Original Report

Image-Based Fluidic Sorting System

for Automated Zebrafish Egg Sorting
into Multiwell Plates
Siegfried F. Graf,1 Sebastian H€
otzel,2 Urban Liebel,2 Andreas Stemmer,3
and Helmut F. Knapp1*
1 
Microfluidics & Liquid Handling, Centre Suisse d’Electronique et de Microtechnique,
Alpnach, Switzerland
2
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology,
Karlsruhe, Germany
3
Nanotechnology Group, Department of Mechanical and Process Engineering,
ETH Zurich, Zurich, Switzerland

their experiments and create results with a higher

Keywords:
toxicity testing,
T he global demand for the reduction of animal testing
has led to the emergence of Zebrafish eggs/larvae as
model organisms to replace current adult animal testing
statistical relevance. ( JALA 2011;16:105–11)

cell sorting, in, for example, toxicity testing. Because of the egg size
automated, (diameter 1.6 mm) and the relatively easy maintenance of
multiwell plate,
Zebrafish farms the eggs also offer high-throughput
Zebrafish,
screening (HTS). However, the current bottleneck for
danio rerio
HTS is the cost-efficient placing of individual organisms
into single wells of a multiwell plate (MWP). The system
presented here is capable of storing, sorting, and placing
individual organisms in a highly reproducible manner. In
about 11 min a complete 96-MWP is filled, which
corresponds to about 8 sec per egg. The survival rate of
fertilized transgenic and wild-type eggs was comparable
to the one of the control (control 6.7%, system 7.6%).
Furthermore, it was also possible to place dechorionated
eggs into individual wells. The results demonstrate that
the cost efficient system works gentle and reliable
enough to disburden scientists from the exhausting and
monotonous job of placing single eggs into single wells,
such that they can concentrate on the scientific aspects of
*Correspondence: Helmut F. Knapp, Ph.D., Microfluidics & Liquid

Handling, Centre Suisse d’Electronique et de Microtechnique,
CH-6055, Alpnach, Switzerland; Phone: þ41.416.727.523;
Fax: þ41.416.727.500; E-mail: helmut.knapp@csem.ch
2211-0682/$36.00
Copyright 
c 2011 by the Society for Laboratory Automation and
Screening
doi:10.1016/j.jala.2010.11.002
INTRODUCTION
Manipulation of single cells or single organisms is
a fast growing market not least because the REACH
initiative was started in 2007. The goal of the
REACH initiative is to test each compound (from
which more than 1 ton is manufactured or imported
in Europe) for its toxicity.1 At the same time, the
number of vertebrate mammals used for toxicity test-
ing should be reduced. The two previous facts call for
high throughput and ethically justifiable toxicity
screening methods. In recent years, fertilized Zebra-
fish eggs established themselves as a model system
and are accepted as an alternative to mammalian tes-
ting.2e4 Another large advantage is that fertilized
Zebrafish eggs up to 5 days after fertilization are
not subject to the same regulatory requirements as
adult mammals. Further advantages are the egg’s
transparency and the fact that development occurs
entirely outside the mother’s body. Finally, Zebrafish
facilities are relatively easy to maintain and because
of the size of an egg (diameter !1.6 mma, see
Fig. 1), dispensing them in up to 384-well plates is
possible.5
a
http://www.neuro.uoregon.edu/k12/FAQs.html.

JALA April 2011 105

Original Report
Figure 1. A. Fertilized (except top right) Zebrafish eggs after 7 h,
(B) different batch with fertilized Zebrafish eggs after 11 h. Two
white eggs are dead. Scale bar indicates 10 mm for both images.
In current toxicity tests, fertilized Zebrafish eggs are dis-
pensed into multiwell plates (MWPs). Test compounds with
different dilutions are pipetted to the individual fertilized Ze-
brafish egg in the single wells. The MWP is then periodically
imaged and analyzed.6 The large amount of data is locally or
remotely stored and analyzed. Finally, fast search engines al-
low one to retrieve the collected data.
In summary, a complete line-up of systems and techniques
from different disciplines is required to perform a low cost
high throughput toxicity test as shown below:
1. Inexpensive and efficient Zebrafish facilitydto get a large
amount of eggs;
2. Inexpensive sorting systemdto place individual eggs into
a MWP;
3. Inexpensive lab robotic systemdto handle the plates and
deliver test compounds into wells;
4. Powerful automated imaging systemdto periodically
observe the eggs quality;
5. Efficient data storage/handling;
6. Efficient image analysis systemdto analyze images; and
7. Efficient search algorithmsdto retrieve results.
106 JALA April 2011
Of the list above, many of the items listed are available.
More specifically, inexpensive, efficient Zebrafish facilities
can be established and maintained by following certain
guidelines.7 Lab robots and powerful automated imaging
system are commercially available although they are still
expensive. Efficient data storage and data distribution is pos-
sible but also an expensive task. Systems for image and visu-
alization analysis8 are available, their performance however
corresponds to the available computer power. There are also
several search engines available such as Harvester,9 which
combine results from several other search engines and data-
bases. The main remaining bottleneck is the inexpensive sort-
ing system.
Currently, most of the Zebrafish eggs are placed into well
plates manually. Two skilled technicians can fill 30 96-well
plates in about 3 h, which corresponds to 12 min per plate on
the average. Unfortunately, this highly monotonous task is
prone to errors and can typically not be performed by a single
person for more than 3 h. Another example comes from Stern
et al.10 who screened about 16,000 compounds in 16 weeks,
while 5000 larvae were collected weekly. They were quality
controlled and then 20 larvae were manually transferred with
a chemical spatula in each well of a 48-well plate. Several at-
tempts have been made to automate the singularization of Ze-
brafish eggs or larvae into MWPs. A possibility is to use
a robotic pick and place approach for sorting eggs from a petri
dish into the well plate. One such system is the CellBot (Fully
Automated Single Cell Handling Platformb) from the Centre

Suisse d’Electronique et de Microtechnique, where a delta ro-
bot, capable of several cycles per second, is combined with
a fully motorized inverted light microscope (iMic from Till
Photonics, Germany). The robot and the microscope move
above and beneath the sample platform, which is stationary.
Because the petri dish and the well plate are never moved dur-
ing the sorting process, this arrangement prevents sloshing
of the sample liquid. The inverted light microscope scans the
petri dish and checks for eggs, if one is found, this egg is
removed by a pipette tool attached to the delta robot and
placed into the well plate. Unfortunately, such a pick and place
system is costly, rather slow, and uses a large portion of lab
space. Further options are the COPAS and BioSorter (both
from Union Biometrica, Holliston, MA), which are specially
designed for sorting of large biological entities (up to
1500 mm). These systems are capable of singularizing Zebra-
fish eggs into 96-well plates in about 2 min.11 However, these
systems were designed for high throughput flow cytometry
and are therefore equipped with costly optics, not necessary
for simply singularizing large entities into MWPs. Addition-
ally, both systems are not capable of checking the quality of
the entities sorted, such as shape, specific features, or damage.
The system presented here, named ZebraFactor, is an
inexpensive sorting device for large biological entities, such
as Zebrafish eggs and larvae, Xenopus oocytes, pollen, and
cell clusters. Sorting is done using a vision system taking
b
http://www.csem.ch/docs/Show.aspx?id¼7824.
 Original Report

complete images of the entities, combined with fast vision al-

gorithms capable of identifying a set of characteristics of an
entity in real time as they are moved past the vision system.
Identified single entities can then be extracted from the sort-
ing system and transferred to a container, such as an individ-
ual well in a well plate, or to a subsequent device. Here,
we will demonstrate the direct sorting and individualization
of Zebrafish eggs into the wells of a 96-well plate. Moreover,
the system also has been used for sorting and feeding
Xenopus oocytes into an automated microinjection module,
a development by us not presented here (XenoFactor: Fully
Automated Microinjection Systemc).
In the following, we present the sorting system in combi-
nation with our WellPlateFeeder (see Fig. 2).
Figure 2. System overview of ZebraFactor consisting of the
WellPlateFeeder (left) and the CellSorter (right) connected by tub-
ing. Black bar indicates 50 mm.
SYSTEM
Hardware The vision system installed along the fluidic path consists
The ZebraFactor as shown in Figure 2 consists of two of one or two inexpensive complementary metal oxide semi-
main units named CellSorter and WellPlateFeeder. The Cell- conductor cameras and a light emitting diode array for illu-
Sorter is used to sort single entities such as Zebrafish eggs or mination. Additionally, excitation and emission filters can be
larvae from the suspension, whereas the WellPlateFeeder introduced for using fluorescence signals for the identifica-
places the single entities into a well of the MWP. The actions tion of fluorescently labeled entities. In case of transparent
of the CellSorter and the WellPlateFeeder are synchronized samples such as Zebrafish egg or larvae, a one-camera setup
to ensure correct feeding of entities into the well plate. is sufficient (as in Fig. 2), whereas for opaque samples such as
After introducing the egg suspension into the CellSorter, Xenopus laevis oocytes or in case where the user is interested
the suspension is continuously moved by our novel concept in the sample surface, a two-camera setup would be used (as
of using a sliding and static ring as shown in Figure 3. The in Fig. 4). The two cameras would be placed opposite to each
two rings define a circular fluidic channel for the egg suspen- other, which enables observing the whole surface of an en-
sion. The sliding ring is rotated by an electro motor and drags tity. This configuration allows the system:
along the suspension buffer because of viscous drag forces. Ad- 1. To inspect the whole surface of any kind of sample (opa-
ditionally, a small friction force acts on the chorion of the egg. que and transparent);
The sum of drag and friction forces results in rolling of the egg 2. To inspect a cell compartment of a transparent sample;
along the fluidic channel and past the cameras positioned to and
visualize a portion of the channel. This concept works most 3. To inspect fluorescence signals.
efficiently with objects larger than 200 mm (Xenopus laevis
oocyte are up to 1300 mm, Zebrafish eggs are up to 1600 mm), The ZebraFactor presented here is simply equipped with
and allows moving cells continuously without destroying one camera and a light emitting diode-array illumination
them. Additionally this concept offers: without using any excitation and emission filters because
the processed Zebrafish eggs were not labeled.
1. To store eggs until their usedcontinuous motion avoids
adhesion;
2. To observe eggs over and over againdin connection with
the vision system;
3. To deliver eggs on demand to a subsequent system; and
4. To load the CellSorter with additional eggs on the fly.d
To remove a single egg from the CellSorter, buffer is redir-
ected inside the channel and, by a gentle push, moves the egg
to the WellPlateFeeder, or any other subsequent system such
as the microinjection module mentioned above. In any case,
light barriers are used to control the process of moving the
egg from the CellSorter to the subsequent system.
c
http://www.csem.ch/docs/Show.aspx?id¼6247. Figure 3. Schematic of fluid and object motion as generated by
d
On the fly means, the system does not need to be stopped to add additional the sliding ring concept. The egg is driven by drag and friction
cells. forces and rolls along the base of the sliding ring.

JALA April 2011 107

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The software to control the hardware is designed to store

the analyzed image together with the batch and well plate
number and well plate location. Further, egg collection time,
stock number, genotype, and sorting time can be added to
the XML database. These data allows one to keep track of
the egg from breeding to the single larva analysis.

Graphical User Interface

The graphical user interface (GUI) is kept as simple as
possible. In the standard view, the GUI offers a ‘‘Fill/Empty
system’’ button, ‘‘Start/Stop,’’ and ‘‘Save image.’’ Informa-
Figure 4. CellSorter from Figure 2 with a two-camera configu- tion about the batch number and the well plate number is
ration to sort opaque cells. shown in the top left corner as illustrated in Figure 5. Addi-
tionally, a virtual MWP shows which wells are already filled
with samples. In the idle mode the upper of the two images
The incorporated vision algorithm is based on a neuronal
on the GUI shows the live image, whereas the lower one
network, which allows the user to set and teach sorting and
remains black. In the running mode, the upper image of
singularization criteria. With this kind of approach, the user
the two images shows the analyzed egg. If a suitable egg is
can simply singularize eggs or larvae by size and shape or
found and the system is ready for removal, a second image
even sort by complex criteria, which currently can only be
is taken just before removal and is shown in the lower image
done manually. In summary, the vision algorithm enables:
of the GUI (see Fig. 6). Additional tabs such as ‘‘Trouble-
1. To simply singularize single eggs or larvae from a suspen- Shoot’’ and ‘‘Extra’’ are inserted into the GUI to assist if
sion (e.g., by size and shape); the system malfunctions or if manual steps are required
2. To sort eggs or larvae by complex criteria from a mixed (see Fig. 6).
suspension (e.g., fertilized or unfertilized, damaged or
healthy.); and VALIDATION
3. To easily teach the vision algorithm a new set of character-
istics for sorting. The system described above was validated with fertilized
Zebrafish eggs. In the following, the methods and the results
The WellPlateFeeder is designed as a gantry robot, that is, are presented.
the MWP is stationary and the feeding tool is moved from
one well position to the next. The system is designed such
that enough space is available to pick the well plate by a robot
and place it onto, for example, an automated microscope for
later analysis. The feeding tool consists of two pinch valves
driven by electro motors. This allows one to keep the valves
open or closed without any energy consumption and heat
dissipation. To decide when to open or close the valves,
a light barrier is placed between the valves. If the signal of
the light barrier is interrupted, the first valve passed by the
egg closes and the second valve opens. A dispensing pump
is then used to dispense the amount of buffer needed to trans-
fer the egg into the 96-well plate. The current system is opti-
mized for 96-well plates. However, the same technique can be
used to dispense single eggs into a 384-well plate or several
eggs into 1 well of a 96-well plate.
The complete system is designed such that with little effort
it can be automatically primed, which takes less than 3 min. Figure 5. Graphical User Interface of the ZebraFactor: on the top
To clean the system at the end, the buffer is removed auto- left the batch number and corresponding well plate number are
matically within less than 2 min. Finally, the user has to indicated. The overall number of buttons is kept low for simplicity:
clean the sorting part to avoid calcinations and salt crystals, ‘‘Start’’ to start the process; ‘‘Light’’ to observe; what is going on in
which takes about 8 min. It is also possible to flush the sys- the sorter; ‘‘Save Image’’ to capture an image; ‘‘New MWP’’ to
tem with pure or diluted ethanol for sterilization. Priming get a new MWP; ‘‘Next Pos’’ to move the WellPlateFeeder to the
and cleaning only needs to be performed at certain intervals, next MWP position; ‘‘Fill System’’ to fill the system; ‘‘Load Settings’’
for example, before and after filling many well plates in if the settings file was changed. Two additional tabs ‘‘TroubleShoot’’
a day’s work. and ‘‘Extra’’ are available for more complex tasks.

108 JALA April 2011

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Figure 6. A. Screen shot taken after three eggs were placed. The first image shows the egg when it was detected and analyzed, the lower
image shows the egg just before the removal. Below the ‘‘Save Image’’ button, information about the egg diameter in pixels, the detection
score, and the processing time in milliseconds are given. On the bottom left, the past time is shown. B. The tab ‘‘TroubleShoot’’ contains
solutions to problems: ‘‘PrimeP1’’ is used to remove bubbles from the pump P1; ‘‘BubbleRem’’ is used to remove bubbles; and ‘‘Unclog-
ging’’ is used to unclog the system. C. The tab ‘‘Extra’’ contains buttons to control the individual devices such as pumps and valves.

Method Petri dishes (Semadeni, Part No 5647) and MWPs

Adult wild-type and transgenic Zebrafish were raised at
the Karlsruhe Institute of Technology following standard
fish care and maintenance protocols.12
Zebrafish eggs from different batches were collected
directly after fertilization. One-half of the eggs were kept in
a petri dish for the control. The other half were used in the
ZebraFactor to automatically fill 96-well plates. We followed
the procedure described by Lammer et al.,6 except that eggs
were dispensed at approximately 4 h instead of 3 h postferti-
lization (hpf). The time for each run was measured and the
errors noted. Finally, the survival rate was counted approx-
imately 18 h after fertilization.
Additionally, for some fertilized eggs the chorion was
removed with tweezers before the sorting.
(Semadeni, Part No 6233) were clean but not sterile. Buffer
used for all the processes was embryo buffer E3þ.13 To han-
dle the eggs, a disposable plastic pipette was used (Semadeni,
Art No 2292).
MWP filling took place at room temperature (about
23  C); however, the incubation was done at 28.8  C.
Results
To verify the system, five runs with transgenic (see Table 1)
and two runs with wild-type (see Table 2) Zebrafish eggs were
made. In Figure 7 dispensed single Zebrafish eggs in individual
wells of a MWP are shown.
In an additional experiment, after modifying the search
parameters, sorting of dechorionated embryos was possible.

Table 1. Results for transgenic Zebrafish eggs automatically placed by the ZebraFactor into 96-well plates, compared with control with
similar amount of eggs kept in a petri dish

Amount of Dead after 18 hpf hpf at placing Errors Time for placing
Line transgenic embryos (entity) (entity) (%) (hpf) (entity) (hh:mm:ss)
Control 449 30 6.7 ca. 4
Plate 1 98 10 10.2 ca. 4 5 00:12:35
Plate 2 96 9 9.4 ca. 4 0 00:12:23
Plate 3 96 5 5.2 ca. 4 0 00:17:24
Plate 4 97 8 8.2 ca. 4 1 00:11:06
Plate 5 97 5 5.2 ca. 4 5 00:12:41

Total 484 37 7.6 11 01:06:09

Average 96.8 7.4 7.6 ca. 4 2.2 00:13:14

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Table 2. Results for wild-type Zebrafish eggs automatically placed by the ZebraFactor into 96-well plates, compared with control with
similar amount of eggs kept in a petri dish

Amount of Dead after 18 hpf hpf at placing Errors Time for placing
Line wild type embryos (entity) (entity) (%) (hpf) (entity) (hh:mm:ss)
Control 201 24 11.9 ca. 4
Plate 1 111 11 9.9 ca. 4 10 00:15:55
Plate 2 99 14 14.1 ca. 4 10 00:12:54
Total 210 25 11.9 20 00:28:49
Average 105 13 12.0 ca. 4 10 00:14:24

Twenty-four embryos with total three errors (none or two
embryos in one well) were dispensed into wells. The placing
into the well plate was gentle enough to avoid crushing the
yolk bag as shown in Figure 8. All embryos survived (sur-
vival count after 100 hpf).
DISCUSSION AND CONCLUSION
The need for toxicity testing of compounds is increasing
because the REACH initiative in Europe was launched in
2007. Numerous publications show that fertilized Zebrafish
eggs and larvae are good model organisms for toxicity testing
and can substantially reduce the amount of vertebrate mam-
mals used for such tests. However, for efficient large scale
toxicity testing, automated solutions are required. Firstly,
lab automation systems to handle liquids and well plates
are necessary. Secondly, software and hardware solutions
to efficiently store, distribute, and analyze the large amount
of data collected from the screens are needed. One remaining
bottleneck in the whole screening process remains by getting
the eggs or larvae from the breeding tank into a processable
format such as the standardized 96- or 384-well plate.
Our sorting device for singularizing eggs from a suspension
into a 96-well plate, named ZebraFactor, consisting of the
CellSorter and WellPlateFeeder, fills a 96-well plate at an
average of 13 min, which corresponds to about 8 sec per egg.
The surviving rate is approximately the same as in the control
group (wild type: ZebraFactor: 7.6% vs Control: 6.7%; trans-
genic: ZebraFactor: 12.0% vs Control: 11.9%). Furthermore,
only by changing some parameters in the software, dechorio-
nated embryos could be dispensed with a 100% survival rate.
Until today, lab personal was only able to manually fill 96-
well plates 3 hs a day before exhaustion lowered their through-
put and quality. Now, it is possible to use the ZebraFactor to
perform this monotonous work with the same constant quality
24 hrs a day, 7 days a week. Moreover, the personnel can pre-
pare and run the experiments while the ZebraFactor is filling
further well plates. Thus, the automated sorting system pre-
sented here is expected to enable toxicity screening with several
factors higher throughput than today.
Several factors limit the minimal time of a cycle: (1) trans-
fer distance and transfer speed between the CellSorter and
the WellPlateFeederdthe shorter the distance or the faster
the transfer speed, the shorter the cycle time; (2) pressure
wave resulting from pinch valves in the interfacedthe faster
the valve switches the higher the pressure wave traveling

Figure 7. Single Zebrafish eggs dispensed into individual wells of Figure 8. Dechorionated embryo after placement into a multi-
a multiwell plate with the ZebraFactor. well plate using the ZebraFactor.

110 JALA April 2011


through the system gets, which can led to malfunctioning of
the CellSorter; and (3) speed of the WellPlateFeeder to move
from one well to the nextdthe faster the WellPlateFeeder the
shorter the cycle time.
As mentioned above, an average automated filling of
a 96-well plate with the ZebraFactor takes 13 min, which re-
sults to a cycle time per egg or well of 8 sec. We strongly be-
lieve that this time can be reduced to 4 sec, by making
adaptions to the design of the fluidics and the control soft-
ware. This would enable filling of a 96-well plate in about
6 min. We assume that the cycle time to fill into a 384-well
plate will be even shorter because of smaller displacement
distances.
In near future, our system presented will not only be capa-
ble to sort into 96-well plates, but also into 384-well plates or
to dispense 1e4 larvae into a single well of a 96-well plate.
Because of the small size of the CellSorter, it could also be
directly combined with an inverted microscope, such that the
embryos are directly placed into a well plate mounted on the
microscope. Finally, the CellSorter can also be combined
with an automated microinjection system and then with
a WellPlateFeeder. This would allow one to perform knock-
down analysis and much more.
A video of the performance of the ZebraFactor can be
viewed at http://www.youtube.com/watch?v¼1ZtMQ-dI52E.
ACKNOWLEDGMENTS
The authors thank the Neuhauss group at the University Zurich, especially
Kara Dannenhauer, for their constant support with fresh Zebrafish eggs.
Competing Interests Statement: The authors certify that they have no relevant
financial interests in this manuscript.
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