertainty in this
value.
42
Without this information, the probe particles size
typically has been estimated through light-scattering analysis
of a representative sample of similar spheres, with an uncer-
tainty no smaller than the samples polydispersity, which
typically is on the order of a few percent.
A. Analyzing a discretely sampled trajectory
The experimentally measured trajectory is sampled at discrete
times t
n
=nt
0
, where n is an integer and t
0
is the cameras shut-
ter interval. The trajectory thus consists of a sequence of posi-
tions, r
n
= r(t
n
)
n
, each of which includes a measurement
error
n
. Unlike conventional particle tracking,
4
holographic
tracking yields an estimate for the localization error at each posi-
tion. This additional information will prove useful in obtaining
Boltzmanns constant from a single-particle trajectory.
We assume that the uncertainty in the measurement time
t
n
is small enough to be ignored; for a conventional video
camera, the inter-frame jitter is typically less than 5 ns.
Similarly, we assume that the cameras exposure time is suf-
ciently short that blurring due to the particles motion can
be ignored.
30,4345
In the experiments we report, the cam-
eras exposure time is set to 100 ls. Whether this is suf-
ciently fast to avoid blurring can be established once the
particles measured trajectory has been analyzed.
A complete data set consists of N samples, from which the
mean-squared displacement at lag time Dt = st
0
can be esti-
mated as
Dr
2
s
=
1
N
s
X
N
s
1
n=0
jr
(n1)s
r
ns
j
2
; (11)
28 Am. J. Phys., Vol. 82, No. 1, January 2014 Krishnatreya et al. 28
where N
s
= bN=sc is the integer part of N/s. The plots in Fig.
4(c) show the mean-squared displacements for each of the
three components of the trajectory in Fig. 4(a) computed
using Eq. (11).
The statistical variance in the estimate for Dr
2
s
is
r
2
s
=
1
N
s
1
X
N
s
1
n=0
[Dr
2
s
jr
(n1)s
r
ns
j
2
[
2
: (12)
The variance can be computed for each of the Cartesian
components of r(t) independently. The error bars in Fig. 4(c)
are computed in this way.
Whereas statistical uctuations can either increase or
decrease the estimated value of Dr
2
s
relative to its ideal value,
measurement errors only increase the apparent positional
uctuations. To see this, imagine that the particle was truly
stationary at the origin of the coordinate system, so that r
n
=
n
for all values of s. The true mean-squared displacement
vanishes in this thought experiment. The apparent mean-
squared displacement, however, is
Dr
2
s
=
1
N
s
X
N
s
1
n=0
j
(n1)s

ns
j
2
=
1
N
s
X
N
s
1
n=0
[
2
(n1)s

2
ns
[
2
N
s
X
N
s
1
n=0

(n1)s

ns
: (13)
If the measurement errors are random, the second term in
Eq. (13) vanishes. If we further assume that the measurement
errors have the same statistical distribution during the entire
experiment, we can dene an apparent mean-squared distri-
bution associated specically with measurement errors by
2d
2
=
1
N
s
X
N
s
1
n=0
[
2
(n1)s

2
ns
[; (14)
which is independent of the measurement interval s.
Measurement errors therefore systematically increase the
mean-squared displacement computed from a trajectory.
Rather than incorporating measurement errors into the
error bars in Dr
2
s
, we instead modify the Einstein-
Smoluchowski relation to account for the measurement-
related offset by writing
Dr
2
s
= 2d
2
2d D(st
0
) v
2
(st
0
)
2
: (15)
In this case, only the statistical uncertainties r
2
s
contribute to
the error estimate for each value of s.
Fits of the data in Fig. 4(c) to Eq. (15) yield values for the
tracking error in each of the coordinates of
x
= 368 nm;

y
= 468 nm, and
z
= 1168 nm. These values are statisti-
cally consistent with the average position errors obtained
from tting to the individual images, which are 0.6 nm,
0.6 nm, and 4 nm, respectively. The dynamical values
obtained from Eq. (15) might reasonably be expected to be
larger because they also incorporate tracking errors due to
mechanical vibrations in the apparatus.
B. Statistical cross-checks
Analyzing motions in each coordinate independently per-
mits useful tests for potential experimental artifacts.
Hydrodynamic coupling to the sample containers glass
walls, for example, increases hydrodynamic drag by an
amount that depends on height above the wall
38
and thereby
reduces the observed diffusion coefcient. This was one of
the principal sources of error in Perrins particle-tracking
experiments. Because hydrodynamic coupling has a larger
effect in the axial direction,
38,40,46,47
agreement between in-
plane and axial diffusion coefcients can be used to verify
that this source of error is negligibly small. Non-uniform
drift due to unsteady uid ow in a poorly sealed sample
similarly would appear as inconsistencies among results for
the three coordinates.
The single-component values are related to the overall
mean-squared displacement by Dr
2
s
= Dx
2
s
Dy
2
s
Dz
2
s
. If
the system is homogeneous and isotropic, the sphere should
diffuse equally freely along each axis so that Dx
2
s
= Dy
2
s
= Dz
2
s
= Dr
2
s
=3. Similarly, statistical uncertainties in these
estimates also should agree, giving r
2
x;s
= r
2
y;s
= r
2
z;s
= 3r
2
s
.
For the data set in Fig. 4, ts to Eq. (15) yield D
x
= 0:301560:0042 lm
2
=s; D
y
= 0:300960:0046 lm
2
=s, and
D
z
= 0:292460:0044 lm
2
=s. As anticipated, the three diffu-
sion coefcients are consistent with each other to within their
estimated uncertainties. Similarly, the statistical variances in
the mean-squared displacements all are consistent with each
other for each measurement interval s.
Agreement among the single-coordinate measurements is
consistent with the assumptions that hydrodynamic coupling
to the bounding surfaces can be ignored and that any nonuni-
form drifts are small. Limits on the axial range set in Sec. IV
therefore appear to have been chosen appropriately, and the
sample cell appears to have been stably sealed and well equili-
brated. We are justied, therefore, in estimating the particles
free diffusion coefcient as the average of the single-
component values: D = 0:298660:0025 lm
2
=S.
C. Independent sampling and greedy sampling
Equation (11) yields an estimate for Dr
2
s
based on a subset
fr
0
; r
s
; ; r
sN
s
g of the measurements in the trajectory starting
from the rst measurement in the series. An equivalent esti-
mate could be obtained by starting from the jth measurement
Dr
2
s;j
=
1
N
s;j
X
N
s;j
1
n=0
jr
(n1)sj
r
nsj
j
2
; (16)
with j < s and where N
s;j
= b(N j 1)=sc. Each such esti-
mate is based on a statistically independent sampling of the
particles trajectory distinguished by its starting index j.
It is tempting to treat each value of Dr
2
s;j
as a separate mea-
surement of the mean-squared displacement and to combine
them to get
Dr
2
s
=
1
s
X
s1
j=0
Dr
2
s;j
; (17)
thereby improving statistical sampling by a factor of

s
p
.
This choice makes use of all of the available data and there-
fore is known as greedy sampling.
The statistically independent samples that go into the
greedy estimate for Dr
2
s
are not, however, statistically inde-
pendent of each other. To see this, consider that a trajectory
starting at r
0
and advancing to r
s
after s time intervals shares
29 Am. J. Phys., Vol. 82, No. 1, January 2014 Krishnatreya et al. 29
s 1 time steps of diffusion with the equivalent trajectory
starting at r
1
and advancing to r
s1
. Such correlated esti-
mates nevertheless can be combined according to Eq. (17),
provided that particular care is taken to account for their cor-
relations.
3
If the underlying trajectory consists of statistically
independent steps, as in the case of a freely diffusing sphere,
then greedy sampling can benecially reduce the variance in
the estimate for the mean-squared displacement without
biasing its value.
3
For the present data set, greedy sampling
yields D = 0:298260:0008 lm
2
=s.
It is important to note that greedy sampling can be risky.
If the underlying trajectory is correlated, then greedy sam-
pling can bias the apparent mean-squared displacement.
Such correlations can occur, for example, if the particles
diffusion coefcient depends on its position, or if the particle
is subject to a force that varies with position or time. To
avoid such biases, we adopt the statistically independent esti-
mator described in Eq. (11), which is equivalent to Dr
2
s;0
from Eq. (16).
VI. MEASURING BOLTZMANNS CONSTANT
Given the foregoing results, Boltzmanns constant can be
estimated using Eq. (10):
k
B
=
6pga
p
D
T
: (18)
For the radius of the particle, we use the average a
p
= 0:80260:011 lm of the values obtained in all of the ts.
From this and the previously discussed values for g, D, and
T, we obtain k
B
=(1.387 60.021) 10
23
J/K, which agrees
to within half a percent with the IUPAC accepted value of
(1.3806488 60.0000013) 10
23
J/K.
This result compares favorably with conventional video mi-
croscopy measurements.
5,7,8
The holographic approach, more-
over, requires only a single trajectory of a single sphere, rather
than an average over multiple measurements on multiple
spheres. Holographic microscopys large axial range keeps the
particle in view for a long-term measurement despite diffusion
and sedimentation. By eliminating uncertainty in the particles
size, holographic microscopy also eliminates the need to aver-
age over the size distribution. Other potential sources of error,
such as non-steady drift and hydrodynamic coupling to the
walls, can be ruled out by comparing results for motion along
independent directions. Constancy in the monitored value for
the particles refractive index similarly excludes thermal and
instrumental drifts as potential sources of error.
The two most important sources of error in the present mea-
surement are statistical uncertainty and variations in the meas-
ured value for the particles radius. The former can be
addressed by acquiring more data; the latter could be reduced
by improving the illuminations uniformity to minimize uncor-
rected background variations and thus to avoid position-
dependent variations in the spheres apparent size. The ultimate
limit on this measurements precision is likely to be set by
uncertainty in the samples temperature through its inuence on
the materials refractive indexes and on the waters viscosity.
VII. CONCLUSIONS
Through holographic video microscopy of a single
micrometer-diameter polystyrene sphere diffusing freely in
water, we have obtained a value for the Boltzmann constant
of k
B
=(1.387 6 0.021) 10
23
J/K. This measurement
took advantage of holographic microscopys ability to track
the spheres motion in three dimensions with high precision
and to measure the spheres radius. Comparably good results
were obtained with repeated measurements on the same
sphere and with measurements on other spheres.
Because no effort was made to match the solvents density
to the spheres, these holographic tracking measurements
also revealed the particles slow sedimentation under gravity.
From the sedimentation velocity and the holographic mea-
surement of the spheres radius, we were able to estimate the
spheres density. Comparing this value with the accepted
value for bulk polystyrene then suggests that the sphere is
porous with a porosity of roughly 1%. This value is consist-
ent with the porosity estimated from the spheres refractive
index, which also was obtained from the same holographic
microscopy data. Consistency among these measurements
lends condence to the result for the Boltzmann constant,
which is indeed consistent with the presently accepted value.
More generally, this measurement highlights the capabil-
ities of holographic video microscopy for particle tracking
and particle characterization. Comparatively simple apparatus
can be used to track individual colloidal particles over large
distances with excellent precision, while simultaneously offer-
ing insights into particle-resolved properties that are not avail-
able with any other technique. These capabilities already have
been applied to measure the microrheological properties of
complex uids,
42
to characterize variations in colloidal par-
ticles properties,
48,49
and to perform precision measurements
in statistical physics.
50,51
Indeed, these capabilities should be
useful for a host of other experiments in soft-matter physics,
physical chemistry, and chemical engineering.
ACKNOWLEDGMENTS
This work was supported in part by a grant from Procter &
Gamble and in part by the PREM program of the National
Science Foundation through Grant No. DMR-0934111.
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