www.landesbioscience.com Organogenesis 211
ReseaRch papeR: IMaGING
Organogenesis 5:4, 211-216; October/November/December 2009; © 2009 Landes Bioscience
Live optical projection tomography
ReseaRch papeR: IMaGING
Optical projection tomography (OPT) is a young 3D imaging
technology.1 OPT was created to fill a gap (1–10 mm across) in
the range of specimen sizes encompassed by existing 3D imaging
techniques.2,3 An overview of the standard OPT methodology,
the consequences of using light for computed tomography (rather
than, for example, X-rays or electron beams), and the basics of
the reconstruction and visualization approaches can be found in
It will take more time to explore all potential biomedical appli-
cations of OPT, yet OPT has already had a significant impact in
the field of organogenesis. Three main reasons can be identified:
(1) OPT computes tomographic reconstructions and provides 3D
global visualization in both transmission and fluorescent modes,
a clear advantage for elucidating the anatomical and molecular
complexity of organogenesis;5 (2) the size and optical proper-
ties of many organs or embryonic organ rudiments are suitable
for OPT (especially for the mouse, the main biomedical animal
model); and (3) OPT can play multiple imaging “roles” that can
all benefit organogenesis research: virtual histology, 3D morphol-
ogy, gene expression 3D pattern, pinpointing labeled cells, atlases
of development, genetic screens and phenotyping mutants, and
analysis of disease models.6
After summarizing the current applications of OPT 3D imag-
ing (especially on fixed and cleared mouse embryonic specimens),
the bulk of the review will be devoted to the examination of the
most recent successes in extending OPT technology into the field
of live imaging of organogenesis.7 Live OPT, in which the OPT set
up and ex vivo organ culture were made compatible, has exciting
potential as a molecular imaging approach to track tissue move-
ments and dynamic gene expression in 3D over time. We will
end discussing challenges ahead, in particular for approaching
le milieu intérieur8 inside the tomograph. The goal is to describe
organogenesis quantitatively and faithfully in order to generate
an accurate computational modeling of organ development and
to tackle new problems in organogenesis.
Current Applications of OPT: 3D Imaging of Fixed
and Cleared Specimens
Since its implementation,1 OPT has mostly been used on fixed
specimens after optical clearing in order to reduce photon scatter-
ing. The range of species successfully imaged now includes human,9
mouse,10,11 chick,12 reptile species,13 zebrafish,14 Drosophila15 and
Arabidopsis.16 Mouse embryos are routinely imaged at E9.5
up to E12.5,17 and, for older specimens, organs in isolation are
scanned, for example, the developing limb18 or brain19 at E14.5.
The Edinburgh Mouse Atlas Project (EMAP) has also performed
purely anatomical scanning (of autofluorescence) for a few whole
embryos up to an age of ∼E15.17 Thus, all the organogenesis stages
of mouse development have been imaged with OPT. Younger and
smaller embryos can also be imaged by OPT, but because previ-
ous techniques (such as confocal microscopy) were already able
to capture these smaller specimens, they have not been consid-
ered an important application for OPT itself. Successes with big-
ger specimens such as whole organs taken from the adult mouse
have also now been reported, for brain,20,21 pancreas,20 kidney5
and lungs.22 This opens up exciting new applications such as
preclinical disease research, and accordingly, the whole pancreas
Jean-François colas and James sharpe
EMBL-CRG Systems Biology Program; Centre for Genomic Regulation; UPF; Barcelona, Spain; Istitució Catalana de Recerca i Estudis Avançats; Barcelona,
Key words: blood circulation, conceptus culture, ex vivo, ex utero, heartbeat, limb bud, live imaging, milieu intérieur, optical
Abbreviations: D in 2D, 3D, 4D, dimensional; DOT, diffuse optical tomography; E in E9.5, E10.5, E12.5, E14.5, E15, E16,
embryonic day; FMT, fluorescence mediated tomography; GFP, green fluorescent protein; LOOCC, live OPT opened conceptus
culture; NOD, non-obese diabetic; OPT, optical projection tomography; SPIM, selective plane illumination microscopy
Correspondence to: Jean-François Colas and James Sharpe; Email: firstname.lastname@example.org and email@example.com
Submitted: 09/02/09; Revised: 09/06/09; Accepted: 10/26/09
Previously published online: www.landesbioscience.com/journals/organogenesis/article/10426
Optical projection tomography (OpT) is a technology ideally suited for imaging embryonic organs. We emphasize here
recent successes in translating this potential into the field of live imaging. Live OPT (also known as 4D OPT, or time-lapse
OpT) is already in position to accumulate good quantitative data on the developmental dynamics of organogenesis, a
prerequisite for building realistic computer models and tackling new biological problems. Yet, live OPT is being further de-
veloped by merging state-of-the-art mouse embryo culture with the OpT system. We discuss the technological challenges
that this entails and the prospects for expansion of this molecular imaging technique into a wider range of applications.
212 Organogenesis Volume 5 Issue 4
line of research has been pursued to combine OPT technology
with ex vivo organ culture techniques.7 The organ chosen was
the developing limb bud of the mouse embryo since it is visually
accessible (unlike the heart for example, which is concealed by
other tissues) and is about 1 mm in size, therefore allowing rea-
sonable transmission of photons.
A life-support chamber was designed to be compatible with
the rotary stage required for OPT and with the growth of the
limb bud (Fig. 3A). This chamber required reliable tempera-
ture control, the ability to avoid evaporation from the growth
medium, and a method of supplying gas (in particular oxygen). A
particularly important but difficult issue to solve was the need for
controlling the angle of the organ explant with respect to the axis
of rotation while the specimen was already inside the tomograph,
without the user opening the chamber—as this would impair the
careful temperature control. A micromanipulator was designed
with six mechanical degrees-of-freedom (including three transla-
tions, one simple rotation and a 2D tilting surface) all control-
lable from outside the life-support chamber (Fig. 3B). Owing to
these new hardware and the development of control software, a
series of experiments for quantifying aspects of limb development
The goal of the first experiment was to build up a dynamic
picture of limb growth by tracking the tissue-level movements
of surface ectoderm. A method for creating fluorescent land-
marks and tracking their 3D movements over time was devel-
oped. The specimen was imaged from 200 angles (every 1.8°)
at 15-minute intervals in both fluorescent and transmission
modes—the former to track the artificial landmarks and the
latter to measure the overall shape of the organ. The datasets
that were produced on tissue movements were both global—i.e.,
capturing the whole developing organ, rather than subregions—
and dynamical. These dynamical measurements provided both
rates of tissue displacement over time (Fig. 1A–C) and rates
of surface expansion (Fig. 1D–F). Quantitative maps on tis-
sue movements were extracted: a tissue velocity map (Fig. 1C)
showing for the first time that a twisting motion is involved in
normal limb development, and a map of regional expansion
rates across the limb bud (Fig. 1E and F) showing a surprising
degree of spatial variation.
A second major achievement of live OPT was made in the
realm of molecular imaging. Live OPT was able to monitor a
dynamically changing 3D gene expression pattern throughout
the volume of a living tissue.7 The same model system as above
was used, the mouse limb bud developing in culture, but this
time the signal tracked over time was the signal emitted by the
green fluorescent protein (GFP) reporting for transcriptional
activities of the gene Scleraxis deep inside the mesenchyme of
the organ rudiment.25 Live OPT imaging of the GFP fluores-
cence revealed how the expression domain of Scleraxis changes
its shape and size in 3D over time (reviewed in ref. 7). It was the
first direct 4D observation of dynamic spatial patterning of the
mesenchymal tissue in a mammalian organ. Therefore a useful
compromise between imaging depth and imaging resolution can
also be found with the molecular modality of live OPT to extract
new and significant 4D biological information.
imaging was performed quantitatively to compare the mass of
β-cell tissue in normal versus diabetic specimens, using the NOD
mouse model.20 A related type of soft tissue, but with a very dif-
ferent application, is human biopsy material. Although still at the
exploratory phase, early tests look encouraging.6
Extending the OPT Technology into the Field of Live
The incentive is always strong for adapting a technological
advance made in “static” imaging—such as OPT—to its “live”
counterpart. Capturing the dynamics of a process in a single liv-
ing individual over time often yields important information not
obtainable from a time series of fixed samples. In the domain
of optical tomography, recent years have seen the development
of approaches—such as diffuse optical tomography (DOT) or
fluorescence mediated tomography (FMT)—for producing 3D
images of entire living adult mice.23 In these cases, photons
must pass through 1 to 2 cm of living heterogeneous tissue, so
the signals emerging are unavoidably weak and highly scattered
and can only be reconstructed into fairly low-resolution images.
Nevertheless, these data revealed biologically useful information,
highlighting that a useful compromise always exists between our
desire for high spatial resolution and the desire for longitudinal
studies that capture the dynamics of a single individual over
time.23 The number of scattering events a photon will experience
depends on both the opacity of the tissue and the thickness of
tissue to penetrate. If useful information can be obtained using
FMT from scattered photons traveling through centimeters of
living tissue, it therefore follows that imaging through millime-
ters of living tissue should provide higher resolution and could
be useful for certain experiments. This is despite the fact that
this resolution will nevertheless be significantly lower than when
the sample is fixed and cleared (by which scattering is reduced to
almost zero). For these reasons, it has been worth extending OPT
technology into the field of time-lapse imaging—it again fills an
optical imaging gap here for living specimens between optical
sectioning techniques on the one hand (such as confocal micros-
copy and SPIM24) and diffuse optical tomography approaches
on the other (such as DOT and FMT). The goal therefore is to
image small living specimens 1 to 2 mm across.
Live OPT 4D Imaging of Mouse Organogenesis
Ex Vivo in Organ Culture
Although a range of animal model systems exists whose embryos
or organ explants grow happily on a microscope stage at room
temperature (e.g., the anamniotes zebrafish and Xenopus),
rodents are the primary research models for biomedical research.
Unfortunately, with placental mammals, time-lapse microscopy
with adequate resolution is not easily compatible with embryos
developing in normal conditions, especially at post-implantation
stages of organogenesis as it involves explantation and dras-
tic environmental changes. This is even more delicate with 3D
imaging (compared to 2D time-lapse) as it often requires longer
and potentially phototoxic scans. Thus a particularly challenging
www.landesbioscience.com Organogenesis 213
protocol is worth finding because: (1) 6 hours of normal growth
is not quite enough for embracing many developmental processes;
(2) preserving the integrity of the embryo could lead to imaging
of developmental events for which there are no existing organo-
typic culture method available; (3) it is likely that most existing
organotypic culture models cause subtle divergence from natural
development due the absence of systemic influences such as the
cardiovascular system, and that preserving blood circulation27 may
be critical for faithful quantitative recording of organogenesis;
(4) the main strength of the mouse model is in its molecular
genetics and so, to take full advantage of investment into subtle
germ line modifications (such as cell- or tissue-specific fluores-
cent protein labeling of deep tissues), it is a sensible endeavor to
develop less invasive and durable live imaging technology.
Reducing the thickness of tissue to penetrate in order to
improve resolution while keeping in vivo-like conditions has
been so far attempted in two different manners: “in utero” after
laparotomy and exposure of embryo’s head at E16, or using whole
embryo culture. The former requires the mother to be immobi-
lized on the microscope stage for imaging28 which is difficult to
combine with an OPT approach which requires the specimen to
rotate. Whole embryo culture offers little help if any at organo-
genesis stages over ex vivo explant culture. In fact, it mostly leads
to new problems as it requires rolling the embryo in a bottle
A third, proof-of-concept experiment was also performed on
a different sample: the head of a 9-day-old mouse embryo from a
transgenic line expressing GFP driven by the control elements of
the Pax6 gene.26 Figure 2 shows that good tomographic recon-
structions through the head were achieved highlighting Pax6-
GFP expression in the developing eye and brain. During the
6-hour time-lapse, virtual sections through the eye were able to
record the early stages of lens induction and the resulting mor-
phogenic shape changes. Thus, live OPT is not restricted to the
study of limb development, but could become a general tool for a
number of different model systems.
Live OPT 4D Imaging of Organogenesis Ex Utero
in Conceptus Culture
As mentioned above, useful biological information as gained with
live OPT using organ culture could not have been obtained in vivo
stricto sensu. In vivo, before reaching the mouse conceptus, pho-
tons would have to penetrate millimeters of adult opaque tissues
and only low resolution images can be reconstructed. Therefore,
imaging improvements beyond the 6 hours of normal organo-
genesis achieved in the limb bud explants will most likely come
from a protocol which is less disruptive to the embryo, but which
still minimizes the amount of tissue in the photon path. Such
Figure 1. Dynamic global data on surface tissue movements as obtained with live OpT imaging of cultured limb buds. The results of experiments
using surface fluorescent microspheres as landmarks to track ectodermal movements are shown. (A and B) Shape of the limb bud ectodermal surface
at time 0 of two different developmental stages (a and B) and arrows (3D velocity vectors) representing the movements made during 6 hours by each
tracked microsphere. (C) Global tissue movements over the surface of the limb bud representated by a velocity vector field (red arrows) produced
by radial basis function interpolation of the B vector set (green arrows). Blue asterisks indicate double-inverted vortex flows in which tissue rotates
around two almost fixed points. (D–F) Isotropic surface expansion rates as estimated by calculating the increase in area for triangulated regions.
(D) Triangulation is based on fluorescent landmarks. (E and F) Two different views of a triangle expansion diagram. The rate of expansion during
6 hours is calculated for each triangle. each triangle is colored according to the rate at which it expanded. (G) color coded scale bar indicating the
percentage area increase over 6 hours (reviewed in reference 7).
214 Organogenesis Volume 5 Issue 4
to facilitate tomography of the limb bud and improve
transfer of gases and nutrients. This procedure ful-
filled its first goal as it enabled healthy development of
the limb bud to go beyond the previous 6-hour limit,
allowing for long-term tomographic recording in
the transmission mode of limb bud growth (Fig. 3).
Moreover, the conceptus could be kept alive for up to
36 h—as judged by heart beating, and blood flowing
in the embryo proper (especially in the marginal vein
of the limb bud) and in the two extraembryonic cir-
cuits of the placenta and the yolk sac (Fig. 3). The lat-
ter implies that many events of organogenesis are now
potentially accessible to 4D imaging during the 10th
and 11th day of mouse embryo development. Because
this method had been specifically developed for cul-
turing and imaging the conceptus inside the live OPT
scanner, it was given an acronym: LOOCC (Live
OPT Opened Conceptus Culture) that underscores
the mutual dependency of these two technologies.
Live OPT Imaging of Organogenesis:
The cardiovascular system is the first organ system at
work in vertebrate embryos. In mouse embryo, circula-
tion develops and sustains growth through the coordi-
nated emergence of cardiac function, a vascular network
and maturing red cells between the 8th and 10th day of
gestation,31 i.e., at the onset of organogenesis. The heart
starts beating during the 8th day of gestation and by the
10th day reaches a rate of 200 beats per minute.31 The
cardiovascular system is therefore a source of embry-
onic motion during all stages of organogenesis, even
before the appearance of first embryonic muscular contractions
during the 12th day.32
Thus, efforts to preserve an intact circulatory system for ex utero
imaging as in LOOCC come with a reward: records recapitulate
more faithfully natural organogenesis. Indeed, organ rudiments
such as the limb bud are early and highly vascularized.33,34 The
blood flow is clearly visible in the marginal vein of limb buds of
10-day embryo developing in LOOCC (Fig. 3E). However, there
is also a fundamental drawback: heartbeat and blood flow induce
motions that greatly complicates 4D image processing especially
with the fairly long exposure time required for fluorescence signal
acquisition during each tomographic scan. There is the risk, while
gaining in healthier and longer development, to lose spatial resolu-
tion attained with organ culture approaches. It is crucial though
to accept and cope with this negative impact of heartbeat-induced
motions to overcome limitations on longitudinal recording of the
dynamics of normal organogenesis. Thus, the next challenge for
live OPT will be to work out the conditions for 4D fluorescence
imaging that cope with motion artifact caused by heartbeats. The
ultimate goal is to track fluorescent signals in transgenic concepti
using live OPT, as was done previously in organ explants. Technical
improvements to the imaging and computational processing have
to be explored to correct for these movements.35,36
which in principle precludes time-lapse imaging. Attempts have
been made for static imaging of cultured embryos but the results
concern at best the first day of organogenesis and are so far in
2D.29 Moreover, development of highly-vascularized structures,
such as the limb buds, is deficient in this arrangement. A third
option, imaging organogenesis while culturing the complete
mouse conceptus (embryo + extraembryonic membranes) has to
our knowledge never been explored. Our previous submerged,
“semi-static” arrangement of culture inside the OPT chamber as
designed for limb bud explant live imaging could be advanta-
geous for culturing concepti in many ways. The minimal-sup-
port rotary arm using tungsten needles would insure that the
conceptus suspended in the medium above the perfluorodeca-
lin layer (Fig. 3C) is free of mechanical pressure, is rotated and
properly oxygenated and already fully integrated into a 4D imag-
ing system. Moreover, keeping the yolk sac and placenta intact
was likely to provide some growth advantage for the embryo.30
However, it was not known whether the conceptus would survive
the many steps of the surgical procedure, and whether any imag-
ing gain could be obtained. Preliminary exploration of this new
approach illustrates a potential route for the future. Mouse con-
cepti of 10 days of age were cultured inside the live OPT scanner.
Extra-embryonic membranes of the conceptus were windowed
Figure 2. Live OpT imaging of embryonic head. early stages of lens induction
revealed within a single specimen by live OpT 4D imaging of a pax6-GFp mouse
embryo (embryonic day 9.5). (a and B) Two of the raw projection images, from
which 3D reconstructions were calculated. (c) The red and white lines through this
panel indicate the positions of virtual sections shown in the remaining panels. (D)
The virtual section highlighted by the red line in (c). Both the prospective retina and
adjacent ectoderm are expressing Pax6-GFP. (E–G) Virtual sections corresponding
to the two white lines in (c). (e) illustrates the two epithelial layers (ectoderm on
left, prospective retina on right) highlighted by Pax6-GFP expression in (F). (H–J)
similar virtual sections after 6 hours in in-vitro culture. The shape of the prospective
retina has changed—it now bulges inwards, due to the growth of the induced lens
which can also be seen expressing pax6-GFp. adapted from reference 7.
www.landesbioscience.com Organogenesis 215
Besides computational improvements, progress may also
come from better cameras, better labels such as in transgenic
mice carrying new-generation fluorescent proteins37 or from
other fields such as similar live imaging research on different
mouse stages38 or on organogenesis in different species.39-41 Also,
extending LOOCC beyond the 11th day of embryonic devel-
opment to access further organogenesis stages should benefit
from research on blood circulation in the conceptus,30,42 culture
media used for whole embryo culture and refinements in the
hardware of the live tomograph itself. In parallel, it is likely
that progress will be made in developing organotypic culture
Figure 3. Live OpT conceptus culture (LOOcc). The layout (a) and design features (B) of the live OpT time-lapse apparatus are shown. specimen
to be cultured is pinned to the mount while in the dissecting dish and protected during transport into the imaging chamber by the plastic capsule.
Three liquids are placed into the imaging chamber: perfluorodecalin to supply oxygen (white), medium (yellow), and mineral oil to prevent evaporation
(orange); adapted from ref. 7. (C–G) Raw OPT projections of specimens developing in LOOCC. (C) Zoom-out view on a conceptus showing essential
features of the technique allowing for blood to circulate throughout the embryo, the yolk sac and the placenta. (D and E) In face view of exposed limb
bud popping out the window made in the yolk sac. (E) Zoom in view of the red inset in (D) highlighting blood cells circulating in the marginal vein of the
limb bud, as observed in real time recording. Red arrowheads point to circulating blood cells in (E) the marginal vein or in (F and G) yolk sac vessels.
(F and G) Growth of a forelimb bud during 30-h of LOOcc. t0 is time 0 of the imaging period. specimen was imaged from 400 angles (every 0.9°) at
20-minute intervals in transmission mode.
models43 allowing for live OPT imaging of many developmen-
tal events. It is through the combination of these explorations
that the range of applications of live OPT will expand and
that a more complete representation of organogenesis will be
We wish to show appreciation for Dr. Marit Boot’s pioneering
contributions to the development of the live OPT approach.
Work is supported by CRG, ICREA, the Spanish Ministry of
Science (MEC) and European Community.
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