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References 275
and the experimental approaches are still quite invasive and represent tentative
measures. Recently, Rajian et al. employed a known-spectrum optical contrast agent
for fluence compensation in tissue-mimicking phantoms [51]. This noninvasive
method holds the potential for in vivo fluence compensation.
11.5.2
High-Speed Imaging for Clinical Applications
As shown in Figure 11.13, OR-PAM has demonstrated cross-sectional imaging
of human cutaneous microcirculation with high spatial resolution and adequate
penetration depth (up to 1 mm in the cuticle region, data not shown). However, the
pronounced motion artifact during the data acquisition prevented us from getting
good volumetric images. The rates of raster scanning, laser repetition, and data
acquisition must be improved to facilitate an effective clinical translation.
In conclusion, PAT has potentially broad applications in microcirculation
imaging, yet much effort still needs to be invested to mature this technology.
We are looking forward to seeing PAT fully ensconced among mainstream micro-
circulation imaging technologies.
Animal Ethics
All experimental animal procedures were carried out in conformance with the
laboratory animal protocol approved by the School of Medicine Animal Studies
Committee of Washington University in St. Louis.
Acknowledgments
The authors appreciate Prof James Ballard’s close reading of the manuscript.
This work was sponsored by National Institutes of Health Grants R01 EB000712,
EB000712A2S1, R01 EB00071207S2, R01 EB008085, R01 CA113453901, U54
CA136398, and 5P60 DK02057933. Prof Wang has financial interests in
Microphotoacoustics, Inc. and Endra, Inc. However, neither supported this work.
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279
12
Fluorescence and OCT Imaging of Microcirculation in Early
Mammalian Embryos
Irina V. Larina, Mary E. Dickinson, and Kirill V. Larin
Proper formation of the heart and vasculature relies on a complex balance
between genetic signaling events and mechanical stimuli [1–3]. Primary genetic
defects that affect cell differentiation or tissue patterning can lead to alterations in
cardiac function and blood flow, producing complicated secondary defects. On the
other hand, defective blood flow can secondarily affect signaling pathways within
cells that are required for normal development. Therefore, to characterize cardio-
vascular development and to understand the nature of cardiovascular congenital
abnormalities, it is important to take into account both genetic and mechanical
factors, such as blood flow and heart contractions.
Since mammalian embryos develop in utero, currently available imaging tech-
niques do not allow embryonic imaging and hemodynamic analysis in vivo with
high spatial and temporal resolution. To overcome this limitation, live static mouse
embryo culture protocols have been developed to allow live imaging at the embry-
onic stages from the 6.5 to about 10.5 dpc [4]. The embryos can be maintained in
culture for over 24 h to provide an access for optical imaging while the heart is
beginning to beat and remodeling into a chambered pump, blood circulation is
establishing, and vasculature is forming and remodeling.
This chapter describes how two optical approaches, confocal microscopy of vital
fluorescent reporters and optical coherence tomography (OCT), can be used for
live imaging of microcirculation during development in cultured mouse embryos.
Confocal microscopy combined with fluorescent protein reporter lines gives the
advantage of performing live imaging with subcellular resolution. We describe
mouse transgenic lines, which can be used for hemodynamic analysis by tracking
individual blood cells as well as for visualization of the developing vasculature.
While confocal microscopy allows for exceptional spatial resolution, its imaging
depth is limited to about 200–300 µm. This limitation can be addressed by using
OCT (as described in Chapter 10), which allows 3D imaging millimeters into tissue,
although at the expense of lower spatial resolution (2–10 µm). Here we describe
how OCT can be applied for hemodynamic analysis in deep embryonic vessels
during development.
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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280 12 Fluorescence and OCT Imaging of Microcirculation in Early Mammalian Embryos
12.1
Mouse Embryo Manipulations for Live Imaging
In mice, blood cells and endothelial cells are first detected in blood islands of
the embryonic yolk sac at 7.5 dpc. Formation of the embryonic heart starts at
about the same time. By about 8.5 dpc, the embryonic heart tube starts to beat
thereby circulating plasma through the vascular system. It takes a few hours for the
heartbeat to become strong enough to pick up blood cells fromthe blood islands into
the circulation. Within the next 24 h, shear stress generated by the circulating blood
cells facilitates remodeling of the vascular plexus into more mature hierarchical
circulatory system. At the same time, the heart tube expands, loops, forms chambers
and valves, and by about 14.5 dpc, it remodels into a four-chambered functional
pump with appropriate inflow and outflow. Mouse embryo manipulation and static
embryo culture protocols described below allow live imaging for over 24 h and
were optimized for 7.5– 10.5 dpc stages. Since these stages correspond to the most
structural rearrangements of the yolk sac vasculature and the heart, the described
methodology provides an opportunity for hemodynamic analysis and time-lapse
imaging during critical stages of development to understand dynamic aspects of
cardiovascular formation.
For timed pregnancies, mating pairs are set overnight and checked for the
presence of a vaginal plug every morning (a presence of the plug is taken as 0.5
dpc). At the desired stage, the female is sacrificed and the embryos are dissected
out of the uterus with the yolk sac intact. The dissection medium consists of
DMEM/F-12 (Invitrogen), 10% fetal bovine serum (FBS) (Invitrogen), and 10 mM
penicillin/streptomycin (Invitrogen). The medium should be freshly prepared
(∼50 ml per pregnant female) and preheated to 37
◦
C. The dissection station is
also preheated and maintained at 37
◦
C using a custom-made heater box and a
conventional heater. Freshly dissected embryos are transferred to the 37
◦
C5%
CO
2
incubator for recovery for at least 30 min.
It is critically important to maintain the temperature of the imaging stage at
37
◦
C throughout the duration of the imaging session. For fluorescence imaging,
this can be achieved with a custom-built incubation system fitted around the
scope, although it was found that the embryo culture technique is more robust
if a commercial incubation system is used (such as from Carl Zeiss, Inc.) since
it provides precise control of the temperature and CO
2
and is equipped with a
humidifier. For OCT imaging, we position the whole scanning head of the OCT
system inside the commercial 37
◦
C, 5% CO
2
incubator.
For fluorescence imaging, mouse embryos can be cultured in glass bottom
culture dishes (MatTek), which have a glass cover slip attached to the microwell in
the bottom of the dish. Alternatively, Lab-Tek 2-well chambers with glass cover slip
bottoms (Nunc) can be used. The glass bottom culture dishes use about 3 ml of the
culture medium, while the Lab-Tek chambers use about 2 ml. The glass cover slip
in the bottom of the chambers allows for imaging with inverted microscopes. For
the OCT imaging, the embryos are cultured in 30 mm culture dishes with about
5 ml of medium.
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12.2 Imaging Vascular Development and Microcirculation Using Confocal Microscopy 281
If embryo culture experiments are limited to a few hours, embryos can be
imaged in the DMEM/F-12 (Invitrogen) medium prepared for embryo dissection
as described above. However, for longer imaging sessions (such as visualization of
vascular plexus formation, remodeling, and endothelial cell tracking), the culture
medium should be supplemented with rat serum as follows: two parts of the
DMEM/F12, one part of the rat serum, supplemented with penicillin/streptomycin
(Invitrogen). Even though the rat serum is commercially available through different
sources, we prefer extracting the rat serum in the laboratory. Using the commer-
cially available serum resulted in very inconsistent results, probably caused by the
traces of anesthetics used during blood extraction as well as variations in serum
extraction and handling. On the other hand, home-made rat serum consistently
allowed us to maintain embryos in static culture for over 24 h.
For rat serum preparation, adult male Sprague Dawley rats are used. Usually, 30
rats provide about 50–100 ml of serum. It is critically important to use ether for
rat anesthesia, because it evaporates easily from the serum, while other anesthetic
agents remain in the serum and can affect embryonic growth and survival. Blood is
collected from an anesthetized rat through the dorsal aorta exposed by abdominal
incision into a Vacutainer blood collection tube (BD Biosciences) using Vacutainer
blood collection set (BD Biosciences). After the extraction, the tube is placed on ice
for up to 40 min during blood extraction from other rats. The tubes with blood are
centrifuged at 1300 g for 20 min, and the supernatant (blood serum) is collected by
pooling. If the supernatant looks pink in some tubes, it is because of lysis of the red
blood cells; discarding those tubes is recommended. Theserum is centrifuged again
at 1300 g for 10 min to get rid of the remaining blood cells, and the remaining super-
natant serum is collected by pooling. The serum should be heat-inactivated at 56
◦
C
for 30 min with the lid unscrewed to allow ether evaporation. For further ether evap-
oration, the tubes are left overnight at 4
◦
C with lids unscrewed, then filtered using
0.45 µm filter (Nalgene
), and stored in 1 ml aliquots at −80
◦
C for up to one year.
One of the major limitations for successful experiments is a sample drift over
time caused by the embryo growth as well as possible floating and rotation. To
prevent excessive movement and floating of the embryo, a part of decidua can be
left attached to the embryo during the dissection or, alternatively, a piece of a hair
can be placed over the embryo to keep it in the same position.
The described mouse embryo manipulation and static culture protocols have
been successfully used for live imaging and hemodynamic analysis using confocal
microscopy of transgenic embryos with vital fluorescent reporters [2, 5, 6] as well
as OCT [7, 8].
12.2
Imaging Vascular Development and Microcirculation Using Confocal Microscopy of
Vital Fluorescent Markers
Time-lapse confocal imaging of vital fluorescent reporter in transgenic animals is
a powerful tool to analyze early development and research cellular consequences of
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282 12 Fluorescence and OCT Imaging of Microcirculation in Early Mammalian Embryos
genetic manipulations [9–12]. Several transgenic mouse lines have been generated
that express fluorescent proteins in specific tissues of the embryo, for example,
ε-globin-GFP expressing green fluorescent protein (GFP) in primitive erythrob-
lasts, Flk1-H2B::EYFP expressing nuclear yellow fluorescent protein (YFP) in the
endothelial cells, and Oct4-GFP expressing GFP in primordial germ cells. Live
imaging of transgenic embryos carrying fluorescent markers crossed to different
mutant lines expands the possibilities in studying molecular mechanisms and
signaling events regulating embryonic development.
A transgenic fluorescent reporter mouse model useful for hemodynamic analysis
in early embryos was generated by Dyer et al. [13]. These animals carry GFP
under control of ε-globin promoter, which drives GFP expression in primitive
erythroblasts. Blood cells brightly labeled with the GFP are first detected in the
blood islands of the embryonic yolk sac before the beginning of the heartbeat. A
few hours after the beginning of the heartbeat (about 8.5 dpc), when the plasma
flow is stronger, the blood cells leave the blood islands and join the circulation.
These events can be directly studied using this reporter in live embryo culture.
Using ε-globin-EGFP transgenic mouse line, expressing GFP in embryonic blood
cells it is possible to visualize the initiation of blood formation, to quantify early
circulation events, and to characterize mutants with circulation defects [2, 5].
Classical point-scanning confocal microscopy is too slow to record the motion
of the rapidly moving blood cells. To overcome this limitation, several groups
have used a line-scanning approach where, instead of the full-frame imaging, the
scanning is performed along a designated line, either along or perpendicular to
the vessel [5, 14, 15]. If the scanning line is set perpendicular to the vessel, the
circulating blood cells are crossing the line, the number of scans and time it takes
for each cell to go across the line can be recorded. The length of the cell trace
divided by time reveals the velocity. If the scanning line is set parallel to the flow,
the blood cells are moving along the line and their velocity can also be measured.
These techniques provide valuable hemodynamic measurements in live vessels
and are still widely used, although they are time consuming since only a single line
recording can be performed at a time.
The recent development of fast-scanning, line-based confocal microscopes pro-
vide the opportunity for full-frame real-time imaging of rapid dynamic events. If
the brightness of the fluorescent marker permits, the Zeiss LSM 5 LIVE confocal
microscope allows one to acquire 512 × 512 pixel images at the rate of up to 120fps.
This is about 100 times faster than using regular standard confocal microscope.
Since the blood flow in early embryos is in the range of millimeters per second,
the dynamics of all blood cells in the field of view can be followed with superior
temporal and spatial resolution for detailed hemodynamic analysis.
To visualize and analyze microcirculation in the embryonic yolk sac, image
processing software Imaris (Imaris 5.0.3, Bitplane) can be used. Figure 12.1 shows
an example of blood flow imaging in cultured ε-globin-GFP embryos using a
Zeiss LSM 5 LIVE fast-scanning confocal microscope and Imaris processing. The
imaging is performed at 10.5 dpc when the yolk sac vasculature is remodeled into
a network of large vessels with high flow and branching from them are smaller
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12.2 Imaging Vascular Development and Microcirculation Using Confocal Microscopy 283
(
a
)(
b
)(
c
)
Figure 12.1 Live imaging of microcir-
culation in the embryonic yolk sac of
Tg(ε-globin-GFP) mice at 10.5 dpc with
fast-scanning confocal microscopy. (a) A
frame from a time lapse acquired at 60 fps.
Green dots represent individual circulating
blood cells labeled by the GFP. (b) Spots
corresponding to the individual blood cells.
(c) Tracks formed by the moving blood cells
over 50 frames. Different colors correspond
to different time points according to the
scale.
vessels with lower flow. An image sequence (512 × 512 pixels) was acquired at
60 fps using 10 × magnification. Figure 12.1a shows a representative frame from
the time lapse with green dots correspond to individual blood cells circulating
through the yolk sac vasculature. To visualize and analyze the microcirculation
in the embryonic yolk sac, each distinguishable GFP-labeled cell in the focal
plane was automatically associated with a spot, as shown in Figure 12.1b. This
procedure was performed in Surpass mode of the Imaris software using the ‘‘Add
new spots’’ function in the Objects window (we usually set minimal diameter
of the detected spots to 7 µm). The number of nuclei-associated spots can be
adjusted by changing the threshold for spot identification to include or exclude
the cells that are partially out of focus. Once the spots are generated, they can be
automatically tracked via the ‘‘Tracking’’ function in the Spots menu (it is best
to use the ‘‘Autoregressive Motion’’ algorithm and set the ‘‘maximum distance’’
traveled between the neighboring images as 10 µm). Figure 12.2c shows tracks
representing the dynamics of the GFP-labeled blood cells over the course of 50
frames. The color of the tracks changes from blue to white according to the
timescale as shown on the bar. Quantitative statistics of the spot movements can
be extracted for further analysis to reveal the heart rate and blood flow profiles in a
variety of vessels at different phases of the cardiac cycle.
To visualize the developing embryonic vasculature, two transgenic mouse lines
are particularly useful, namely, Tg(Flk1-myr::mCherry) and Tg(Flk1-H2B::EYFP).
In both transgenic lines, the fluorescent proteins are expressed in the embryonic
endothelium and endocardium [6, 16]. In the Tg(Flk1::myr-mCherry) line, the
fluorescent protein mCherry is fused to a myristoylation motif (myr), which allows
for membrane localization of the fluorescent protein and, therefore, outlines
the structure of the vasculature and reveals cellular morphology and cell–cell
boundaries between the endothelial cells. In the Tg(Flk1-H2B::EYFP) line, the
expression of the YFP is localized to the nuclei of the endothelial cell to allow
analysis of endothelial cell migration, division, and apoptosis. Intercrossing these
markers allows identifying each endothelial cell within a vessel to reveal the
distribution of individual cells within each vessel segment.
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284 12 Fluorescence and OCT Imaging of Microcirculation in Early Mammalian Embryos
(a) (b)
(c) (d)
100 µm
200 µm
50 µm
20 µm
Figure 12.2 Live imaging of the develop-
ing vasculature in Tg(Flk1-myr::mCherry)
X Tg(Flk1-H2B::EYFP) mice using confo-
cal microscopy. (a) Premature vascular
plexus of the embryonic yolk sac labeled
by mCherry (red) and EYFP (yellow) at
8.5 dpc. (b) Remodeled yolk sac vascu-
lature at 10.5 dpc. (c) Vascular plexus of
the 8.5 dpc yolk sac at 63 × magnification
in homozygous Tg(Flk1::myr-mCherry) ×
Tg(Flk1::H2B-EYFP) embryos. The boundaries
between the endothelial cells are clearly out-
lined by the mCherry. (d) The vasculature
is labeled by mCherry and EYFP while the
blood cells are labeled with GFP (green) in
Tg(Flk1-myr::mCherry) × Tg(Flk1-H2B::EYFP)
× Tg(ε-globin-GFP) mice at 8.5 dpc. Yellow
arrows indicate some of the EYFP-labeled
nuclei of the endothelial cells; green arrows
indicate GFP-labeled blood cells.
The fluorescence of mCherry and EYFP is first detectable at 7.5 dpc (also known
as the early headfold stage) in the blood islands of the yolk sac. At this stage, the
fluorescence is relatively dim and is only detectable in homozygous embryos. At
8.0 dpc, the markers label the vascular plexus as it develops in the embryonic
yolk sac. By 8.5 dpc, the vascular plexus is brightly outlined by the mCherry with
clearly distinguishable YFP-labeled nuclei (Figure 12.2a). Both markers remain in
the endothelial cells as the vascular plexus of the embryonic yolk sac undergoes
remodeling into a more mature circulatory system consisting of larger vessels that
branch into progressively smaller vessels. Figure 12.2b shows the remodeling yolk
sac vasculature at 10.5 dpc, labeled by the mCherry and the EYFP. Membrane
boundaries between endothelial cells are distinguishable in some cells at high
magnification; for example, Figure 12.2c shows a fragment of an 8.5 dpc yolk sac
homozygous for both fluorescent markers at 63 × magnification. In the embryo
proper, the markers are coexpressed from 8.5 dpc until late gestationthroughout the
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