Content uploaded by Taeseok Daniel Yang
Author content
All content in this area was uploaded by Taeseok Daniel Yang
Content may be subject to copyright.
Full-field and single-shot quantitative
phase microscopy using
dynamic speckle illumination
Youngwoon Choi, Taeseok Daniel Yang, Kyoung Jin Lee, and Wonshik Choi*
Department of Physics, Korea University, Seoul 136-701, Korea
*Corresponding author: wonshik@korea.ac.kr
Received April 8, 2011; revised June 2, 2011; accepted June 3, 2011;
posted June 3, 2011 (Doc. ID 145548); published June 22, 2011
We developed an off-axis quantitative phase microscopy that works for a light source with an extremely short spatial
coherence length in order to reduce the diffraction noise and enhance the spatial resolution. A dynamic speckle
wave whose coherence length is 440 nm was used as an illumination source. To implement an off-axis interferometry
for a source of low spatial coherence, a diffraction grating was inserted in the reference beam path. In doing so, an
oblique illumination was generated without rotation of the wavefront, which leads to a full-field and single-shot
phase recording with improved phase sensitivity of more than a factor of 10 in comparison with coherent illumina-
tion. The spatial resolution, both laterally and axially, and the depth selectivity are significantly enhanced due to the
wide angular spectrum of the speckle wave. We applied our method to image the dynamics of small intracellular
particles in live biological cells. With enhanced phase sensitivity and speed, the proposed method will serve as a
useful tool to study the dynamics of biological specimens. © 2011 Optical Society of America
OCIS codes: 110.6150, 180.3170, 100.3175.
The speckle wave generated by the illumination of a co-
herent wave on random media is an interesting subject
due to its intriguing optical properties and its advantages
in holographic imaging for metrology purposes [1,2]. In
general, the speckle wave has been considered an obsta-
cle for imaging in astronomy and life sciences [3,4], but
many studies have proved that the speckle wave can be
used to improve imaging performance. In [5], a unique
propagation property of the speckle wave was used to
retrieve the phase of the wave . In coherent imaging, such
as digital holographic microscopy and quantitative phase
microscopy, either static or dynamic speckles have been
used to reduce diffraction noise [6–9]. The short spatial
coherence length of dynamic speckle waves has enabled
rejection of the interference caused by unwanted reflec-
tions [8,9]. In addition, short autocorrelation length, both
axially and laterally, has provided confocal equivalent
sectioning and enhanced spatial resolution [7]. How-
ever, some care has to be taken to use a partially coher-
ent source for interferometry. The sample and reference
paths need to be carefully matched to form a high-
contrast interference image, which is a prerequisite for
phase imaging. In addition, phase modulation should be
applied either in time (on-axis) or in space (off-axis) to
reconstruct a complex electric field image from the inter-
ference image. Conventional off-axis interferometry, in
which a mirror in the reference beam path is tilted, will,
however, fail to generate interference of uniform con-
trast since physical rotation of the wavefront degrades
the cross correlation of the reference beam to the sample
beam [7]. In this sense, so-called phase-shifting interfero-
metry [10] is a straightforward solution and, thus, has
been used in most of studies. However, the requirement
of multiple recordings in time inevitably slows down the
data acquisition speed and thereby limits its application
to dynamical systems.
In this Letter, we present off-axis dynamic speckle
interferometric microscopy (ODSIM), which features
full-field and single-shot quantitative phase imaging with
improved phase sensitivity, spatial resolution, and depth
selectivity. The key idea in this development is the use of
a diffraction grating in the reference beam path that
makes it possible to generate fine interference fringes of
high contrast across the entire field of view for a light
source of extremely low spatial coherence. We demon-
strate imaging of live cell dynamics in which detailed
motions of intracellular particles are visualized. The
improved sectioning ability is also demonstrated in com-
parison with the coherent illumination method.
Fig. 1. (Color online) (a) Experimental setup and (b), (c) the
advantage of using a diffraction grating. (a) Off-axis Mach–
Zehnder interferometer. D, diffuser mounted on an electric
motor (not shown); BS1 and BS2, cube beam splitters; L1, L2,
and TL, lenses with focal lengths of 250, 250, and 200 mm, re-
spectively; C, condenser lens; OL, objective lens; G, diffraction
grating; A, iris diaphragm. For the conventional tilting a mirror
method, the zeroth-order diffracted beam is selected by A and
tilted by BS2. (b) Interference image taken without the diffrac-
tion grating. (c) Image taken with the grating in place. Only a
4μm×48 μm section of the entire field of view (36 μm×48 μm)
is shown in (b) and (c) to save space. Scale bar, 5μm.
July 1, 2011 / Vol. 36, No. 13 / OPTICS LETTERS 2465
0146-9592/11/132465-03$15.00/0 © 2011 Optical Society of America
Our experimental setup [Fig. 1(a)] is an off-axis inter-
ferometric microscope that uses a dynamic speckle field
as an illumination source. An He–Ne laser illuminates a
rotating diffuser (DG20-1500-H2, Thorlabs, Inc.) to gener-
ate a speckle field, which is then split by a beam splitter
(BS1) into sample and reference beams. In ordinary
Mach–Zehnder interferometry, it is difficult to form a
high-contrast interference image due to the low spatial co-
herence of the speckle waves. In our setup, the optical
configurations for the sample and reference arms are
matched to deliver almost the same speckle waves to the
detector plane. Specifically, each arm has a condenser
lens (Nikon 1.4 NA oil) for illumination, an objective lens
(Olympus 1.4 NA oil), and a tube lens to deliver the
sample image to the camera (Dalsa, Genie M1024) with
a magnification of 100. The difference between the two
arms is that a sample in a medium is positioned in the sam-
ple beam path while only the blank medium is placed in
the reference arm to match the path length. For the align-
ment purpose, the diffuser is initially set to be stationary
and the speckle patterns coming through the two arms are
matched by carefully positioning various optical elements
in the reference arm. Then the diffuser, mounted on a dc
electric motor, is rotated and the resulting interference
image is recorded with the camera, whose exposure time
is set to be longer than the period of the rotation. As a re-
sult, wide intensity variation in the static speckle field is
eliminated and the intensity distribution is made uniform.
In order to obtain a quantitative phase image from a
single interference image, an off-axis interferometry is
implemented in which fine interference fringes have to
be generated by oblique illumination of a reference beam
with respect to the sample beam. When the spatial period
of the fringes is comparable to or smaller than the diffrac-
tion-limited spot, the electric field of a sample beam can
be reconstructed by means of Hilbert transform up to the
diffraction-limited resolution [11]. As mentioned earlier,
the conventional approach of tilting a mirror in the refer-
ence beam path results in an uneven contrast of interfer-
ence across the field of view [Fig. 1(b)]. The physical tilt
of the wavefront makes the reference speckle wave dif-
ferent from that of the sample beam except at the axis
of the tilt. As a result, the cross correlation between the
two beams is reduced and the interference contrast
decreases away from the center where the axis of the tilt
is positioned. There is about 25% contrast reduction
per 10 μm on average. To maintain cross correlation, a
diffraction grating (Edmund Optics, Ronchi ruling,
40 lines=mm) is inserted in the reference beam path at
the conjugate plane to the camera and only the first-order
diffracted beam is selected by an iris diaphragm (A).
Then, the reference beam becomes oblique with respect
to the optic axis without physical rotation of its wave-
front. The spatial filtering process just adds a linear
spatial phase ramp to the existing speckle fields, which
makes interference fringes. The choice of the diffraction
grating is such that the period of the grating at the camera
plane is 25 μm, which is smaller than the diffraction-
limited spot of 28 μm at the same plane. This condition
ensures that the spatial resolution of the reconstructed
image is at least as good as that of the diffraction limit.
As can be seen in Fig. 1(c), the fringe contrast is now
almost uniform across the entire view field.
It is important to reduce the spatial coherence of
the speckle illumination since it is strongly related to the
attenuation of diffraction noise and enhancement in spa-
tial resolution. The diffraction from dust particles, optics,
and other parts of a sample located farther away from the
focal plane than the coherence length will not interfere
with the signal of interest. The coherence length is deter-
mined by the size of a speckle wave illuminating the con-
denser lens. We set the opening of an aperture located at
the back of the condenser lens such that the condenser
lens has an effective NA of 0.8. Then the coherence
length is measured to be 440 nm from the autocorrelation
width of multiple static speckle fields, which is quite
close to 483 nm as predicted by Van Cittert–Zernike the-
orem. If the sample and reference arms are perfectly
identical, the magnitude of the cross correlation is ex-
pected to be the same as that of the autocorrelation.
However, due to the aberration caused by the diffraction
grating, the cross correlation is reduced to 45% of the
autocorrelation in our experiment, which is still suffi-
cient to obtain a high-contrast image.
With improved interference images, quantitative phase
imaging of a live biological cell is performed. A microglia
cell immersed in a culture medium is placed in the sample
plane. Microglia cells extracted from a rat brain are incu-
bated for seven days in a culture medium of Dulbecco’s
modified eagle medium with 10% fetal bovine serum
and then plated on a coverslip in a density of 50 cells=
mm2for observation. The interference image taken with-
out the diffuser is shown in Fig. 2(a), where the diffraction
noise due to the floating particles in the culture medium
Fig. 2. (Color online) Quantitative phase imaging of a live cell
using ODSIM: (a) raw interference image, (c) processed quan-
titative phase image, and (e) numerically simulated DIC image
for the coherent illumination. (b), (d), and (f) Same as (a), (c),
and (e) but with dynamic speckle illumination. The insets in (a)
and (b) are zoom-in images at the background by three folds.
Scale bar, 10 μm. Color bar, phase in radians. Media 1 shows the
dynamics of the intracellular particles in the same cell. Images
are taken with a frame rate of 2fps.
2466 OPTICS LETTERS / Vol. 36, No. 13 / July 1, 2011
and other sources in the optics distorts the interference
fringes. With the ODSIM, the fringes are extremely clean
due to the significant reduction of noise [Fig. 2(b)]. The
interference images are processed to acquire quantitative
phase images using Hilbert transform. As can be seen in
Fig. 2(d), tiny vesicles inside the cell are clearly visible in
the ODSIM image. In the case of coherent illumination, the
diffraction noise masks the particles with small phase
shifts [Fig. 2(c)]. The phase sensitivity is estimated by cal-
culating the standard deviation of the phase variation in
the area of 8μm×8μm void of the cell. With the use of
dynamic speckle illumination, the background phase
noise is reduced from 160 to 14 mrad, which is equivalent
to an optical path length of 1:3nm. This is less than twice
the shot-noise-limited phase noise of 7:9mrad. Using the
single-shot recording ability of the phase image, a movie of
the same cell is recorded (Media 1). The data acquisition
speed is limited only by the camera frame rate. As can be
seen in Media 1, the vigorous motion of small particles is
clearly observed, which was not possible with the coher-
ent illumination. In addition, by numerically processing
the acquired phase image into a differential interference
contrast (DIC) image to emphasize the field gradient, nu-
clear membrane and internal structures of the cell are
clearly visible in ODSIM [Fig. 2(f)]. For the coherent illu-
mination case, diffraction noise is emphasized and masks
cellular structures [Fig. 2(e)]. These results demonstrate
full-field and single-shot phase recording of the ODSIM
with improved phase sensitivity. The ability of our techni-
que in tracking intracellular particles paves the way for
studying microrheology of biological cells without admin-
istrating external agents [12,13].
We now validate the sectioning ability of the ODSIM
originated from the short autocorrelation of the speckle
wave. Multiple 10-μm-sized polystyrene beads (Polybead,
Microsphere) are prepared following the arrangement
shown in Fig. 3(a). The medium is index-matching oil
with a refractive index of 1.56 (Cargille Labs). In conven-
tional off-axis interferometry, the beads located away
from the objective focus are visible, with their bound-
aries exhibiting diffraction rings [Figs. 3(b) and 3(c)].
In contrast, when the ODSIM is used, each layer is visible
at the same time [Fig. 3(d) and 3(e)]. This clearly shows
the sectioning ability of the ODSIM. Only those beads lo-
cated at the focus plane are visible, and each bead shows
a clean boundary. In this recording, it is to be noted that
the objective lens in the reference arm is scanned in con-
junction with the scanning of the focus in the sample arm
in order to maintain the cross correlation. The pattern of
a speckle field varies as the focus plane is scanned.
Therefore, constructive interference occurs only for the
depth matched to the reference arm, which is the origin
of the sectioning. In an additional experiment, the axial
width of the point spread function is determined to be
968 38 nm by measuring the phase profile of a 350 nm
bead along the axial direction. This is quite close to the
theoretically expected axial correlation length of the
speckle, which is λ=NA2¼980 nm. The lateral width of
the same bead is also measured to be 343 8nm. This
is approximately the same as the diffraction limit of
350 nm, but a little bit better than the correlation length
of 440 nm. This will be due to the use of a higher NA
objective lens than the effective NA of a condenser lens.
Both axial and lateral resolutions are about twice better
than those of the coherent illumination.
In conclusion, we developed an off-axis interfero-
metric microscopy that works with extremely short
spatial coherence sources. Our method offers enhanced
phase sensitivity, depth selectivity, and spatial resolution
in comparison with a conventional off-axis method that
uses coherent sources. We believe that the ODSIM will
offer opportunities to investigate nanoscale motion of
biological specimens at the full speed of a camera.
This research was supported by the Basic Science Re-
search Program through the National Research Founda-
tion of Korea (NRF) funded by the Ministry of Education,
Science and Technology (MEST, 2011-0005018 and 2011-
0016568), the National R&D Program for Cancer Control,
Ministry of Health & Welfare, South Korea (1120290), the
Korea Science and Engineering Foundation (KOSEF,
R17-2007-017-01000-0), and a Korea University grant.
References
1. J. W. Goodman, Speckle Phenomena in Optics: Theory and
Applications (Roberts, 2007).
2. P. Jacquot and J. M. Fournier, in Interferometry in Speckle
Light: Theory and Applications: Proceedings of the Inter-
national Conference (Springer, 2000), pp. 25–28.
3. J. E. Baldwin, C. A. Haniff, C. D. Mackay, and P. J. Warner,
Nature 320, 595 (1986).
4. A. E. Desjardins, B. J. Vakoc, G. J. Tearney, and B. E.
Bouma, Opt. Express 14, 4736 (2006).
5. P. F. Almoro, G. Pedrini, P. N. Gundu, W. Osten, and S. G.
Hanson, Opt. Lett. 35, 1028 (2010).
6. Y. Park, W. Choi, Z. Yaqoob, R. Dasari, K. Badizadegan, and
M. S. Feld, Opt. Express 17, 12285 (2009).
7. M. G. Somekh, C. W. See, and J. Goh, Opt. Commun. 174,
75 (2000).
8. M. C. Pitter, C. W. See, and M. G. Somekh, Opt. Lett. 29,
1200 (2004).
9. F. Dubois, M. L. N. Requena, C. Minetti, O. Monnom, and
E. Istasse, Appl. Opt. 43, 1131 (2004).
10. K. Creath, Prog. Opt. 26, 349 (1988).
11. T. Ikeda, G. Popescu, R. R. Dasari, and M. S. Feld, Opt. Lett.
30, 1165 (2005).
12. D. Wirtz, Annu. Rev. Biophys. 38, 301 (2009).
13. Z. Wang, L. Millet, M. Mir, H. F. Ding, S. Unarunotai, J.
Rogers, M. U. Gillette, and G. Popescu, Opt. Express 19,
1016 (2011).
Fig. 3. (Color online) Depth selectivity of ODSIM. (a) Sche-
matic diagram for beads in the sample plane. (b) and (c) Phase
images taken without the diffuser with the objective foci lo-
cated at the upper and lower dashed lines, respectively. (d) and
(e) Same as (b) and (c) but with the use of the diffuser. The
objective lens in the reference arm is also adjusted to match
the speckle waves at each focus. Color bar, phase in radians.
Scale bar, 10 μm.
July 1, 2011 / Vol. 36, No. 13 / OPTICS LETTERS 2467
