As Featured in

Featuring work in fish-on-a-chip technology from the Centre As featured in:
for Nano-biosensors in the School of Laboratory Medicine at

Hubei University of Chinese Medicine.
Title: Fish-on-a-chip: microfluidics for zebrafish research
We provide a critical review of recent progress on zebrafish

manipulation, imaging and phenotype readouts of external stimuli
using microfluidic tools and discuss the challenges that confront
these promising fish-on-a-chip technologies.
See Fan Yang, Guo-Jun Zhang et al.

Lab Chip, 2016, 16, 1106.
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Fish-on-a-chip: microfluidics for zebrafish

research
Cite this: Lab Chip, 2016, 16, 1106
Fan Yang,*a Chuan Gao,a Ping Wang,b Guo-Jun Zhang*a and Zuanguang Chenc
High-efficiency zebrafish (embryo) handling platforms are crucially needed to facilitate the deciphering of
the increasingly expanding vertebrate–organism model values. However, the manipulation platforms for
zebrafish are scarce and rely mainly on the conventional “static” microtiter plates or glass slides with rigid
gel, which limits the dynamic, three-dimensional (3D), tissue/organ-oriented information acquisition from
the intact larva with normal developmental dynamics. In addition, these routine platforms are not amenable
to high-throughput handling of such swimming multicellular biological entities at the single-organism level
and incapable of precisely controlling the growth microenvironment by delivering stimuli in a well-defined
spatiotemporal fashion. Recently, microfluidics has been developed to address these technical challenges
via tailor-engineered microscale structures or structured arrays, which integrate with or interface to func-
Received 12th January 2016, tional components (e.g. imaging systems), allowing quantitative readouts of small objects (zebrafish larvae
Accepted 16th February 2016
and embryos) under normal physiological conditions. Here, we critically review the recent progress on
zebrafish manipulation, imaging and phenotype readouts of external stimuli using these microfluidic tools
DOI: 10.1039/c6lc00044d
and discuss the challenges that confront these promising “fish-on-a-chip” technologies. We also provide
www.rsc.org/loc an outlook on future potential trends in this field by combining with bionanoprobes and biosensors.
1. Introduction genetics, chemical biology, disease modeling and drug

screening for its several unique merits.1–4 First, unlike the
The zebrafish (Danio rerio), as a typical small multicellular large-size rodent animal models such as mice, zebrafish is
model organism, has become increasingly significant in the only vertebrate model organism that can be cultivated in
multiwell plates or microfluidics, enabling high-throughput
a
School of Laboratory Medicine, Hubei University of Chinese Medicine, 1 screening, akin to the small invertebrate organisms such as
Huangjia Lake West Road, Wuhan 430065, China. the fruit fly (Drosophila melanogaster) and soil-dwelling nem-
E-mail: [email protected], [email protected];
atodes (C. elegans).5–11 Second, the egg-laying period is short
Fax: +86 27 68890259; Tel: +86 27 68890259
b
School of Basic Medicine, Hubei University of Chinese Medicine, 1 Huangjia
(∼one week), and the egg yield is high. One female fish can
Lake West Road, Wuhan 430065, China spawn tens to hundreds of eggs in a hatch, which is signifi-
c
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China cant for high-throughput screening that often requires a pool
Fan Yang received his B.S. (2005) Chuan Gao received his Ph.D. in
in clinical medicine from Hubei Genetics from National Univer-
University of Chinese Medicine and sity of Singapore in 2010. He
his Ph.D. in Pharmaceutical Analy- then had his postdoctoral train-
sis from Sun-Yat Sen University in ing in Fox Chase Cancer Center,
2011. Prior to becoming an associ- Philadelphia, and University of
ate professor at Hubei University of California, San Francisco. Cur-
Chinese Medicine, he was a post- rently, he is working on genetics
doctoral fellow at Shanghai Insti- and oncology in zebrafish as an
tute of Applied Physics (SINAP), associate professor in Hubei Uni-
Chinese Academy of Sciences (CAS). versity of Chinese Medicine.
His research interests are in micro-
Fan Yang fluidics, zebrafish-based drug Chuan Gao
screening and biosensors.
1106 | Lab Chip, 2016, 16, 1106–1125 This journal is © The Royal Society of Chemistry 2016
View Article Online
Lab on a Chip Critical review
of larvae with identical developmental stage, and much humans reach up to approximately 70% that makes this
more cost-effective than the rodent counterparts. Third, the small animal (including the transgenic species) an excellent
embryo grows fast.12 For example, the heartbeat starts at 22 model for studying human disease mechanism and progres-
hours post-fertilization (hpf), and the cardiovascular circula- sion.15 These inherent and unique properties endow this
tory system is fully developed and functional at 36 hpf.13 multicellular organism a special appeal to biomedical
Within 72 hpf, the embryo can grow into an early larva fields and greatly fuel the research in genetics, neurobiol-
with well-developed organs.12 Such a rapid developing pro- ogy, developmental biology, disease modeling and drug
cess can dramatically reduce the period of pharmaceutical screening.15–20

toxicity evaluation and accelerate drug discovery. Fourth, Although the research on the zebrafish-based organism
the optical transparency of the body, especially at embryo model has achieved remarkable progress, the manipula-
stage, facilitates the imaging of its developmental dynamics tion platforms customized for these small animals are in-
for better understanding of the fundamental vertebrate sufficient and still confined to microplates or a thin sheet
developmental biology. Importantly, the morphological of glass slide.21 These are usually coated with agarose to
abnormities or alterations can be easily identified via position zebrafish either with the aid of anesthetics for a
the common optical microscope. This is extremely beneficial motionless “insensible” state or by means of steel tweezers
for toxicity and teratogenicity evaluation in which high- for oriented immobilization.22 The frequent and excessive
throughput phenotype examination is necessary.14 More sig- personnel interference not only requires tedious and time-
nificantly, the genetic similarity between zebrafish and consuming manual processes but also easily damages the
fragile body, resulting in the zebrafish developing under
an abnormal condition. This may invalidate the imaging
results. Moreover, these routine platforms often function
Ping Wang obtained his M.D. in with low efficiency in handling a large pool of zebrafish
Geriatrics from Hubei University simultaneously, particularly at the single-organism level.
of Chinese Medicine in 1998. He Furthermore, visualization of target organs of interest in
is a full professor and chief of zebrafish is generally blocked by organs such as the eyes
the Institute of Geriatrics in Hu- and the yolk sac or obscured by the skin pigmentations.
bei University of Chinese Medi- Therefore, a high-efficiency multifunctional device to si-
cine, where his research interests multaneously handle these small multicellular organisms
include the development of novel in a high-throughput fashion while precisely controlling
geriatrics models for unraveling their orientation and physiological microenvironment
disease mechanisms and drug would be the key issue in next-generation zebrafish-di-
screening. rected biomedical research such as phenotypic screening.23
Fortunately, emerging biological micro-electromechanical
Ping Wang systems (bioMEMS), particularly the soft lithography-based
microfluidics, have demonstrated great potential to become a
Guo-Jun Zhang received his Ph. Zuanguang Chen received his

D. in medicine from Tongji Medi- Ph.D. in Analytical Chemistry
cal School, Huazhong University from Sun-Yat Sen University in
of Science and Technology, in 2000. He currently works as a
1999. He was then a postdoc- professor at the School of Phar-
toral fellow at Wuhan Univer- maceutical Sciences in Sun-Yat
sity, Institute for Physical High Sen University, where his re-
Technology (Jena, Germany), search interests are focused on
and Waseda University. After- analytical instrumentation,
wards, he became a visiting lec- microfluidics-based drug screen-
turer in 2004, a visiting associ- ing in C. elegans and zebrafish,
ate professor in 2005 at Waseda and biosensors.
Guo-Jun Zhang University, and a member of the Zuanguang Chen
technical staff at the Institute of
Microelectronics (Singapore). He currently works as a professor at
Hubei University of Chinese Medicine with research interests in
the fabrication of nanomaterial-based FET biosensor and its ap-
plication in disease diagnostics.
This journal is © The Royal Society of Chemistry 2016 Lab Chip, 2016, 16, 1106–1125 | 1107
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Critical review Lab on a Chip
critical and indispensible technological platform for zebrafish zebrafish transport, iii) perfusion and dynamic culturing of
manipulation.6,13,24 zebrafish, and iv) on-chip microinjection and electroporation.
The last decade witnessed a rapid growth of microfluidics Current microfluidic technologies for zebrafish handling are
in cell culturing, stimulating, sorting, trapping and summarized in Table 1.
capturing.25–34 This came about through the micrometer di-
mension of the flow channel (1–100 μm) perfectly matching
the cell size (∼10 μm), while the photolithography process 2.1. Zebrafish entrapment
can generate hundreds to thousands of paralleled microstruc- In comparison to cell immobilization, zebrafish trapping is
tures in a single exposure. Thus, multiple cell tasks can be unique for the following reasons. First, unlike several lines of
handled simultaneously for elucidating diverse molecule/cell cells that can spontaneously attach to the culturing substrate
level events. Following this trend, microfluidics has also or wall for growth and migration in a static physiological en-
proven to be advantageous for studying the models of large- vironment,34 zebrafish frequently swim in water, especially
size tissues or organs (above microscale), such as organ-on-a- the hatched larva. Despite being motionless at the early de-
chip, by measuring their dynamic responses under precisely velopmental stage (less than 24 hpf), the embryo body begins
tunable local stimuli.35–37 These unique characteristics fur- to wobble regularly as the tail stretches to detach from the
ther extend microfluidics to larger multicellular organisms yolk (more than 24 hpf).12 Second, the cell surface expresses
(zebrafish embryo, ∼700 μm in diameter) for developmental abundant (antibody) protein molecules that often have paired
dynamics studies and phenotype readouts in drug and toxic- ligand biomolecules to bind.50 Hence, an efficient functional
ity screening.14,24,38 It should be noted that, recently, numer- cell-recognition surface can be easily tailored to capture the
ous tailor-engineered microfluidic devices have been used to target cell.27,51 The fish embryo, however, is protected by a
accelerate and facilitate zebrafish-oriented research by their chorion structure, and its surface has no evident antibody-
capability of processing all the demanded tests in like biomolecules to dock the embryo in a “cell-like” manner.
parallel.39–41 Several related reviews so far have reported Third, both the “bulky” embryo and the hatched larva require
zebrafish processing, either introducing a general scope of a dynamic three-dimensional immobilization strategy given
multiple small multicellular organisms (e.g. C. elegans, the site-specific accessible probing (e.g. imaging) and varied
zebrafish and Drosophila)4,13,23,35,39 or centering on zebrafish- motility during different developmental stages. In addition,
based drug screening.24 However a comprehensive and criti- the embryo is heavier than the small-size cell, which allows
cal introduction of fish-on-a-chip technology is necessary. In the embryo to dock simply by self-gravity. However, this way
this review, we focus on how microfluidics facilitates the is unsuitable for the motile embryo and the well-developed
zebrafish-oriented biomedical investigation, including on- larva. Therefore, the zebrafish immobilization strategy de-
chip fish manipulation, imaging and phenotypic readout. mands taking developmental dynamics into consideration
Further, we will make a brief discussion on future outlook re- due to the whole growth spanning from the “static” embryo
garding microfluidics-based zebrafish research incorporating to the vibrant larva.
advanced technologies or functional structures, such as The routine positioning of zebrafish (embryo) requires te-
nanoprobe-mediated optical imaging,42 biosensors,43–44 DNA dious and multi-step manual manipulation with forceps or
nanostructures and genome editing strategies.45–49 similar tools based on agarose substrate, and even with the
help of anesthetics.52,53 This method can damage its vital
2. Zebrafish manipulation on signs ascribed to either the imposed extra stress or narcotism
microfluidic chip to the tested organism. Additionally, the planar glass slide
fails to perform large-scale paralleled assays and lacks precise
Controlling the target zebrafish with high precision and fa- control of uniform body orientation during high-throughput
vorable orientation is a prerequisite for high-resolution imag- imaging. Therefore, a better immobilizing approach is highly
ing within a specific temporal–spatial condition. However, desirable. To this end, a typical physiological mounting tech-
the swimming larva poses a great challenge to handle if we nique has been adopted to trap the embryos in a fluorinated
want to explore the time- and site-specific information from ethylene propylene (FEP) tube (inner diameter of 0.8 mm) via
the growing body. For example, phenotype-based drug low concentrations of either agarose (0.1%) or methylcellu-
screening requires high-throughput transport and trapping of lose (3%).54 The organic microtube has a matched refractive
a large number of larval fish, and a dynamic culturing micro- index to water, allowing for strengthened immobility and
environment is necessary to ensure an excretion-free physio- high imaging quality. This approach also provides accurate
logical growing condition. In addition, the orientation of physiological relevance by maintaining normal cardiac func-
zebrafish is critical to accurately perform organ- or site- tions and avoids the need for anesthetizing drugs such as
specific microinjection and electroporation. Microfluidics is tricaine, which may cause cardiac dysfunction due to its inhi-
an ideal technological platform to address these problems be- bition of muscle contraction.54 However, this multilayer
cause of its microscale flow-through, flexible microstructure mounting technique may influence the embryonic develop-
design and multiplexing capability. Specifically, microfluidic ment because the agarose used (if at high concentrations)
devices enable i) highly efficient entrapment of zebrafish, ii) can lead to severe growth restrictions and developmental
View Article Online
Table 1 Present microfluidic technologies for zebrafish research
Trapping
Trapping Channel unit (fish
principle materials Fish age and type amount) Imaging system Stimuli Ref.
Agarose or Fluorinated Transgenic Single Selective plane illumination Tricaine 54
methylcellulose ethylene TgĲkdrl:GFP), microscopy (SPIM)
propylene TgĲmyl7:GFP)
embryo
Funnel aperture PDMS 5 hpf Single Microscopy (non-specific) MIF, retinoic acid, BMPs, Noggin 55
dechorionated
embryo
Hydrodynamic PDMS-glass 4 hpf embryo; 16 Array Fluorescence stereomicroscope VEGFR inhibitor AV951 56
and suction hpf transgenic (>20) with a DS-U2/L2 camera
force fli1a:EGFP embryo
Hydrodynamic, PMMA 16 hpf transgenic Array (20) Stereomicroscope with a Leica VEGFR1-3 inhibitor AV951, 57
suction force fli1a:EGFP embryo DFC295 camera; fluorescence VEGFR2/PDGFR inhibitor Sunitinib,
and gravity stereomicroscope with a DS-U2/L2 broad-range Bcl-2 inhibitor TW37,
camera or infrared camera biphenolic Akt inhibitor Honokiol
Vacuum Non-specific Embryo Array Stereomicroscope with Fluorescent dyes, ntl-MO 58
suction (560) microrobots, fluorescence
microscope
Restriction and Polystyrene, 3–5 dpf larva, 3 dpf Single Light-scanning confocal Neutrophil attractant (LTB4), PI3K 59
surface-tension PDMS–glass transgenic mpx: and array microscope inhibitor LY294002
driven dendra2 larva (>25)
pumping
“Fish-Trap” PDMS–glass 4–6 dpf transgenic Array Inverted fluorescence microscope, Glutamate and tricaine; neurotoxin: 40
(Hydrodynamic elavl3:GCaMP5G resonant scanner confocal MVIIA, Vc1.1, SO-3, Mr1.8–1,
focusing) and Fli1 larva imaging with CCD camera Mr1.8–2, nitrendipine, Con-T[M8Q],
HS-10
Gravity and Glass 1–4 hpf embryo Array Inverted microscope with digital Doxorubicin, 5-fluorouracil, 14
microstructure (21–28) camera cisplatin, ascorbic acid
Gravity and Polyurethane 8 hpf embryo, 48 Array Line-scanning confocal imaging Valproic acid (VPA) 64
microstructure –PDMS–glass hpf transgenic (>8) with two cameras
TgĲcmlc2:dsRed)
and SqET4
embryos
Gravity and Borosilicate 8 hpf embryo Array — Ethanol 65
microstructure glass (>100)
Gravity and PDMS–glass 3 hpf embryo and Array Inverted microscope with digital Aminophylline 38
microstructure 72 hpf larva (210 camera
embryos
and 210
larvae)
Capillary PDMS–glass Embryo Single — Methylene blue (MB) and 62
suction rhodamine B (R)
Manual placing PDMS–glass 3 hpf embryo and Single Upright and inverted microscope Trypan blue, Texas red, GFP-DNA, 78
24 hpf GFP-mRNA
dechorionated
embryo
Droplet Teflon tube 4–6 hpf embryo Array 260 Microscope with a camera SDS, CuCl2 96
encapsulation
Hydrodynamic PDMS-glass 7 dpf larva Single Microscope with digital camera Hypoxia 95
drag
Gravity and 3D printing 1.5 hpf and 24 hpf Array Stereoscopic fluorescence 3D printing polymers 97
microstructure polymers (fli1a:EGFP) (>120) microscope with digital camera
embryo
Agarose PMMA 5 dpf transgenic Single Two-photon microscope with fast Citric acid; L-proline 88
HuC:GaCMP3 larva infrared camera
Hydrodynamic PMMA 6 hpf embryo Array (21) Miniaturized USB polarization Copper sulfate, phenol, ethanol, 102
force microscope caffeine, nicotine, dimethyl
sulfoxide
Fin-clipping PDMS 36 hpf embryo, Array (33) Non-specific microscope — 98
transgenic Ga14
embryo
Microvalve PDMS–glass 36 hpf embryo, Array (24) Phase contrast and fluorescence Cyclopamine 94
transgenic 8xGli: microscopy
GFP and flk:GFP
embryo
View Article Online
Table 1 (continued)
Trapping
Trapping Channel unit (fish
principle materials Fish age and type amount) Imaging system Stimuli Ref.
Gravity and PDMS–glass 3 hpf embryo Array Inverted microscope with digital Lead (Pb2+), copper (Cu2+) 93
microstructure (240) camera
defects. Therefore, an agarose-free immobilization strategy of an upstream flow that contains embryonic organisms into
will be more attractive. For example, a microfluidic bioreactor the subsequent traps in each row. Such specially designed
with an aperture structure was designed to immobilize microstructures allow embryo immobilization and develop-
zebrafish embryo for local site-specific stimulation.55 The ment until 72 hpf (a hatched time-point for normal zebrafish
chip contains two polydimethylsiloxane (PDMS) layers, the embryonic development),12 and then they would fail because
top layer with a funnel-shaped open aperture to entrap the the well-developed larvae can freely swim out of the original
embryo and the bottom microchannel layer delivering fluidic traps (Fig. 1B). In order to immobilize more embryos, a high-
stimulants (Fig. 1A). By submerging in a Petri dish filled with density microarray has been created with several ordered
fish water (E3), the chip allows the immobilized embryo bot- microtraps per row, which achieves a high docking efficiency
tom to be exposed to the test compounds, which flow under- of zebrafish embryos based on hydrodynamic force.
neath and are transported by a gravity-driven pump (Fig. 1A). Using this powerful hydrodynamic force, the same group
In this way, the viability of the dechorionated embryo could developed a multi-layer microfluidic device for efficient im-
be documented for six days. Moreover, the transparent PDMS mobilization and real-time analysis of in vivo angiogenesis ki-
device allows for automated imaging and is amenable for netics in transgenic zebrafish embryos.57 In this design, a
phenotypic screening via access ports to administer infec- unique three-dimensional microtrap array was engineered via
tions and treatments to the entrapped embryos. This ap- four thermally bonded polyĲmethyl methacrylate) (PMMA)
proach circumvents the use of agarose for embryo fixation sheets, which consisted of a main loading channel and sev-
and thus prevents the possible developmental defects arising eral interconnected suction channels, embryo trapping
from it. In spite of the fact that such an agarose-free device microstructures and a heating component (Fig. 1C). Aside
functions well as a proof of concept, it is inherently limited from the active forces of hydrodynamics and suction, a pas-
in scaling-up capabilities. A truly high-throughput application sive self-gravity of the embryo body also plays an important
cannot be achieved as the embryos need to be pipetted man- role in highly efficient embryo docking. In this work, the
ually one by one into the arrayed apertures. Such manual embryonic entities array pre-stored in plastic tubing was
handling can also damage the fragile embryos and eventually first transported along the main loading channel by a piezo-
lead to arbitrary orientation of the trapped embryos, which is electric ultrasonic pump (Fig. 1C). As they passed over the
detrimental to the uniformity of the site-specific test. trapping well, the combined sinking force between gravita-
Fortunately, an automated embryo (i.e. fruit fly) position- tional sedimentation and low-pressure suction enabled the
ing strategy based on hydrodynamic micromechanical trap- multicellular embryo docking in series (Fig. 1C). Such a syn-
ping can be easily adapted to address these issues.6 Inspired ergistic effect would be more effective for sequential high-
by this well-established approach, Wlodkowic et al. presented throughput trapping of zebrafish embryos at the single-
a miniaturized hydrodynamic embryo-trapping array for au- organism level. As the first embryo occupied a microcavity,
tonomous and serial immobilization of zebrafish embryos, the second would directly dock into the next trapping room,
enabling uniform drug microperfusion and high-resolution maintaining one embryo per trap room. In addition, the au-
imaging without manual handling.56 The chip has several tomated delivery design and the U-shaped heating manifold
functional microstructures to accomplish high-efficiency em- can effectively control the beneficial temperature for on-chip
bryo trapping in arrays, where the main serpentine channel culturing of the immobilized embryos.
is responsible for embryo loading and medium perfusion. It should be noted that suction force is a reliable and effi-
The loaded embryos are dragged into an array of micro- cient embryonic immobilization strategy. To better demon-
mechanical traps by hydrodynamic force and the suction strate this strong active mechanical force, a vacuum-based
force arising from narrow suction channels that interconnect embryo-trapping method as a case in point has been
the embryo-trapping rooms with the main loading channel presented for fully automated zebrafish microinjection.58
(Fig. 1B). This channel configuration fully synergizes hydro- This strategy is particularly effective for entrapping a large
dynamic force from the main s-shape channel and the ade- number of zebrafish embryos. For instance, the arrayed
quate dragging force through the connected suction channels through-holes (∼400 μm in diameter), evenly spaced and pat-
to enable sequential docking of individual embryos. Because terned within an embryo-holding microdevice, were
each microtrap can hold only one fish embryo, the docked connected to a backside microscale suction channel that was
embryos thus serve as plugs to increase flow resistance across directly interfaced with a vacuum source (Fig. 1D). Upon
the occupied trap (Fig. 1B). This, in turn, leads to redirection manually dispersing a batch of embryos onto this hole-
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Fig. 1 Illustration of present microfluidic technologies for zebrafish entrapment. (A) The funnel device consists of a gravity-driven pump, a funnel
layer and a microchannel layer. The embryo is immobilized in the funnel and fluid in the microchannel below is separated from the fluid above
(reproduced from ref. 55 with permission from Mary Ann Liebert, Inc., publishers). (B) Photographs of a device mounted on an 11.25 degrees tilted
angle stage used to perform automatic trapping and uniform drug microperfusion. Microphotograph showing a six row section of the device with
completely trapped zebrafish embryos (top inset). Images of developing zebrafish embryos (16 hpf) up to 72 hours (bottom inset). (C) 3D chip with
four integrated modules for in vivo analysis of angiogenesis kinetics: (i) the microperfusion channel, (ii) a linear trap array, (iii) a suction manifold,
and (iv) a heating manifold. Cross section of the immobilized embryos inside the trapping array (inset) (reproduced from ref. 57 with permission
from Elsevier). (D) Vacuum-based embryo holding device (top). Embryos are immobilized on individual through holes via a negative pressure, en-
abling fully automated microinjection. Picture (bottom) of a device (565 holes). (E) Image of ZEBRA device fabricated in polystyrene (top) and
PDMS. Schematic and image of 4 dpf zebrafish, which is first taken into a pipette tail first (head first, see bottom) for horizontal alignment and then
loaded into the device head first (tail first, see bottom) for vertical alignment via passive pumping (middle). Scale bars represent 0.5 mm. (F) Set-up
for imaging a larva in a microfluidic “Fish-Trap” channel for real-time brain-wide mapping at lateral or dorsal view (left). Scale bar, 100 μm. De-
tailed design (top view) of the “Fish-Trap” microfluidic chip (right). (E, F) Reproduced from ref. 59 and 40, respectively, with permission from The
Royal Society of Chemistry.
View Article Online
patterned holding device, a negative sucking pressure (2–7 a design was critical for on-demand immobilization and ori-
inHg) was employed to rapid immobilization, with each entation of the zebrafish larva. In their design, the larva
through-hole trapping a single embryo in several seconds. adopted two major orientations, lateral and dorsal, which
The suction strength depending on this pressure level proved depended mainly on the dimensions of the restricted portion
effective in trapping zebrafish embryos without damage. No- of the tapering channel, 150 μm height for lateral position
tably, this facile immobilization approach is compatible with and 150 μm width for dorsal position, whereas the width and
high-throughput automatic microinjection for embryos. height remained the same. Similar to the trapping principle
In contrast to the immobile embryo that has uniform reported by the Wlodkowic group,56 here, as a larva flowed
spherical geometry enclosed in a chorion structure, a well- through Ch.B in a tail-forward format, it could dock into the
hatched larva features active swimming, random orientation first trapping channel acting as a plug due to relative low
and a dynamic complex shape. This poses particular chal- flow resistance. In this way, the in situ flow resistance of the
lenges for real-time and site-specific information readout be- occupied channel is increased dramatically to redirect the
cause of the problems associated with larva immobilization bulk flow, and this allows the subsequent larva to bypass the
and rapid developmental dynamics. To solve this problem, a occupied room and enter the next empty channel in se-
low-cost, agarose-free on-chip mounting technique was quence. Despite these progress steps, there is still lack of a
presented, which allowed either lateral or dorsal views of the design that can span the diverse developmental stages to en-
immobilized target fish (Fig. 1E).59 This microfluidic device, able fish immobilization from immotile embryo to dynamic
called Zebrafish Entrapment By Restriction Array (ZEBRA), larva.
can be fabricated with either polystyrene or PDMS, which
contains optimal channel dimensions to position zebrafish
of 3–5 days post fertilization (dpf). To better entrap the larva, 2.2. Zebrafish delivery
channels with a range of heights (0.25–1.0 mm), widths (0.4– Aside from immobilization, safe and undamaged transport of
1.2 mm) and varied constriction widths between 0.3 mm and zebrafish (embryos) to desired locations is another important
0.6 mm were designed and optimized. The two ends of the manipulation process. Practically, precise positioning or en-
middle-narrow channel were connected respectively to an trapment of zebrafish requires a well-established delivery sys-
oval input (6 mm by 3 mm) and a circular output port (diam- tem, and meanwhile a high-throughput transport is particu-
eter, 8 mm). Finally, the optimal single ZEBRA device has a larly necessary to accelerate the embryo handling and
straight channel (0.8 mm wide; 0.5 mm tall) with a 0.3 mm- zebrafish-based drug development process. To meet such re-
wide constriction portion for single zebrafish immobilization. quirements, several special microfluidic platforms toward
Such a “fish clamp” design can be easily extended to a multi- transport of zebrafish have been created. For example, Son
ZEBRA device for trapping multiple swimming zebrafish, and et al. reported a two-plate droplet-based “digital” microfluidic
the lateral or dorsal view can be precisely controlled by the technology for on-chip transporting of zebrafish embryos via
tail-first or head-first loading. Another advantage of ZEBRA an electrowetting-on-dielectric (EWOD) mediated electrome-
device is the extremely simple operation by using passive chanical force.61 Specifically, an embryo-incorporated droplet
surface-tension-driven pumping to quickly position zebrafish migrated within a 1.5 mm gap between two plates (Fig. 2A).
in a predictable array.60 The top plate serves as a common ground electrode, whereas
Another promising alternative for larva immobilizing is to the bottom plate contains electrodes beneath a dielectric
build a tailored entrapment microstructure for holding layer for governing the transfer of droplets. The embryo drop-
zebrafish at desirable orientations in a rapid and repeated let needs 0.5 to 1 s to move from one electrode to the adja-
manner. Recently, Shi et al. developed such a versatile “Fish- cent electrode by applying a voltage of approximate 100 Vrms
Trap” technology for real-time mapping of brain-wide neural and 8 kHz that was higher than the voltage used for a small
activity in tens of drug-responsive animals by high- yeast-containing droplet. In order to realize normal embryo
throughput immobilizing and orientating awake larval transport, relatively large addressed electrodes with a size of
zebrafish in an automatic, gel-free and anesthetic-free fash- 5 × 10 mm or 3 × 3 mm were employed. During transport,
ion.40 The core structure of the “Fish-Trap” microfluidic chip the embryo appeared to spin in the droplet and to be “reluc-
consisted of two side-by-side horizontal flow channels (Ch.B tantly” dragged to the next electrode as a last element in the
and Ch.C, both 0.8 mm wide and 0.5 mm high) (Fig. 1F). The droplet due probably to its high density. Despite exposure to
two channels were bridged by a series of short vertical chan- temporal direct current and Joule heating, the embryo
nels (0.4 mm wide and 0.5 mm high) that facilitate fish water remained viable and evolved normally after transfer to a stan-
pumping into Ch.B to increase the hydrodynamic flow focus- dard zebrafish culture medium. Interestingly, such an auto-
ing on trapping channels, each of which has a tapering mated electro-actuated transport of well-defined droplets can
microstructure for trapping larval fish at specific develop- be extended to remove the chorion of an embryo, namely
mental stages (4–6 dpf). This 9.0 mm-long trapping channel dechorionation, which was an important procedure to facili-
was directly connected to Ch.B with a curved inlet to prevent tate electroporation, microinjection and transfection. Two in-
potential damage to larva, whereas its rear end has a 150 μm dependent droplets (20 μL), one containing an embryo and
narrow restricted region only adapted to fix a larva tail. Such the other filled with digestive Pronase, were transported to
View Article Online

Fig. 2 Two examples of present microfluidic technologies for zebrafish transport. (A) Schematic view of an electrowetting device (cross section)
in which a droplet containing a zebrafish embryo is confined within two plates and the dielectric layer (left). The movement of a 5 mm diameter
droplet containing a zebrafish embryo (24 hpf) (right, top). Droplet-based dechorionation of a zebrafish embryo (2 hpf) (right, bottom). The embryo
and digestive reagent were combined on the center electrode (inset 1). Chorion separated from the embryo in 2 h (inset 2) (reproduced from ref.
61 with permission from The Royal Society of Chemistry). (B) Larvae are automatically loaded to the system from either reservoirs or multiwell
plates and flow through a photodetection system including two LEDs and one high-speed photodiode (PD), which discriminates the passage of a
larva from air bubbles and debris. After the larva is transported and trapped at a designated position in a microscale capillary, two stepper motors
rotate the capillary along its axis of rotation, facilitating the organ-oriented imaging (reproduced from ref. 41 with permission from Nature Publish-
ing Group).
collide and incorporate into an integral big droplet. After larva moves into a refractive-matched capillary and is posi-
peeling off the chorion within 2 h, the dechorionated embryo tioned in the optical field, allowing subsequent imaging and
can be moved to a designated site and separated from the microsurgery in a required favorable orientation (dorsal, lat-
chorion debris (Fig. 2A). eral or even any angle) aided by rotation of two stepper mo-
Although droplet-based embryo transport is effective, the tors; iii) the post-surgery larva can be dispensed back to the
driven force to transport the embryo requires digital electron- culture multiwells. Note that the entire transport process was
ics that relies on complex circuit design and multi-step finely tuned by the cooperation among fluidic valves, re-
photolithography of electrode (array). In addition, a large- stricted region of the capillary and programmed syringe
scale simultaneous transport of embryo-incorporated droplets pumping force. The core component of this transport system
without crosstalk is challenging. Thus, a direct, droplet-free has been extended to improve the throughput of the previ-
high-throughput delivery of zebrafish to the designated posi- ously reported “Fish-Trap” technology.40 Briefly, an integrated
tions would be more attractive. Yanik and coauthors demon- larva-transporting system, composed of a syringe pump, cap-
strated a high-throughput vertebrate automated screening illary, fish-handling component and electronic valves, was ca-
technology (VAST) that automatically guided larvae trans- pable of loading and sending larvae into the trap array. To
porting from reservoirs or multiwell plates to a series of better transport the larva, a slightly wide capillary with inner
downstream pipelines (Fig. 2B).41 For example, i) a photo- diameter of 1.0 mm was chosen, which allowed smooth and
detection system first discriminates the passage of a larva injury-free delivery of the fish through the tube by eliminat-
from air bubbles and debris; ii) after photodetection, the ing the possibility of body twisting. Moreover, a direction-
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switching loop was incorporated to ensure either tail-forward transported an embryo to the required position mainly by
or head-forward transport in the flow-through process. suction from an external syringe.62 First, a loading tube
A high-efficiency transport of zebrafish is also critical for connected to the embryo inlet port was brought in close prox-
high-quality microinjection. The Selvaganapathy lab imity to the target embryos in a Petri dish, which facilitated
presented a PDMS-based microinjection device that the loading process. Then, by applying suction to a syringe in
Fig. 3 Microfluidics-based zebrafish perfusion and dynamic culturing. (A) A three-layer microfluidic chip including a concentration gradient gener-
ator (CGG) and an embryonic inlet array for embryo dynamic culturing and perfusion, which aims to evaluate the toxicity of drugs on zebrafish de-
velopmental dynamics (left). The microphotographs of modeling the perfusion process in the embryo culturing room. The scale bar is 300 μm
(middle). The on-chip development of zebrafish embryos (4 hpf) until 72 hpf (right) (reproduced from ref. 14 with permission from AIP Publishing
LLC). (B) Another microfluidic perfusion platform allowing zebrafish dynamic culturing and live imaging during embryogenesis, which consists of
three parts: inlet layer drawing into the gradient generator, fish tanks and waste channels ending in outlets (left). Side view of the chip (right). (C)
Schematic of a 32-well chip with pre-heated water circulating for non-invasive perfusion and culturing. Buffer enters the chip via inlet (bi) and out-
let (bo) ports and circulates through feeder channels. Each well has a total of 6 connections (3 inflow, 3 outflow). Scale bar, 1 cm. Magnified view
of zebrafish embryo in a 2.0 mm well (right, top) and schematic drawing showing the levels of the z-planes and x–y coordinates and their relation
to the inlet channels (blue) and outlet channels (orange) (right, bottom). (B, C) Reproduced from ref. 64 and 65, respectively, with permission from
The Royal Society of Chemistry.
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connection with the embryo outlet port, one selected target imaging during embryogenesis.64 The perfusion system
embryo will be inhaled into the loading tube automatically. also featured an embedded concentration gradient genera-
Instantaneously, this tube is raised to prevent a next possible tor that connected with eight fish tanks to position em-
embryo loading while ensuring continuous perfusion of fish bryos with one animal per tank (Fig. 3B). This prototype
buffer to reduce environmental shocks that may affect viabil- design was somewhat similar to our device,14 especially in
ity. Following this way, batches of embryo array inside the the perfusion format, both providing continuous gradient
channel, with predetermined spacing between each target an- perfusion to the trapped embryos. They used an oxygen-
imal, can be transported safely to the injection site. Once in- permeable and removable polyurethane membrane as a lid to
jection is completed, the injected embryo is transported into seam the embryo inlet, which easily produced air bubbles,
the appropriate viable or waste reservoir still driven by the rather than the employment of a simple open chamber in
suction. our design, which facilitated bubble removal and oxygen ex-
change.64 To further facilitate the perfusion, the inner sur-
face of the microchannel was made hydrophilic to minimize
2.3. Perfusion and dynamic culturing of zebrafish potential air bubbles. Note that both micro-perfusion devices
A normal growth environment for zebrafish is dynamic are able to accommodate and ensure normal embryo develop-
macroscopic water filled with oxygen, which is beneficial for ment at early stages (<92 hpf, Fig. 3B) due mainly to the con-
timely dilution of excreta and replenishment of culture me- stant and uniform provision of medium, which minimizes
dia. In contrast, a traditional static platform like a plastic additional stresses to the vulnerable embryos during
multiwell plate or Petri dish is incompatible with the require- gastrulation.
ments of dynamic culturing. In particular, the periodic aspi- Prior to our gradient-perfusion design, Wielhouwer et al.
ration and replacement of buffer are extremely invasive, in- had developed another microfluidic flow-through system by
ducing stress to early-stage vulnerable embryos. Also, several providing non-invasive culture conditions and accessibility to
other concerns regarding static exposure to zebrafish (eggs) aid the normal dynamic growth of zebrafish embryos
need to be considered. One typical problem is the irreversibly (Fig. 3C), which could yield a survival rate of 100% after 5
gradual reduction of the exposed solution because of evapo- days of culture.65 This microdevice also consists of three
ration and nonselective adsorption, which leads to layers of bonded (borosilicate) glass, but it features two inde-
uncontrolled changes in concentration and pH of the original pendent fluidic modes: an in-built microheating channel for
solution. To address this issue, a modified 24-well micro- circulating preheated water to ensure the desired tempera-
plate-based flow-through system has been developed that can ture for embryo growth and a solution channel for continu-
alleviate but not totally eliminate the problem.63 Unfortu- ous dynamic perfusion of on-chip embryos. In this device,
nately, this method brings other obstacles such as depleting the embryo-culturing chamber has a size (2.5 mm in diame-
large volumes of solutions, which is neither environment- ter) much smaller than that of wells in conventional 96-well
friendly nor inexpensive, especially for toxic or rare test sub- microtiter plates (6 mm in diameter), whereas it is big
stances. To solve this problem, we devised a microfluidic ar- enough for a well-hatched larva to swim freely, and such a
ray device for microscale gradient flow-through perfusion small culture volume is beneficial for saving valuable com-
and dynamic culturing of embryos to evaluate the toxicity of pounds. Of note, each chamber connects three inlet and
drugs on zebrafish developmental dynamics (Fig. 3A).14 This three outlet channels that permit on-chip flow-through cul-
chip is composed of three layers with the middle sandwiched turing without local blocking from either the embryo itself or
layer containing seven embryo culture chambers that were shed debris (Fig. 3C). This design together with parallel ar-
mechanically micromachined to place and co-culture several rangement of culturing wells significantly reduced the possi-
embryos in each well. Embryos were manually transferred ble cross-contamination between wells. In addition, the au-
into the culturing chambers by exposing to the constant gra- thors made a detailed investigation of the relationship
dient perfusion from upstream distributed medium, which between the perfusion rate (0.5–6 μL per well per minute)
could be rapidly and efficiently mixed in less than 100 sec- and embryo survival on this micro-perfusion chip. They
onds (Fig. 3A, middle). Such a design facilitated removal of found that a flow rate of 2–4 μL per well per minute yielded a
the discharge waste or debris from the organism by tilting higher survival rate than either too high or too low perfusion
the chip at around 20 degrees. Under this dynamic culturing rates, implying that sufficient oxygen was supplied in this
condition, the target embryos obtained the same well- flow-rate range. Unfortunately, a similar generation of air
developed morphology and stage-specific features as that of bubbles occurred under the lid as presented in a previous re-
the control (Fig. 3A right); meanwhile the whole organogene- port,64 which would reduce the advantages of dynamic perfu-
sis obviated the possible mechanical damage from frequent sion. To avoid the confined bubbles, they used a novel
manual pipetting. Nevertheless, the fabrication of our first- perfusion-channel design with outlets higher than inlets to
generation fish-chip was somewhat complicated and uneco- enable automatic air bubble removal and solution circula-
nomical, requiring glass etching with hydrofluoric acid (HF). tion. Finally, this design contributed to a high survival rate
Later, Choudhury et al. described another microfluidic perfu- (100%) through parallel arrangement of wells with minimal
sion platform allowing zebrafish dynamic culturing and live cross-contamination and blocking debris.
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Obviously, the on-chip high survival rate and normal de-

velopment of the zebrafish benefits from the naturally dy-
namic microenvironment supplied by the perfusion-style
microfluidics, which can in a timely manner remove the
harmful excretions and refresh the solution with sufficient
oxygen exchange. That is to say, the dynamic perfusion struc-
ture is the key to designing a microfluidic platform enabling
zebrafish research involving trans-stage development. How-

ever, a common limitation of these innovative designs is the
repetitive manual dispensing of each embryo into the minia-
turized culturing chamber and even followed by manual
sealing of the open chamber with air-permeable membranes
or plastic plugs.38,64
2.4. On-chip microinjection and electroporation

Active transferring of foreign materials, such as nucleic acids
(e.g. DNA, RNAi), proteins, nanoparticle conjugates and phar-
maceutical agents, into cells or embryos plays important
roles in transgenics, signal transduction pathway, in vitro fer-
tilization and drug development.45,66–70 Nevertheless, well-
established chemical or biological approaches often suffer
from complex design and synthesis of biomolecule-carriers
and the ensuing cellular barriers or degradation. To over-
come this trouble, physical means such as microinjection
and electroporation, which can insert a microneedle or create
a transient and ultrasmall pore on the cellular membrane to
introduce carrier-free molecules into the cell or embryo body,
would be an ideal alternative.71–75 For example, DNA injec-
tion has been used to establish transgenic zebrafish lines
and the loss-of-gene-function model in the embryo.76 How-
ever, an efficient transport of exogenous biomolecules into
zebrafish embryo via active physical introduction still poses
several challenges: (i) high-efficiency transport and immobili-
zation of embryos to facilitate mechanical injection (ii) with
high frequency (for ultrashort-time opening of membrane)
Fig. 4 Microscale zebrafish injection and electroporation. (A) A
and (iii) high-precision control of each dosage release. To micropipette tip penetrates the embryo and deposits materials at a
address these challenges, Wang et al. presented a micro- pre-set destination in a specified volume (left). Micropipette retracts
robotic system that is capable of injecting a large number of out of the embryo (right). (B) Zebrafish embryo is loaded and
embryos in a short time window, featuring full automation, immobilized by suction. The needle is actuated by the compliant de-
formation of the PDMS substrate by an external microactuator.
high-speed sample immobilization and high survival rates
Electroosmotic pumping of methylene blue solution into the embryo
(Fig. 4A).58 After array patterning of embryos on a holding and retracted from the embryo (reproduced from ref. 62 with permis-
microdevice aided by a negative suction pressure, a micropi- sion from The Royal Society of Chemistry). (C) Embryos were placed
pette tip was finely moved to a switching point that indi- on top of the patterned anode. The PDMS boundary created a cham-
cated the embryo boundary and global structure, penetrat- ber which contained the dye solution. A second electrode on top of
the embryo acted as the cathode, allowing electrode-shaped patterns
ing the chorion and depositing materials (fluorescent dyes)
of extracellular compounds delivered into the embryo. White light im-
at the desired location of the cytoplasm center (defined as age of ‘M’ shaped design (right, top) and fluorescent image of the ‘M’
the combination of the yolk and the cell portion of a pattern (right, bottom) (reproduced from ref. 78 with permission from
zebrafish embryo). After completing the microinjection, the Springer).
micropipette tip was withdrawn out of the embryo to a spe-
cific position above the contact point with 1.4 mm for sub-
sequent injections. To increase the embryonic injection effi- extracellular material into targeted cytoplasm or nucleus
ciency, two microrobots were operated in parallel without without cell and reagent restriction, the use of pressure-
an off-line process. driven flow limits the minimum injection needle size (0.2–0.5
Although this typical capillary microinjection technique μm) that requires high pressures for dosing. The restriction
has original advantages as a simple and direct way to inject on the tip diameter is critical to successful injection, because
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smaller tips lead to growing viability as they minimize the survival rate (∼90%) of 24 hpf dechlorinated embryos was
damage exerted on cells of embryo during injection. To this achieved after electroporation by introducing GFP-DNA and
end, a non-pressure-driven reagent delivery approach, ionto- GFP-mRNA. Such a high survival rate suggests minimal dam-
phoresis, with a small-size needle tip would be an excellent age to the dechorionated embryos under the imposed electri-
means of injection.62 This approach overcomes the limita- cal pulses. Importantly, the pulse amplitude combined with
tions on tip size due to its independence of needle geometry, patterned multiple electrodes allows simultaneous delivery of
whereas the slow delivery, dependence on ionic properties, different molecules into the developing embryo.
difficult quantitation and requiring operator-mediated pro- Despite this progress, the local electroporation of
cess make iontophoresis impractical for clinical research, es- zebrafish embryos in parallel with high efficiency and re-
pecially in requiring a statistically significant sample size. To duced cell damage remains a challenge. Fortunately, we may
address these issues, Selvaganapathy et al. presented a novel find solutions to overcome this hurdle inspired by a recently
PDMS-based embryonic microinjection system in a micro- developed microdevice with a dielectric guide-based design,
fluidic format with precise electroosmotic dosage, which was which achieved localized electroporation with enhanced effi-
fully scalable and maintains the advantages of capillary ciency and minimal cell injury by using mouse embryo as an
microinjection, and circumvented the restrictions (Fig. 4B).62 organism model to study morphogenesis.79 Most importantly,
By exploiting the compliance and flexibility of soft PDMS as a this design can be easily extended to a variety of microscale
simplified microinjection strategy, this microdevice realized a embryological entities (e.g. zebrafish embryos) in segregated
fully integrated high-throughput injection to zebrafish em- developmental stages.
bryos. In particular, a special channel structure with compli-
ant deformations was designed to actuate the integrated 3. Imaging of zebrafish
microneedle, and then another advantage, electroosmotic
flow (EOF) was used to transport reagents with non-pulsating Microscopic imaging is a powerful tool to enable direct obser-
flow and precise electrical dosage control driven by potentials vation of micrometer or sub-micrometer-scale tiny morphol-
between 5 V and 25 V. This EOF-based microinjection en- ogies (phenotype) and molecular events (e.g., fluorescent
ables precise individual dosing of embryos via direct electri- probe tracking) both in cells and in multicellular
cal feedback that can be used to identify the location of the organisms.80–83 In contrast to cells, which have a small size
microneedle inside the organism body. Therefore, the use of (∼10 μm), thin and flat “2D” morphology, as well as static
electroosmotic reagent delivery may give rise to more favor- and transparent properties, relatively bulky zebrafish (em-
able and accurate results for on-chip embryonic injections. bryo, ∼700 μm) are characteristic of rapid dynamic develop-
Electroporation is another physical approach to deliver ex- ment, unpredictable locomotion, sub-millimeter thickness of
ogenous molecules into cells or embryos via the electric per- the 3D specimen and the presence of multiple tissue types
meation of the plasma membrane.77 Unlike microinjection and organs. These features pose significant challenges for
relying on a small-size needle tip, electroporation employed real-time, 3D and site-specific imaging. The reasons are i) the
an external electric field as a transmembrane potential to sharp morphological alterations of zebrafish embryos in early
transiently and reversibly break down cell membranes development requiring a time-lapse imaging system to track
in vitro. Instead of a direct current (dc) field that often gener- the real-time feedback of minimal developmental progres-
ates bubbles from water electrolysis and Joule heating associ- sion, which is a necessary practice, specifically during a drug
ated with the required high field strength, pulsed or alternat- evaluation process to study long-term developmental toxicity
ing current (ac) was preferentially applied to produce the effects; ii) the frequent twisting and the well-hatched swim-
field necessary for electroporation. Moreover, high-density ming larva resulting in uncontrollable orientations that make
microscale electrode array has been engineered to reduce the real-time and site-specific imaging extremely difficult and iii)
in-between gap of the electrode to minimize the overall volt- the thickness of multicellular embryonic zebrafish at sub-
age requirement. Particularly, a local and positioned electro- millimeter scale reducing the optical spatial resolutions,
poration with patterned delivery of foreign triggers is appeal- which will be further deteriorated with on-going accumula-
ing and significant for studying developmental biology in tive pigmentation, although partial tissues or organs of
several organism models. For example, a microfabricated zebrafish appear transparent.84,85 Therefore, a rational and
electroporator was engineered to deliver extracellular com- complementary combination of advanced microfluidic ma-
pounds (e.g., trypan blue, Texas red, GFP-DNA, GFP-mRNA) nipulation technology with a well-matched imaging system
into zebrafish embryo with specific patterns in localized re- toward real-time, spatially resolved characterization of
gions (Fig. 4C).78 By depositing a thin layer of patterned plati- zebrafish and embryo is highly desirable. To this end, Yanik
num electrode on a glass substrate, single square pulses of et al. employed a rotatable capillary to immobilize the target
10–20 V (0.20–0.40 kV cm−1) and 50–100 ms pulse width were zebrafish and facilitate it at the viewing field of an optical
sufficient to create transient pores on embryos to transfer imaging system, which allows the use of a high-numerical-
molecules between the late blastula period (3 hpf) and early aperture water-immersion objective because of the similar re-
pharyngula period (24 hpf). In this way, trypan blue and fractive index between capillary texture and water (Fig. 5A).41,86
Texas red were initially transferred into the yolk and a high However, imaging a specific phenotype often requires a favorable
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Fig. 5 Zebrafish imaging. (A) A multifocal confocal head with an EM-CCD camera and a second large-area CCD are used for high-speed confocal
and wide-field fluorescence imaging, respectively. A high-speed CCD camera connected to the inverted microscope allows zebrafish screening at
cellular resolution in three dimensions. (B) An epifluorescence two-photon microscope images neural activity while pulse-like stimuli are delivered
to a zebrafish larva. An IR-sensitive camera images tail behavior from below. (C) A resonant scanner imaging system recording of brain-wide neural
activity from larval zebrafish using the “Fish-Trap” technology. (A, C) Reproduced from ref. 86 and 40, respectively, with permission from The Royal
Society of Chemistry. (D) Photograph of the microfluidic device interfaced with ESEM (left) and a photograph taken with an internal camera of the
ESEM and depicting a chip-based device inside the ESEM chamber placed on a motorized x–y Peltier cooling stage (right) (reproduced from ref. 89
with permission from John Wiley and Sons).
accessing angle, especially when the thick tissues and skin and position the larva's head at the center of the field of
pigments of zebrafish would make the target phenotype view, aided by a fast camera and an automated image-
obscured. For example, a clear visualization of the midline processing algorithm. To image the target larva, both an
crossing of the Mauthner motor neuron axons can only be upright high-resolution water-immersion objective and an
realized by observation from the hindbrain.41 By introduc- inverted air objective have been employed, which allows
ing capillary two-end stepper motors, the immobilized high-resolution confocal imaging and wide-field fluores-
larva can be oriented to facilitate imaging and can also be cence imaging, respectively. Based on these advanced im-
rapidly reoriented to render visualization of organs or tis- aging systems, they achieved high-throughput in vivo
sues from multiple angles. Prior to this controllable orien- zebrafish screening at cellular resolution in three
tation, a three-axis stage needs to automatically regulate dimensions.
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Although the desired imaging portion of the confined fluorescence microscope with a tandem scanner can directly
larva can be easily accessed via finely rotating the capillary, map the spiking-induced calcium dynamics because brain-
only a single orientation exists in each rotation. Meanwhile, wide neurons in the larva have been genetically encoded with a
the one-dimensional tubular structure only places several fish calcium indicator. Notably, the fluorescence-trackable calcium
in a linear arrangement. This could reduce the feasibility to transients associated with neural activity from either a local
simultaneously observe multiple larvae with desired orienta- specific brain region or global brain can be recorded with
tions using a wide-field imaging system to record much more single-cell resolution at a 10 Hz frame rate. Such a high re-
images, enabling better statistical analysis. Recently, a micro- sponse frequency is the key to realize high-quality imaging of
device, called ZEBRA, has been designed to immobilize larvae calcium transients. In addition, this imaging system allows
with desired orientations via multichannel entrapment struc- long-term (36 hours) live imaging of the laterally immobilized
tures with different widths or lengths.59 Importantly, this de- larva, and importantly, the occasional tail-shaking interference
sign is compatible with the wide-field microscopy system that can be easily removed by using a simple registration algorithm.
enables simultaneous imaging of a batch of zebrafish with These advantages are critical to enable high-throughput and
selected phenotype from either the dorsal or the lateral view. real-time brain-wide imaging.
Nevertheless, the optical scattering will become fairly intense In contrast to fluorescence microscope imaging, like con-
as the visualization depth in zebrafish tissues is over a few focal imaging that usually offers flat and reconstructed im-
hundred micrometers. As a result, the resolution and image ages, environmental scanning electron microscopy (ESEM)
quality of the routine optical microscopy degrade rapidly, in- can provide detailed 3D surface topographical information.
cluding confocal microscopy that is often used for high- This is particularly appealing for the phenotype characteriza-
resolution 3D in vivo imaging. tion of zebrafish. Note that ESEM imaging is performed in a
In contrast to confocal microscopy, two-photon micros- gaseous atmosphere under low vacuum mode, which is ame-
copy has a deeper tissue penetration and imaging depth, nable to biological specimen imaging without complex
which renders it a powerful tool to image the nervous system staining. However, conventional SEM imaging requires a
in zebrafish.87 This is because two-photon microscopy uses high-vacuum environment and multiple preparative proce-
ultrafast pulsed lasers to emit sufficient photon densities for dures for bio-specimens such as dehydration, fixing and
high-efficiency multiphoton absorption that produces fluores- staining with gold–palladium deposition. These requirements
cence signals. Candelier et al. designed an open-ended two- greatly limit the potentially intended high-throughput pheno-
photon imaging microfluidic chip performing neuronal activ- typical analysis and prevent further screening of morphologi-
ity recordings on agarose-restrained zebrafish larva that was cal features. Recently, a proof-of-concept microfabricated pro-
chemically stimulated with unprecedented spatial and con- totype allowed open-access larvae trapping and interfacing
centration accuracy and reproducibility (Fig. 5B).88 This fish- with the ESEM system (Fig. 5D).89 This was the first evidence
chip was integrated with two-photon functional imaging and showing that microfluidic devices could be successfully
high-speed video recording for simultaneous monitoring of coupled with ESEM imaging to provide rapid entrapment of
the motor behavior and brain activity. In order to record mo- larvae and subsequent high-resolution imaging of micro- and
tor behavior, a microscope coupled to a fast-responsive infra- nanoscale morphological features. First, a semispherical
red (IR) camera was positioned below the microfluidic chip. microwell array was engineered to hold and align the yolk of
By placing the larva-containing chip under an epifluorescence dechorionated zebrafish larvae. Prior to ESEM image acquisi-
two-photon microscope, this fast IR-sensitive camera can im- tion, the larvae need to be temporarily anaesthetized to in-
age the neuronal activity and simultaneously monitor the tail hibit the intrinsic body-twitching movements. Following the
wobbles from below via transparent PDMS. In combination mentioned procedures, a larvae-containing open-access chip
with GCaMP strains (transgenic zebrafish larva expressing a was inserted to and interfaced with the ESEM chamber. How-
genetically encoded calcium indicator, GCaMP) and two- ever, the evaporated tissue fluids from the living larva would
photon calcium imaging, they found different brain circuit cause intense deformations of the fish body, which signifi-
activity patterns induced by the stimuli of opposite hedonic cantly degrade the live imaging. Ultimately, paraformalde-
values. hyde (PFA)-treated specimens were employed because the tis-
Although ultrafast pulsed laser-based two-photon micros- sue structures cross-linked by PFA were strong enough to
copy is capable of visualization in deep tissues of zebrafish, avoid deformation as they were exposed to the intense pres-
several weaknesses still exist, such as low scan speed and sure in the ESEM chamber. In this way, rapid ESEM imaging
high cost, which significantly limits high-throughput assay of the trapped fish array was performed with high resolution.
and imaging of the fast physiological processes (e.g., neuro- Aside from the sophisticated and expensive imaging setup,
nal activity monitoring via Ca2+ imaging). Recently, a “Fish- the cost-effective and portable imaging system is also attrac-
Trap” microfluidic device coupled into a resonant scanner tive, particularly for on-site visualization of zebrafish pheno-
imaging system was used for high-throughput mapping of types to interrogate the pharmaceutical effects. For acute tox-
brain neuronal activity in awake and stimuli-responsive icity evaluation of lead compounds, the local or global
zebrafish using the transgenic line elavl3:GCaMP5G (Fig. 5C).40 phenotype alteration can both qualitatively and quantitatively
As immobilized in the channel with a dorsal orientation, a reflect the drug candidate's developmental toxicity and
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teratogenicity. Importantly, these simple phenotypic alter- damage on the chemical-exposed embryos. However, several
ations can be easily resolved via common optical microscopy. disadvantages limit its further application. For example, the
For instance, we employed routine inverted microscopy to sloped chip that facilitated the removal of waste can block
quantitatively assess the embryonic toxicity of some clinical the real-time imaging of the embryonic development. Addi-
popular drugs by recording a series of typical phenotypic fea- tionally, the glass-based chip texture is biologically incompat-
tures.14 In combination with a standard morphological scor- ible, to some extent, with a live system that requires oxygen
ing system, such a phenotype-based microscopic readout exchange. Accordingly, we designed an air-permeable PDMS
would be more reliable and appealing. Moreover, image sen- chip to evaluate the developmental toxicity and teratogenic
sors, such as charge-coupled devices (CCD) and complemen- effect of an anti-asthmatic agent, aminophylline (Apl), on em-
tary metal oxide semiconductors (CMOS), coupled to portable bryos and larvae, simultaneously.38 The chip contains two
light-emitting diodes (LEDs) as light source has greatly con- CGG units, one for embryo and the other for larva, with three
tributed to the development of field-portable on-chip imaging repeated culture chambers, which entrap the fish in se-
technologies, like the cell-phone based microscope.90–92 This quence enabling a reasonable statistical analysis. At the end
mobile-phone mounted microscope has been broadly used to of the fish transfer process, the top open inlet will be sealed
image cell samples in either bright field mode or fluorescent by insertion of a transparent PMMA cylinder plug while leav-
mode using a self-carried camera and a liquid crystal display ing the last chamber with a hollow plug connected to a Tef-
screen to capture and show the images. Such a portable and lon tube for removal of waste. This consecutive open design
multifunctional miniaturized imaging device will be an ideal can remove the dead embryo easily and enables a precise and
platform for future zebrafish research in drug discovery. continuous real-time analysis. Upon continuous exposure to
Apl, real-time imaging shows that it dose-dependently im-
pairs the development of organs and tissues. The degree of
fish injury was characterized by measuring the heart rate,
4. Phenotypic readout of zebrafish survival rate, body length and hatching rate of the embryos.
responses to microscale external Although the microscale CGG unit has a great advantage to
stimuli yield gradient testing substances, it is confined to single com-
pound or compound combination types, which fails to simul-
Site-specific stimulation in combination with high-resolution taneously provide a comprehensive gradient profile of indi-
imaging is essential to enable qualitative and quantitative vidual and collective testing candidates. To solve this
phenotypic readout of zebrafish responses to external stimuli. problem, we engineered a disc-shaped concentration gradient
Indeed, rapid and synchronous stimulation on zebrafish is generator (DCGG) that aims to rapidly generate a multiplexed
also critical to accelerate the safety assessment of lead com- gradient pattern, such as a single Pb2+ gradient, single Cu2+
pounds and environmental pollutants. However, common gradient and single combined (Pb2+ and Cu2+) gradient
platforms such as the microplate and glass slide are techni- (Fig. 6B).93 Based on this design, a collective gradient profile
cally infeasible to provide precise manipulation of external of multiple substances can be formed to enable the evalua-
stimuli at high spatial–temporal resolution or on-line rapid tion of the dynamic developmental toxicity and teratogenicity
serial dilution of the test substances. In addition, these con- of Pb2+ and Cu2+ on embryos. We found that Pb2+ or Cu2+
ventional platforms are difficult to carry out flow-through could induce deformity and cardiovascular toxicity in
multi-tasks in a debris-free context that is beneficial to the zebrafish embryo and more severe toxicity caused by the
normal growth of zebrafish. Thus, there is an urgent need to combination of the two metal ions.
develop novel zebrafish handling and stimulating systems to As compared to multiple embryo monitoring, single-
address the above-mentioned concerns. organism level drug testing can provide accurate and high
To this end, we first describe a microfluidic array system spatial–temporal information to local organs or tissues.
using a phenotype-based whole-organism model to evaluate Huang et al. developed an easily accessible and automated
the developmental toxicity and teratogenicity of pharmaceuti- microfluidic device to monitor the dynamic effect of cyclo-
cals, which offers continuous stimulation to the entrapped pamine on intersegmental blood vessels (ISV) and somite de-
zebrafish embryos using gradient test substances produced velopment by using transgenic zebrafish embryo as an in vivo
by an upstream rapid concentration gradient generator reporter model (Fig. 6C).94 This device comprises an array of
(CGG) (Fig. 6A).14 Gradient stimuli were delivered in a contin- open-access culture chambers to accommodate zebrafish em-
uous or developmental stage-specific manner to induce dy- bryos with one egg per chamber. Each chamber is 4 mm in
namic developmental toxicity on embryos. The toxicity of diameter with a small volume of about 400 μL, suitable for
doxorubicin on zebrafish eggs was quantitatively assessed via culturing a single embryo. The multi-layer soft lithography
heart rates, and its teratological effects were characterized by permits the chip a two-layer configuration design, a fluidic
other phenotypic features, such as pericardial impairment, layer and a control channel layer. The crossover of these two
deformations of tail fin and notochord, and SV–BA distance/ layers of channels forms monolithic microvalves and the
body length (Fig. 6A, bottom). By scoring the teratogenic se- cross-section of fluidic channels is much smaller than the
verity, we precisely evaluated the time- and dose-dependent size of the embryos, ensuring that the entrapped embryos
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Fig. 6 Phenotypic readout of zebrafish responses to microscale external stimuli. (A) The generated drug gradients from chambers (1 to 7) to
stimulate the trapped embryos. Magnified section of a single chamber structure (top). Side view of a 5-dpf normal larva marked with pericardium,
tail fin (area between the red and white dotted line), notochord (white rectangle), sinus venosus (SV), and bulbus arteriosus (BA). Body length signi-
fied by a white oblique line. (B) A disc-shaped gradient generator and 24 fish tanks named C1–C24 (top). Typical morphological abnormalities of
embryos exposed to Pb2+ and Cu2+ (bottom). (A, B) Reproduced from ref. 93 and 14, respectively, with permission from AIP Publishing LLC. (C) The
zebrafish embryo culture chip with medium refreshment for monitoring the dynamic effect of cyclopamine on ISV and somite development (left).
The bright-field and fluorescence images of zebrafish that were treated with cyclopamine (right) (reproduced from ref. 94 with permission from
The Royal Society of Chemistry). (D) Sketch of the stimulant flow in the device during the three stationary states: at rest, during injection of SA and
during injection of SB (left). Neuronal responses to chemical stimulation (right).
stay inside the wells as fluids flow through the chamber. easy using pipettes, it cannot offer dynamic responses from
Such a design allows “withdraw–refill” cycles to efficiently re- microenvironment stimuli with a high spatial–temporal reso-
place the exposed culture or testing medium with fresh me- lution, which is beneficial in site-specific phenotypic
dium within 10 seconds, which is critical in long-term analysis.
zebrafish culture and drug treatment. By using transgenic Recently, Candelier et al. reported a microfluidic chip ca-
zebrafish as the reporter, they identified the blocking effect pable of delivering high spatial resolution and pulsed (milli-
of cyclopamine on the Hh signal pathway via imaging the second-level switching rate) chemical stimuli to a single
typical phenotypes of somite and ISV. Although in this multi- agarose-restrained zebrafish larva with its face right in front
layer device the open-access design makes embryo transfer of the delivery channel (Fig. 6D).88 By combining the
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advantage of precise control over microscale fluid flow with especially in fish immobilization that is a critical step to facil-
the likelihood of recording simultaneous behavioral and neu- itate subsequent in vitro or in vivo assay using a physiologi-
ronal activity on zebrafish, the neuronal and motor responses cally intact animal model. This intact model can reflect the
to acute chemical stimuli have been studied. Consequently, most real responses to external stimuli and finally turn into
the stimuli of opposite hedonic values (citric acid and easily discernible phenotypic events. Notably, the combina-
L-proline) lead to different circuit activity patterns by imaging tion of biocompatible soft PDMS materials with a flexible
the related phenotypes, such as recording of tail movements. channel-structure design endows zebrafish an unstressed
Notably, the duration of the stimulus (50–500 ms) and the physiological growing environment and enables the gel-free
near-millisecond switching rate can be precisely controlled in and anesthesia-free immobilization of larvae in multiple ori-
this work, which is the key to realizing high spatiotemporal entations (e.g., lateral, ventral or dorsal view). By precisely
and acute stimulus on the restrained zebrafish. tuning the tailor-engineered microtraps and hydrodynamic
In addition to studying stimuli responses of chemicals or (suction) forces, reversible trap and release could be realized,
drugs using the microfluidic zebrafish model, it is also ideal which allows the possible on/off-chip gene-sequencing,
to interrogate the physiological and behavioral effects on the genotyping98 or other complicated bioassays. Thus, a trend
embryos to microenvironment hypoxia. To demonstrate this of “fish-on-a-chip” could potentially develop a new-
advantage, a custom microfluidic device was presented, generation of an “all-in-one” platform for on-chip larva
which precisely controlled a broad range of hypoxic and trapping, culturing, exposing and analyzing. However, a
normoxic conditions to stimulate a spatially confined larva great challenge lies in how to create a trans-stage (ranging
without anesthesia.95 A computer-controlled gas mixture al- from immotile embryo to swimming larva) immobilization
lows high-frequency switching (2 second timescale) between structure or approach to finely regulate the animal trap-
diverse oxygen concentrations in the medium around the par- ping mode. To overcome this hurdle, the synergized
tially immobilized larva. These merits enable direct observa- magnet-controlled manipulation system with highly effi-
tion of multiple behavioral responses of zebrafish to different cient hydrodynamic trapping would be an ideal means by
levels of imposed acute hypoxia including the behavior magnetically labeling and controlling of the animal body.24
change within 15 seconds following the alteration of oxygen Such a highly controllable strategy, if further inspired by
content in the exposed medium. 3D printing microstructures,99–101 would largely benefit
Beyond dynamic stimulation, the “fish-on-a-chip” platform site-specific orientation and phenotype screening.
is also ideal for recording static exposure to the microscale On the other hand, microfluidics is capable of ordered
confined zebrafish larvae. Funfak et al. introduced a long Tef- and directional transport of zebrafish to the designated loca-
lon tube by inserting an air bubble between embryo- tion for oriented or large-scale immobilization, which facili-
containing droplets to construct fluid-segment microreactors tates organ-specific and high-quality wide-field imaging of
for studying the zebrafish behavior, embryonic development multiple animal bodies simultaneously at the single-
and toxicity screening.96 This was the first generation of a organism level. This is critically important for future on-chip
fish-on-a-chip platform, suggesting the potential of micro- high-throughput drug screening and environmental toxicity
fluidic zebrafish models for chemical screening. By incorpo- evaluation. However, a smart and easy to use zebrafish-
rating chemicals, such as a membrane-damaging anionic sur- transport system is crucially needed to enable future high-
factant (SDS), into small volume droplets, different growth end applications. For example, we can interface a bubble-
effects in the static culture condition have been observed via spaced zebrafish delivery tube to a well-defined “fish-trap”
documented phenotypic images. This fluid-segment tech- structured array, guiding ordered trapping in sequence via
nique is compact and cost-effective to house zebrafish larvae, gravity and hydrodynamic force.44,102 By patterning an
but it limits the access to larvae and prevents exchanges be- electrochemical electrode array (e.g., transparent ITO
tween media, chemicals and oxygen. Apart from static screen- electrode) in the trapping room under the trapped animals,
ing in droplets, this non-dynamic embryonic toxicity assay we can electrochemically sense and at the same time obtain
can be adapted to assess the biocompatibility of 3D printing fluorescence images of the extra-embryonic molecule events
polymers by quantifying the toxicity of the 3D printed micro- associated with developmental dynamics or oxygen consump-
fluidic device.97 Based on the recorded key developmental tion confined in the micro-compartment.103
phenotypes, the biocompatibility of four commercially avail- Of note, the highly controllable flow-through pattern and
able 3D printing materials has been evaluated, and the re- inherently air-permeable textures (PDMS) of microfluidics
sults show that they are highly toxic to the embryos and even provide an excellent perfusion and dynamic culturing micro-
deadly. environment for zebrafish embryos. Such a dynamic perfu-
sion mechanism can, in a timely manner, remove the
Concluding remarks and future excretive metabolites and effectively replenish the tested sub-
perspectives stances and oxygen. Nevertheless, current microfluidic ap-
proaches fail to effectively and automatically remove the dead
Microfluidics has demonstrated unique advantages over fish bodies in a minimally damaged fashion as exposed to
other techniques in zebrafish (embryo) manipulation, deadly environmental toxicants, which would be taken into
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consideration in the future design of a dynamic perfusion 21475034, 81130064). Dr. Ping Wang thanks the New Products
microsystem. of TCM Senile Diseases Co-innovation Center of Hubei for the
In addition, by interfacing tailored on-chip microneedles support. We appreciate Mr. Qiu Gao (SUNY Buffalo) and Miss.
or electrodes to microfluidics, foreign molecules could be Lisa Ramirez (SUNY Albany) for their assistance in grammati-
precisely delivered into the fish body via a mechanical trans- cal correction and language proofreading.
membrane mechanism governed by microinjection or electro-
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Featuring work in fish-on-a-chip technology from the Centre As featured in:

for Nano-biosensors in the School of Laboratory Medicine at

Title: Fish-on-a-chip: microfluidics for zebrafish research

We provide a critical review of recent progress on zebrafish

See Fan Yang, Guo-Jun Zhang et al.

Fish-on-a-chip: microfluidics for zebrafish

1. Introduction genetics, chemical biology, disease modeling and drug

Lab on a Chip Critical review

cess can dramatically reduce the period of pharmaceutical screening.15–20

Guo-Jun Zhang received his Ph. Zuanguang Chen received his

Critical review Lab on a Chip

Lab on a Chip Critical review

Table 1 Present microfluidic technologies for zebrafish research

Critical review Lab on a Chip

Lab on a Chip Critical review

Critical review Lab on a Chip

Lab on a Chip Critical review

Critical review Lab on a Chip

Lab on a Chip Critical review

Critical review Lab on a Chip

Obviously, the on-chip high survival rate and normal de-

zebrafish research involving trans-stage development. How-

2.4. On-chip microinjection and electroporation

Lab on a Chip Critical review

Critical review Lab on a Chip

Lab on a Chip Critical review

Critical review Lab on a Chip

Lab on a Chip Critical review

Critical review Lab on a Chip

Lab on a Chip Critical review

transfer can transport any extracellular molecules of interest

Critical review Lab on a Chip

26 E. K. Sackmann, A. L. Fulton and D. J. Beebe, Nature, 53 J. Nordgreen, F. M. Tahamtani, A. M. Janczak and T. E.

Lab on a Chip Critical review

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