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14.2 Micro-Computed Tomography 325
is theoretically limited in spatial resolution to about 2 mm [35]. This is due to a
combination of factors, most notably the finite range of the positron (distance be-
fore collision with an electron). When used in conjunction with micro-CT imaging,
this problem is mitigated somewhat. Thus, high spatial resolution data, related to
functional processes, is achievable through the use of a joint imaging modality
such as micro-CT/PET. An example, combined micro-CT/PET system is displayed
in Figure 14.8.
Figure 14.9 shows coregistered small-animal micro-CT/PET images [11].
Through the use of a novel radioligand tracer, tumor vasculature was noninvasively
assessed in terms of how the vasculature responds to different treatments.
Thus, the use of a multimodality imaging technique allows an enhancement for
understanding various aspects of physiology and, in this case, an understanding of
how different therapeutic strategies affect tumor vasculature.
14.2.6.2 Micro-Single Photon Emission Computed Tomography
Single photon emission computed tomography (SPECT) is the combination of both
conventional radionuclide imaging and image reconstruction from projections.
Conventional radionuclide imaging uses position-sensitive radiation detectors
to measure X-ray photon emission from the three-dimensional distribution of
Tumor
Tumor
Bladder
(a)
(
b
)
100
%
100
%
0%
0%
Figure 14.9 (a,b) Micro-CT image data (200 µm voxel size)
is here shown coregistered with micro-PET data. High spa-
tial resolution data, which is related to functional processes,
is achievable through the use of the joint imaging modalities
of micro-CT/PET. (Source: Figure modified from Ref. [11]).
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326 14 Studying Microcirculation with Micro-CT
radioactivity of radiolabeled biomarkers. This information forms a two-dimensional
projection image. In SPECT, an image reconstruction method is applied to multiple
2D projection images to form a 3D image.
A commercially available combined nano-CT/SPECT imaging system is shown
in Figure 14.10. Wietholt and colleagues [38] used micro-CT/SPECT fusion (regis-
tered) images to study circulation in rat lungs. Figure 14.11 shows the registered
images for both the control group and a group of rats that had been subjected to
a surgically induced occlusion of the left pulmonary artery. These transaxial slices
(utilizing both micro-CT and micro-SPECT image data) allow the perfused regions
within the specimen to be explored. In the case of the group of rats that had a sur-
gical occlusion induced, the accumulation of the radiotracer occurred exclusively
in the right (nonoccluded) lung of the animal, whereas the left (occluded) lung was
not perfused via the pulmonary circulation.
A limitation of micro-SPECT is the low spatial resolution (in comparison to
that of micro-CT imaging). Recent improvements in detector technologies and the
incorporation of coated aperture (pinhole) geometry have allowed resolutions down
to 1 mm to be achieved [39]. In addition to supplying high spatial resolution data,
micro-CT data is also used as an attenuation correction for micro-SPECT data,
which improves, for example, the uniformity of myocardial perfusion images [40]
and thus allows micro-SPECT data to be useful quantitatively (not just qualitatively
as merely a visualization tool). Thus, the full potential of SPECT functional imaging
is realized by unambiguously assigning tracer uptake to organs or other anatomical
features [41].
Figure 14.10 Combined nano-CT/SPECT imaging system
offered by Bioscan (Washington, DC). This system uses a
coded aperture to generate the scan data used for tomo-
graphic reconstruction.
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14.2 Micro-Computed Tomography 327
10 mm
10 mm
10 mm
10 mm
(a) (b)
(c) (d)
Figure 14.11 Transaxial slice through
micro-CT/SPECT registered images for con-
trol rat group (a,b) and surgically occluded
left pulmonary artery rat group (c,d), for
both a left carotid artery injection (a,c) and
a femoral vein injection (b,d). These transax-
ial slices (utilizing both micro-CT as well
as micro-SPECT image data) allow the per-
fused regions within the specimen to be
explored and related to anatomic structure.
In the case of the group of rats that had a
surgical occlusion induced, the accumula-
tion of the radiotracer occurred exclusively in
the right (nonoccluded) lung of the animal,
whereas the left (occluded) lung is not per-
fused via the pulmonary circulation. (Source:
This figure was modified from Ref. [38]).
14.2.6.3 Digital Subtraction Angiography
Combined micro-CT and digital subtraction angiography (DSA) have been incor-
porated into a single system recently [42]. Utilizing a blood-pool contrast agent
(Fenestra VC), micro-CT imaging was used to assess the 3D vascular architecture
of the tumor, while DSA imaging was used to assess tumor perfusion. The DSA
data provides perfusion maps that can be combined (spatially coregistered) onto
the micro-CT image data. An example from a combined micro-CT/DSA study
is shown in Figure 14.12 [43]. The combination of micro-CT with DSA allows
for morphological (in terms of accurately assessing 3D vascular architecture) and
functional data (in terms of assessing tissue perfusion).
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328 14 Studying Microcirculation with Micro-CT
3.05
3.01
2.97
2.92
2.88
0.34
0.34
0.33
0.33
0.33
0.98
0.97
0.96
0.95
0.94
tMIP of DSA rBV
rBF rMTT
Figure 14.12 Maximum intensity projec-
tion (MIP) image and three perfusion maps
(with a spatial resolution of 100 µm): rela-
tive blood volume (rBV), relative blood flow
(rBF), and relative mean transit time (rMTT)
are shown for a combined micro-CT/DSA
study of the fibrosarcoma tumor in a rat.
A conventional contrast agent, Isovue 370,
was used in the DSA study and provided
complementary functional information to
the high spatial resolution micro-CT image
data. (Source: This figure was modified from
Ref. [43]).
14.2.6.4 Histology
One of the most common microscope-based imaging methods in use is histology,
in which thin sections of tissue are imaged. Specific regions (such as particular
cells) can be enhanced by specific stains that color particular regions. Histology is an
inherently destructive imaging method, requiring bloodless biopsies of tissue to be
extracted from a specimen. Micro-CT images have been shown to be able to acquire
information somewhat similar to some aspects of histology (for example, micro-CT
was shown to be feasible for the study of coronary arterial walls ([44], Figure 14.13),
which is beneficial since micro-CT offers the possibility for nondestructive testing,
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14.3 Specimen/Animal Preparation 329
5
1
3
2
4
6
2
3
1
6
4
(a) (b)
Figure 14.13 Advanced fibrocalcific
atherosclerotic lesions were imaged by both
micro-CT and histological sectioning (micro-
scopic images of thin tissue samples). (a)
Transverse micro-CT image. (b) Conventional
histological image. The bar at the bottom
right is 0.5 mm in length. Micro-CT is able
to provide many anatomical structures with
the same amount of detail as histology. This
is beneficial since micro-CT provides fully
3D images, nondestructively. (1) Eccentric
neointimal thickening, (2) boundary between
media and adventitia, (3) adventitia, (4) cal-
cification, (5) paraffin used to store coronary
artery, and (6) vessel lumen. (Source: This
figure was modified from Ref. [44]).
as well as analysis of much larger 3D tissue biopsies (where the histological
sectioning process induces deformations in the samples). The information from
histological sections can also be used to enhance micro-CT data by looking at cell
differentiation in tissue.
14.3
Specimen/Animal Preparation
Unfortunately, the walls and contents of vasculature provide little inherent contrast
from surrounding soft tissues for X-ray imaging. Specimen preparation is therefore
an important step in micro-CT microcirculation studies so that the vasculature in
the image can be visualized. To allow vascular visualization, contrast agents are
injected into the vascular lumens, thereby causing the X-ray to be significantly
attenuated by the vasculature. The considerations in specimen/animal preparation
and contrast agent usage differ depending on the study.
14.3.1
Ex Vivo – Casting
Using a partially polymerized methyl methacrylate plastic (typically Batson’s No. 17
corrosion casting compound), casts of vascular networks can be created. The details
of this standard casting technique were outlined by Gannon [45]. The details of a
recent study [46] where the cast of a rabbit’s kidney vasculature was made and im-
aged by micro-CT are described next in order to outline the relevant steps involved.
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330 14 Studying Microcirculation with Micro-CT
A one-year-old female New Zealand white rabbit weighing 2.2 kg was euthanized
by intravenous injection. Next, the animal’s thoracic cavity was exposed by a ventral
incision and the renal artery was cannulated. Heparinized saline solution was
perfused through the vasculature using a three-way valve. This was performed to
prevent blood clots from forming. Note that the perfusion pressure is an important
consideration. Here, a pressure of 100 mmHg was used and 20–40 mmHg would
have been used if performing venous perfusion (to prevent vessel wall rupture).
Once blood was cleared from the tissue, the valve was switched to perfuse a freshly
prepared Batson’s No. 17 solution. The Batson’s solution was allowed to polymerize
within the animal’s tissue. With the specimen in an ice bath, the polymer took
2–3 h to cure. The use of an ice bath helps slow down the polymerization process
that helps to minimize cast distortion during this exothermic reaction.
Once fully polymerized, the tissue was immersed in a 25% potassium hydroxide
solution for 24 –48 h. This solution corrodes away the tissue leaving only the
polymerized plastic that represents the vascular tree lumens. The cast may then
be imaged by micro-CT. The method of casting is clearly destructive but has the
benefit of removing the surrounding tissue, which can cause potentially significant
background noise in the subsequent micro-CT image. An image of both the created
cast and the representative micro-CT image is depicted in Figure 14.14 [46].
14.3.2
In Situ – Contrast Agent Enhancement
In order to imagethe vascular lumens withinintact specimens, a contrastagent such
as Microfil is frequently used [1, 46 –48] (latex- and gelatin-based contrast agents are
also available [5]). The procedure is performed as discussed in the previous subsec-
tion; however, the Batson’s No. 17 corrosion casting compound is replaced by the
contrast agent. A contrast agent, such as Microfil, allows for the analysis of intact
vasculature because of its high X-ray attenuation (2500–5000 HU) so that the per-
fused vasculature can easily be delineated from surrounding tissue. In contrast, the
4321
500 µm
(
a
)(
b
)
Figure 14.14 (a) Vascular cast of a whole rabbit kidney
(ruler is in centimeters). (b) 3D volume rendering of a por-
tion of the vascular cast, imaged by micro-CT (35 µm voxel
size). (Source: Modified from Ref. [46]).
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14.3 Specimen/Animal Preparation 331
2 mm
(
a
)(
b
)(
c
)
2 mm
Figure 14.15 Micro-CT image data (20 µm
isotropic voxels) of the vascular bed of a rat
heart, which was filled with a contrast agent
(Microfil) and was imaged in situ,ishere
displayed by different visualization meth-
ods. In panel (a), cross-sectional slices are
shown for two different gray scale window-
ing levels. In panel (b), a maximum intensity
projection (MIP) of the micro-CT data of
the same vascular bed is displayed. Using a
region-growing segmentation method, a fully
connected vascular tree can be segmented,
shown in red in panel (c). The vasculature
in the image that are displayed as gray are
either from the right myocardial arteries or
are disconnected vessel segments due to the
segmentation process or low contrast agent
concentration.
corrosion casting method (discussed in the previous subsection) has relatively low
attenuation (0–1500 HU) and thus requires that the surroundingtissue be removed.
Micro-CT image data (20 µm isotropic voxels) of the vascular bed of a rat
heart, which had the coronary artery filled with a contrast agent (Microfil), then
embedded in wax and imaged in situ, is displayed by different visualization
methods in Figure 14.15. In panel (a), cross-sectional slices are shown for two
different grayscale windowing levels. The windowing level assigns the color black
to any pixel in the image with a CT value less than the lower threshold and the
color white to any pixel with CT value greater than the upper threshold. In the
top panel, the windowing level thresholds are [200 15 000], and, in the bottom
panel, the windowing level thresholds are [ –3000 15 000]. Pixels with CT values in
the intermediate range are shown on a gray scale. Images such as these can be
used, for instance, to manually define regions of interest within the images as well
as measure various distances between different anatomical features. In panel (b),
an MIP of the micro-CT data of the same vascular bed is displayed. It is shown
here that the vasculature is easily delineated from the surrounding tissue because
of the vasculatures’ far higher CT values. Using a region-growing segmentation
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332 14 Studying Microcirculation with Micro-CT
method, a fully connected vascular tree can be segmented, shown in red in panel
(c). The vascular tree in this case is visualized by a technique known as volume
rendering. In this case, the surface of the vessel tree is visualized and rendered
with shading in order to give the structure a three-dimensional look. Properties
of this segmented vessel tree such as the total vessel volume and surface area can
then be measured, and local geometrical properties such as interbranch segment
diameters and branching angles can be quantified.
Other contrast agents, such as osmium tetroxide (OsO
4
), have also been used in
micro-CT vascular research. Zhu et al. [49] showed how visualization of coronary
arterial wall structure, early lesion formation, and changes in vascular permeability
could be detected for the vasculature of porcine hearts by using OsO
4
as the
contrast agent (Figure 14.16). First, the hearts were placed in formaldehyde
immediately after harvesting. The unbound formaldehyde was then flushed from
Normal
HC
(a) (b)
(
d
)
(
c
)
Histolo
gy
Micro-CT
1000 µm
Figure 14.16 Representative myocardial immunohistochem-
istry of vascular endothelial growth factor (VEGF, a) and cor-
responding micro-CT image (b) of the same section. Note
the similar distribution of VEGF (red, c) and perivascular
OsO
4
(white, d). (Source: From Ref. [49]).
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14.3 Specimen/Animal Preparation 333
the vasculature by infusion of the coronary arteries with a 60 ml 0.9% saline, 10 ml
1% OsO
4
solution. This solution was hand injected in 1 ml increments into the
artery every 6 min for 1 h. Finally, a portion of the distal epicardial coronary artery,
which included a piece of the attached ventricular myocardium, was then sectioned,
prepared, and scanned by micro-CT. The use of OsO
4
acts as a sort of cellular
staining (e.g., its density distribution allows for some differentiation of the cells
within vessel walls and myocardium).
14.3.3
In Vivo
The long scanning times of in vivo experiments (between 0.5 and 2 h) require
intravascular contrast agents to either not leave the blood pool rapidly or be
continuously infused into the vasculature [5]. Since contrast agents used for in vivo
studies can be administered multiple times to the same animal, the possibility for
longitudinal micro-CT studies exists.
Using a conventional iodinated vascular contrast agent (which is encapsulated
within polyethylene glycol-stabilized liposomes), intravascular space was shown
to be suitably enhanced for more than 3 h [50]. Iodinated triglycerides contain-
ing lipophilic cores of oil-in-water lipid emulsions (offered by Fenestra) provide
long-lasting visualization of vascular systems with micro-CT. Kong et al. [51] used
Pluronic F127 and a naturally iodinated compound (Lipiodol) to successfully form
radiopaque nanoreservoir structures.
Shown in Figure 14.17 are the cerebral arteries of a mouse, which have been
imaged by micro-CT in vivo [52]. Imeron 300, a conventional contrast agent, was
used, which thereby allowed the cervical, extra-, and intracranial arteries and veins
to be evaluated. With a reconstructed voxel resolution of 22 µm, vessels with a
diameter
>
50 µm were successfully visualized.
14.3.4
Use of Probes
14.3.4.1 Nano-/Microspheres
As an example, microsphere embolization study [53], control, and
‘‘microsphere-simulated’’ embolization samples were obtained from a par-
ticular animal model. In this sense, the embolized specimens differed from the
control in that an injection of nonradioactive microspheres into the left anterior
descending artery was performed. Following the microsphere injection, the animal
was euthanized and the hearts harvested. Next, the coronary arteries were infused
with Microfil polymer at 100 mmHg infusion pressure and a nominal 2 cm
3
trans-
mural biopsy of the myocardial region was collected after the Microfil injection.
A micro-CT scanner was then used to generate the 3D specimen image data.
Comparison between control and embolized specimens was recently used [54]
to study the volume loss of vessels below the size of the microspheres. This study
essentially characterized the vascular consequences of microsphere embolization.
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334 14 Studying Microcirculation with Micro-CT
IPV
SOV
STV
IMV
EJV
BA
SCA
PCA
MCA
ICA
ACA
PCA
SCA
BA
ICA
0.24 mm
(
a
)(
b
)
Figure 14.17 Volume-rendered images of
the cerebral arteries of a mouse imaged by
micro-CT in vivo. Imeron 300, a conventional
contrast agent was used, which thereby al-
lowed the cervical, extra-, and intracranial
arteries and veins to be evaluated. With a
reconstructed resolution of 22 µm, vessels
with a diameter of 50 µm were successfully
visualized. (a) Overview of the neurocra-
nium with intracranial arteries and extracra-
nial veins. (b) Magnification of the basal
cerebral arteries with a white measurement
bar placed over the ICA. Labels are ACA, an-
terior cerebral artery; MCA, middle cerebral
artery; ICA, internal carotid artery; PCA, pos-
terior cerebral artery; SCA, superior cerebellar
artery; BA, basilar artery; IPV, infrapalpebral
vein; SOV, supraorbital vein; STV, superficial
temporal vein; IMV, internal maxillary vein;
and EJV, external jugular vein. (Source: This
figure is from Ref. [52]).
Microsphere embolization has shown how the perfusion defects due to blocked
vasa vasorum could be studied for coronary vessel walls [6]. Also, local perfusion
measurements were quantified in rat kidney vasculature [21] and pig myocardium
[55] utilizing the method of microsphere embolization.
14.4
Image Analysis
A great challenge for the researcher studying microcirculation by micro-CT imag-
ing is the postprocessing of the reconstructed image data. At the foundation
of this challenge is the overall goal of accurately quantifying the microvascular
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