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12.3 Live Imaging of Mammalian Embryonic Development and Circulation with OCT 285
vasculature of different embryonic tissues, such as the trunk, skin, brain, and eye.
During late gestation, expression of myr::mCherry and H2B::EYFP decreases in
major blood vessels but remains in some smaller vessels throughout postnatal life.
Endothelial markers mCherry and EYFP can be combined with the blood-specific
GFP marker. Figure 12.2d shows an image acquired with fast-scanning confocal mi-
croscopy from anembryonic yolk sacat 8.5 dpcwhere the Flk1-drivenmyr::mCherry
and the H2B::EYFP are combined with the GFP expressed in the embryonic blood
cells. Although the imaging was performed at 30 fps, the vasculature and the blood
cells are clearly outlined. While the excitation and emission spectra of the GFP and
the EYFP spectra overlap significantly, they can be distinguished. In the example
shown, the EYFP and the EGFP were excited at 488 nm, while the mCherry was
excited at 532nm simultaneously. The emission was detected using only two chan-
nels: BP 500–525 and 560 –675. The EGFP fluorescence is detected entirely by the
first channel, and the EYFP fluorescence is mostly detected by the first channel.
However, since its emission spectrum is shifted toward longer wavelengths relative
to the EGFP, there is some signal detected in the second channel as well. The
mCherry is only detected by the second channel. This difference can be used
to distinguish between the fluorophores. Because the ε-globin-EGFP transgene
allows one to track individual blood cells and perform detailed hemodynamic
analysis, this combination of labels provides an opportunity to study the correla-
tion between hemodynamic and structural changes during vascular remodeling,
thereby allowing a better understanding of the roles that blood flow and shear
stress play in vascular formation and how disturbing these processes contributes
to cardiovascular birth defects and diseases.
12.3
Live Imaging of Mammalian Embryonic Development and Circulation with OCT
While fluorescence microscopy allows visualizing microcirculation and endothelial
morphology with subcellular resolution, its imaging depth is limited to about
200 µm. In the cultured mouse embryo, this imaging depth restricts the imaging to
the yolk sac and very superficial layers of the embryo immediately underneath the
yolk sac, while deeper vessels remain inaccessible for this methodology. Also, the
imaging plane of the confocal microscope is always perpendicular to the scanning
beam: as a result, if a blood vessel is not lying within the imaging plane, the blood
cells cannot be tracked even with fast-scanning microscopy, since it would require
changing of the focal plane position and would not be fast enough to follow the
cells. For the yolk sac vasculature, it is not a problem since it is relatively flat and
is parallel to the imaging plane; however, for other vascular structures, which do
not meet this requirement, fluorescence microcirculation imaging is challenging.
These limitations can be addressed by using OCT. OCT is 3D imaging modality
with the capability to image 2–3 mm into soft tissue with a resolution of 2–20 µm
[17, 18]. OCT has significant advantages over other methods such as laser scanning
microscopy, which has limited imaging depth, or ultrasound biomicroscopy and
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286 12 Fluorescence and OCT Imaging of Microcirculation in Early Mammalian Embryos
micro-MRI, which allow deep tissue imaging but with lower spatial resolution,
which makes OCT an ideal imaging technique for studying early embryonic mouse
development.
In the experiments described here, we used modified swept source optical
coherence tomography (SS-OCT) based on the Thorlabs SL1325-P16 system
with output power P = 12 mW at central wavelength λ
0
= 1325 nm and spec-
tral width λ = 110 nm. The scanning rate over the full operating wavelength
Allantois
Yolk sac
Extraembryonic
cavity
Extraembryonic
vasculature
Amniotic
cavity
Ectoplacental
cavity
Amnion
Amnion
200 µm
500 µm
1 mm
200 µm
Pericardial sac
Headfold
Outflow tract
Ventricle
Embryonic
yolk sac
Embryonic yolk sac
Heart
Head
Head
Tail
Tail
Branching vessels
Yolk sac
Spine
Limb
(a) (b)
(
c
)(
d
)
Figure 12.3 Live structural imaging of early mouse embryos
with SS-OCT. 3D reconstructions of live cultured embryos
at (a) 7.5 dpc, (b) 8.5 dpc, (c) 9.5 dpc, and (d) 10.5 dpc.
(Adapted from Ref. [19].)
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12.3 Live Imaging of Mammalian Embryonic Development and Circulation with OCT 287
range is 16 kHz, resulting in the A-scan acquisition and processing rate of
16 kHz. The scanning head of the OCT system was positioned inside the com-
mercial cell culture incubator set at 37
◦
C, 5% CO
2
. The dissection station was
heated and maintained at 37
◦
C using custom-made heater box and conventional
heater.
The embryos were handled as described earlier in this chapter. Figure 12.3a –d
shows examples of 3D reconstructions of live mouse embryos at different
developmental stages acquired with SS-OCT. The reconstructions provided suffi-
cient details about embryonic structures, as labeled on the panels.
Implementation of Doppler processing in OCT facilitates the acquisition of both
structural and velocity information with the same spatial and temporal resolution
[20]. If the direction of the blood flow is known, blood flow at each pixel can be
reconstructed according to the formula [21]
ν =
ϕ
2n
2π
λ
τ cosβ
(12.1)
where ϕ is a Doppler phase shift calculated between successive A-scans, n is a
refractive index, τ is time between A-scans, and β is an angle between the flow
direction and the laser beam. An angle β can be calculated from structural 2D
and 3D data sets acquired from the embryos. In the experiments presented here,
refractive index was taken as n = 1.4.
Figure 12.4 shows an example of hemodynamic analysis in a yolk sac vessel and
the dorsal aorta deep within the 9.5 dpc mouse embryo [7], when the blood flow
is well established. Structural (Figure 12.4a) and Doppler (Figure 12.4b) images
of part of the yolk sac and the embryonic trunk were taken at 256 A-scans per
frame. Strong Doppler signals were detected from two regions. Different colors
indicate opposite directions of the flow in these structures. The Doppler phase
shift was measured and blood flow velocity profiles were reconstructed along
the lines shown in Figure 12.4a corresponding to the yolk sac vessel and the
dorsal aorta (Figure 12.4c,e). The angle between the direction of the flow and
the scanning beam required for the velocity calculation was determined from the
structural data sets acquired from the embryo. As expected, the velocity profiles
across the vessel have a parabolic shape. The peak flow velocity across the vessels
was plotted versus time (Figure 12.4d,e) and reveals the periodicity of the cardiac
cycle, which can be used to calculate the heart rate of the embryo as well as to study
hemodynamic changes during the heartbeat. The results of yolk sac measurements
made with this system were similar to values previously measured by fast-scanning
confocal microscopy [5], while the dorsal aorta flow profiles are unique for this
imaging technique and demonstrate the advantage of the Doppler SS-OCT over
the confocal microscopy to detect blood flow in deep tissues, suggesting that
Doppler SS-OCT is an effective way to make blood flow measurements in early
embryos.
Hemodynamic analysis with Doppler OCT can also be performed at early stages
of circulation while blood flow is being established[8]. Figure 12.5a shows a
structural image of the dorsal aorta within the 8.5 dpc embryo and corresponding
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288 12 Fluorescence and OCT Imaging of Microcirculation in Early Mammalian Embryos
Yolk sac vessel
Yolk sac vessel
0
1.0
1.5
2.0
2.5
3.0
3.5
12345
Time (s)
Peak blood flow velocity (mm s
−1
)
Dorsal aorta
Dorsal aorta
200 µm
Yolk sac
Blood
vessel
Embryonic
trunk
Yolk sac vessel
0
1
2
3
(a) (b)
(c)
(d)
501234
Time (s)
Peak blood flow velocity (mm s
−1
)
0
1
2
3
4
5
6
7
8
Dorsal aorta
9
(f)
−60 −40 −20
0204060
Distance from the vessel center (µm)
Blood flow velocity (mm s
−1
)
0 ms
185 ms185 ms
290 ms290 ms
(e)
Distance from the center of dorsal aorta (µm)
Blood flow velocity (mm s
−1
)
0 ms
74 ms
185 ms
0
1
−1
−40 −20
2
3
4
5
6
7
8
02040
Dorsal aorta
Figure 12.4 Live hemodynamic analysis with
Doppler SS-OCT in cultured mouse embryo
at 9.5 dpc. (a) Structural image of 9.5 dpc
embryo showing fragments of a yolk sac
and an embryonic trunk. (b) Corresponding
color-coded Doppler SS-OCT image show-
ing strong signals produced by blood flow
in the yolk sac vessel and a dorsal aorta.
(c,e) Blood flow velocity profiles and (d,f)
dynamics of the peak blood flow velocity in
a vessel of the yolk sac and the dorsal aorta,
respectively, measured along the lines shown
in (a) at different phases of the heartbeat
cycle. (Adapted from Ref. [7].)
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12.3 Live Imaging of Mammalian Embryonic Development and Circulation with OCT 289
(a)
(c)
(
d
)
(b)
1.3 mm s
−1
−0.2 mm s
−1
−2.3 mm s
−1
5.3 mm s
−1
8
6
4
2
0
−2
−4
6
4
5
2
3
1
0
−1
−3
−100 −50
−2
Blood flow velocity (mm s
−1
)
Time (s)
0123456
velocity (mm s
−1
)
0 50 100
Distance from the center of the dorsal aorta (µm)
Systole
R
2
= 0.832
Diastole
R
2
= 0.558
(mm s
−1
)
8.5
0
−8.5
20 µm
100 µm
Figure 12.5 Hemodynamic analysis with
SS-OCT in 8.5 dpc cultured mouse embryos
from individual circulating blood cells. (a)
Structural and corresponding color-coded
Doppler velocity images acquired at different
phases of the heartbeat cycle. Green corre-
sponds to an absence of cells or zero veloc-
ity. Individual blood cells are distinguishable
in the dorsal aorta. (b) Magnified view of the
same area showing the Doppler signal from
single cells as well as a small group of cells.
(c) Average blood flow velocity as a func-
tion of time in the corresponding area of the
dorsal aorta. Each data point corresponds to
the Doppler OCT velocity measurement from
an individual cell. The data points were re-
gressed using parabolic fit. (d) Blood flow
velocity profiles. Each data point corresponds
to the Doppler OCT velocity measurement
from an individual cell. The data points were
regressed using parabolic fit. (Adapted from
Ref. [8].)
Doppler velocity maps acquired at different phases of the heartbeat cycle from the
same area of the embryo just a few hours after the beginning of heartbeat when
blood circulation first begins. At this stage, the majority of blood cells are still
found in the blood islands with limited numbers of circulating erythroblasts [2].
The Doppler velocity images were taken at 512 A-scans per frame at 25 fps. Doppler
shift signal from a small group of cells as well as individual circulating blood
cells is clearly distinguishable in the images (Figure 12.5a,b). Doppler velocities
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290 12 Fluorescence and OCT Imaging of Microcirculation in Early Mammalian Embryos
from all individual detectable blood cells in Figure 12.5a were measured for each
acquired Doppler time frame in the time lapse. Figure 12.5c shows an average
blood flow velocity plotted versus time. Dynamics of the blood flow velocity in
time reveal the pulsatile nature of the flow and allow analyzing hemodynamic
changes during the heartbeat. The heart rate (about 2 beats per second) correlates
well with previously reported measurements at this embryonic stage, and the flow
velocity values acquired in the dorsal aorta are similar to published blood flow
measurements in the yolk sac at the same embryonic stage [5], supporting the
physiological relevance of these data.
Blood blow velocity profiles across the vessel at different phases of the heartbeat
cycle were reconstructed by analyzing individual time frames and measuring the
Doppler velocity shift from each visible blood cell and the distance of the blood
cell to the vessel wall. The data points for each time frame were regressed using
parabolic fit. Figure 12.5d shows the data points and the corresponding fits for
different phases of the cardiac cycle. Even though the profiles were reconstructed
from very limited number of cells, it is clear that cell velocity is greater in the
center of the vessel than near the vessel wall, and the measurements fit well with
the parabolic profile suggesting that laminar flow is present. These measurements
demonstrate that SS-OCT can provide sensitive spatially resolved hemodynamic
measurements at the earliest stages of blood circulation.
12.4
Summary
Since the major function of the heart is to pump blood, the pattern of blood
flow is an essential parameter for characterization of cardiodynamics. In addition,
mechanical stimuli from the blood flow and heart contraction are as important for
proper formation of the cardiovascular system as are molecular signaling events.
For these reasons, live hemodynamic analysis in early mammalian embryos is a
critically important tool in studying cardiovascular birth defects. In this chapter, we
described mouse embryo manipulation and static culture protocols, which allow
live optical imaging and hemodynamic analysis during early stages of development.
We discussed two optical approaches for microcirculation analysis in early mouse
embryos, namely, fast-scanning confocal microscopy of vital fluorescent reporters
and Doppler OCT. These approaches provide complementary analyses: while
confocal microscopy in combination with vital fluorescent reporters can be used for
subcellular analysis of vascular development and hemodynamics in the embryonic
yolk sac and superficial embryonic tissues right underneath the yolk sac with
subcellular resolution, OCT can be used for dynamic 3D structural embryonic
imaging and blood flow analysis deep within the embryonic circulatory system
with resolution of single cells. These complementary approaches can be applied
in a wide range of embryonic studies and can potentially answer many interesting
questions about the formation of the cardiovascular system and the nature of
congenital abnormalities.
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References 291
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293
13
High Frequency Ultrasound for the Visualization and
Quantification of the Microcirculation
F. Stuart Foster
13.1
Introduction
Aberrant microcirculatory morphology and hemodynamics are signatures of many
human diseases such as cancer, macular degeneration, diabetes, psoriasis, and
many others. The need for noninvasive means to visualize these processes in
biomedical research and clinical practice is compelling. In preclinical research,
detailed knowledge of microvascular patterning and hemodynamics in mouse
models of disease contribute to the quantification of both the normal physiology
and that of disease progression. The systematic investigation of new therapies and
combinations of therapies can be conducted to inform the development of strategies
suitable for clinical investigation. From the clinical point of view, imaging tools
for the microcirculation offer the potential to characterize disease progression and
determine response to therapy enabling timely modifications to the therapeutic
strategy. Imaging findings may also carry prognostic information that is important
for patient management. In this chapter, microcirculation imaging is explored
in the context of cancer, although the concepts are equally applicable in other
microvascular disease areas.
For many years, the importance of angiogenesis in cancer has been clear and
the rationale for the development of targeted antiangiogenic and antivascular ther-
apeutics well articulated. In the past decade, targeting of the vascular endothelial
growth factor (VEGF) pathway has shown clinical benefit in patients with metastatic
colorectal cancer, advanced nonsmall cell lung cancer, hepatocellular carcinoma,
metastatic breast cancer, and renal cell carcinoma. However, the results of antian-
giogenic therapies have been modest in terms of survival benefit, and the cracks in
our understanding of how these drugs work – and sometimes fail to work – are now
evident. Most prominent is the lack of understanding of the mechanisms of action
of the drugs, the inability to identify which patients might ultimately benefit from
treatment, the lack of effective biomarkers to predict and track treatment response,
and the development of resistance to therapy. There is an urgent need to better
understand the biological underpinnings of targeted therapeutics and to design
better approaches to combine therapies in such a way as to minimize resistance
Microcirculation Imaging, First Edition. Edited by Martin J. Leahy.
2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.
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294 13 High Frequency Ultrasound for the Visualization and Quantification of the Microcirculation
effects. Few studies have been carried out which take advantage of a noninvasive
biomarker of vascular response both in preclinical models of targeted therapy and
in patients: this chapter focuses on the development of micro-ultrasound for this
role.
Ultrasound imaging systems are found in every radiology and cardiology depart-
ment in the world and are the basis of hundreds of millions of clinical imaging
studies each year [1]. The maximum imaging frequencies of clinical systems are
typically 12–15 MHz, and they can provide resolution on the order of 300 µm
at the higher end of this spectrum. In preclinical imaging, where the imaging
target is often a mouse or rat, this is a serious limitation because the structures
under investigation are often so small that they cannot be adequately resolved.
Examples of mouse structures that fall into this category include cardiac, cancer,
and neuromicrocirculations. To address this deficiency, a new ultrasound techno-
logy referred to as micro-ultrasound has been developed in the 15–80 MHz range.
In this chapter, relevant background on the physics of ultrasound are summarized
and the implementation of mechanical and linear array-based micro-ultrasound
systems are described with examples of applications in the assessment of the
microcirculation in mice.
13.2
Ultrasound Fundamentals
The general theory of ultrasound imaging has been well explored by many authors
[2, 3]. In general, design factors are determined by three principle considerations:
resolution, maximum imaging depth, and imaging frame rate. Not surprisingly,
these factors cannot be considered independently but must be approached through
a series of compromises. All ultrasound imaging systems rely on the propagation
of mechanical (ultrasonic) waves from a transducer into tissue and the subsequent
interactions of these waves with mechanical discontinuities in the tissue. At each
discontinuity, a small reflected wave (scatter) called an echo is created and its energy
is detected by the transmitting transducer. The range, z, of the scattering structure
is determined by the time of flight of the ultrasound to and from the target via the
simple equation
z =
ct
2
(13.1)
where c is the speed of sound. The propagation of ultrasound in a medium implies
a traveling wave for which the wavelength, frequency, and speed of sound are
governed by the wave equation
∂
2
p
∂t
2
= c
2
∇
2
p (13.2)
where p represents acoustic pressure and c is the speed of sound and represents
the second-order spatial derivative (Laplacian) operating on pressure. On the basis
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