The average concentration of marine copepods has been

estimated at about one per liter, making them the most

numerous multicelled animals on earth (Boxshall, 1998).
These micro-crustaceans form major links between the
plankton world and nektonic organisms. The rate and
specicity of their feeding can control phytoplankton
population growth, while copepods themselves are an
important source of food for sh, euphausiids and
coelenterates (Mann and Lazier, 1996). Along with food
availability and predator pressure, the copepod population
dynamics are affected by swimming behavior and the types of
ow elds they generate to capture food, which is species
dependent (Strickler, 1982). The quantity of food depends on
the volume of water they can scan and their ability to locate
prey (Koehl, 1984). Locomotion and the generation of feeding
currents increase the encounter rates with food, and can
enhance the copepods detection ability by stimulating
escapes in motile prey (Yen and Strickler, 1996). Alternately,
motionless sinking can make the copepod less conspicuous to
predators or prey. Within the species, advertisement is
important for reproduction (Doall et al., 1998; Yen and
Strickler, 1996). Because copepods sense their environment
utilizing antennules studded with arrays of setae that respond
to uid deformation, the ow eld that a swimming animal
generates is a lens through which the copepod views its
surroundings. Knowledge about this ow eld is important for
understanding the impact of swimming behavior on the
success of a copepod in locating food, avoiding predators and
nding mates.
Current knowledge is largely based on video observations,
including a Schlieren system for visualizing wakes (Strickler,
1977), high-speed cinematography for understanding how food
particles are captured and feeding currents are generated
(Koehl and Strickler, 1981; Strickler, 1984), and three-
dimensional (3-D) video tracking of free-swimming copepods
in particle elds (Paffenhfer et al., 1995; Strickler, 1985). The
latter combines particle tracks at different times to observe
two-dimensional (2-D) ow elds in lateral and dorsal views.
More recently, Particle Image Velocimetry (PIV) has been
used to map instantaneous 2-D ow elds around tethered
specimens (van Duren et al., 1998). However, since ow elds
around copepods are 3-D, unsteady and vary with swimming
speeds and orientations (Bundy and Paffenhfer, 1996;
Strickler, 1982; van Duren et al., 1998), 3-D measurements are
3657 The Journal of Experimental Biology 206, 3657-3666
2003 The Company of Biologists Ltd
doi:10.1242/jeb.00586
Digital in-line holography is used for measuring the
three-dimensional (3-D) trajectory of a free-swimming
freshwater copepod Diaptomus minutus, and
simultaneously the instantaneous 3-D velocity eld around
this copepod. The optical setup consists of a collimated
HeNe laser illuminating a sample volume seeded with
particles and containing several feeding copepods. A time
series of holograms is recorded at 15Hz using a lensless
2Kx2K digital camera. Inclined mirrors on the walls of the
sample volume enable simultaneous recording of two
perpendicular views on the same frame. Numerical
reconstruction and matching of views determine the 3-D
trajectories of a copepod and the tracer particles to within
pixel accuracy (7.4m). The velocity eld and trajectories
of particles entrained by the copepod have a recirculating
pattern in the copepods frame of reference. This pattern
is caused by the copepod sinking at a rate that is lower
than its terminal sinking speed, due to the propulsive force
generated by its feeding current. Consequently, the
copepod sees the same uid, requiring it to hop
periodically to scan different uid for food. Using
Stokeslets to model the velocity eld induced by a point
force, the measured velocity distributions enable us to
estimate the excess weight of the copepod (7.210
9
N), its
excess density (6.7kgm
3
) and the propulsive force
generated by its feeding appendages (1.810
8
N).
Movies available on-line
Key words: digital in-line holography, particle image velocimetry,
copepod, Diaptomus minutus, ow.
Summary
Introduction
The three-dimensional ow eld generated by a feeding calanoid copepod
measured using digital holography
Edwin Malkiel
1
, Jian Sheng
1
, Joseph Katz
1,
* and J. Rudi Strickler
2
1
Johns Hopkins University, Department of Mechanical Engineering, N. Charles Street, Baltimore, MD 21218, USA
and
2
Great Lakes WATER Institute, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53204, USA
*Author for correspondence (e-mail: Katz@jhu.edu)
Accepted 7 July 2003
3658
essential. Considering the importance of copepods in the
aquatic and marine food webs, and 30 years of related
hypotheses, it seems worthwhile to investigate whether digital
cinematographic holography may break barriers and allow us
to test these hypotheses.
Unlike video microscopy, holography maintains the same
lateral resolution over a substantial depth (Vikram, 1992). This
advantage has led to the development of several submersible
holography systems for studying plankton, starting with a
sample volume of a few ml (Carder et al., 1982), to samples
of one liter and above (Katz et al., 1999; Malkiel et al., 1999;
OHern et al., 1988; Watson et al., 2001). The latter utilize
pulsed lasers and emulsions as recording media.
Cinematographic holography was introduced for laboratory
research decades ago (Heinger et al., 1978; Knox and Brooks,
1969), but difficulties in acquiring and processing data resulted
in limited applications. Recent development in digital imaging,
which simplies the acquisition, and computing power, which
enables numerical reconstruction, has led to renewed interest
in cinematic holography (Kebbel et al., 1999; Owen and
Zozulya, 2000; Xu et al., 2001). The limited resolution of
digital imaging, which is at least an order of magnitude lower
than that of holographic emulsions, restricts us to in-line
holography. This technique (details follow) maximizes the
fringe spacing. Because the reference beam typically passes
through the sample volume, it becomes increasingly degraded
with increasing particle concentration. Consequently, the
reconstructed images are noisier and the maximum particle
concentration is lower compared to off-axis holograms (Zhang
et al., 1997).
Holographic PIV is the only technique to date that can
measure a 3-D instantaneous velocity distribution over a nite
volume (Barnhart et al., 1994; Pu and Meng, 2000; Sheng et
al., 2003; Tao et al., 2002; Zhang et al., 1997) at a resolution
of millions of vectors. The velocity is obtained by recording
two exposures of a ow eld seeded with microscopic
particles, and measuring the displacement of these particles.
However, the depth coordinate of a reconstructed particle is
less accurate than the lateral coordinates, severely reducing the
ability to estimate the corresponding velocity component. This
problem has been solved by recording two inclined holograms,
using each for determining a 3-D distribution of two velocity
components, and matching the two sets to obtain the 3-D
velocity. Utilizing emulsion and off-axis holography, Tao et al.
(2002) measured 136130128 3-D velocity vectors in a cubic
sample with sides of about 45mm. By inserting an inclined
mirror in the path of the illuminating beam inside the test
facility, the incident and reected beams create two views that
can be recorded on the same emulsion (Sheng et al., 2003).
Using off-axis holography, this method enables measurement
of particle locations to within 7m, and resolves about
200particlesmm
3
. We adopt this approach, but use digital in-
line holography to record a time series of a free-swimming
copepod and the ow eld surrounding this animal. To our
knowledge, this is the rst time that digital holographic PIV
has been implemented as a tool for simultaneous observation
of the animals behavior and measurement of the complex ow
around it.
Materials and methods
Digital in-line holography is a two-step process, consisting
of rst recording an in-line hologram on a digital recording
medium, and then numerical reconstruction. The recording
process entails back-illuminating a sample volume lled with
objects by a coherent light source. A diffraction pattern is
generated as a result of interference between the illuminating
beam and light scattered from the objects. Coherence of the
light source is important in order to enhance the visibility (or
contrast) of the interference pattern, which conveys
information on the shape and location of an object in space. As
shown in Fig.1 we use a standard, single mode, 3mW
HeliumNeon laser, which generates a CW (continuous wave)
polarized beam of red light (632.8nm) with a coherence length
far beyond that required for digital in-line holography (which
is on the order of 1cm). The beam is spatially ltered and
expanded to cover at least the entire test section. Collimating
the expanded beam enables recording of the diffraction
patterns at a constant magnication. This nonessential choice
simplies the numerical reconstruction by avoiding a
calibration procedure to determine the origin of the
illumination source.
The expanded beam illuminates an 80ml sample volume
constructed of antireection-coated windows attached to two
supporting prisms. First-surface mirrors are attached to the
inclined side walls of the test chamber to direct the illumination
beam through the test section. These mirrors provide two
perpendicular views of objects located in regions where the
incident and reected beams overlap (cf. Fig.2). One view is
created by light that is rst reected from the mirror and then
incident on the object, while the other (mirrored view) is
generated by light incident on the object and then reected off
the mirror. The two views are recorded onto a single recording
plane, but they are laterally separated. During reconstruction,
the rst view appears in focus at its original location, while the
second perpendicular view is a mirror image (right side of
Fig.2). Use of two mirrors (Fig.1) doubles the triangular
overlapping volume without necessitating an increase in
recording area.
E. Malkiel and others
Fig.1. Optical setup for digital in-line holography and test section.
HeNe Laser
Kodak ES 4.0
2Kx2K; 15 fps
First surface mirror
Overlap region
Test section CCD camera Light source
ND
filter
Spatial
filter
Collimating
lens
32 mm
3659 Three-dimensional copepod ow eld
The interference patterns are recorded on a lensless
Megaplus ES 4.0 digital camera, which has a 20482048 pixel
CCD sensor with a 7.4m pitch, providing a 15mm15mm
eld of view. The in-line system minimizes the angle between
the light scattered from the objects and the remaining reference
beam. Consequently, the fringe spacing is maximized (Vikram,
1992), overcoming the limited resolution of the CCD. The
CCDs interline transfer capability eliminates the need for a
shutter. Because the experiment involves recording moving
objects (a copepod and seed particles), the exposure time is
adjusted to be short enough to prevent the smallest resolvable
fringe spacing from smearing. Here, the velocity does not
exceed 4mms
1
(540pixelss
1
), and the electronic shuttering
of the camera (0.1ms exposure time) is sufficient to reduce the
movement during exposure to much less than 1pixel. Faster
ows (~1ms
1
) would require higher power, even pulsed
lasers, to generate the energy required for recording a
hologram. Images have been recorded at the maximum frame
rate of the present camera, 15 frames per second, buffering 10s
segments (150frames) to the RAM of the computer hosting the
image acquisition card.
Our subject animal, a female Diaptomus minutus Lilljeborg
1889, was freshly caught in Lake Michigan and transported to
Baltimore in a Dewars jar. The test section was lled with
artesian well water from the Pryor Street well in Milwaukee.
It was seeded with monodisperse, 20m diameter polystyrene
spheres (specic gravity 1.05) at a concentration of 4mm
3
.
Although denser seeding would improve the spatial resolution
of the ow, it would also reduce the signal-to-noise ratio of
reconstructed images. Image acquisition to RAM was started
when the copepod appeared in the eld of view. Successful
image sequences, which captured the copepod in a region with
two views, and were sufficiently far from the wall (>4mm),
were stored. Fig.3 is a sample digital in-line hologram
containing two interference patterns (shadows) of the same
copepod, along with the very faint diffraction patterns of the
seed particles.
Digital reconstruction regenerates the original light intensity
distribution at any desired distance from the recording plane.
The amplitude of light U(x,y,z) at any point in the
reconstruction volume is a superposition of light reaching this
point from an array of source wavelets distributed over the
entire recording plane (the hologram). Using the
FresnelHuygens Principle (Hecht, 2002) with a paraxial
approximation,
Here, (,) are coordinates of the hologram, indicates the
area of the entire hologram, indicates convolution and
k=(2/), where is the reconstruction wavelength. If is set
equal to the recording wavelength, the volume is reconstructed
at its original depth. The intensity of the light, I(x,y,z), is
calculated from I(x,y,z)=UU*, where U* is the complex
conjugate of U. Use of the paraxial approximation is justied
because we record predominantly forward scattered light.
Furthermore, due to the limited resolution of the recording
medium, the camera can resolve only near forward
fringes. Equation1 is actually a 2-D convolution integral
parameterized by z, the distance between the hologram and the
reconstruction plane. The kernel h
z is the 3-D eld generated
by an innitesimal point source located at (x,y,z). The
discretized version of this convolution is performed over the
entire hologram for each desired depth. Because of the large
convolution kernel and image size, the integration is more
efficiently performed in Fourier space. After calculating the
U(x,y,z) =
hZ(x,y) = exp . [(x)
2
+ (y)
2
] j

I(,) h
Z
(x,y) dd = I h
Z
,
exp(jkz)
jz
k
2z

'

;
)
(1)
Mirror
(reconstructed)
Mirrored view
Original
view
Fig.2. Recording and reconstruction of an object near a mirror.
Fig.3. A sample digital hologram containing two views of the same
swimming copepod in a seeded test section. Scale bar, 1mm.
3660
2-D Fourier transforms of both the recorded image and the
convolution kernel, they are multiplied. Then, an inverse
Fourier transform provides the intensity distribution in the
desired plane. A similar approach is presented in Milgram and
Li (2002), where further details on the mathematics can be
found. We typically reconstruct 45 planes separated by
0.2mm.
The purpose of the next phase of the analysis is to match the
in-focus perpendicular views of particles in order to determine
their exact locations in space. Recall that a single view can
provide lateral positional accuracy to within a pixel (7.4m)
in the lateral plane, but is substantially less accurate in the axial
direction (0.5mm). These views are laterally separated (as in
Fig.3). An automated procedure for matching the two views
in densely seeded ows recorded using off-axis holography (on
emulsion) is outlined in Sheng et al. (2003). There, the array
of reconstructed images is thresholded, the particles are
segmented into 3-D volumes, and then reduced to line
segments that pass through the barycenters of these volumes
and have lengths corresponding to their axial extent. The
intersection of two perpendicular line segments determines the
3-D coordinates of the particle centroid. The analysis requires
the calibration of the location and orientation of the mirror,
achieved by manually matching corresponding views of
several reference particles. The calibrated mirror orientation is
used to transform (ip) the mirrored views onto their original
locations in space. For two views of the same particle to be
matched, we require that the perpendicular segments are less
than a particle diameter away from each other. The 3-D
coordinates of the particle centroid are approximated as the
midpoint of the shortest line segment connecting the views.
This approach did not work while analyzing the present in-
line digital holograms. The primary reason was excessive
background noise resulting from the reference (illuminating)
beam passing through the sample volume. It should work with
less heavily seeded ows and/or an optical setup with a
separate reference beam that does not pass through the sample
volume. The limited resolution of the digital camera prevented
the implementation of off-axis holography, which is
characterized by much ner fringe spacing. Consequently, we
have used two semi-manual techniques to obtain the 3-D
locations of particles and velocity elds.
The rst task is to identify particles and distinguish them
from background noise. Three successive exposures are
superposed, and elongated traces, or three closely spaced spots
resulting from the displacement of particles, are identied. This
approach has turned out to be a very effective tool for
distinguishing between real particles and speckle noise. Based
on the location of these traces, we estimate (computationally)
the most likely location of the mirrored view. The linear
transformation used for this calculation is calibrated by
matching corresponding views of the copepods extremities,
which can be easily identied. The mirrored view lies along an
almost horizontal line that corresponds to the depth uncertainty
of the rst view. If we nd three spots (or elongated traces)
along this line, the two views of the same particle are matched.
At the present particle concentration, having more than one
match is very unlikely. If we do not nd the second view of a
particle along the expected line, for example, when it is located
in the shadow of the copepod, the unmatched trace is
disregarded.
When the second view is found, the program proceeds to
measure the particle displacement in each of the two views,
using cross-correlation of the intensity distribution, generated
by the three exposures. Details on the cross-correlation
procedures, including methods of achieving sub-pixel
accuracy, are discussed by Roth and Katz (2001), Roth et al.
(1999) and Sridhar and Katz (1995). The lateral displacement
in each of the views and the average vertical displacements are
combined into a 3-D velocity vector, positioned at the particle
mean location. The uncertainty in velocity of individual
particles is about 0.05mms
1
.
Extended particle tracks in the vicinity of the copepod are
more easily identied. Superposing sets of ten exposures near
the plane of the copepod in each view, and placing them side-
by-side, allows identication of corresponding tracks based on
their elevation at a given time. This identication is veried by
comparing subsequent sets of exposures. Combining the sets
generates the 3-D trajectory of the particle. Even when one of
the views is partially blocked by the copepod, it is usually
possible to match the segments that are not blocked and then
interpolate them to estimate the trajectory in the blocked
section. The endpoints of segments are stored and used for
measuring the velocity along the path of the particle. In regions
with relatively high velocity, the tracks appear as a series of
dots. In this case, individual traces are used for measuring the
velocity as discussed before.
Results
Fig.4 shows reconstructed perpendicular views of the
copepod and seed particles, computed from the hologram
shown in Fig.3. Note that the two views are reconstructed at
different depths. Clearly, digital reconstruction of in-line
holograms can resolve ne details of the copepod structure.
Sample instantaneous distributions of 3-D velocity vectors in
the vicinity of this copepod in the ambient and copepod
reference frames are presented in Fig.5A and B, respectively.
The graphic representation of the copepod has the correct
scales. Vectors that are located within the central two thirds
span of the antennules are black; vectors outside of this span
are gray. A recirculating ow pattern is evident in the copepod
frame of reference, which sinks at an average speed of
0.29mms
1
. Similar sets of 3-D velocity distributions are
obtained from each pair of reconstructed holograms.
The reconstructed images from 15 sequences consistently
show that the copepod sinks for several seconds. It then
executes a short hop upwards and resumes sinking (see
supporting movie). As it sinks, the copepod generates a feeding
current by moving its feeding appendages. The present 15Hz
recording rate may not be sufficient for following the (high
frequency) motion of the appendages, but different phases of
E. Malkiel and others
3661 Three-dimensional copepod ow eld
their motion are discernible (Fig.4). The high-speed ow
generated by the appendages (Fig.5A) is most evident in the
ventral region of the copepod (z<79.5mm), extending to its
anterior and posterior regions. The feeding current generates a
reaction force that propels the copepod. This vertically directed
force acts against the copepods excess weight (weight minus
buoyancy) and drag, reducing its sinking rate compared to the
terminal speed (speed with no feeding current).
The recirculating ow pattern generated by the copepod is
clearly demonstrated in Fig.6 by combining 130 reconstructed
images, shifted in time, so that the image of the copepod is
xed. Since the copepod velocity is constant during this period,
the shift applied to each image is a linear function of time.
Blurring of the copepod occurs due to slight variations in
sinking speed (<5%), and motion of the appendages. Streaks
generated by the seed particles are clearly evident in both
Fig.5. Measured instantaneous velocity near the copepod (A) in the ambient frame of reference, and (B) in the copepod frame of reference.
78
80
8
6
4
1 mm s
1
z (m
m
)
x
(
m
m
)
78
80
8
6
4
1 mm s
1
z (m
m
)
x
(
m
m
)
12
10
8
y

(
m
m
)
12
10
8
y

(
m
m
)
A
7.3
5.3
x B
7.3
5.3
x
Fig.4. In-focus, numerically reconstructed, dorsal (A) and lateral (B) views of the same swimming copepod, from the hologram of Fig.3. s,
setae on antennule; f, feeding appendages; p, tracer particle (there are many). Inserts show the feeding appendages in up-stroke (top) and down-
stroke (bottom) positions. Scale bar, 1mm.
3662
views. Above the copepod, several particles are drawn towards
the center of the copepod. Below and to the sides of the
copepod, particles are ejected downwards, subsequently
looping around and migrating upward (relative to the
copepod), some of them touching its antennules. Quantitative
data on the trajectory and velocity along the path of selected
particles are presented in Figs7 and 8. The velocity peaks in
a narrow domain near the tips of the appendages, reaching
3.6mms
1
, i.e. 12.5 times the sinking velocity. We use these
data to estimate the propulsive force generated by the feeding
appendages.
Excess weight and propulsive force
The particles slightly beyond the reach of the antennules
circumvent the copepod, creating a closed streamline pattern,
without a separated wake. This pattern is characteristic of low
Reynolds number ows (ReL=UL/, U and L being
characteristic velocity and length scales, respectively, and ,
the kinematic viscosity of the liquid), such as Stokes ows with
Re<<1, or Oseen ows with Re~1 (Pozrikidis, 1992). The
present Re, based on the sinking velocity and prosome length,
is 0.29. Based on the diameter of the recirculation zone, Re
increases to 1.2. Thus, the present ow lies in the transition
region between Stokes and Oseen ows. Here we use the
simpler Stokes ow in order to estimate the magnitude of the
forces produced by the copepod.
The horizontal (u) and vertical (v) velocity components
induced by a vertical point force (Stokeslet) in Stokes ow
(Jiang et al., 2002c; Pozrikidis, 1992) are:
where f is the force, is the liquid viscosity, x and y are
coordinates, and r is distance from the origin. The Stokeslet
describes the far-eld ow realistically, but neglects the fact
that the object has a nite size. The far-eld net effect of the
copepod on the surrounding ow, its excess weight (w
excess),
can be modeled as a Stokeslet.
The ow pattern generated by a Stokeslet in an absolute
frame of reference is illustrated in Fig.9A, with
Uref=wexcess/8L. A recirculating ow pattern appears in a
frame of reference moving downward at a fraction of Uref
(Fig.9B). For the recirculation to form, there must be a
residual downward velocity component (jet) below the object
in its own reference frame. This ow can only be generated
by a propulsive force. Thus, in reducing its sinking speed and
generating a propulsive feeding current, the copepod creates
a recirculating pattern that extends slightly beyond its
antennae. This combination of sinking and feeding is not
discussed in detailed conceptual and numerical analyses of the
forces acting on a swimming copepod under various
v = + and ,
f
8L
(y/L)
2
(r/L)
3
1
r/L
(2)

,
u =
f
8L
xy/L
2
(r/L)
3

,
E. Malkiel and others
Fig.6. Particle streaks in the copepod reference frame obtained by combining 130 appropriately shifted reconstructed images. In all cases the
dorsal and lateral views are in focus.
3663 Three-dimensional copepod ow eld
conditions (Jiang et al., 2002b,c). They include cases of
stationary bodies producing feeding currents (hovering or
conceivably tethered) and freely sinking bodies, both of which
do not generate recirculating patterns.
The data can be used for estimating wexcess. In a reference
frame sinking at vsink, the vertical velocity component vanishes
at y0=wexcess/4vsink. Estimating y0 as half the distance
between the points with zero velocity in Fig.6 (y0=2mm), and
since vsink= 0.29mms
1
, one obtains wexcess=7.210
9
N. With
a volume of 1.110
10
m
3
(determined following Chojnacki,
1983), the estimated excess density of the copepod is
6.7kgm
3
(1006.7kgm
3
). This value falls in the range
measured by Svetlichny (1980) and Knutsen et al. (2001). The
same analysis can be performed in any frame, including one
y
x
z
4
Speed
(mm s
1
)
2
0
Fig.7. Selected particle tracks (16) in 3 dimensions. AA, see inset
in Fig.8.
4
3
2
1
0
0
0
0
0
4 2
Time from capture region (s)
S
p
e
e
d

(
m
m

s

1
)
0 2 4
0.2
0.15
0.1
0.05
0.25 0 0.25
z (mm)
w

(
m
m

s

1
)
0.5
0
Fig.8. Speeds of selected particles (16) approaching feeding
appendages. Inset: Horizontal velocity component w near tail along
line AA of Fig.7. z axis origin at center of mass.
10
A B
5
0
5
10
10 5 0
x/L
u/U
ref
=1
y
/
L
5 10 10 5 0
x/L
u/U
ref
=1
5 10
Fig.9. Flow eld generated by a Stokeslet (see Equation3) at (A) the absolute reference frame and (B) the reference frame sinking at 0.33Uref.
3664
without recirculation, by measuring the velocity and distance
between points above and below the copepod with the same
velocity.
One can also estimate the propulsive force, P, generated by
the feeding appendages. Averaging v of a Stokeslet at y=0 over
a certain radius, R, one obtains v =f/(4R). Based on Figs78,
there are two regions with a radius of 200m and characteristic
peak velocity of v =3.6mms
1
in the vicinity of the feeding
appendages. Combining the two regions, P=2(4Rv ), i.e.
P=1.810
8
N, 2.5 times higher than wexcess. At a constant
sinking velocity, P must balance the sum of wexcess and the drag
force (Jiang et al., 2002c). Since the relative velocity around
the copepod is downward (Figs58), so is the drag, requiring
P to be larger than wexcess. Thus, the estimated drag is about
1.5 times the excess weight. The propulsive force also
generates a moment (negative x direction), since it is applied
at about 30m in front of the centerline of the prosome, based
on the location of maximum v . The magnitude of this moment
is about 5.410
13
Nm. To overcome this moment and avoid
tumbling, the copepod turns its tail, creating a velocity bias in
the positive z direction (see insert in Fig.8). Turning the ow
creates a reaction force and a moment in the positive x
direction. Based on the average velocity bias (~0.1mms
1
,
Fig.8), over a radius of 250m (half the tail length), the force
is 0.610
9
N. Multiplying it by the distance to the center of
mass of the copepod (1mm), the estimated moment is
610
13
Nm. Thus, the moment generated by the tails
redirection of uid is of sufficient magnitude to compensate
against the moment of the propulsive force, allowing the
copepod to maintain a relatively vertical orientation.
Discussion
The discussion is in two parts. The rst compares
holography to other 3-D imaging techniques, and the second
focuses on the behavior of a copepod.
Comparison with other techniques
It is useful to compare the capabilities and difficulties of
digital in-line holography with other 3-D particle tracking
techniques, such as Stereo PIV and 3-D particle tracking with
multiple cameras. 2-D PIV (Bartol et al., 2003; Drucker and
Lauder, 2001; van Duren et al., 1998; Wilga and Lauder, 2002)
provides high density data, but only in a plane. Typical stereo
PIV (Nauen and Lauder, 2002; Prasad, 2000) provides all three
velocity components, but still only in a plane. It also requires
elaborate calibration procedures. Existing scanning PIV
(Brucker, 1997) and potentially future stereo-scanning PIV,
provide data in multiple planes at different times at the cost of
added complexity. All are unsuitable for following the 3-D
trajectories of swimming organisms.
Alternatively, multiple pinhole photography (Kieft et al.,
2002; Maas et al., 1993; Moroni et al., 2003; Ott and Mann,
2000; Stuer et al., 1999; Virant and Dracos, 1997) and its
variants (Pereira and Gharib, 2002) can be used for 3-D
tracking of particles. The increased depth of focus required by
this method is inherently coupled to reduced resolution and the
need for bright illumination. Conversely, in holography the
image resolution does not have to be compromised. Fine
details, e.g. setae and swimming appendages can be imaged at
any depth. Furthermore, in pinhole images, the elongated
traces of particles extend in depth through the entire volume
of interest. Consequently, matching of perpendicular views can
only be performed at low particle concentrations. Multiple
camera systems partially overcome this effect and can provide
as many as 1600 vectors per time step, and may track as many
as several hundred particles over extended periods (Virant and
Dracos, 1997). Such systems require extensive calibration and
relatively complex processing algorithms. In in-line
holography, the elongated traces extend in depth less than
1mm, enabling matching of views at concentrations as high as
several particles per mm
3
(i.e. 13500 in the present
overlapping volumes). The associated calibration process is
also straightforward. On the other hand, one of the
shortcomings of in-line holography, as shown in this paper, is
the noise generated in a great part by deterioration of the
reference beam. This problem can be partially resolved by
using a separate reference beam that does not pass through the
sample volume. A separate reference beam should also allow
a much higher seeding concentration, but at the cost of added
complexity to the optical setup. When using high-resolution
emulsions, instead of a digital camera, we have successfully
reconstructed 200 particles per mm
3
. Another shortcoming of
holography is inherent to the use of coherent light, which
makes the system more sensitive to the quality of windows and
variations of density within the sample volume.
As shown in this paper, digital holographic PIV is a
relatively simple, but powerful tool for analysis of 3-D
copepod (or other organism) dynamics and its interaction with
its local environment, be it with other organisms or the local
3-D ow eld.
Copepod behavior
While generating the feeding current (Strickler, 1982, 1984)
the mouthparts, studded with chemoreceptors (Friedman and
Strickler, 1975), scan the water owing by them. When the
presence of a food particle is perceived, additional movements
of the mouthparts capture the particle and bring it to the mouth
(Koehl and Strickler, 1981; Strickler, 1984, 1985). Sensory
setae on the stretched out antennules (Fig.4; Huys and
Boxshall, 1991) are mechanoreceptors (Strickler and Bal,
1973), as well as chemoreceptors (Bundy and Paffenhfer,
1993). These receptors enlarge the volume of water scanned
for food (Jiang et al., 2002a; Strickler, 1985). Particles that do
not smell good enough are either actively rejected after
capture by the mouthparts, or passively rejected (allowed to
pass without being captured). For a stationary hovering
copepod, these rejected particles remain in the water below the
copepod, and are not entrained into the feeding current again
(Strickler, 1982).
The recirculating pattern in Figs58 is, to our knowledge,
the rst report of a multi-encounter feeding pattern in calanoid
E. Malkiel and others
3665 Three-dimensional copepod ow eld
copepods. The combination of feeding and sinking results in
particles being drawn toward the copepod with its feeding
current, passively rejected and then recirculated. The
recirculation is interrupted aperiodically, after 8.7s in the
present example, when the copepod hops. During a hop, the
copepod jumps about 0.5mm upward to a position where its
mouthparts are just below the stagnation point of the previous
recirculation zone. If it were not for the hop, the copepod would
never encounter new particles, due to the closed recirculation
pattern. The recirculation allows the copepod to taste some of
the particles that have passed near the mouthparts once more,
this time with different, perhaps even more sensitive
chemoreceptors on its antennules. Considering that the 20m
polystyrene, spherical particles are mechanically desirable but
chemically undesirable, this additional sensing by the sensors
on the antennules ensures that the animal does not forfeit
potentially good food. The available sequences of holograms
suggest that the timing between hops is sufficient for a rejected
particle to reach the antennule (about half the recirculation
cycle). We speculate that once the copepod senses, using its
antennules, a particle that has already been tasted and rejected
(and is still not acceptable), it hops to another volume to look
for different food. These assertions require testing by altering
the properties of the particles, e.g. replacing them with desirable
food, and observing the behavior of the copepod.
This work was supported by the National Science
Foundation. J.R.S. dedicates this work to Owen Phillips for
his support at a crucial moment years ago.
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