3242 RESEARCH REPORT
TECHNICAL PAPER
Development 139, 3242-3247 (2012) doi:10.1242/dev.082586
© 2012. Published by The Company of Biologists Ltd
Multilayer mounting enables long-term imaging of zebrafish
development in a light sheet microscope
Anna Kaufmann*, Michaela Mickoleit*, Michael Weber* and Jan Huisken‡
SUMMARY
Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging
of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited
by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in
conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically
quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of
fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance
between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for
fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and
provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques
applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other
small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging
optical imaging techniques, such as optical projection tomography (OPT).
INTRODUCTION
Fluorescence microscopy is an invaluable tool for in vivo studies
of developmental processes. However, excessive light exposure in
commonly employed techniques, such as confocal, two-photon or
spinning disc systems, causes phototoxicity and fluorophore
bleaching, and only short intervals of developmental processes can
be imaged. By contrast, in light sheet microscopy, such as selective
plane illumination microscopy (SPIM) (Huisken et al., 2004) or
digital scanned laser light sheet fluorescence microscopy (DSLM)
(Keller et al., 2008), the sample is illuminated with a thin laser light
sheet in the focal plane of the detection objective and imaged at
high speed with a sensitive camera. The energy input into the
sample is minimized, making light sheet microscopy ideally suited
for time-lapse imaging of biological processes over long periods of
time (Huisken and Stainier, 2009; Weber and Huisken, 2011), such
as heart function and morphogenesis in zebrafish (Scherz et al.,
2008; Arrenberg et al., 2010), neuronal cell migration in C. elegans
(Wu et al., 2011), or cell lineage tracing during lateral line
formation in zebrafish (Swoger et al., 2011).
Imaging a developing organism over many hours or a few
days requires careful optimization of the mounting technique. In
SPIM and DSLM, sample preparation is radically different from
the dish or slide mounting used for conventional microscopy.
Immersing a vertically oriented sample in an aqueous solution
facilitates rotation of the fragile sample without deformation and
provides unobstructed views from all angles (Huisken and
Stainier, 2009).
The optimal mounting for imaging developmental processes
should provide enough space for the sample to grow, while keeping
it in a fixed position. For in vivo imaging in SPIM and DSLM,
specimens are commonly embedded in a low-melting-point agarose
cylinder (Huisken et al., 2004; Keller et al., 2010). The transparent
agarose matches the refractive index of water (1.33) and biological
tissue, and concentrations of 1.0-1.5% provide enough mechanical
stability to reproducibly move the sample. However, such agarose
embedding is only suitable for specimens that do not alter in shape
or size during the experiment, such as Drosophila embryos
(Huisken and Stainier, 2009; Preibisch et al., 2010).
The translucent zebrafish embryo is ideal for imaging vertebrate
development (Godinho, 2011). Since embryo length increases by a
factor of four within 3 days (Kimmel et al., 1995), time-lapse
imaging of embryos embedded in a rigid scaffold, such as agarose,
leads to severe growth restrictions and developmental defects (Fig.
1A,A⬘) (Keller et al., 2008; Keller et al., 2010). Thus, the potential
of long-term imaging with light sheet microscopy is challenged by
the lack of physiological mounting techniques.
We have evaluated the development of zebrafish embryos in
different mounting media and tested their suitability for SPIM. We
provide mounting protocols for the first days of zebrafish
development for light sheet microscopy and other long-term imaging
techniques. Mounting the sample in low concentrations of agarose
or methylcellulose in refractive-index-matched fluorinated ethylene
propylene (FEP) tubes ensures both immobility and growth of the
embryo as well as optimal image quality from all sides.
MATERIALS AND METHODS
Zebrafish lines
Max Planck Institute of Molecular Cell Biology and Biology and Genetics,
Pfotenhauerstr. 108, 01307 Dresden, Germany.
*These authors contributed equally to this work
‡
Author for correspondence ([email protected])
Accepted 14 June 2012
Zebrafish (Danio rerio) adults and embryos were kept at 28.5°C and were
handled according to established protocols (Nüsslein-Volhard and Dahm,
2002) and in accordance with EU directive 2011/63/EU as well as the
German Animal Welfare Act. Transgenic lines Tg(kdrl:GFP) (Jin et al.,
2005) and Tg(myl7:GFP) (Huang et al., 2003) were used. Tnnt2
morpholino injection was performed as described (Sehnert et al., 2002).
DEVELOPMENT
KEY WORDS: Zebrafish, In vivo imaging, Time-lapse microscopy, Mounting, Light sheet microscopy, SPIM
Mounting for long-term imaging
RESEARCH REPORT 3243
Fig. 1. Influence of different
mounting media on growth and
immobility. (A,A⬘) Zebrafish embryo
[Tg(myl7:GFP)] embedded in 1.5%
agarose from 24-48 hpf exhibits a short
crooked tail (arrowhead) and disturbed
morphology of the heart (arrow).
(B)Immobility, heart rate and edema
after incubation from 24-72 hpf in
different concentrations of Tricaine.
Superimposed time-lapse images of one
embryo are shown for four
concentrations (pictograms 1-4). 0%
immobility refers to movement in E3
(control); 100% immobility corresponds
to 200 mg/l Tricaine. Tricaine
concentrations as low as 133 mg/l result
in immobilization of the fish without
altering the heart rate. Edemas were
observed with concentrations as low as
100 mg/l. (C)Immobility and growth in
different agarose concentrations after
embedding embryos from 24-72 hpf. At
0.1% agarose, both immobility and
normal growth were achieved.
(C⬘)Immobility and growth in different
methylcellulose concentrations after
embedding embryos from 24-72 hpf. At
2.5-3.0% methylcellulose, both
immobility and growth were satisfactory.
Mean ± s.d., n6. Dashed lines
represent empirical fits for better
Cleaning of FEP tubes
Heart rate measurement in Tricaine
FEP tubes (Bola, S1815-04, refractive index 1.338, inner diameter 0.8 mm,
outer diameter 1.6 mm) were flushed with 1 M NaOH (Merck), placed in
0.5 M NaOH and ultrasonicated for 10 minutes. They were flushed with
double-distilled H2O and with 70% ethanol. Tubes were then placed in
70% ethanol and ultrasonicated for 10 minutes. Finally, tubes were flushed
with and stored in double-distilled H2O. All solutions had been degassed
and filtered using a syringe filter (Millex-HV PVDF 0.45 m).
Embryos at 24 hpf were placed in 96-well plates with different
concentrations of Tricaine and incubated for 48 hours. Movies were acquired
at 72 hpf using an Olympus SZX16 microscope and a Panasonic Lumix G2
camera. The average heart rate was determined by manual counting over a
period of 8 seconds.
Low-melting-point agarose (Sigma) and methylcellulose (Sigma) were
diluted in E3 (Nüsslein-Volhard and Dahm, 2002). For embedding in 1.5%
(w/v) agarose, embryos were placed in liquid agarose, drawn into a glass
capillary with Teflon-coated plungers (BRAND) and extruded after
polymerization. For mounting in liquid media, FEP tubes were used as an
enclosure. Embryos were placed in the mounting medium and drawn into
the tube with a needle (100 Sterican, blunt) and syringe. The tube was
plugged with solid 1.5% agarose. For long-term time-lapse imaging, the
tube was coated with 3.0% methylcellulose containing 200 mg/l Tricaine
(Aldrich). Methylcellulose was slowly drawn into the tube and out again.
The coated tube was rinsed once with E3 containing 200 mg/l Tricaine.
Immobility and growth measurements
Embryos at 24 hours postfertilization (hpf) were dechorionated and placed,
one embryo per well, in 96-well plates (Nunc) filled with mounting
medium. Control embryos were placed in E3 or 200 mg/l Tricaine. Each
well was imaged every hour for 48 hours at 28.5°C using a Zeiss Axiovert
200M microscope. Embryo length was determined at 24 and 72 hpf using
Fiji software (Schindelin et al., 2012) and compared with that in E3.
Immobility was measured with Fiji by superimposing all images of the
same well and subtracting the minimum from the maximum intensity
projection. The mean gray value of the resulting image is an indicator of
the movement of one embryo. Immobility in E3 and Tricaine was
considered 0% and 100%, respectively.
Embryos at 24 hpf were mounted in FEP tubes with 15 l E3, 0.1% agarose
or 3.0% methylcellulose, which were placed in a reaction tube filled with E3.
Every 12 hours, six embryos were carefully removed from the tube, imaged
and measured (Zeiss Axiovert 200M, Fiji). A different set of embryos was
used for each time point to prevent damage of the embryos. Additional
images were taken at 72 hpf (Olympus SZX16, AVT Stingray camera).
Selective plane illumination microscopy
A multidirectional SPIM (mSPIM) setup was used as previously described
(Huisken and Stainier, 2007). Long-term time-lapse images of developing
embryos were acquired using a Leica HCX APO 10⫻/0.3W objective,
Coherent Sapphire 488 nm laser and Andor iXon 885 camera. Embryos
were imaged every 10 minutes or incubated at 28.5°C and imaged every 6
hours. Fluorescence and transmission images were projected using
maximum intensity and Gaussian-based stack focuser algorithms (Fiji plugin by J. Schindelin, University of Wisconsin-Madison), respectively.
Optical quality
Fluorescent beads (Estapor F-Y050, 500 nm, 1:2000 dilution) were imaged
by mSPIM (Leica HCX APO 20⫻/0.5W objective, Chroma LP575 emission
filter) in 1.5% agarose, 1.5% agarose in FEP tubes, and 1.5% agarose in glass
capillaries (BRAND). Six samples per mounting condition were imaged in
the center of each cylinder (147 planes, 0.5 m step size) and cropped to the
central 25%. Beads were detected in three dimensions with subpixel
localization (Preibisch et al., 2010), registered and averaged. Lateral x and y
and axial z line profiles were measured in the central slice of the resulting
DEVELOPMENT
Mounting
Growth in FEP tubes
3244 RESEARCH REPORT
Development 139 (17)
image stack using Fiji. In MATLAB (MathWorks), a Gaussian fit was
performed on the measured intensity profile and the full width at half
maximum (FWHM) was computed. Intensity traces in x, y and z for all beads
extracted from one sample per mounting condition were plotted.
RESULTS AND DISCUSSION
Because embedding in rigid agarose restricts the growth and
morphogenesis of zebrafish embryos (Fig. 1A,A⬘), we examined
alternative, physiological mounting methods. Embryos were
mounted in different concentrations of agarose, methylcellulose
and Tricaine at 24 hpf, and their development was monitored until
72 hpf. Growth and immobility were determined as criteria for
proper development and spatial confinement for precise imaging.
visualization.
Fig. 2. Influence of tube enclosure on growth. (A)Kinetics of
embryonic growth during 2 days in E3, 0.1% agarose, 3.0%
methylcellulose, with and without FEP tubes. (B,C)Length (B) and
morphology (C) of 72-hpf zebrafish embryos from A. Mean ± s.d., n6.
*, P<0.01.
Influence of different mounting media on growth
and immobility
Zebrafish embryos were examined in 0-1.5% agarose (Fig. 1C) and
0-3% methylcellulose (Fig. 1C⬘) from 24-72 hpf in order to
determine the concentration that balances growth and immobility.
These mounting media have already been used in various
microscopy techniques (Renaud et al., 2011), are available in most
laboratories, do not expose the sample to heat or mechanical stress
during the mounting, and their refractive index is comparable to
that of water. Agarose allows unperturbed growth of the embryos
at a concentration as low as 0.1% (Fig. 1C) with an immobility of
80%. Agarose concentrations higher than 0.4% completely inhibit
growth of the embryo. These results indicate that the commonly
employed concentrations of agarose are non-physiological. In the
case of methylcellulose, 85% immobility and 68% growth were
achieved at a concentration of 3%. However, in contrast to solid
1.5% agarose, the viscous 0.1% agarose and 3.0% methylcellulose
require an enclosure to hold the specimen in the microscope.
a tube of FEP to be a promising support. FEP is well suited for
imaging aqueous samples from all sides because of its matching
refractive index of 1.338. FEP tubes have been used for short-term
observations of zebrafish embryos in fluorescent widefield
microscopy (Petzold et al., 2010) and optical projection
tomography (OPT) (Bassi et al., 2011; McGinty et al., 2011).
We chose FEP tubes with an inner diameter matching the size of
the fish, but still allowing for proper growth over a few days, and
evaluated the development of embryos mounted in our media of
choice. The FEP enclosure had no influence on the shape of the
embryonic growth curve (Fig. 2A), and the differences in length at
the end of our experiments were minor (Fig. 2B). Embryos
mounted in a tube filled with E3 or 0.1% agarose grew 8% less
than freely swimming siblings, possibly owing to reduced oxygen
supply or a lack of movement. When 3% methylcellulose was used
as mounting medium, the tube enclosure had no significant effect
on growth. The morphology of the embryos suggests that
development was delayed but not otherwise impaired (Fig. 2C).
Embryos were raised and no difference in the survival rate was
observed. We conclude that FEP tubes are physiological enclosures
for non-rigid mounting media.
Influence of FEP tubes on development
In conventional microscopy, glass slides provide a platform for
imaging from one side. For light sheet microscopy, an enclosure
for vertical mounting and multiview imaging is needed. We found
Optical quality of FEP tubes
To quantify the optical performance of FEP tubes, fluorescent
beads were embedded in 1.5% agarose inside a tube and imaged
in SPIM. For comparison, beads were also imaged in a glass
DEVELOPMENT
Long-term side effects of Tricaine
Zebrafish embryos can be immobilized mechanically by embedding
in a rigid scaffold or chemically by applying anesthetizing drugs such
as Tricaine, which inhibits muscle contractions (Frazier and
Narahashi, 1975). Tricaine concentrations ranging from 100 mg/l
(Tsutsui et al., 2010) up to 750 mg/l (Truong et al., 2011) have been
used as sedative, although as little as 30 mg/l Tricaine can influence
embryonic development (Kimmel et al., 1995). Tricaine suppresses
the contraction of not only skeletal muscles but also of smooth and
cardiac muscles, resulting in reduced heart rate (Muntean et al.,
2010) and affecting developmental processes that are influenced by
hemodynamics (Culver and Dickinson, 2010).
We examined heart rate and morphology (Fig. 1B) after
exposing embryos to Tricaine from 24-72 hpf. We used Tricaine
concentrations of up to 800 mg/l, which was lethal after 2 days. At
concentrations below 200 mg/l the heart rate fluctuated by less than
10%, which is consistent with a previous study (Craig et al., 2006).
However, heart edemas were observed at concentrations as low as
100 mg/l (Fig. 1B, lower part). Thus, for long-term imaging, we
recommend Tricaine concentrations that are as low as possible, not
exceeding 200 mg/l or even 100 mg/l for cardiac studies. This
concentration guarantees 80% immobilization on our scale.
Additional stabilization of the sample is needed to achieve accurate
and reproducible imaging over time.
Mounting for long-term imaging
RESEARCH REPORT 3245
A
B
Objective
Light
Ligh
ht shee
sheet
Lateral (x)
unge
er
Plunger
F
EP tu
u
FEP
tube
(c
coated
d inside)
(coated
pillaryy
Glass capillary
Fluorescent
Fluo
res
e cent
bead
A
g s 0.1%
garos
Agarose
Agarose 1.5%
Zebrafish
Z
ebraf
Zebrafish
Axial (z)
A
gaross 1.5%
Agarose
Lateral (y)
C
No
enclosure
D
14
No enclosure
FEP tube
Glass capillary
1 μm
12
FEP
tube
FWHM (μm)
n = 82
n = 103
Lateral (y)
Axial (z)
Lateral (x)
Glass
capillary
n = 115
6
4
Fig. 3. Optical quality of FEP mounting.
(A)Schematic of mounting in 1.5% agarose
(left) and multilayer mounting in 0.1% agarose
in coated FEP tubes (right). (B)Principle of SPIM
and direction of axes for the analysis of optical
quality. Not to scale. (C)Maximum intensity
projections of averaged images of beads (n,
number of beads) in 1.5% agarose without
enclosure, in an FEP tube and in a glass
capillary. (D)Full width at half maximum
(FWHM). Mean ± s.d., n6. (E)Developing
zebrafish embryo [Tg(kdrl:GFP)] embedded in
0.1% agarose in a coated FEP tube during SPIM
time-lapse (supplementary material Movie 1).
Images resolve even the finest structures, e.g.
the cell extensions forming the parachordal
vessels (arrowheads).
2
0
Lateral (x)
Lateral (y)
Axial (z)
E
Tg(kdrl:GFP), 24 hpf
51 hpf
96 hpf
Time-lapse imaging in FEP tubes
Combining the results from the individual experiments, we have
established protocols for the multilayer mounting of embryos for
imaging their development over many days. In a direct
comparison with a previous method using 1.5% agarose, our
techniques constitute a major improvement. Whereas
traditionally embedded embryos were unable to undergo
morphological changes such as outgrowth of the head and died
at 50 hpf (supplementary material Fig. S2), our mounting
techniques allowed normal development.
To demonstrate the superior physiological and optical
performance of the sample mounting technique, transgenic
Tg(kdrl:GFP) embryos were embedded in 0.1% agarose enclosed
in FEP at 24 hpf and imaged with SPIM for 3 days. The tubes had
been coated with 3% methylcellulose before embedding to prevent
adhesion of the tail to the tube. Transmission and fluorescence
images show that development was undisturbed and finest detail in
the developing vasculature was resolved (Fig. 3E; supplementary
material Movie 1).
Recommended mounting techniques
The ideal mounting conditions depend on the organ of interest,
the developmental stage and the duration of the experiment
(Fig. 4). Agarose at 1.5% represents a quick and stable
mounting medium for acquiring a snapshot, and fiducial
markers for multiview reconstructions can easily be added
(Preibisch et al., 2010) (supplementary material Movie 2).
Optionally, the sample can be enclosed in FEP for extra
stability and distortion-free high-speed rotations. For long-term
imaging, we suggest the use of 3% methylcellulose or 0.1%
agarose in an FEP tube to ensure proper growth and
immobilization of the embryo. Methylcellulose should be used
when imaging for up to 1 day and for Tricaine-sensitive organs,
such as the heart, as it provides stronger immobilization
(supplementary material Movie 3). Otherwise, we recommend
0.1% agarose with 200 mg/l Tricaine as mounting medium in
an FEP tube coated with 3.0% methylcellulose. This multilayer
mounting ensures immobilization of the embryo over several
days with only minor influences on development
(supplementary material Movie 1).
DEVELOPMENT
capillary and without an enclosure. For each condition, intensity
profiles of beads were plotted (supplementary material Fig. S1).
They showed little variance and were averaged to quantify
aberrations caused by the surrounding material (maximum
intensity projection in Fig. 3C). FEP does not distort the bead
image compared with no enclosure, whereas a glass capillary
causes severe aberrations (Fig. 3D). Therefore, FEP is well suited
as an enclosure for non-rigid mounting media in light sheet
microscopy and other microscopy systems.
3246 RESEARCH REPORT
Development 139 (17)
During early stages of development, prior to 24 hpf, the embryo
can be left in the chorion, which has no influence on the optical
quality when imaged by SPIM. The embryos develop normally in
the chorion and therefore the rigidity of the mounting medium is
irrelevant (supplementary material Movie 4).
Outlook
Light sheet microscopy is a promising technique for non-invasive
imaging of fragile samples over long periods of time, which up to
now has been limited by insufficient mounting techniques. This
study provides techniques for mounting zebrafish and potentially
other small vertebrates, unleashing the full potential of SPIM for
long-term imaging. Protocols for light sheet microscopy will
become even more important with the release of commercial
microscopes. The mounting protocols presented here have also
been successfully tested on the first commercial multiview light
sheet microscope (Zeiss). Our results will also be applicable to
other multi-angle imaging techniques, such as tomography (e.g.
OPT). We have shown that dedicated sample mounting techniques
are a key component in the application of current and future
developments in microscopy.
Acknowledgements
We thank S. Preibisch and C. Perez-Campos for evaluating the bead images; J.
Schindelin for the Fiji plug-in; B. Borgonovo for help with the FEP tube
cleaning; and H.-O. K. Lee and D. Richmond for comments on the manuscript.
Funding
Our work is funded by the Max Planck Society and the Human Frontiers
Science Program (HFSP).
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.082586/-/DC1
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Mounting for long-term imaging