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We used environmental scanning electron microscopy (ESEM) and energy-dispersive X-ray (EDX) microanalysis to study
the microstructure topography and elemental composition of a cross section of a giant human cardiac calculus after cutting
it in half. The giant cardiac calculus was isolated from an 80-year-old male patient after invasive open heart surgery. After
treatment with formalin, the yellow-brown fist-shaped calcareous wet mass, 50 mm × 45 mm × 30 mm in size, became a
brown-yellow hard calculus and was surrounded by a thick white tissue layer. The dissection of the whole cardiac calculus
into two halves clearly revealed a dense layer on the encrusted surface and a bread-like texture of the internal structure of
the calculus. The cross-sectional ESEM micrograph of the cardiac calculus illustrated that different agglomerates were
deposited on the uneven petrous surface. The EDX spectra and elemental chemical mapping revealed that C and O were
the major elements on the surface of the different irregular, near-spherical agglomerates with trace amounts of N, Na, P,
and Ca, whereas the petrous surface showed higher amounts of C, O, P, and Ca, as well as trace amounts of N and Na.
These elemental compositions were consistent with the surface microstructure analysis results, which showed that the
agglomerates were mainly composed of cholesterol, and the petrous surface was mainly composed of calcium
hydroxyapatite, as confirmed by our previous Raman spectroscopic detection. This study indicates that ESEM–EDX
microanalysis may directly provide useful information about microstructural characteristics and the spatial distribution of
elements on a cross-sectional surface of a halved cardiac calculus.
Keywords: cardiac calculus; ESEM; EDX; microanalysis; morphology; element; mapping; cholesterol; hydroxyapatite
1. Introduction
Scanning electron microscopy (SEM) is a well-known, powerful imaging research tool and has superior performance
for visualizing the surface topography and composition of a sample. As conventional SEM requires samples to be
imaged under high-vacuum conditions, one of the limitations of SEM is that it cannot be applied to wet materials and
biological samples. To examine the natural state of wet samples via imaging, Danilatos first introduced the
environmental scanning electron microscope (ESEM), which is capable of imaging non-conductive materials in their
natural state under low-vacuum conditions [1-3]. ESEM allows the direct examination of hydrated or dried specimens
without a conductive metallic coating. The application of ESEM could have advantages in the study of biological and
biomedical specimens due to a reduction in sample preparation time [4-6]. Donald has introduced and discussed the
ESEM technique and its key advantages [7].
Energy-dispersive X-ray (EDX) spectroscopy is an analytical technique used for elemental analysis or chemical
characterization of a sample. It is also used for chemical composition microanalysis in conjunction with advanced SEM
to provide high-resolution imaging, semi-quantitative elemental analysis, and qualitative X-ray elemental maps for
investigating the architecture and composition of different samples [8-9]. Over the past decade, ESEM equipped with
EDX has been extensively used to rapidly and accurately examine the morphology and elemental chemical composition
of wet materials [10-12]. ESEM–EDX has several advantages over other sample analysis methods, including (a) direct
measurement of the concentration of major elements (>0.1 wt.%) in samples in situ; (b) measurement of any available
cations and anions above the atomic weight of Be; (c) determination of opaque minerals; and (d) rapid sample change
over and the fast acquisition of analytical spectra [13]. ESEM–EDX provides a potentially useful complement to ESEM
and not only gives a useful microscopic picture of the sample surface but also combines this information with an
overview of the elemental composition of the sample surface.
Calcification in biological systems is a complex pathological phenomenon in which insoluble compounds or salts are
deposited in different soft tissues [14-15]. These insoluble salt deposits gradually accumulate and aggregate into
clusters, which cause a loss of flexibility and pliability in soft tissues, resulting in sclerosis, thickening, and/or
deterioration [16-17]. Abnormal or inappropriate biomineralization in soft tissues is a vital structural or functional
factor that causes many chronic diseases due to inflammation caused by mineral crystals [18-19]. In fact, almost all the
tissues, including the brain, eye, teeth, salivary gland, heart, breast, liver, gall bladder, vessels, urinary tract, skin,
muscle, joints, and other parts of the body, are easily calcified [14-15]. It has been reported that the prevalence and
incidence of tissue calcification have been increasing worldwide in recent years [20-21].
Lithiasis is a very important medical issue because many adult populations suffer from different stone diseases that
may be asymptomatic or symptomatic [20-22]. Cardiovascular calcification commonly occurs in the aortic valve of the
heart or in the coronary arteries [23-25]. However, there is rare evidence for calculus formation in the human cardiac
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cavity. Recently, we reported a giant cardiac calculus that was isolated from an 80-year-old male patient after invasive
open heart surgery. This calculus had occupied the left atrium of his heart and had possibly been present for a long
duration. Analysis with a portable fibre-optic Raman analyzer revealed that localized deposits of cholesterol, calcium
hydroxyapatite, and protein were heterogeneously distributed on the surface of the whole cardiac calculus and the cross-
sectional surface of the halved cardiac calculus [26-27]. In this study, the ultrastructural topography and elemental
composition of the cross-sectional surface of the halved cardiac calculus were investigated by ESEM–EDX
microanalysis to thoroughly visualize and examine its microstructural features.
3. Results
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A B
Fig. 1 Morphological features of the whole (A) and the halved (B) human cardiac calculus.
A B
50 μm 10 μm
10 μm
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4. Discussion
Pathological calcification is the abnormal tissue deposition of calcium salts, together with smaller amounts of different
elements and other mineral salts in a wide variety of disease states, particularly in aging populations [14-17, 29]. In the
Fig. 3 The surface topographies of cardiac calculus (A, B) along with the qualitative
results of the elemental mapping (C, D) and the EDX spectra of two selected locations.
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5. Conclusions
A giant, fist-shaped cardiac calculus was isolated from an older male patient after invasive open heart surgery. ESEM–
EDX microanalysis was successfully used to examine the surface topographic features and elemental chemical
composition of the cross section of the giant cardiac calculus. Numerous irregular, near-spherical agglomerates that
appeared on the petrous surface of the halved cardiac calculus were clearly observed by ESEM. In addition, ESEM–
EDX analysis showed that these agglomerates contained major amounts of the elements C and O, with trace amounts of
N, P, and Ca, and that higher amounts of the elements C, O, Ca, and P were markedly present on the petrous surface.
Acknowledgements This study was partially supported by research grants from Taiwan Association of Cardiac Vascular Surgery
Research, Taipei, Taiwan, ROC and National Science Council, Taipei, Taiwan, ROC.
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