Optical Sectioning Deep Inside Live Embryos by Selective Plane

Illumination Microscopy
Jan Huisken et al.
Science 305, 1007 (2004);
DOI: 10.1126/science.1100035

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 REPORTS
proved resolution, LSM suffers from two ma-
Optical Sectioning Deep Inside jor limitations: a limited penetration depth in
heterogeneous samples and a marked differ-

Live Embryos by Selective Plane ence between the lateral and axial resolution.
We developed selective plane illumina-
tion microscopy (SPIM), in which optical
Illumination Microscopy sectioning is achieved by illuminating the
sample along a separate optical path or-
Jan Huisken,* Jim Swoger, Filippo Del Bene, Joachim Wittbrodt, thogonal to the detection axis (Fig. 1 and
Ernst H. K. Stelzer* fig. S1). A similar approach in confocal
theta microscopy has been demonstrated to
Large, living biological specimens present challenges to existing optical improve axial resolution (10 –12). In SPIM,
imaging techniques because of their absorptive and scattering properties. the excitation light is focused by a cylin-
We developed selective plane illumination microscopy (SPIM) to generate drical lens to a sheet of light that illumi-
multidimensional images of samples up to a few millimeters in size. The nates only the focal plane of the detection
system combines two-dimensional illumination with orthogonal camera- optics, so that no out-of-focus fluorescence
based detection to achieve high-resolution, optically sectioned imaging is generated (optical sectioning). The net
throughout the sample, with minimal photodamage and at speeds capable effect is similar to that achieved by con-
of capturing transient biological phenomena. We used SPIM to visualize all focal LSM. However, in SPIM, only the
muscles in vivo in the transgenic Medaka line Arnie, which expresses green plane currently observed is illuminated and
fluorescent protein in muscle tissue. We also demonstrate that SPIM can be therefore affected by bleaching. Therefore,
applied to visualize the embryogenesis of the relatively opaque Drosophila the total number of fluorophore excitations
melanogaster in vivo. required to image a 3D sample is greatly
reduced compared to the number in con-
Modern life science research often requires either by detection through a pinhole (con- focal LSM (supporting online text).
multidimensional imaging of a complete focal LSM) (8) or by exploitation of the GFP-labeled transgenic embryos of the
spatiotemporal pattern of gene and protein nonlinear properties of a fluorophore (mul- teleost fish Medaka (Oryzias latipes) (13)
expression or tracking of tissues during the tiphoton microscopy) (9). Despite the im- were imaged with SPIM. In order to visu-
development of an intact embryo (1). In
order to visualize the precise distribution of
developmental events such as activation of Fig. 1. (A) Schematic of the sample
chamber. The sample is embedded in a
specific genes, a wide range of processes, cylinder of agarose gel. The solidified
from small-scale (subcellular) to large- agarose is extruded from a syringe
scale (millimeters), needs to be followed. (not shown) that is held in a mechan-
Ideally, such events, which can last from ical translation and rotation stage.
seconds to days, will be observed in live The agarose cylinder is immersed in
and fully intact embryos. an aqueous medium that fills the
chamber. The excitation light enters
Several techniques have been developed the chamber through a thin glass win-
that allow mapping of the three-dimensional dow. The microscope objective lens,
(3D) structure of large samples (2). Gene which collects the fluorescence light,
expression has been monitored by in situ dips into the medium with its optical
hybridization and block-face imaging (3). axis orthogonal to the plane of the
Techniques that provide noninvasive (opti- excitation light. The objective lens is
sealed with an O-ring and can be
cal) sectioning, as opposed to those that moved axially to focus on the plane of
destroy the sample, are indispensable for fluorescence excited by the light
live studies. Optical projection tomography sheet. In a modified setup, for low-
can image fixed embryos at high resolution magnification lenses not corrected for
(4 ). Magnetic resonance imaging (5) and water immersion, a chamber with four
optical coherence tomography (6 ) feature windows and no O-ring can be used.
In this case, the objective lens images
noninvasive imaging, but do not provide the sample from outside the cham-
specific contrasts easily. and resolution ber. det., detection; ill., illumination;
In optical microscopy, green fluorescent proj., projection. (B to E) A Medaka
protein (GFP) and its spectral variants are used embryo imaged with SPIM by two dif-
for high-resolution visualization of protein lo- ferent modes of illumination. Lateral
calization patterns in living organisms (7). [(B) and (C)] and dorsal-ventral [(D)
and (E)] maximum projections are
When GFP-labeled samples are viewed, op- shown. In (B) and (D), the sample was
tical sectioning (which is essential for its illuminated uniformly, i.e., without
elimination of out-of-focus light) is obtain- the cylindrical lens, as with a conven-
able by laser scanning microscopy (LSM), tional widefield microscope. There is
no optical sectioning. The elongation
of fluorescent features along the de-
European Molecular Biology Laboratory (EMBL), tection axis is clearly visible in (D). In
Meyerhofstra!e 1, D-69117 Heidelberg, Germany. contrast, selective (select.) plane illu-
*To whom correspondence should be addressed. E- mination [(C) and (E)] provided optical sectioning because the cylindrical lens focused the
mail: [email protected] (J.H.) and [email protected] excitation light to a light sheet. Both image stacks were taken with a Zeiss Fluar 5", 0.25
(E.H.K.S.) objective lens.

www.sciencemag.org SCIENCE VOL 305 13 AUGUST 2004 1007

 REPORTS
alize the internal structure, we imaged the embryos. We routinely image live Medaka the yolk cell. We imaged transgenic
transgenic line Arnie, which expresses GFP and Drosophila embryos over periods of up Medaka Arnie embryos and show a recon-
in somatic and smooth muscles as well as in to 3 days without detrimental effects on struction of the inner heart surface (Fig.
the heart (14 ). A 4-day-old fixed Arnie embryogenesis and development. To dem- 3A) derived from the data set shown in Fig.
embryo [stage 32 (15)] is shown in Fig. 1. onstrate the potential of SPIM technology, 2. This reveals the internal structure of the
SPIM was capable of resolving the internal we investigated the Medaka heart, a struc- heart ventricle and atrium. In a slightly
structures of the entire organism with high ture barely accessible by conventional con- earlier stage, internal organs such as the
resolution (better than 6 #m) as deep as 500 focal LSM because of its ventral position in heart and other mesoendodermal deriva-
#m inside the fish, a penetration depth that
cannot be reached using confocal LSM (fig. Fig. 2. A Medaka embryo (the same as
S6). The axial resolution in SPIM is deter- in Fig. 1) imaged with SPIM and pro-
mined by the lateral width of the light cessed by multiview reconstruction
sheet; for the configuration shown in Fig. 1, (figs. S2, S3, and S6 and movies S1 and
the axial extent of the point spread function S2). (A) Overlay of a single stack (green)
(PSF) was about 6 #m, whereas without the and the fusion of four data sets (red and
green). (B) Dorsal-ventral and (C) lateral
light sheet it was more than 20 #m (sup- maximum intensity projections of the
porting online text). fused data. The high resolution
Any fluorescence imaging system suf- throughout the entire fish allows iden-
fers from scattering and absorption in the tification of different tissues: rgc, retinal
tissue; in large and highly scattering sam- ganglion cells; so, superior oblique; io,
ples, the image quality decreases as the inferior oblique; ir, inferior rectus; sr,
superior rectus; im, intermandibualaris;
optical path length in the sample increases. hh, hyohyal; rc, rectus communis; dpw,
This problem can be reduced by a multi- dorsal pharyngeal wall; fad, fin adduc-
view reconstruction, in which multiple 3D tor; fab, fin abductor; sm, somitic me-
data sets of the same object are collected soderm; tv, transverse ventrals. The
from different directions and combined in a stack has a size of 1201 by 659 by 688
postprocessing step (16–18). The high- pixels (1549 #m by 850 #m by 888
#m).
quality information is extracted from each
data set and merged into a single, superior
3D image (supporting online text). One
way to do this is by parallel image acqui-
sition, using more than one lens for the
detection of fluorescence (18).
We collected SPIM data for a multiview
reconstruction sequentially by generating
multiple image stacks between which the
sample was rotated. Sample deformations
were avoided with a rotation axis parallel to
gravity (Fig. 1). In contrast to tomographic
reconstruction techniques [such as those in
(4)], which require extensive processing of
the data to yield any meaningful 3D informa-
tion, rotation and subsequent data processing
are optional in SPIM. They allow a further
increase in image quality and axial resolution Fig. 3. A Medaka heart imaged with
compared to a single stack, but in many cases SPIM (movies S3 and S4). (A) Sur-
a single, unprocessed 3D SPIM stack alone face rendering of the heart taken
provides sufficient information. from the data shown in Fig. 2. The
We performed a multiview reconstruction heart has been cut open computa-
tionally to make the internal struc-
with four stacks taken with four orientations ture visible. hv, heart ventricle; ha,
of the same sample (figs. S2 and S3). Com- heart atrium. (B) Schematic repre-
bination of these stacks (supporting online sentation of a Medaka embryo at
text) yielded a complete view of the sample, stage 26 of development (13), 2
$1.5 mm long and $0.9 mm wide. In Fig. 2, days post-fertilization. Three opti-
the complete fused data set is shown and the cally sectioned planes are indicated.
At this stage, ventral structures such
most pronounced tissues are labeled. The de- as the heart are deeply buried in the
crease in image quality with penetration yolk sphere. d, dorsal; v, ventral; a,
depth is corrected by the fusion process. It anterior; p, posterior; y, yolk; ey, eye.
yielded an increased information content in (C) Optical section of an Arnie em-
regions that were obscured (by absorption or bryo showing the eye and the optic
scattering in the sample) in some of the un- nerve labeling and the dorsal part of
the heart ventricle. on, optic nerve.
processed single views. (D) Optical section showing the
The method of embedding the sample in heart ventricle chamber and the
a low-concentration agarose cylinder is dorsal wall of the heart atrium. (E)
nondisruptive and easily applied to live Optical section showing the atrium chamber.

1008 13 AUGUST 2004 VOL 305 SCIENCE www.sciencemag.org

 REPORTS
tives are deeply buried in the yolk sphere, es, the embryo was unaffected and completed context of morphogenesis. Heart function
under the body of the embryo (Fig. 3B). In embryogenesis normally. and development can be precisely followed
Fig. 3, C to E, three optical sections at In summary, we present an optical wide- in vivo using SPIM in Arnie transgenic
different depths illustrate GFP expression field microscope capable of imaging pro- embryos. Because of its speed and its au-
in the muscles of the living heart. Fast tein expression patterns deep inside both tomatable operation, SPIM can serve as a
frame recording (10 frames per s) allows fixed and live embryos. By selective illu- tool for large-scale studies of developing
imaging of the heartbeat (movies S3 and mination of a single plane, the excitation organisms and the systematic and compre-
S4); similar imaging has previously only light is used efficiently to achieve optical hensive acquisition and collection of ex-
been demonstrated at stages when the heart sectioning and reduced photodamage in pression data. Even screens for molecules
is exposed and by cooling the embryo to large samples, key features in the study of that interfere with development and
reduce the heart rate (19). embryonic development. The method of regeneration on a medium-throughput scale
To demonstrate that SPIM can also be sample mounting allows positioning and seem feasible. SPIM technology can be
used to image the internal structures of rela- rotation to orient the sample for op- readily applied to a wide range of organ-
tively opaque embryos, we recorded a time timal imaging conditions. The optional isms, from whole embryos to single cells.
series (movie S5) of the embryogenesis of the multiview reconstruction combines inde- Subcellular resolution can be obtained in
fruit fly Drosophila melanogaster (Fig. 4). pendently acquired data sets into an opti- live samples kept in a biologically relevant
GFP-moesin labeled the plasma membrane mal representation of the sample. The environment within the organism or in cul-
throughout the embryo (20). Even without implementation of other contrasts such as ture. Therefore, SPIM also has the po-
multiview reconstruction, structures inside scattered light will be straightforward. The tential to be of use in the promising fields
the embryo are clearly identifiable and trace- system is compact, fast, optically stable, of 3D cultured cells (21) and 3D cell
able. Stacks (56 planes each) were taken au- and easy to use. migration (22).
tomatically every 5 min over a period of 17 SPIM is well suited for the visualization
hours, without refocusing or realignment. of high-resolution gene and protein ex- References and Notes
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Fig. 4. Time-lapse imaging of Drosophila melanogaster embryogenesis. Six out of 205 time points for providing the Drosophila samples. The beating-
acquired are shown (movie S5). At each time point, 56 planes were recorded, from which two (at heart data was recorded by K. Greger.
depths of 49 #m and 85 #m below the cortex) are shown. No multiview reconstruction was
necessary. The optical sectioning capability and the good lateral resolution are apparent. Despite Supporting Online Material
www.sciencemag.org/cgi/content/full/305/5686/1007/
the optically dense structure of the Drosophila embryo, features are well resolved at these depths
DC1
in the sample. For this figure, the images were oriented so that the illumination occurs from below. Materials and Methods
This results in a slight drop in intensity and clarity from the bottom to the top of each slice. SOM Text
Nevertheless, the information content across the embryo is nearly uniform, and the overall Figs. S1 to S6
morphogenetic movements during embryonic development can be followed. The images were References and Notes
normalized to exhibit the same overall intensity, thus compensating the continuous production of Movies S1 to S5
GFP-moesin. We took 205 stacks at 5-min intervals with a Zeiss Achroplan 10", 0.30W objective
lens (56 planes per stack at 4-#m spacing) for 11,480 images in total. 6 May 2004; accepted 15 July 2004

www.sciencemag.org SCIENCE VOL 305 13 AUGUST 2004 1009