Optical micro-tomography “OPenT” allows the study of large toadfish Halobatrachus didactylus embryos and larvae

Abstract

Batrachoidids, which include midshipman and toadfish are less known among embryologists, but are common in other fields. They are characteristic for their acoustic communication, and develop hearing and sound production while young juveniles. They lay large benthic eggs (>5mm) with a thick chorion and adhesive disk and slow development, which are particularly challenging for studying embryology. Here we took advantage of a classical tissue clearing technique and the OPenT open-source platform for optical tomography imaging, to image a series of embryos and larvae from 3 to 30mm in length, which allowed detailed 3D anatomical reconstructions non-destructively. We documented some of the developmental stages (early and late in development) and the anatomy of the delicate stato-acoustic organs, swimming bladder and associated sonic muscles. Compared to other techniques accessible to developmental biology labs, OPenT provided advantages in terms of image quality, cost of operation and data throughput, allowing identification and quantitative morphometrics of organs in larvae, earlier and with higher accuracy than is possible with other imaging techniques.

Full text

Optical micro-tomography “OPenT” allows the study of large toadfish Halobatrachus
didactylus embryos and larvae
Pedro M. Felix
a
, Ania Gonçalves
b
, Joana R. Vicente
c
, Paulo J. Fonseca
d,e
, M. Clara P. Amorim
c
,
José L. Costa
a,e
, Gabriel G. Martins
b,d,
⁎
a
MARE – Marine and Environmental Sciences Centre, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisboa, Portugal
b
Instituto Gulbenkian de Ciência, R. Quinta Grande, 6, 2780-156 Oeiras, Portugal
c
MARE – Marine and Environmental Sciences Centre, ISPA - Instituto Universitário, 1149-041 Lisbon, Portugal
d
cE3c – Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
e
Departamento de Biologia Animal, Centro de Biologia Ambiental, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
abstractarticle info
Article history:
Received 4 July 2015
Received in revised form 21 February 2016
Accepted 4 March 2016
Available online xxxx
Batrachoidids, which include midshipman and toadfish are less known among embryologists, but are common in
other fields. They are characteristic for their acoustic communication, and develop hearing and sound production
while young juveniles. They lay large benthic eggs (N 5 mm) with a thick chorion and adhesive disk and slow
development, which are particularly challenging for studying embryology. Here we took advantage of a classical
tissue clearing technique and the OPenT open-source platform for optical tomography imaging, to image a series
of embryos and larvae from 3 to 30 mm in length, which allowed detailed 3D anatomical reconstructions non-
destructively. We documented some of the developmental stages (early and late in development) and the anat-
omy of the delicate stato-acoustic organs, swimming bladder and associated sonic muscles. Compared to other
techniques accessible to developmental bio logy labs, OPenT provided advantages in terms of image quality,
cost of operation and data throughput, allowing identification and quantit ative morphometrics of organs in
larvae, earlier and with higher accuracy than is possible with other imaging techniques.
© 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords:
Toadfish
Inner ear
Swim bladder
Development
OPenT
Optical tomography
1. Introduction
Fishes are the largest extant group of vertebrates and exhibit a tre-
mendous diversity of features and adaptations (Nelson, 2006), includ-
ing many homologous to vertebrate tetrapods (e.g. Bass et al., 2008).
The study of embryonic development presents a unique opportunity
to investigate those homologies. Most of what we known of fish embry-
ology derives from work on the model organisms zebrafish and medaka
(Kimmel et al. 1995; Iwamatsu, 2004), whose transparent and small
embryos are easily studied with conventional microscopy. Fish embryos
vary considerably both in size and shape (Richardson et al., 1997), with
zebrafish and medaka falling at the small end of the spectrum. On the
other end, larval stages (even of the two small species), are too large
for conventional microscopy and are still studied resorting mostly to
histological sectioning (e.g., Sabaliauskas et al., 2006). (See Table 1.)
Morphomics and a rekindled interest in detailed anatomical studies
have recently gained prominence in developm ental biology, after
mesoscopic imaging by “Optical Projec tion Tomography” or “Light-
Sheet Microscopy”, were introduced to embryology by Sha rpe et al.
(2002) and Huisken et al. (2004), respectively. Both techniques proved
valuable to study embryos of model organisms, in ways that were not
possible with conventional microscopy; for example, light-sheet
micro scopy is well suited for imaging the early develo pment of
live zebrafish and drosophila embryos (Huisken et al., 2004; Keller
et al., 2008), and opti cal tomography for 3D imaging large embry os
(Bryson-Richardson and Currie, 2004; Ruparelia et al., 2014). The
open-source community has already provided “DIY” solutions based on
hardware and software which, for the most part, are already familiar to
developmental biologists (Pitrone et al., 2013; Gualda et al., 2013). A
question some labs are facing is whether these techniques, in simpler
open-source forms are “worth the trouble”, in other words, whether
they provide better results than those obtained with existing techniques,
and effectively solve the difficu lties of large non-model organisms.
Fishes from the Batrachoididae family, which include midshipman
and toadfish, are less familiar to developmental biologists, but widely
used in ecotoxicology and ethology (Caçador et al., 2012), in bioacous-
tics (Ric e et al., 2011; Vasconcelos et al., 2012) and neurophysiology
(Bass et al., 2008; Vasconcelos et al., 2011; Elemans et al., 2014).
Batrachoidids lay large benth ic eggs (N 5 mm) with a thick chorion
and adhesive disk, and the embryos develop rather slowly (Arora,
1948; Dovel, 1960; Britz and Toledo-Piza, 2012), making them less ame-
nable for ontogenetic studies. They are characteristic for their acoustic
communication, and although it is known that hearing and sound
Mechanisms of Development xxx (2016) xxx–xxx
⁎ Corresponding author at: Instituto Gulbenkian de Ciência, R. Quinta Grande, 6, 2780-
156 Oeiras, Portugal.
E-mail addresses: pmfelix@fc.ul.pt (P.M. Felix), gaby@igc.gulbenkian.pt (G.G. Martins).
MOD-03395; No of Pages 6
http://dx.doi.org/10.1016/j.mod.2016.03.001
0925-4773/© 2016 Elsevier Ireland Ltd. All rights reserved.
Contents lists available at ScienceDirect
Mechanisms of Development
journal homepage: www.elsevier.com/locate/mod
Please cite this article as: Felix, P.M., et al., Optical micro-tomography “OPenT” allows the study of large toadfish Halobatrachus didactylus embryos
and larvae, Mechanisms of Development (2016), http://dx.doi.org/10.1016/j.mod.2016.03.001

production develop early (Vasconcelos and Ladich, 2008; Alderks and
Sisneros, 2011; Vasconcelos et al., 2015) the details on the ontogeny
of the associa ted anatomical structures remain largely unknown.
Those structures are too minute and delicate for micro dissection, and
yet too large and deep inside the larvae to be accessible by conventional
micro scopy; furthermore, since some of the structures are cavities
(e.g., the contents of the otic capsule) they cannot be properly dissected
out, and are best studied intact in toto. Having obtained a collection of
Halobatrachus didactylus (the Lusitanian toadfish) embryos at several
stages with sizes ranging from 3 to 30 mm in length, we took advantage
of a classical tissue clearing technique and a custom-built “OPenT ” -
optical tomography sc anner, based on the Open SpinMicroscopy
platform (Gualda et al., 2013), to gain insight into the anatomy and de-
velopment of the stato-acoustic organs, swimming bladder and associ-
ated sonic muscles, and highlight the potential of optical tomography
as a prime tool for developmental biologists.
2. Results & discussion
The H. didactylus embryos were first visible only betwee n 10–
12 days post-fertilization (dpf) as a 2.8–2. 9 mm lon g streak with an
engorged rostral end. After the second week, embryos reached N 3mm
in length, and showed a neural tube, otic and optic vesicles, pectoral
fin buds and overt segmentation of paraxial mesoderm (Fig. 1C), with
15–20 somites; none of the major organ systems were yet recognizable
at this stage. As a way of comparison, this was merely equivalent to a
zebrafish 17 h post-fertilization (Kimmel et al., 1995). Up to this stage,
the embryos were too small and positioned far from the centre of the
large yolk mass, to allow optimal imaging with optical tomography.
They could be imaged with confocal microscopy (Fig. 1), but that re-
quired excising the embryo from the yolk sac and acquiring Z-stacks
in multiple adjacent fields (followed by 3D-image stitching) using a
10× objective, otherwise the embryo's natural curvature did not pro-
vide access to the limited working distance of high-quality objectives;
confocal imaging wit h low NA (e.g. 4× magnification) objectives did
not provide images properly resolved in depth.
After the first two weeks, and throughout organogenesis, embryos
could no longer be imaged with confocal microscopy and only OPenT
provided images of the whole embryo and its internal anatomical
details (Figs. 1, 2), with magnifications of 0.3–3 × at the detector
plane, a range of magnifications not available on conventional confocal
microscopy setups. Large-scale (i.e., low magnification) laser-scanning
imaging with a “macro confocal” allowed imaging of a lateral fi
eld-of-
view closer to that of OPenT, but with significantly limited axial field-
of-view and resolution, when imaging 30 dpf embryos. Though th e
lateral resolution was high (at the em bryo surface), the 3D dataset
was highly anisotropic, showing low axial resolution and light penetra-
tion when compared to images obtained with OPenT (not shown).
After the first four weeks of development, all H. didactylus embryos
had hatched and most organ systems were already visible. These larvae
were developmentally equivalent to a 60 h pec-fin stage zebrafish
(Kimmel et al., 1995), though almost 3× larger. Our observations of ex-
ternal anatomy were similar to those described for the oyster toadfish
Opsanus tau by Tracy (1959) and Dovel (1960). Our use of OPenT
allowed the reconstruction and analysis of both the external and inter-
nal 3D anatomy in situ of H. didactylus, without the need to dissect or
section embryos or larvae, up until free-living forms 3 months old
(N 20 mm long; Fig. 1), often allowing identification of organs earlier
than was detected using a dissecting stereoscope. This demonstrates
that OPenT is a useful technique to study large embryos and larvae,
allowing detailed morphological studies up to late stages, covering the
full mesoscopic range.
Measurements obtained from 3D datasets, allowed us to follow
the embryo's natural curvature and determine the full length of the
body and head. The body grew progressively from the second week on-
ward at a pace of 0.147 mm/day (~6 μm/h), slowing down at the end of
the second month (Fig. 2C). This rate of growth is comparable to that
previously reported for O. tau (Tracy, 1959), and considerably slower
than that reported for Danio rerio which grows at a rate of 125 μm/h
during embryogenesis and at 20 μm/h during larvagenesis (Kimmel
et al., 1995). By the end of the second month, the yolk mass had been
consumed (Fig. 1) and larvae begun feeding and swimming freely. In-
terestingly, the head of H. didactylus (measured as the distance from
tip of mouth to level of pectoral fin bud) grew constantly throughout or-
ganogenesis and larvagenesis (Fig. 2C), which contrasts with zebrafish
and medaka whose heads' length practically does not increase during
organogenesis and early larvagenesis (as per figures in Kimmel et al.,
1995; Iwamatsu, 2004). A disproportionately large head is a feature of
most vertebrate embryos and early larvae, and is lost during
larvagenesis as the body lengthens at a fast pace. Our observations
suggest that the disproportionately large head patent in juvenile and
adult Batrachoidids, may be a neotenic trait, instead of a morphological
characteristic that develops secondarily.
Because of the interest of H. didactylus as a model organism for stud-
ies of communication we then focused our observations on the anatom-
ical details of the stato-acoustic organs. We found that the semicircular
canals (SCCs) + sacullae and swim bladder were well visible before the
end of the first month (Fig. 2). Using OPenT 3D reconstructions we were
able to identify and precisely measure these structures earlier than was
possible using a stereoscope and dissection of fresh larvae. Before the
Table 1
Comparison of optical techniques used to image H. didactylus embryos.
Advantages Disadvantages
Stereoscope –Allows “fresh”/live embryos
(i.e., not fixed)
–Fast screening
–Fluorescence not required
–Natural colour images
–Inexpensive and widely available
–No 3D imaging
–Limited internal anatomy
–Low contrast/resolution
Confocal (micro & macro) –3D imaging at cell resolution
–High-contrast/resolution
–Widely available
–Requires embryo fixation + clearing
–Requires 3D stitching of whole embryos
–Impractical to image larvae whole
–Anisometric resolution; especially limiting in “macro” variant
–Limited imaging in depth, even with macro confocal
–Expensive to buy and operate
Optical μTomography (OPenT) –3D imaging of larvae (3–30 mm)
–3D in fluorescence & brightfield modes
–Isometric resolution
–High contrast images
–Fast screening of whole anatomy
(even considering image processing steps)
–Inexpensive to build and operate
–Requires embryo fixation + clearing
–Limited resolution in early stages (before organogenesis)
–Requires more experience with image processing
–Commercially unavailable (has to be custom built, e.g., OPenT) — as of 2015
2 P.M. Felix et al. / Mechanisms of Development xxx (2016) xxx–xxx
Please cite this article as: Felix, P.M., et al., Optical micro-tomography “OPenT” allows the study of large toadfish Halobatrachus didactylus embryos
and larvae, Mechanisms of Development (2016), http://dx.doi.org/10.1016/j.mod.2016.03.001

end of the first month, the ovoid-shaped otic vesicle had transformed
into a complex assembly of cavities which, when reconstructed in 3D re-
sembled a typical inner ear of fish, with three yet incomplete SCCs, and
the saccule and lagena (Fig. 2A). At 50 dpf, the three SCCs were
completely formed (i.e., their lumen was continuous), and a utricule
was now well individualized (Fig. 2B). The length of the otic capsule
also increased linearly from the second week onwards until the end of
the second month (Fig. 2D). The swim bladder first appeared as a min-
ute sac (collapsed dorso-ventrally) appended to the gastro-intestinal
tract dorsally (Fig. 2A), although the sonic muscles were not yet discern-
ible; this was not noticeable with stereoscopy, even after dissecting the
larva. Later, at 50 dpf the swim bladder had differentiated into two well
individualized sacs, each with an associating sonic muscle which ap-
peared as two cell masses positioned medially t o the swim b ladder
(Fig. 2B). The growth of the swim bladder also seems to be linear during
the first 2 months of development.
The datasets obtained with this work will be made available publicly
through the “Haeckaliens” online database (www.gabygmartins.info/
research/haeckaliens), and the segmented organs of two specimens
can be inspected in 3D interactively with the 3D figure is supplementary
materials using the standalone Acrobat reader application (Adobe).
Video 1 shows a sequence of coronal, sagittal and axial sections of 30
and 46 dpf embryos, and a 3 month-old larva. The 3D reconstructions
of these organs allowed measurements with a nominal resolution of
2.75 μm (empirical resolution, measured as “half-width-at-half-
maximum” of small features, was of 5 μm; for the largest larvae, nominal
resolution was 5 μm, and empirical 10 μm).
Compared to other techniques accessible to developmental biology
labs, OPenT provided clear advantages both in terms of image quality,
cost of operation and data throughput. Though images of confocal mi-
croscopy are better resolved when imaging early embryos (i.e., before
the onset of organogenesis), they require excision of embryos and
acquisition of multiple high-magnification Z-st acks + 3D-stitc hing,
which is impractical once organogenesis is underway and organ sys-
tems begin assembling.
Other contenders to OPenT are X-ray micro-Computed Tomography
(μCT) and micro-Magnet ic Res onance Imaging (μMRI), which we
did not test. However, OPenT image details and speed acquisitions
were one order of magnitude smaller than those typical of μMRI and
similar to μCT, and cost of installation/operation two orders of magni-
tude lower than μCT, its closer contender. One further advantage of
OPenT for developmental biologists, besides the access ibility of the
Fig. 1. A) 12 dpf H. didactylus embryo, fixed, dechorionated and imaged with: fluorescence stereoscopy (top inset), confocal microscopy (middle) and OPenT (bottom). The embryo was
excised from the egg before imaging with the stereoscope and confocal microscope. B) Similar embryo imaged“fresh” with side-illumination and a colour camera coupled to a stereoscope;
because of the curvature, the embryo cannot be imaged completely in a single field-of-view. C) 3D reconstruction of a 16 dpf embryo imaged with optical tomography; because of their
volume and curvature around the yolk sac, these embryos could no longer be properly imaged in 3D with confocal microscopy. D) 3D reconstruction of a 26 dpf embryo imaged with
optical tomography. By this stage most organ systems had formed and internal anatomical details could easily be studied (see Fig. 2); these were not visible in images obtained with a
“macro” confocal. E) 42 dpf embryo. F) Larva at 3 months, already free-swimming and completely devoid of yolk. Total length = 24.76 mm. See Supp material “Movie 1” for slices and
details of internal anatomy of embryos depicted in D), E) and F).
3P.M. Felix et al. / Mechanisms of Development xxx (2016) xxx–xxx
Please cite this article as: Felix, P.M., et al., Optical micro-tomography “OPenT” allows the study of large toadfish Halobatrachus didactylus embryos
and larvae, Mechanisms of Development (2016), http://dx.doi.org/10.1016/j.mod.2016.03.001

Fig. 2. 3D reconstruction of H. didactylus embryos showing left and dorsal views, internal anatomy and segmented organs of interest: Semi-circular canals (opaque blue), swim-bladder
(opaque orange), central nervous system (CNS; transparent blue), and gastro-intestinal tract (GI; transparent orange), sonic muscles (red). A) 26 dfp. Embryo total length = 8.6 mm
B) 50 dfp embryo; total length = 20.6 mm. Embryos are drawn to scale. B) After hatching at 50 dpf, the swim-bladder (orange) now consists of two separate chambers, and the sonic
muscles (red) are already forming. C) Graph showing increase in length of whole body and head length. D) Graph showing growth of otic vesicle/capsule and swim bladder, measured
as the an tero-posterior length (see diagrams in A) and B)). CNS = central nervous system, SB = Swim bladd er, SM = sonic mus cle, SCC = s emi-circu lar canal. See also
Supplementary figure in the form of an interactive 3D pdf image.
4 P.M. Felix et al. / Mechanisms of Development xxx (2016) xxx–xxx
Please cite this article as: Felix, P.M., et al., Optical micro-tomography “OPenT” allows the study of large toadfish Halobatrachus didactylus embryos
and larvae, Mechanisms of Development (2016), http://dx.doi.org/10.1016/j.mod.2016.03.001

technique, is the similarity of sampl e pr eparation and imaging
protocols.
The accessibility to image organs deep inside embryos/larvae with-
out having to physically dissect them is important to create visual 3D
maps of the location and shape of these organs, and to understand
how they are formed during development. It is especially important in
the case of inner-body cavities, such as SCCs or the otic vesicles, which
cannot be easily dissected out and are best studied in toto. Following
with precision the development of hearing and sound organs allows
parallels with studies of ontogenesis of behaviour of acoustic communi-
cation and its role in social interactions. It is also useful for studies
involving the physiology of hearing and sound production, namely to
define the ages at which these organs are formed and function and for
the proper positioning of electrodes for stimulation. It has potential
application for developing optogenetics methods which rely on precise
location of tissues/cells of interest in deep tissues. OPenT can easily cope
with large embryos, of which H. didactylus is a particularly challenging
example, but also large larval stages of more common species, which
are typically not used because of their size, especially zebrafish larvae
beyond the first days of development.
One particular aspect of the whole imaging and analysis workflow
that remains a challenge for all these techn iques is the automatic
segmentation of anatomy. In our case, due to the isometric nature of re-
constructed tomograms, image stacks typically contained hundreds-to-
thousands of optical sections, and manual or semi-automatic segmenta-
tion, though cumbersome and with low through put, still was more
dependable than even machine learning tools such as those of WEKA
segmentation or Ilastik (Hall et al., 2009; Sommer et al., 2011). We facil-
itated the semi-automatic segmentation process by su btracting from
the reconstructed 3D tomogram a derivative (e.g. a Sobel edge detection
or Gr adien t Magnitude); interestingly this procedure also alleviated
some of the star-like and beam-hardening artefa cts that ca n occur
with reconstruction of optical CT images.
Another standing challenge to researchers is the difficulty in pre-
senting real 3D imagery along the traditional manuscript format. For
that, we have explored accessible online tools which require only limit-
ed computer expertise from life-scientists. This included the prepara-
tion of interactive 3D-pdf illustrations as in Ruthensteiner and Heß
(2008), an example of which is presented in the supplementary mate-
rials, and the use of simple online tools for dissemination of OPenT 3D
datasets such as those used in the “Haeckaliens” online database.
3. Materials & methods
3.1. Egg/embryo collection
Fertilized eggs were colle cted in an intertidal zone of a sandy
beach in a public-restricted area (38° 41′ 41″ N, 9° 2′ 55″ W, Montijo,
Portugal), and took place d uring the breeding season (June–July
2013). Artificial nests were placed 2 m apa rt and their inner surface
was coated with plasti c sheets to facilitate egg collection. Territorial
males spontaneously occupied these nests and the females, attracted
by the vocalizati on of males, entered the n est to spawn, after which
the eggs were fertilized. This “semi-natural” approach did not allow us
to determine the exact date and time of fertilization, so we also collected
males and females and kept them, in loco,inpoolswithartificial nests;
in this case eggs were collected immediately after fertilization and we
could record the first stages of development. These eggs were treated
similarly to those of the intertidal area and used to determine the
exact age of the latter. Collected eggs were then transported to facilities
at Lisbon University and kept in aquaria under controlled conditions:
salinity of 22 and average temperature of 19 °C (±1), with constant aer-
ation. To follow and register embryonic and larval development, a sam-
ple of four eggs was collected randomly from the batches at pre-set time
intervals: every 12 h for the first four days; every 24 h for the following
five days; every 48 h for the next 10 days; and every 96 h until the onset
of the juvenile stage. Embryos/larvae were anesthetized with MS222
and in vivo images were collected using a Leica DFC 280 digital camera,
coupl ed to a Leica MZ6 stereomicroscope and the Leica Application
Suite v4.1. Later, all larvae were exposed to a lethal dose of MS222, for
further sample preparation and 3D imaging.
3.2. Sample preparation, 3D image acquisition, analysis and presentation
For 3D imaging (optical tomography & confocal microscopy), em-
bryos were fixed in 4% form alin for 24 h at 4 °C, washed extensively
with PBS and H
2
O at room temperature, and then slowly dehydrated
through a series of methanol soluti ons up to 100%. This dehydration
step, required for tissue clearing, was gradual to minimize tissue defor-
mations and anisometric shrinkage. When observed fresh under a ste-
reoscope, the eggs measured an average diameter of 6.19 ± 0.39 mm,
and after dehydration they had shrunk an avera ge 9.4% (5.66 ±
0.31 mm); all measurements presented herein do not account for this
shrinkage.
Dehydrated embryos were then transferred to BABB (benzyl
alcohol:benzyl benzoate 1:2) until the tissues became completely trans-
parent, which required 1–5 days. Cleared embryos were glued with
cyanocrylate to the tip of a metal rod which was magnetically attached
to a stepper-motor axis, and imaged using a custom-built OPenT optical
tomography scanner as described in Gualda et al. (2013, 2014), which,
along with the officia l website includes all detai ls on how to build
the system (https://sites.google.com/site/opensp inmicroscopy/). For
image acquisition, we used a DMK 2Mpixel camera (The Imaging Source
Inc.) mounted on an Infinitube 1× lens + lens tube assembly (Infinity
Optics), and captured the fluorescence of whole embryos while rotating.
To obtain different magnifications we changed the tube length in
infinity space behind the objective, adjusting it so the embryo image
completely filled the CCD detector. Tissue auto-fluorescence was ob-
tained by excitation with a 470 nm LED source (+470 ± 20 nm excita-
tion filter) , and collected after a LP510nm emission filter at infinity
space in the lens tube. A total of 1600 projections were acquired for a
360° turn using micromanager and the OpenSpin plugin (Gualda et al.,
2013). The projection dataset was then pre-processed by noise filtering
and alignment of the rotation axis using FIJI (Schindelin et al., 2012;see
more details also in Gualda et al., 2013), and su bsequently, back-
projection reconstructed with the free software NRecon (Skyscan
Inc.). This produced an isometric 3D stack of sections from which the
internal anatomical details and 3D reconstructi ons could be studied
(see Supp Material movie1). For comparison purposes, Z-stacks were
also acquired with confocal imaging, either a Leica SP5 confocal using
a 10 × 0.3NA objecti ve (early stage embryos) or a zoom macro-
confocal Leica LSI (30 dpf).
The 3D stacks were then processed and segmented (internal organs
digitally “labelled”) using Fiji and Amira v5.3 (FEI Inc.). To facilitate
semi-automatic segmentation, in some cases we computed a derivative
of the original stack (“gradient magnitude” or “Sobel edge detection”),
which was then subtracted from the original stack. After segmentation,
each organ was turned into a 3D model, exported as Wavefront “obj” file
and later imported into Simulab composer V4 (Simlab soft) to prepare
the 3D PDF interactive illust ration (Fig. 2). Movie 1 was prepared
using FIJI. The whole body, head, inner ear and swim bladder were mea-
sured with both FIJI or Amira, using 3D reconstruction of the whole vol-
ume or of free-angle slices, and measured as antero-posterior lengths. In
SCCs we considered this to be the linear distance between the anterior-
most end of anterior SCC and the posterior-most end of the posterior
SCC. The full body length and head were measured from the tip of the
nostril to the posterior end of the tail; when the embryo presented a
curvature we followed the body's mid axis using a spline. The head-
body separation was limited at the level of insertion of the finbuds,or
in earlier stages at the caudal end of the otic vesicles. The measurements
presented are an average of 2–3 embryos at the stages mentioned.
5P.M. Felix et al. / Mechanisms of Development xxx (2016) xxx–xxx
Please cite this article as: Felix, P.M., et al., Optical micro-tomography “OPenT” allows the study of large toadfish Halobatrachus didactylus embryos
and larvae, Mechanisms of Development (2016), http://dx.doi.org/10.1016/j.mod.2016.03.001

Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.mod.2016.03.001.
Acknowledgements
The authors acknowledge the help of the personnel of the Air Force
Base No. 6 in Montijo (Portugal) for allowing egg collection in their mil-
itary facility, Manuel Vieira, Joana Amado, and Daniel Alves for help dur-
ing the sampling ca mpaigns, Nuno Martins and Hugo Pereira of the
Advanced Imaging Unit of IGC for help with imaging and discussions,
and Leica for the use of a the LSI macro confocal for test purposes. This
study was funded by Science and Technology Foundation, Portugal
(project PTDC/MAR/118767/2010 and the strategic project UID/MAR/
04292/2013 granted to MARE, and PEst-OE/MAR/U10199/2014).
References
Alderks, P.W., Sisneros, J.A., 2011. Ontogeny of auditory saccular sensitivity in the plainfin
midshipman fish, Porichthysnotatus. J. Comp. Physiol. A. 197 (4), 387–398.
Arora, H.L., 1948. Observations on the Habits and Early Life History of the Batrachoid Fish,
Porichthysnotatus Girard. Copeia. pp. 89–93.
Bass, A.H., Gilland, E.H., Baker, R., 2008. Evolutionary origins for social vocalization in a
vertebrate hindbrain–spinal compartment. Science 321 (5887), 417–421.
Britz, R., Toledo-Piz a, M., 2012. Egg surface structure of the freshwater toadfish
Thalassophryneamazonica (Teleostei: Batrachoididae) with information on its distri-
bution and natural habitat. Neotropical Ichthyol. 10 (3), 593–599.
Bryso n-Richardson, R.J., Currie, P.D., 2004. Optical projection tomography for sp atio-
temporal analysis in the zebrafish. Methods Cell Biol. 76, 37–50.
Caçador, I., Costa, J. L., Duarte, B., Silva, G., Medeiros, J. P., Azeda, C., …&Costa,M.J.(2012).
Macroinvertebrates and fishes as biomonitors of heavy metal concentration in the
Seixal Bay (Tagus estuary): which species perform better? Ecol. Indic., 19, 184–190.
Dovel, W., 1960. Larval development of the oyster toadfish, Opsanus tau. Chesap. Sci. 1
(3–4), 187–195.
Elemans, C.P., Mensinger, A.F., Rome, L.C., 2014. Vocal production complexity correlates
with neural instructions in the oyster toadfish (Opsanus tau). J. Exp. Biol. 217 (11),
1887–1893.
Gualda, E.J., Vale, T., Almada, P., Feijó, J.A., Martins, G.G., Moreno, N., 2013.
OpenSpinMicroscopy: an open-source integrated microscopy platform. Nat. Methods
10 (7), 599–600.
Gualda, E., Moreno, N., Tomancak, P., Martins, G.G., 2014. Going "open" with Mesoscopy: a
new dimension on multi-view imaging. Protoplasma 251 (2), 363–372.
Hall, M., Frank, E., Holmes, G., Pfahringer, B., Reutemann, P., Witten, I.H., 2009. The WEKA
data mining software: an update. ACM SIGKDD Explor. Newsl. 11 (1), 10–18.
Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J., Stelzer, E.H., 2004. Optical sectioning
deep inside live embryos by selective plane illumination microscopy. Science 305
(5686), 1007–1009.
Iwamatsu, T., 2004. Stages of normal development in the medaka Oryzias latipes. Mech.
Dev. 121, 605–618.
Keller, P.J., Schmidt, A.D., Wittbrodt, J., Stelzer, E.H., 2008. Reconstruction of zebrafish
early embryonic development by scanned li ght sheet micros copy. Science 322
(5904), 1065–1069.
Kimmel, C.B., Ballard, W.W. , Kimmel, S.R., Ullmann, B., Schilling, T.F., 1995. Stages of
embryonic development of the zebrafish. Dev. Dyn. 203 (3), 253–310.
Nelson, J.S., 2006. Fishes of the World. John Wiley & Sons.
Pitr one, P.G., Schindel in, J., Stuyvenberg, L., Preibisch, S., Weber, M., Elicei ri, K.W.,
Tomancak, P., 2013. OpenSPIM: an open-access light-sheet microscopy platform.
Nat. Methods 10 (7), 598–599.
Rice, A.N., Land, B.R., Bass, A.H., 2011. Nonlinear acoustic complexity in a fish ‘two-voice’
system. Proceedings of the Royal Society of London B: Biological Sciences,
rspb20110656.
Richardson, M.K., Hanken, J., Gooneratne, M.L., Pieau, C., Raynaud, A., Selwood, L., Wright,
G.M., 1997. There is no highly conserved embryonic stage in the vertebrates: implica-
tions for current theories of evolution and development. Anat. Em bryol. 19 6 (2),
91–106.
Ruparelia, A.A., Simkin, J.E., Salgado, D., Newgreen, D.F., Martins, G.G., Bryson-Richardson,
R.J., 2014. The Quail Anatomy Portal. Database, 2014, bau028.
Ruthensteiner, B., Heß, M., 2008. Embedding 3D models of biological specimens in PDF
publications. Microsc. Res. Tech. 71 (11), 778–786.
Sabaliauskas, N.A., Foutz, C.A., Mest, J.R., Budgeon, L.R., Sidor, A., Gershenson, J., Joshi, S.,
Cheng, K.C., 2006. High-throughput zebrafish histology. Methods 39 (3), 246–254.
Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch,
S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.Y., White, D.J., Hartenstein, V.,
Eliceiri, K., Tomancak, P., Cardona, A., 2012. Fi ji: an open-source platform for
biological-imageanalysis.Nat.Methods9(7),676–682.
Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A., Hecksher-Sørensen, J., Davidson, D., 2002.
Optical projection tomography as a tool for 3D microscopy and gene expre ssion
studies. Science 296 (5567), 541–545.
Sommer, C., Straehle, C., Koethe, U., & Hamprecht, F. (2011). Ilastik: Interactive learning
and segmentation toolkit. In Biomedical Imaging: From Nano to Macro, 2011 IEEE In-
ternational Symposium on Biomedical Imaging (ISBI). pp. 230–233.
Tracy, H.C., 1959. Stages in the development of the anatomy of motility of the toadfish
(Opsanus tau). J. Comp. Neurol. 111 (1), 27–81.
Vasconcelos, R.O., Ladich, F., 2008. Development of vocalization, auditory sensitivity and
acoustic communication in the Lusitanian toadfish Halobatrachusdidactylus. J. Exp.
Biol. 211 (4), 502–509.
Vasconcelos, R.O., Sisneros, J.A., Amorim, M.C.P., Fonseca, P.J., 2011. Auditory saccular
sensitivity of the vocal Lusitanian toadfish: low frequency tuning allows acoustic
communication throughout the year. J. Comp. Physiol. A. 197 (9), 903–913.
Vasconcelos, R.O., Carriço, R., Ramos, A., Modesto, T., Fonseca, P.J., Amorim, M.C.P., 2012.
Vocal behavior predicts reproductive success in a teleost fish. Behav. Ecol. 23 (2),
375–383.
Vasconcelos, R.O., Alderks, P.W., Ramos, A., Fonseca, P.J., Amorim, M.C.P., Sisneros, J.A.,
2015. Vocal differentiation parallels development of auditory saccular sensitivity in
a highly soniferous fish. J. Exp. Biol. 218 (18), 2864–2872.
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Please cite this article as: Felix, P.M., et al., Optical micro-tomography “OPenT” allows the study of large toadfish Halobatrachus didactylus embryos
and larvae, Mechanisms of Development (2016), http://dx.doi.org/10.1016/j.mod.2016.03.001