Journal Name Dynamic Article Links ►

Cite this: DOI: 10.1039/c0xx00000x

www.rsc.org/xxxxxx ARTICLE TYPE
C.L.I.P. – Continuous Live Imaging Platform for Direct Observation of
C. elegans Physiological Processes
Jan Krajniaka, Yan Haob, Ho Yi Makb, and Hang Lua,c,d
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5 DOI: 10.1039/b000000x

Direct observation of developmental and physiological changes in certain model organisms over time has
been technically challenging. In the model organism Caenorhabditis elegans, these studies require
frequent or continuous imaging at physiologically benign conditions. However, standard methods use
anaesthetics, glue, or microbeads, which prevent animals from feeding during the experiment. Thus,
10 animals’ normal physiological function may be affected over time. Here we present a platform designed
for dynamic studies of C. elegans. The system is capable of immobilizing only animals’ bodies under
benign conditions and without physical deformation. Simultaneously, animals’ heads remain free to move
and feed for the duration of the experiment. This allows for high-resolution and high-magnification
fluorescent imaging of immobilized and feeding animals. The system is very easy to fabricate, set up, and
15 operate, and should be widely applicable to many problems in development and physiology.

Deeper understanding of the human nervous system is critical for
understanding neurodegenerative diseases, discovering drug
targets, and developing novel treatments. However, studying the
human nervous system presents significant and at times
20 insurmountable experimental complexity. Genetic manipulation
or in vivo live observation of responses to stimuli at cellular and
sub-cellular levels is currently impossible. Many fields of biology
including neuroscience take advantage of model organisms
because many of the molecular and genetic mechanisms are
25 conserved among these species and human1-4. Caenorhabditis
elegans has served as one of the model organisms for studying
the development, function, generation of behavioral responses,
and genetic regulation of the nervous system 5-20. The nematode
is transparent and thus ideal for fluorescence imaging. It is
30 hermaphroditic, and as such has conserved genetics and
development across a population. Additionally, the C. elegans
genome has been sequenced and a variety of genetically
characterized mutants along with gene and RNA expression
manipulating techniques are available to researchers.
35 Thus far, studies in C. elegans have contributed to our
understanding of synaptic development, synaptic vesicle
transport, neuronal signal and regulatory pathways, and the
genetic regulation of these and many other processes4, 18, 21-30.
Among these areas of neuroscience research, dynamic studies of
40 individual animals have become increasingly more relevant.
Dynamic studies can provide much greater detail of a process in
question due to live and continuous observation of individual
animals and the additional data generated. For example,
observation of regenerative properties of neurons after
45 microsurgery, frequency of synaptic vesicle trafficking, or rates
of formation and re-arrangement of synapses have each provided
unique insights into roles and effects of various genes.
Unfortunately, live dynamic studies of C. elegans can be quite
difficult to implement experimentally.
50 The difficulty of dynamic studies lies in the need for frequently
repeated or continuous imaging of animals at high magnification
and resolution over extended periods of time, while maintaining
normal physiological function. High resolution imaging of C.
elegans necessitates complete immobilization. The standard
55 method of immobilization uses anesthetics like sodium azide,
glue, or microbeads on an agarose pad31, 32. This approach
prevents the feeding of animals for the duration of experiment
and can affect physiological processes over extended periods of
time. Lavamisole has been successfully utilized to immobilize
60 animals without affecting the function of the nervous system32.
However, lavamisole treated animals are not able to feed.
Microfluidic methods such as immobilization via cooling, by
compression with a membrane or narrowing channel geometry, or
immobilization via CO have also been showed to immobilize
65 animals sufficiently11, 33-37. However, these immobilization
methods cannot be applied over long periods of time without
affecting physiological processes and also do not allow for
feeding while the animals are immobilized. Hulme et al.38
recently presented a microfluidic system capable of culturing
70 animals for long-term studies. Animals are immobilized for
imaging intermittently via tapering channel geometry and
allowed to feed and grow most of the time. Rhode et al.39 have
presented a system based on a similar principle. Here, however,
animals are immobilized via cooling. We have also recently
75 presented a system capable of culturing and repeatedly imaging
animals. Immobilization for imaging is achieved via a thermo-
reversible sol-gel transition of a solution of the polymer F12740.
The formed gel is capable of immobilizing animals without
affecting normal physiological processes. These three methods

This journal is © The Royal Society of Chemistry [year] [journal], [year], [vol], 00–00 | 1
 Journal Name Dynamic Article Links ►

Cite this: DOI: 10.1039/c0xx00000x

www.rsc.org/xxxxxx ARTICLE TYPE

Fig. 1 Microfluidic device component of the platform. a) The device is a simple array of trapping channels. Animals and solutions are loaded through the
worm loading inlet. From here animals are moved to the 8-channel array. Bodies are immobilized inside the channels with the immobilizing agent,
Pluronic F127 solution (or Sodium alginate/Gelatin mixture) and microbeads. Animals heads are free to move and feed inside the head stop muzzle.
5 Nutrients are delivered to the muzzle through media inlets via symmetric bifurcated flow. b) The device is extremely simple and only has 5 inlets/outlets.
Positioning of animals is done passively via geometry design. The muzzle is sized to only allow the head of an animal to enter. Spacing between pillars
prevents animals’ escape. Bumps in the trapping channel help prevent sinusoidal body movement of animals.

are applicable to long-term processes, where intermittent imaging

with long intervals of feeding in between is sufficient to capture
10 the process dynamics. However, for processes with dynamics
requiring very frequent or continuous imaging, even these
methods are insufficient; animals cannot feed while immobilized
affect physiological processes are affected with extended
immobilization.
15 Here we present a microfluidic platform capable of immobilizing
just the animal body while leaving the head free to move, and
doing so for a prolonged period while we observe physiology in
vivo. The worm body is immobilized at physiologically benign
conditions without any physical deformation. At the same time,
20 the animals are fed via their freely moving heads. This approach
ensures proper physiological function during the experiment. The
platform combines a microfluidic device and an immobilization
agent. The microfluidic component is an array of channels used
to properly position animals (Fig 1). The second component is an
25 immobilization agent for the portion of animals’ bodies in the
trapping channels. The combination allows us to observe
dynamic processes without artifacts and changes caused by
alteration of normal physiological function. The platform is
designed to be extremely simple to prepare for experiments and
30 to not require user manipulation after initial preparation. It also
requires minimal use of external peripheral devices to work and
can be easily applied in any laboratory.
MATERIALS AND METHODS
Device fabrication and setup
35 We fabricated all microfluidic devices using a combination of
microfabrication and soft lithography techniques41, 42. In short,
the design was prepared in AutoCAD 2012; photolithography
chrome masks were prepared by Elvesys (Paris, France). The
mask pattern was transferred to masters of SU8-2025 photoresist
40 (Microchem) spun to 60 µm on Si wafers following standard
protocol by the manufacturer. After developing the resist, the
finished master was treated with tridecafluoro-(1,1,2,2-
tetrahydrooctyl)-1-trichlorosilane silane (UCT Specialties, LLC).
The microfluidic chips were molded from the masters into
45 PDMS. A 5:1 mix of pre-polymer to cross-linker mixture was
poured over the master to a depth of 4 mm, degassed in a
desiccator for 2 hours to allow the PDMS to enter into the small
features, and cured for two hours at 70 ºC. Afterwards, the
individual chips were cut out and fluid interconnect holes were
50 cut. Seven holes are required to operate the device - a
7 gauge hole into the device loading chamber, and a total of six
21 gauge holes. The 21 gauge holes are cut at the loading
chamber flushing outlet, at the device liquid withdrawal outlet, in
the media delivery channel (x2), at the media inlet, and at the
55 stimulant inlet each (Fig 1). The 7 gauge hole is cut at the loading

This journal is © The Royal Society of Chemistry [year] [journal], [year], [vol], 00–00 | 2
 chamber inlet. The whole chip was then cleaned with clear tape
and compressed air, followed by bonding to a glass slide or cover
slip via oxygen plasma treatment for 20 sec (PDC-32G plasma
cleaner).
5 The 21 gauge holes are connected to PE-60 tubing (Micro
Medical Tubing, Scientific Commodities, Inc.) via metal pins for
fluid delivery. All liquid manipulation was performed via syringe
pump (NE-1000, New Era Pump Systems). Liquids were loaded
into the 7 gauge hole directly via micropipeter. The hole serves as
10 an open liquid pseudo-reservoir.
Materials
Solution of sodium alginate (Sigma Aldrich) with gelatin in water
was prepared at 0.75% w/v sodium alginate and 15% w/v gelatin.
Prior to the experiment, the solution was boiled for proper mixing
15 and rapidly cooled to 40 ºC prior to loading. It was applied to the
device at 25 ºC. Solution of Pluronic F127 was prepared at 25%
w/v. All solutions were filtered through a 0.2 um nylon
membrane filter (MF-75, Nalgene) to remove particulates.
Standard nematode buffer solution containing OP50 bacteria was
20 prepared by centrifuging OP50 bacteria suspended in LB
medium, removing the supernatant, and re-suspending the
bacteria in M9 buffer at OD600=1.0.
C. elegans culture, experimental procedure, and microscopy
C. elegans were cultured at 20 °C on standard nematode culture
25 plates with OP50 bacterial lawns, and were suspended in standard
nematode buffer43 for experiments. We used the N2 strain for all
experiments where fluorescence was not required. The hjSi3;
hjSi112 (carrying R01B10.6::GFP and mRuby::DGAT-2)
strain was used for all fluorescence studies.
30 To measure the ability of animals to feed during the experiment,
we measured the pharyngeal pumping rate of animals trapped on
device and animals on standard plates. The rate was quantified by
counting the number of bulb contractions in the pharynx over a
period of 2 minutes.
35 Temperature calibration and measurement in device was
performed by measuring fluorescent intensity of Rhodamine B
(Rho B) and Rhodamine 110 (Rho 110)44. Calibration was
performed using a thermocouple suspended in a large reservoir.
The reservoir was filled with Pluronic F127 solution containing
40 Rho B/Rho 110 at a 1:1 ratio. It was cooled to 4 C and then
placed on the microscope. The fluorescent intensity of each dye
was recorded at 0.5 C intervals. The Rho B/Rho 110 intensity
ratio data was then used to calculate a correlation between
temperature and intensity ratio. Temperature measurement on a Fig. 2 Simple loading procedure. a) Animals are loaded into the device
45 microfluidic device was then performed using the standard 60 via the chamber inlet. They are pulled into and positioned in the trapping
loading procedure. A movie was recorded starting immediately channels by withdrawing liquid from the outlet. b) Next, solution
containing 10 µm microbeads is loaded. This solution is used to remove
after the cooled PF127 solution was loaded into the device to excess animals from the loading chamber. Also, the microbeads
measure temperature changes in the device as they would occur accumulate at the tail and physically immobilize it. c) Then, the
during an experiment. Frames were extracted at each time point 65 immobilizing solution of Pluronic F127 (or alternatively mixture of
50 of interest to measure intensities of each dye and processed using sodium alginate and gelatin) is applied while cooled to 0 ºC (sodium
alginate/gelatin is applied at 25 ºC). The solution warms to ambient
ImageJ. We measured the temperature at the inlet to each
temperature. During this time it crosses through the sol-gel transition
trapping channel. Measurement of sodium alginate/gelatin temperature of 17 ºC and forms the immobilizing gel. d) Lastly, nutrients
solution temperature was performed with a standard 70 are delivered to the animals for feeding through the media inlets via the
thermocouple prior to use. symmetrical bifurcated flow. (Insert) If the alternative immobilizing agent
55 Gel behavior on chip was observed via recordings of PF127 is used, the inlet and outlet used are positioned as shown. Additionally,
zinc gluconate is applied through the loading inlet to trigger the gelation
solution dyed with Rhodamine B. Movies were recorded at each
of sodium alginate component.
stage of the setup and experimental process, frames were

This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 3
 extracted at time-points of interest, and thresholded to determine
the gel-media boundary in the immobilization channels and the
media delivery channel.

SYSTEM OPERATION
5 To design a platform that is easy to use for a variety of
applications in any laboratory, we placed emphasis on simple
fabrication, an extremely simple experimental setup, and minimal
requirements for external peripherals, while maintaining all the
necessary functionality.
10 The device is a single-layer PDMS-based device and has no
active components such valves. After initial setup, the device
does not require any additional input from the researchers. It
consists of a loading chamber (Fig 1), which is used to load all
liquids and animals. The inlet to this chamber is cut with a 7
15 gauge cutter. The inlet is cut to this size to serve as a liquid
pseudo-reservoir on device. From this chamber, animals are
loaded into the array of trapping channels (Fig 1) by withdrawing
liquid through the main outlet (Fig 2). In these channels, their
bodies are immobilized with our immobilizing agent. However,
20 the animals’ heads protrude into an array of pillars, or the
“muzzle” (Fig 1). The array width and length dimensions are
designed to accommodate the head of the animals to the second
pharyngeal bulb. The pillars are spaced 6 µm apart to prevent
animals from escaping. This array serves multiple purposes. First,
25 it stops the forward motion of the animals. This leads to proper
positioning; bodies remain inside the channel, and heads are
positioned in the muzzle. Second, it allows flow to enter the
muzzle area. This facilitates delivery of nutrients and stimulants
to the animals’ heads. These are delivered to the muzzle from the
30 nutrient and stimuli inlets via a symmetric, bifurcated flow (Figs
1, 2).
While the microfluidic chip places the animal in position, we use
an immobilization agent to suppress the body movement. The
immobilizing agent is either a solution of Pluronic F12740 or a
35 solution of sodium alginate with gelatin. Both of these are used in
combination with microbeads. We chose the Pluronic F127
solution because it undergoes a thermo-reversible sol-gel
transition. This transition is rapid, requires a very small
temperature change (<1 ºC), and the desired transition
40 temperature can be easily tuned with concentration. Additionally,
the formed gel does not affect animal physiology and does not
cause physical deformation. The ideal ambient temperature for
using this solution is between 18 to 20 ºC. Sodium
alginate/gelatin solution can be used as an alternative when Fig. 3 Device functional characterization. a) Single head-first animals per
45 ambient temperature control is either difficult, or the temperature 60 channel are loaded efficiently. The average number of properly loaded
is too high (such that it is not feasible to handle PF127). The animals per experiment out of 27 experiments is 4. b-c) Snapshots of the
microbeads are used to physically immobilize animals’ tails and extent of the presence of the Pluronic F127 gel after formation at different
times. At t = 0 sec, the gel is present in the bulk of the channel and
prevent backwards movement. muzzle area. Within 2 minutes, the gel is completely eroded. It remains
The platform is prepared for experimentation by following a 65 stable inside the trapping channels. The gel remains stable inside the
50 simple loading procedure (Fig 2). Briefly, after connecting tubing channels because the streamlines of the bifurcated symmetric flow do not
via metal pins and de-gassing the device, approximately 20 enter the channels. d) The alternative immobilizing solution of sodium
alginate/gelatin cannot be eroded once gel has formed. It has to be
animals in 30 µL of solution are loaded into the chamber
removed prior to gellation. Alginate and gelatin solutions are viscous and
inlet/reservoir. Animals are pulled into the trapping channels and 70 are difficult to remove from the muzzle. The mixture of 15% w/v and
properly positioned by withdrawing liquid through the outlet at 2 0.75% w/v gelatin and sodium alginate respectively is the highest
55 mL/hr (Fig 2a). Next, solution containing 10 µm diameter concentration which can be removed efficiently prior to gellation. Also,
microbeads is applied into the reservoir. This solution is used to the solution is removed fast enough to limit ingestion by the animals.
flush out excess animals from the loading chamber. It is also used

4 | Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year]

Fig. 4 The temperature of Pluronic F127 solution as measured at the inlet
to trapping channels using a fluorescent intensity measurement
technique44. On average, animals are not exposed to temperatures lower
5 than 12 ºC. The lowest temperature occurs in the channels closest to the
outlet of the loading chamber, where fluid exchange is the most rapid.
to deliver microbeads into the trapping channels (Fig 2b). Here,
the microbeads accumulate at the animals’ tails. The beads
physically prevent backward motion and aid in immobilizing the
10 tail.
Then, the immobilization solution is loaded into the device. In
case of the PF127, 40 µL of 25 w/v% solution chilled to 0 ºC is
loaded into the inlet reservoir. The procedure relies on passive
warming to ambient room temperature. While the solution warms
15 to room temperature, it passes the sol-gel transition temperature
at ~17 ºC. This leads to the formation of the gel. We chose to not
include active temperature control components to keep the system
as simple as possible. Active temperature control makes
fabrication more complicated and requires external equipment.
20 This would make using the platform in other laboratories difficult
and potentially costly. Instead, we designed the system and
loading procedure to be functional, simple, and inexpensive.
However, in environments where ambient temperature is not
steady or is too warm, the solution may gel prematurely. In this
25 scenario, a solution of sodium alginate/gelatin can be used. The
same volume of a 0.75% w/v sodium alginate/15% w/v gelatin is
loaded at 25ºC.
In the last step, loading is finalized. Nutrient and stimuli inlets
are opened, so that these may be delivered to the trapped animals.
30 In case of the sodium alginate/gelatin solution, an additional
action is required. Here, a 5% w/v zinc gluconate solution is
applied to the loading reservoir. This provides the Zn2+ ions
required to cross link the alginate. This procedure from start to
finish takes approximately 20 minutes. It is easy to follow and the
35 platform can be set up in any laboratory with a syringe pump
capable of withdrawing liquid.
RESULTS
Loading and Analysis on Gel Behavior
40 The goal of the loading procedure is to provide as many single
head-first animals loaded per channel as possible. The device is
scaled for L4 larvae/young adults. We observed the frequency of
single head-first animal loading over 27 experiments. On average,
the array is loaded with more than 4 animals (median = 5 animals
45 per experiment) at the prescribed loading flow rate of 2 mL/hr
(Fig 3). At higher flow rates, the fraction of head-first animals
This journal is © The Royal Society of Chemistry [year]
decreases significantly. For instance, at 3 mL/hr, the number of
properly loaded animals decreases to below 2 on average. This is
because there is a tendency of animals to orient and swim against
50 the flow. If moderate flow rates are used, the loading procedure
provides a sufficient number of animals per experiment, since
typically high resolution imaging limits the field of view to just
one animal. However, if more are necessary, the array can be
expanded and the loading process can be optimized.
55 The critical step following efficient loading is proper
immobilization. Animals’ bodies have to be immobilized, while
their heads are free to move. However, both immobilization
solutions are allowed to flow through the device simply until they
gel for the sake of simple loading. In the case of PF127, the gel
60 forms everywhere the solution is present (Fig 3). This includes
the area of the muzzle and the bulk of the delivery channel.
However, after flow of nutrients is turned on, the gel is eroded
rapidly. Within 2 minutes (Fig 3), the muzzle and channel areas
are both clear of PF127 gels. Gel inside the trapping channels
65 remains stable and does not get eroded. This is because the
bifurcated symmetrical flow stream lines do not dip into the
channels themselves.
Gelation of sodium alginate/gelatin solution follows different
mechanics. Sodium alginate polymers are cross-linked via
70 binding to Zn2+ ions. These ions diffuse from the loading
reservoir through the loading chamber all the way into the
trapping channels. This process takes approximately 15 minutes.
The sodium alginate component provides stiffness and rigidity.
The gelatin component settles over time as individual gelatin
75 polymers intertwine. At 15% w/v, this process also takes
approximately 15 minutes. The gelatin gel component provides
elasticity and shape retention properties. However, this particular
gel cannot be eroded by flow after gelation. Thus, it has to be
removed from the muzzle prior. This cannot be achieved with
80 reasonable flow rates using the symmetrical flow because of the
high viscosity of the solution. Instead, direct cross flow from the
extra inlet and outlet in the nutrient delivery channel is used.
With this side to side flow, over 90% of the gel solution is
removed from the muzzle within 2 minutes (Fig 3). We chose this
85 specific combination of concentrations for two reasons. First, the
similar gelation times lead to more uniform immobilization.
Second, higher concentration and thus higher viscosity solutions
are more difficult to wash away from the muzzle area. This
concentration is the highest, where a timely removal can be
90 achieved.
Temperature Characterization
The goal of the platform is immobilization without altering
physiology. C. elegans is generally cultured at 15-25 ºC. Because
the immobilization solutions are loaded either chilled (PF127) or
95 prepared by heating (sodium alginate/gelatin) for the sake of
simple setup. We therefore verified that the animals remain
within the physiological range or are exposed to temperatures
outside that range only for a very short time.
We used a previously reported fluorescent intensity temperature
100 measurement technique44. We focused at the inlets of the trapping
channels, as once the solution passed this point it can only get
warmer from the surrounding material. It is also difficult to
measure temperature inside the channels because of accumulation
of dye in the PDMS and presence of animals. The average lowest
Journal Name, [year], [vol], 00–00 | 5
 Journal Name Dynamic Article Links ►

Cite this: DOI: 10.1039/c0xx00000x

www.rsc.org/xxxxxx ARTICLE TYPE

Fig. 5 Characterization of animals’ environment and feeding on device. a) The muzzle design facilitates delivery of nutrients by allowing perfusion of
bacteria. The design also slows flow inside and around the muzzle. We observed this effect qualitatively using fluorescent beads. In the bulk of the
channel during long exposure the beads leave streaks. In the area of the muzzle we can see individual beads. This indicates much lower flow rate, leading
5 to lower shear and higher residence time of bacteria. b) Lower shear and higher bacterial residence time can lead to build-up of bacteria and potential
clogging. We measured the percentage of the area covered by biofilm in the muzzle and bulk of the channel. Over 2 hours (approximate period of a
standard experiment), this effect is negligible. c) Pharyngeal pumping is an excellent indicator of animals feeding properly. Compared to animals feeding
on standard nematode growth plates, the difference is statistically not significant (p>0.1).

temperature at the channel inlet is 13 ºC (Fig 4). This is lower
10 than the 15 ºC physiological range low limit. However, the sol-
gel transition occurs within 20 seconds. This means that the
temperature rapidly reaches 17 ºC. The very short exposure to the
low temperature is thus acceptable.
We measured the temperature of the sodium alginate/gelatin
15 solution using a standard thermocouple. First, the bulk of the
solution has to be rapidly cooled to 40 ºC. After this, 40 µL of the
solution can be withdrawn with a standard micropipeter. This
process further cools the solution to 27 ºC on average. The
solution rapidly equilibrates to ambient temperature after loading
20 because of the small thermal mass and the very high surface to
volume ratio. This means that animals are not exposed to
temperatures outside their physiological range during the
experiment.
Characterization of Nutrient Delivery and Animal Feeding
25 Once animals are loaded and immobilized, the device has to
facilitate proper feeding. The ability to properly feed the animals
is critical for experiments lasting over a period of several hours.
Additionally, animals should not be exposed to high shear within
the muzzles. We qualitatively observed the conditions in the bulk
30 of the delivery channel and in the muzzle area using fluorescent
microbeads. As demonstrated in Figure 5, the speed of the
microbeads is much higher in the bulk than around and inside the
muzzle traps. This leads to lower shear and higher residence time
of bacteria.
35 A possible downside of lower shear and higher bacterial
residence time is clogging from buildup of bacteria and formation
of biofilms. We measured the rate buildup of bacteria inside the
device without animals consuming them. For the period of the
first 4 hours, the build-up, as measured by the average percentage
40 of the surface area inside the muzzles and the total area in the
bulk of the channel covered by bacteria, remains negligibly low
(Fig 5). After 16 hours, the buildup is significantly increased.
However, this device is intended for experiments up to
approximately 4-6 hours, as most processes which require this
45 type of imaging do not last longer. As such, the buildup over such
a long period does not pose a problem.
Next we tested whether the animals themselves actually feed
while immobilized. Pharyngeal pumping is a good indicator of
animals feeding. Animals pump in the presence of food, and
50 pharyngeal pumping leads to crushing of bacteria and the
pumping of nutrients into the gut. . We observed the pharyngeal

This journal is © The Royal Society of Chemistry [year] [journal], [year], [vol], 00–00 | 6
 Fig. 6 Dynamic studies of C. elegans using C.L.I.P. a) While the animals
heads are free to move, the body remains perfectly immobilized. In this
figure, images from three time points are overlaid on top of each other.
5 The animal’s head is in a different position at each time point.
Meanwhile, the body remains immobile. b) We have maintained animals
trapped on device for up to 16 hours. After 16 hours we were still able to
observe expression of fluorescent markers, indicating that physiology has
not been affected. c) We were able to monitor animals over time. We
10 observed the formation and development of lipid droplets labeled with
Seipin:GFP. Over three hours, we observed apparent growth of existing
and formation of new droplets. d) After three hours, the number of
droplets with a large diameter as well as very small diameter droplets has
increased. This indicates growth of existing droplets and formation of
15 new droplets. The box bottom and top represent the 25th and 75th
percentile respectively, the center line is the median, and whiskers
represent the min and max values. e) Over the period of the experiment,
the distribution shifts to indicate appearance of new lipid droplets. Left y-
axis corresponds to the histogram data, while the right y-axis corresponds
20 to the fitted gamma distribution.

pumping rate of animals immobilized on the device as compared

to pumping on plates in normal culture conditions. Immobilized
animals were recorded 30 minutes after loading over a period of 2
minutes. The comparison between animals trapped in the device
25 and animals on standard nematode growth plate with a bacterial
lawn showed no statistical difference (Fig 5).

Immobilization of the Animal Body to Facilitate Long-term

Imaging
The last critical aspect for complete functionality of the device is
30 the ability to image properly immobilized animals. We evaluated
the positional shift of the body of animals immobilized within the
gel while exposed to blue fluorescent light (as animals would be
during experimental conditions). This is relevant because C.
elegans is normally excited by blue light that causes increased
35 body movement 45. For the purpose of this experiment we imaged
animals continuously over a period of 1 minute (Fig 6). Over this
period, when frames from 0 seconds, 20 seconds, and 40 seconds
are overlaid on top of each other, it is clear that the body remains
perfectly immobilized, while the head is free to move inside the
40 muzzle. This result shows the applicability for analysis of cell-
wide fluorescent signals, such as calcium signaling, and for
analysis of signals from networks of neurons. We were able to
maintain animals immobilized in this fashion for up to 16 hours
(Fig 6). At this point we were still able to observe expression of
45 fluorescent markers, indicating normal physiological processes
were not affected.
Next, we imaged animals over a period of 3 hours and observed
the change in size of fluorescently labeled lipid droplets. C.
elegans has lipid droplets in the intestinal cells for fat storage. It
50 is known that genetics and diet have an effect on droplet
morphology and size46-49. We imaged animals expressing
R01B10.6:GFP, which labels a subset of lipid droplets (Y. Hao
and H.Y. Mak, unpublished data) when observed at 40x
magnification (0.5 µm steps in the z-direction). We measured the
55 diameter of the lipid droplets at the start of the experimental
period and at the end of 3 hours. At this magnification on an
epifluorescence microscope, we were only able to detect droplets
larger than 2 µm, while confocal microscopy may allow
resolution of smaller droplets50. Over the 3 hours of continuous
60 feeding on-chip, we observed an increase in size of existing
droplets, and also detected formation of new and small droplets

This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 7
 (Fig 6).
Conclusions
In this work, we have demonstrated the ability of our system to
immobilize animals for continuous live imaging at physiological
5 conditions. We verified that animals can be loaded and
immobilized for experiments and that immobilized animals feed
on a continuous flow of nutrients provided for them for the
duration of experiments. If necessary, the channel array can be
expanded to accommodate a larger number of trapped animals
10 and improve the speed of data acquisition. Most importantly, this
technology allowed us to observe the type of dynamic change
which could previously be observed only statically and is critical
for understanding the true nature of many processes.
Notes and references
17. E. R. Sawin, R. Ranganathan and H. R. Horvitz, Neuron, 2000, 26,
619-631.
18. K. Shen and C. I. Bargmann, Cell, 2003, 112, 619-630.
60 19. L. E. Waggoner, G. T. Zhou, R. W. Schafer and W. R. Schafer,
Neuron, 1998, 21, 203-214.
20. J. A. Zallen, S. A. Kirch and C. I. Bargmann, Development, 1999,
126, 3679-3692.
21. H. A. Colbert, T. L. Smith and C. I. Bargmann, J. Neurosci., 1997,
65 17, 8259-8269.
22. J. G. Crump, M. Zhen, Y. Jin and C. I. Bargmann, Neuron, 2001, 29,
115-129.
23. L. S. Huang, P. Tzou and P. W. Sternberg, Mol. Biol. Cell, 1994, 5,
395-411.
70 24. E. M. Jorgensen, E. Hartwieg, K. Schuske, M. L. Nonet, Y. S. Jin and
H. R. Horvitz, Nature, 1995, 378, 196-199.
25. E. A. Lundquist, P. W. Reddien, E. Hartwieg, H. R. Horvitz and C. I.

15
a
School of Chemical & Biomolecular Engineering, Georgia Institute of Bargmann, Development, 2001, 128, 4475-4488.
Technology, Atlanta, GA 26. S. L. McIntire, R. J. Reimer, K. Schuske, R. H. Edwards and E. M.
b
Interdisciplinary Program in Bioengineering, Georgia Institute of 75 Jorgensen, Nature, 1997, 389, 870-876.
Technology, Atlanta, GA 27. K. Nakata, B. Abrams, B. Grill, A. Goncharov, X. Huang, A. D.
c
Interdisciplinary Program in Bioengineering, Georgia Institute of
20 Technology, Atlanta, GA Chisholm and Y. S. Jin, Cell, 2005, 120, 407-420.
d
The Petit Institute for Bioengineering and Biosciences, Georgia Institute 28. M. Nangaku, R. Satoyoshitake, Y. Okada, Y. Noda, R. Takemura, H.
of Technology, 311 Ferst Drive, N.W., Atlanta, GA 30332-0100. E-mail: Yamazaki and N. Hirokawa, Cell, 1994, 79, 1209-1220.
hang.lu@chbe.gatech.edu; Fax: +1 404 894 4200; Tel: +1
80 29. J. E. Richmond, W. S. Davis and E. M. Jorgensen, Nat. Neurosci.,
404 894 8473
25 † Electronic Supplementary Information (ESI) available: [details of any 1999, 2, 959-964.
supplementary information available should be included here]. See 30. D. Sieburth, Q. Ch'ng, M. Dybbs, M. Tavazoie, S. Kennedy, D.
DOI: 10.1039/b000000x/ Wang, D. Dupuy, J. F. Rual, D. E. Hill, M. Vidal, G. Ruvkun
and J. M. Kaplan, Nature, 2005, 436, 510-517.
1. C. I. Bargmann, Science, 1998, 282, 2028-2033.
85 31. W. R. Schafer, WormBook.
30 2. A. G. Fraser, C. James, G. I. Evan and M. O. Hengartner, Curr. Biol.,
32. S. Shaham (ed.), WormBook.
1999, 9, 292-301.
33. P. B. Allen, A. E. Sgro, D. L. Chao, B. E. Doepker, J. S. Edgar, K.
3. M. O. Hengartner and H. R. Horvitz, Cell, 1994, 76, 665-676.
Shen and D. T. Chiu, J. Neurosci. Methods, 2008, 173, 20-26.
4. M. R. Koelle and H. R. Horvitz, Cell, 1996, 84, 115-125.
34. K. H. Chung, M. M. Crane and H. Lu, Nat. Methods, 2008, 5, 637-
5. Z. Altun-Gultekin, Y. Andachi, E. L. Tsalik, D. Pilgrim, Y. Kohara
90 643.
35 and O. Hobert, Development, 2001, 128, 1951-1969.
35. S. E. Hulme, S. S. Shevkoplyas, J. Apfeld, W. Fontana and G. M.
6. L. Avery, Genetics, 1993, 133, 897-917.
Whitesides, Lab Chip, 2007, 7, 1515-1523.
7. C. I. Bargmann and L. Avery, Methods in Cell Biology, Vol 48, 1995,
36. C. B. Rohde, F. Zeng, R. Gonzalez-Rubio, M. Angel and M. F.
48, 225-250.
Yanik, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 13891-
8. C. I. Bargmann and H. R. Horvitz, Science, 1991, 251, 1243-1246.
95 13895.
40 9. C. I. Bargmann and J. M. Kaplan, Annu. Rev. Neurosci., 1998, 21,
37. F. Zeng, C. B. Rohde and M. F. Yanik, Lab Chip, 2008, 8, 653-656.
279-308.
38. S. E. Hulme, S. S. Shevkoplyas, A. P. McGuigan, J. Apfeld, W.
10. C. I. Bargmann, J. H. Thomas and H. R. Horvitz, Cold Spring
Fontana and G. M. Whitesides, Lab Chip, 2010, 10, 589-597.
Harbor Symp. Quant. Biol., 1990, 55, 529-538.
39. C. B. Rohde and M. F. Yanik, Nat. Commun., 2011, 2.
11. S. H. Chalasani, N. Chronis, M. Tsunozaki, J. M. Gray, D. Ramot, M.
100 40. J. Krajniak and H. Lu, Lab Chip, 2010, 10, 1862-1868.
45 B. Goodman and C. I. Bargmann, Nature, 2007, 450, 63-+.
41. D. C. Duffy, J. C. McDonald, O. J. A. Schueller and G. M.
12. J. M. Gray, J. J. Hill and C. I. Bargmann, Proc. Natl. Acad. Sci. U. S.
Whitesides, Anal. Chem., 1998, 70, 4974-4984.
A., 2005, 102, 3184-3191.
42. M. A. Unger, H. P. Chou, T. Thorsen, A. Scherer and S. R. Quake,
13. J. M. Gray, D. S. Karow, H. Lu, A. J. Chang, J. S. Chang, R. E. Ellis,
Science, 2000, 288, 113-116.
M. A. Marletta and C. I. Bargmann, Nature, 2004, 430, 317-
105 43. S. Brenner, Genetics, 1974, 77, 71-94.
50 322.
44. D. W. Jeong, C. Y. Lee and S. Y. Lee, J. Heat Transf.-Trans. ASME,
14. O. Hobert, I. Mori, Y. Yamashita, H. Honda, Y. Ohshima, Y. X. Liu
2009, 131.
and G. Ruvkun, Neuron, 1997, 19, 345-357.
45. S. L. Edwards, N. K. Charlie, M. C. Milfort, B. S. Brown, C. N.
15. Y. S. Jin, E. Jorgensen, E. Hartwieg and H. R. Horvitz, J. Neurosci.,
Gravlin, J. E. Knecht and K. G. Miller, PLoS Biol, 2008, 6,
1999, 19, 539-548.
110 e198.
55 16. S. Mitani, H. P. Du, D. H. Hall, M. Driscoll and M. Chalfie,
46. K. T. Jones and K. Ashrafi, Dis. Model. Mech., 2009, 2, 224-229.
Development, 1993, 119, 773-783.
47. H. Y. Mak, J. Lipid Res., 2012, 53, 28-33.

8 | Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year]
 48. B. C. Mullaney and K. Ashrafi, Biochim. Biophys. Acta Mol. Cell
Biol. Lipids, 2009, 1791, 474-478.
49. S. B. O. Zhang, A. C. Box, N. Y. Xu, J. Le Men, J. Y. Yu, F. L. Guo,
R. Trimble and H. Y. Mak, Proc. Natl. Acad. Sci. U. S. A.,
5 2010, 107, 4640-4645.
50. N. Y. Xu, S. B. O. Zhang, R. A. Cole, S. A. McKinney, F. L. Guo, J.
T. Haas, S. Bobba, R. V. Farese and H. Y. Mak, J. Cell Biol.,
2012, 198, 895-911.

10

This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 9