3D Cardiac microvessels embolization imaging based on X-ray phase contrast imaging
BioMedical Engineering OnLine
3D Cardiac microvessels embolization imaging based on X-ray phase contrast imaging
Lu Zhang 0
Ke Wang 2
Fei Zheng 1
Xia Li 0
Shuqian Luo 0
0 School of Biomedical engineering, Capital Medical University , Beijing , P.R. China
1 Beijing Friendship Hospital, Capital Medical University , Beijing , P.R. China
2 Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University , Beijing , P.R. China
Background: The treatment of microcirculatory impairment will have great impact if it can be applied to myocardial infarction (MI) patients. The problem is how to study these tiny structures and microphenomenon in heart. Methods: We investigated the visualization of cardiac microvessels embolization by the mean of X-ray phase contrast imaging (XPCI), which is a recently emerged imaging technique. Using the information of X-ray phase shift, it is sensitive to weak absorbing materials. Two MI SD rats were used as the microvessel embolization samples. MI was surgically induced by ligating left anterior descending artery. Imaging was performed 24 hours post-infarct, with barium sulfate as contrast agent. Results: The coronary arteries were visualized with smooth walls and clear edges. The ligated vessels, with the diameter of about three hundred microns, can be clearly distinguished and there were no distal blood flow downstream from these branches. The results indicate that phase contrast imaging can directly demonstrate the distribution of microvessels, and estimate the area of MI. The infarct location was in good agreement with pathological analyses of the models. Conclusions: The advantage of our method is directly observing and evaluating microvessel embolization which simplifies the procedure of diagnoses. Moreover, it is helpful for predicting the prognosis in MI and judging if angiogenesis happens.
X-ray phase contrast imaging (XPCI); Microvessel embolization imaging; Myocardial infarction (MI)
The occlusion of coronary artery is the most common cause of Myocardial Infarction
(MI) . When MI happens, diminished blood supply of one or some coronary arteries
will lead to myocardial ischemia, which will result in myocardial cell damage or death.
To date, MI is still a worldwide disease with high morbidity and mortality. The main
reason of that might be because of the link between the occurrence of MI and
hyperlipidemia, diabetes mellitus, hypertension, smoking, obesity, lack of exercise and job
stress. People, more or less, suffer from these risk factors .
Clinically, reperfusion has proven to be the vital procedure to improve the survival
rate of MI patients. Recent studies have shown that even though after a rapid
reopening of the previously obstructed coronary artery, some distal microvessels remain
ischemia, which is called no-reflow phenomenon [3,4]. This abstruse, but important
phenomenon is frequently happened during reperfusion of myocardial infarction. It
has close relationship with patients chest pain, hemodynamic deterioration, ST-segment
elevation, infarct extensions, ventricular arrhythmias, early congestive heart failure, and
even cardiac rupture [5,6]. Therefore, the reperfusion of microvessels is currently of
significant interest. The treatment of microcirculatory impairment will have greater impact if
it can be applied to MI patients. The problem is how to study these tiny structures and
microphenomenon in heart.
With the development of imaging technique, contrast echocardiography (MCE),
positron emission tomography (PET), and contrast-enhanced magnetic resonance imaging
(MRI) are becoming the most effective method for microvascular assessment. The area
of ischemia can be defined through those methods. But the use of these techniques
have been limited on imaging individual microvessels [7-9]. For the study of myocardial
microcirculation, structure and function have the same importance. However, there are
no ideal methods to realize cardiac microvessel imaging without destroying the original
complete heart. Conventional histological studies only provide an accurate view of
microvessels on cross-sectional specimens in two-dimension (2D). However, in order to
study the cardiac microvessels embolization, three-dimemsional (3D) morphology of
the blood vessel system has to be precisely determined. The 3D reconstruction of
vascular network via a serious of sectioning is very complicated and time-consuming.
Confocal laser scanning microscope (CLSM) has a high imaging resolution as well as 3D
imaging ability. Unfortunately, the sample size should be very small and thin . It is
not suitable for a whole rat heart imaging.
X-ray Phase contrast imaing (XPCI) became a research focus about some decades
ago with the awareness of its high spatial resolution and contrast on imaging soft
tissues [11,12]. It is capable for detecting the direction deviation when X-rays travel
through one object. Compared with clinical used X-ray mechanism, which relies on
beams attenuation, XPCI is more sensitive to density variations in the sample. The
Xrays direction change at the boundaries between different tissues, e.g. the interface
between vessels and surrounding tissues, will be transformed into intensity change in the
final image. The image contrast and resolution have been greatly improved. This
superiority has been exploited for imaging malignant tumor, joint, cerebral vessels, lung
airways, and etc. [13-16].
In order to observe blood vessels, contrast agents are widely used in conventional
Xray examination, including Digital subtraction angiography (DSA), Computed
tomography angiography (CTA) and enhanced CT scanning. Though PCI can provide high
contrast of soft tissues, we used contrast agent in our experiments to highlight
microvessels in rat hearts. In this way, the image will compose more details of vessel
networks due to phase contrast. Moreover, other soft tissues such us cardiac valves and
myocardium can also be detected in the same image.
Accordingly, in this paper, we sort to directly visualize coronary microvessels, micro
embolization and evaluate myocardial ischemia area by means of phase contrast
Two adult normal SD rats, approximately 250 g of body weight each, were anesthetized
by an intraperitoneal injection of chloral hydrate 4.5 ml/kg, tracheal intubated and
connected to a small animal ventilator. MI was induced by ligating of the left anterior
descending (LAD) coronary artery with a 6 absorbable suture through the left fourth
intercostal space. Then we closed the thoracic cavity layer by layer and removed the
In order to observe the micro vascular network, contrast agent was used. But, in our
experiment, the particles of contrast agent should be smaller than the capillaries. At
the same time, the agent cannot migrate through the walls of the vessels. Moreover, it
should be insoluble even though the tissue was fixed in formalin. After a serious of test,
barium sulfate is proven to be the desirable one. To ensure microvessels angiography,
it should be ground into very fine particles prior to use.
24 h after the operation, we anaesthetized the rats again, opened their thoracic cavity
to expose the heart, cut the postcava, ligated the ascending aorta, and then injected the
barium sulfate solution (20% v/v) by a scalp acupuncture, which connected to a 20 ml
syringe, via the aorta root. After the coronary artery turned white, we harvested the
hearts and quickly preserved them in formalin solution. Finally, phase contrast imaging
was performed on these isolated hearts. After imaging, every heart was equally
dissected into four blocks along the long axis of heart and paraffin embedded. 5 m thick
sections were cut and stained with hematoxylin and eosin (H&E).
X-ray Phase contrast imaging
The coronary microvessels were visualized by X-ray phase contrast imaging. The
principle of XPCI is Fresnel diffraction theory . Contrast and resolution are derived
from direct Fresnel diffraction with synchrotron radiation which has good spatial
coherence and high intensity. When the beam traverse the object, X-ray undergo
amplitude attenuation in addition to phase-shifts. After the downstream beam propagates for
a sufficient distance, the phase-shifts are transformed into measurable intensity
variations by means of Fresnel diffraction. Since the phase shift between biological soft
tissues is almost one thousand times greater than the absorption term at photon energies
greater than 10 keV, this technique can greatly improve the image quality of soft
The imaging experiment was performed in Shanghai Synchrotron radiation facility
BL13W beamline. Briefly, it consists of one double-crystal monochromator, one rotation
sample stage and one CCD detector (Figure 1). The high-energy and high parallel
synchrotron X-ray beam first cast on the monochromator crystals (Si 111 or Si 311) which
can be rotated to select beam energy. The outcome beam became single energy X-ray.
The tunable energy range was from 8 to 72.5 keV, with the energy resolution of about
0.5%. In our experiment, it was adjusted to 16.5 keV. This beam then travel through the
imaging object. The direction associated with the intensity of the beam will change due to
refraction, small angle scattering and attenuation of the beam. Subsequently, the
transmitted beam propagated for some distance and then captured by the CCD detector. Just
during this distance, the downstream beam was enhanced by Fresnel diffraction and the
phase modulation was transformed into CCD receivable intensity.
For 2D imaging, the CCD detector we chosen had a resolution of 2048 2048 pixels
with 3.7 m 3.7 m each. The distance between sample and detector was 0.28 m. The
Figure 1 Schematic set-up of the XPCI system and the processing flow chart.
explore time was 3.5 s. For CT experiment, the resolution of the detector was 4008
2672 pixels with 9 m 9 m each. After the sample stage rotated for 180 degree, 1254
projection images were obtained, with an exposure time of 90 milliseconds each. The
distance between sample and detector was 0.52 m. The surface dose was about
2.66 mGy for each projection. Filtered back projection method was used to reconstruct
the cross-section of the sample . The algorithm was implemented via Matlab 2009b
(Mathworks Inc, Natick, MA, USA). Surface rendering later was used for 3D rendering
of the heart by a 3D imaging software (Amira 5.2, Visage Imaging, Berlin, Germany).
The two rats were established as models of acute myocardial infarction. 24 h after
infarction, we started the imaging test. The phase contrast image of the heart is shown in
Figure 2. The vessels in heart were visualized with smooth walls and clear edges. The
ligated vessels can be distinguished and no distal vessels of these branches as pointed by
arrow. For the study of cardiac microvessels, depicting the 3D architecture of the
vessels is real challenging, if we only imagine it from 2D projected images. In our study,
we reconstructed the cross-section of the heart volume, shown in Figure 3. The result
was in good agreement with pathological section. Since the vessels were filled with
contrast agents, they turned dark brown color in pathological sections. For microvessels,
we showed three vessels with diameter less than 100 m in Figure 4. The
corresponding section was found by tissue pathology with similar size and relative position.
Totally, we got 1677 phase contrast imaging slices. From the 3D rendered heart, the
tree-like coronary arteries and the distal small arteries up to tenth level with a diameter
of 15 m were clearly visualized (Figure 5(a)). Since the ligated LAD coronary artery
had no blood supply, the distal vessels were missing in the image. From this 3D model,
we could directly find how many microvessels were blocked, where they were blocked
and roughly identify where the heart muscle was not receiving enough blood supply
(red part in Figure 5(a)). The use of histological stain is a widely accepted method to
estimate the accuracy of phase contrast imaging. We equally divided the heart tissue
into four blocks along the long axis of heart and then got three cross-sections. Three
corresponding H&E staining histological slices were shown as (b), (c), (d) up to down
separately. They were approximately the same as those part shown in Figure 5(a). The
first cross-section was above the ligation position. Therefore, in this part, left and right
coronary arteries existed in the image (Figure 5(b)). For the other two cross-sections,
under the ligation position, left coronary artery was missing but the right was visible
(Figure 5(c), (d)). The infarct area was also confirmed by H&E staining (Figure 6). The
region had ischemic myocardium and with numerous neutrophils (Figure 6).
Our present study demonstrated that microvessel embolism is detectable and visible in
myocardial infarction by phase contrast imaging. By assessing the no-blood-flow vessel,
we have speculated the infarction area.
For myocardial infarction imaging, there are a couple of methods. CTA is widely used
in clinic to evaluate coronary arteries diseases; DSA is currently considered as the
diagnostic standard. Other modalities such as contrast enhanced MR, PET, and ultrasound
are becoming the effective methods for infarction imaging [8,19,20]. In contrast to
mark the ischemia region, direct visualization and quantification of the infarct vessel is
more meaningful. In a number of clinical cases, after relief of the infracted large vessel,
the blood flow of the ischemic tissue was still impeded which lead to poor prognosis.
Till now there is no explicit explanation to this no-reflow phenomenon. Some
researchers tend to believe that it is caused by microvessels obstruction [4,6]. For this
reason, microvessel imaging in MI is gained more importance. Only a limited number
of previous studies have mentioned cardiac microvessels imaging. Kiyooka et al. 
observed coronary capillary by using their unique high-resolution intravital
chargecoupled device video microscopy in two dimension (2D). However, one inherent
limitation of 2D imaging is tissue overlap which make it difficult to distinguish spatial
distribution of blood vessel. Eiji Toyota and associates  reported their work about 3D
visualization of microvessels by synchrotron radiation micro-computed tomography.
But they focused on the phasic change in resistance of microvessels during systole and
diastole not the vessel visualization. Subsequently, Bert Mller et al.  used the same
technique to image microvessels in some selected part. The thinnest vessel was on
capillary level. However, the vessels appeared to be disconnected.
In the study of coronary heart disease, it is very important to directly observe the
anatomic variations and anastomosis of microvessels. Thus, we imaged the whole rat
hearts. Microvessels, such as coronary artery and some other arteries and veins were
shown in our results. The infarct vessel can be directly found in the 3D vessel tree. For
the part without vessel supply, we predict it as the ischemia area. The results were
highly consistent with the histological section which is recognized as the accurate view
The contrast agent we used in our experiment was barium sulfate. It can deposit in
the vessel even after the heart is fixed in formalin solution. The histological result also
proved this: the vessel lumen was completely filled with gray color contrast agent. The
shortcoming is that the blood flow was solidified too fast in microvessels before barium
sulfate contrast agent enter into them. The reason to choose barium sulfate lies in our
in vitro heart imaging experiment. If we can image the heart in vivo, then clinical
soluble contrast agent can be used. However, the most important problem to apply XPCI
to in vivo heart imaging is the imaging speed. It is possible for 2D continuous heart
imaging by XPCI. But for 3D imaging, the fast heart beating is still a big problem. Even some
tiny movement will cause blur in 3D reconstruction of microvessels. In our opinion, the
possible solution may be new high speed CCD and rotation stage.
In conclusion, by using XPCI, it is possible to directly image microvessels in MI model
and quantify the infarct microvessels. The quantitative character of the microvessels
can be directly observed. The microvessel stenosis strongly correlates with clinical
noreflow phenomenon. Phase contrast imaging may become a new way to study MI. In the
next step, we will explore the clinical significance of our findings, especially in no-reflow
This study was supported by the National Natural Science Foundation of China, Grant No. 61227802, 60532090 and
30770593, the 7th Framework Programme of the European Community, Grant Agreement Number PIRSES-GA-2009-269124,
and the Importation and Development of High-Caliber Talents Project of Beijing Municipal Institutions (CIT&TCD201304186).
The authors thank staffs from Shanghai Synchrotron radiation facility BL13W beamline for their kindly assistance
in our experiments.
1. White HD , Chew DP : Acute myocardial infarction . Lancet 2008 , 372 : 570 - 584 .
2. Yusuf S , Hawken S , Ounpuu S , Dans T , Avezum A , Lanas F , McQueen M , Budaj A , Pais P , Varigos J , Lisheng L : Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study . Lancet 2004 , 364 : 937 - 952 .
3. Agati L : Microvascular integrity after reperfusion therapy . Am Heart J 1999 , 138 (Sppl): 76 - 78 .
4. Rezkalla SH , Kloner RA : No-reflow phenomenon. Circulation 2002 , 105 : 656 - 662 .
5. Ndrepepa G , Tiroch K , Fusaro M , Keta D , Seyfarth M , Byrne RA , Pache J , Alger P , Mehilli J , Schmig A , Kastrati A : 5 - year prognostic value of No-reflow phenomenon after percutaneous coronary intervention in patients with acute myocardial infarction . J Am Col Cardiol 2010 , 55 : 2383 - 2389 .
6. Heusch G , Kleinbongard P , Bse D , Levkau B , Haude M , Schulz R , Erbel R : Coronary microembolization: from bedside to bench and back to bedside . Circulation 2009 , 120 : 1822 - 1836 .
7. Lepper W , Belcik T , Wei K , Lindner JR , Sklenar J , Kaul S : Myocardial contrast echocardiography . Circulation 2004 , 109 : 3132 - 3135 .
8. Gao H , Lang L , Guo N , Cao F , Quan Q , Hu S , Kiesewetter DO , Niu G , Chen X : PET imaging of angiogenesis after myocardial infarction/reperfusion using a one-step labeled integrin-targeted tracer 18 F-AlF-NOTA-PRGD2 . Eur J Nucl Med Mol Imaging 2012 , 39 : 683 - 692 .
9. de Waha S , Desch S , Eitel I , Fuernau G , Lurz P , Leuschner A , Grothoff M , Gutberlet M , Schuler G , Thiele H : Relationship and prognostic value of microvascular obstruction and infarct size in ST-elevation myocardial infarction as visualized by magnetic resonance imaging . Clin Res Cardiol 2012 , 101 : 487 - 495 .
10. Smith JH , Green CR , Peters NS , Rothery S , Severst NJ : Altered patterns of Gap junction distribution in ischemic heart disease. An immunohistochemical study of human myocardium using laser scanning confocal microscopy . Am J Pathol 1991 , 139 : 801 - 821 .
11. Wilkins SW , Gureyev TE , Gao D , Pogany A , Stevenson AW : Phase-contrast imaging using polychromatic hard X-rays . Nature 1996 , 384 : 335 - 338 .
12. Hwu Y , Margaritoondo G : Phase contrast: the frontier of x-ray and electron imaging . J Phys D Appl Phys 2013 , 46 : 490301 .
13. Liu Y , Nelson J , Holzner C , Andrews JC , Pianetta P : Recent advances in synchrotron-based hard x-ray phase contrast imaging . J Phys D Appl Phys 2013 , 46 : 494001 - 494013 .
14. Zhu P , Zhang K , Wang Z , Liu Y , Liu X , Wu Z , McDonald SA , Marone F , Stampanoni M : Low-dose, simple, and fast grating-based X-ray phase-contrast imaging . Proc Natl Acad Sci U S A 2010 , 107 : 13576 - 13581 .
15. Heinzer S , Krucker T , Stampanoni M , Abela R , Meyer EP , Schuler A , Schneider P , Mller R : Hierarchical microimaging for multiscale analysis of large vascular networks . NeuroImage 2006 , 32 : 626 - 636 .
16. Siew ML , Wallace MJ , Kitchen MJ , Lewis RA , Fouras A , te Pas AB , Yagi N , Uesugi K , Siu KKW , Hooper SB : Inspiration regulates the rate and temporal pattern of lung liquid clearance and lung aeration at birth . J Appl Physiol 2009 , 106 : 1888 - 1895 .
17. Wu X , Liu H : Clinical implementation of x-ray phase-contrast imaging: theoretical foundations and design considerations . Med Phys 2003 , 30 : 2169 - 2179 .
18. Kak AC , Slaney M : Principles of Computerized Tomographic Imaging. New York: IEEE Press ; 1988 .
19. Ng J , Jacobson JT , Ng JK , Gordon D , Lee DC , Carr JC , Goldberger JJ : Virtual electrophysiological study in a 3-dimensional cardiac magnetic resonance imaging model of porcine myocardial infarction . J Am Coll Cardiol 2012 , 60 : 423 - 430 .
20. Benavides-Vallve C , Corbacho D , Iglesias-Garcia O , Pelacho B , Albiasu E , Castao S , Muoz-Barrutia A , Prosper F , Ortiz-de-Solorzano C : New strategies for echocardiographic evaluation of left ventricular function in a mouse model of long-term myocardial infarction . PLoS One 2012 , 7 : e41691 .
21. Kiyooka T , Hiramatsu O , Shigeto F , Nakamoto H , Tachibana H , Yada T , Ogasawara Y , Kajiya M , Morimoto T , Morizane Y , Mohri S , Shimizu J , Ohe T , Kajiya F : Direct observation of epicardial coronary capillary hemodynamics during reactive hyperemia and during adenosine administration by intravital video microscopy . Am J Physiol Heart Circ Physiol 2005 , 288 : H1437 - H1443 .
22. Toyota E , Fujimoto K , Ogasawara Y , Kajita T , Shigeto F , Matsumoto T , Goto M , Kajiya F : Dynamic changes in three-dimensional architecture and vascular volume of transmural coronary microvasculature between diastolic- and systolic-arrested rat hearts . Circulation 2002 , 105 : 621 - 626 .
23. Mller B , Fischer J , Dietz U , Thurner PJ , Beckmann F : Blood vessel staining in the myocardium for 3D visualization down to the smallest capillaries . Nucl Instrum Methods B 2006 , 246 : 254 - 261 .