6 Imaging
Interface-Sensitive Micro-Imaging Based on X-ray Reflectivity
It is important to understand interfaces since they are omnipresent in nature and control the properties of modernmaterials. Recently, the authors have succeeded in realizing X-ray reflectivity imaging of heterogeneous interfaces inultrathin films. To visualize a wide viewing area, the image reconstruction scheme is employed instead of micro-beamscanning. The technique achieves image contrast by using the difference in reflectivity at each in-plane point in the thinfilm sample. X-ray reflectivity profiles at local positions on the sample can be obtained without using a micro beam, as
equivalent information can be extracted from a series of wide X-ray reflection projection datasets collected as a functionof grazing angle.
The authors have developed the X-ray reflectivity
imaging technique [1-4]. A schematic of the experimental setup at BL-14B is shown in Fig. 1. In the experiment, the X-rays were monochromated to 16 keV by adouble-crystal Si 111 monochromator having an energy
resolution of 10−4. The monochromatic X-rays were collimated to form a thin parallel beam. The primary collimating slit was set at 22.5 m from the wiggler source,
to collimate the beam to 1 mm (horizontal, H) × 8 mm(vertical, V). The incident X-ray intensity was monitored
throughout the experiment by an ionization chamber(D0) set 0.45 m behind the primary slit. In front of theentrance window of D0, a fixed width (100 mm, H) slit
was attached to further cut the beam horizontally; thus,
the final size of the incident beam was 0.10 mm (H) ×8 mm (V). The sample stage, which was set at 0.45 m
downstream from D0, is based on a high-precision goniometer with an accuracy of 0.001º. A rotational motor
is vertically attached to an L-shaped stand fixed on thegoniometer to realize in-plane rotation (-axis in Fig. 1).
The samples were vertically mounted using a sample
holder. The sample holder was equipped with two
manual tilt stages. The parallel beam illuminated around
10 mm [H, the footprint length of the X-rays, is always

long enough to cover the silicon substrate size (10 mm)]
× 8 mm (V) of the sample surface at grazing-incidencegeometry. The reflected X-rays were recorded by an Xray CCD camera (pixel size 6.45 µm) set 0.30 m on thedownstream side of the sample as a one-dimensional
projection image, where the imaging conditions are in
the near-field regime.
An X-ray reflectivity imaging example of buried
heterogeneous interfaces is shown in Fig. 2. Here, apatterned ultrathin film was studied by the imaging approach. The sample was fabricated on a pre-cleaned
silicon substrate by an Eiko DID-5A magnetron sputtering system. Under the top uniform Ti layer the heterogeneous layer consists of two groups of thin films,
as shown in panel a: (i) Au thin films including the topleft polygon and bottom-right rectangle with different
thicknesses; (ii) Ni thin films consisting of the bottomleft thick rectangle, top-right triangle and center-rightthin bar with different thicknesses. Panel b is an X-ray
reflectivity sinogram of the sample at a specific incidence angle corresponding to the wavevector transfer
Qz = 4 sin  /  = 0.0502 Å−1 (  : the grazing incidence
angle in Fig. 1,  : X-ray wavelength), where obvious
sinusoidal patterns are observed.

Figure 1: Schematic of the X-ray reflectivity imaging experimental set-up. VW: BL-14 vertical wiggler; M: monochromator; SL: 2D slit;
D0: ionization chamber, with a horizontal slit (0.05 mm, H); SS: sample stage on a goniometer; D: X-ray CCD camera. Inset is the enlarged
image of SS from the top view: TSs: tilting stages. Reprinted, with permission, from reference [1].

64

HIGHLIGHTS

Figure 2: Typical X-ray reflectivity imaging data. (a) An optical image of a heterogeneous patterned (Au and Ni) ultrathin film sample before
coating with a Ti uniform layer. (b) An X-ray reflectivity sinogram of the sample at Qz = 0.0502 Å−1. (c) A reconstructed X-ray reflectivity image of the sample at Qz = 0.0502 Å−1. (d) Three selected µXR profiles at local positions [as indicated by the same symbols in panel (c)] of thesample extracted from reconstructed X-ray reflectivity images. Reprinted, with permission, from reference [2].

The 1338 pixels are equally binned into 90 pixels(pixel length: 96 µm, 96 µm × 90 = 8.6 mm). The algebraic imaging reconstruction approach is employed toquantitatively reconstruct the X-ray reflectivity image.
Panel c presents the corresponding reconstructed X-ray
reflectivity image of the sample at Qz = 0.0502 Å−1. Although the sample is buried by the top Ti layer, the Au
and Ni patterns are clearly observed by X-ray reflectivityimaging. At this wavevector transfer, the Au patterns
produce higher reflectivity than the Ni patterns, thus giving a contrast between the patterns of the two different
materials.
By scanning grazing angles, a series of X-ray reflectivity images sampled equally over a range of wavevector transfers is collected, thus µXR, which is the X-rayreflectivity profile at every micro-sized pixel, can be extracted. Panel d shows three selected µXR profiles. The
pixel [40, 10] contains only a uniform Ti layer. The µXR
profile confirms this point by displaying a sharp drop at
Qz = 0.042 Å−1 and equal-period interference fringes.
At pixels [40, 80] and [75, 35], the µXR has an intensity
drop around Qz = 0.042 Å−1 and shallow oscillations
below Qz = 0.080 Å−1. Beyond Qz = 0.080 Å−1, the µXR
profile drops sharply and experiences deep oscillations.

The two µXR profiles of Au patterns show different oscillation periods beyond Qz = 0.080 Å−1, indicating thedifferent thicknesses of the patterns.
In the present research, X-ray reflectivity imaging
was achieved by combining -scan and measurementof the 1D intensity profile of reflection projection. It has
been demonstrated that the technique yields interfacesensitive images of ultrathin film samples. The newtechnique enables the non-destructive study of interfaces.

REFERENCES[1] J. Jiang, K. Hirano and K. Sakurai, J. Appl. Phys. 120, 115301(2016).
[2] J. Jiang, K. Hirano and K. Sakurai, J. Appl. Crystallogr. 50,712 (2017) .
[3] K. Sakurai and J. Jiang, J. Surf. Sci. Soc. Japan. (2017) (inJapanese language, in press).
[4] J. Jiang and K. Sakurai, Rev. Sci. Instrum. 87, 93709 (2016).

BEAMLINE
BL-14BJ. Jiang1, 2, K. Hirano3 and K. Sakurai2, 1 (1Univ. of Tsukuba,NIMS, 3KEK-IMSS-PF)

2HIGHLIGHTS

65

6 Imaging
A Novel Strategy to Reveal Latent Liver Abnormalities in HumanEmbryonic Stages from a Large Embryo Collection
The cause of spontaneous abortion of normal conceptuses remains unknown in most embryos because of the difficultyof diagnosing too small embryos. We aimed to reveal latent liver abnormalities using novel phase-contrast radiographic
computed tomography (PXCT). Embryos with liver volumes ≥ 2 SD above or below the mean for the stage of development were screened from 1156 MR images from the Kyoto Collection. Selected embryos were further analyzed by using PXCT. Liver abnormality was detected in 9 embryos by our protocol. Most of such liver abnormality embryos do notsurvive, as liver function becomes essential.
The cause of spontaneous abortion of normal conceptuses remains unknown in most embryos [1] because of the difficulty of obtaining appropriate embryo
materials as well as diagnosing internal abnormalities
in very small embryos. The crown-rump length of anembryo is ranging 23 to 32 mm during the embryonicperiod by Carnegie stage (CS) 23 (about 56–60 daysafter fertilization) [2], by which stage all primordia of theorgans are already provided.
Many factors are involved in spontaneous abortion,
not only the embryonic factor itself but also maternal
health and the intrauterine environment affected by unspecified modulators. Such complicated factors distract
attention from the laborious inspection of embryos, especially when embryos are externally normal.
More than 40,000 embryo and fetal specimens havebeen collected in the Kyoto Collection since 1961 forrevealing the mechanism of congenital anomalies [3].
An MRI database of approximately 1200 well-preserved
human embryos, diagnosed as externally normal, wasacquired in 2000–2005 (the Kyoto embryo visualizingproject) [4] to observe a number of normal human embryos. The database has proven to be useful for research purposes and as a teaching material [5].
Although the MRI database includes only externallynormal embryo specimens, whether the internal organs
were also normal cannot be guaranteed. Consideringthat the cumulative intrauterine mortality rate in normal
conceptuses was estimated at 18% [1], the MRI database includes embryos that have potential abnormalities that would have led to spontaneous abortion. Thus,
detailed observation of the internal organs of embryo
specimens from the database may provide clues tospontaneous abortion in the embryonic and fetal periods. In this connection, we aimed to determine the latent abnormalities that may cause spontaneous abortionby using the MRI database and novel phase-contrast
radiographic computed tomography (PXCT).
The MRI database was screened by using the volume of the liver as the target organ. Embryos with liver66

HIGHLIGHTS

volumes ≥ 2 SD above or below the mean for the stageof development were selected. Embryos with potentiallyabnormal livers were further analyzed by using PXCT.
The PXCT data provide a resolution of ≥ 18 µm/pixel,
which enabled highly sensitive measurement, approximately > 1000 times more sensitive than the conventional radiographic method using absorption contrast [6].
Liver abnormality was detected in 9 embryos after
the procedure of our protocol (Fig. 1), which consistedof hepatic agenesis (2 embryos), hepatic hypogenesis(4), liver lobe defect (1), involvement of the liver to thethoracic cavity by diaphragm herniation (1), and other(1). Three embryos had only liver abnormalities and6 exhibited complications in other organs. The prevalence of liver malformations in CS18 and CS21 in theintrauterine population of externally normal embryos
is approximately 1.7%. Most of such liver abnormality
embryos do not survive, as liver function becomes essential during development [7].

Figure 1: Protocol for detecting latent abnormal embryos that may
result in spontaneous abortion.

Figure 2: Representative embryos with liver abnormality (liver agenesis).
a External view of the embryo at CS21, which shows no obvious external abnormality. b Phase-contrast X-ray computed tomography (PXCT)transverse sections at the level of Th4. No liver (Li) was detected in the plane sections, while the stomach (St) was observed in the midsagittaltransverse sections at Th4. The diaphragm is indicated by arrow heads. Lung (Lu). c Left anterior oblique view of the three-dimensional (3-D)
PXCT reconstruction of the embryo using Amira software (Visage Imaging, Berlin, Germany), demonstrating the locations of all intrathoracic,retroperitoneal, and intra-abdominal organs.
The findings of note were as follows: agenesis of the liver; the stomach was deviated ventrally and cranially; the pancreas (Pa) was deviatedventrally; and the right mesonephros (Ms) and genital ridge (Ge) were absent. Adrenal gland (Ad), metanephros (Mt), and heart (He).

A representative embryo with liver agenesis isshown in Fig. 2. The size and gestational age werewithin the normal range for the embryo at CS 21. Obvious damage to, or anomalies of, the external forms
were not present (Fig. 2a). The liver at CS21 usually occupies a large space in the abdominal cavity, which has
a smooth surface due to the contact between the cranial
surface and the diaphragm, and between the ventral
surface and the abdominal wall [7]. In the present embryos, the liver was not detected in any of the serial
plane sections (Fig. 2b). The locations of all intrathoracic, retroperitoneal, and intra-abdominal organs werereconstructed in three dimensions (Fig. 2c). The absence of the liver had affected the locations of the otherinternal organs, especially the stomach, duodenum, andpancreas. The stomach was observed on the midsagittal line in the Th4 transverse sections, indicating thatthe stomach had deviated cranially and ventrally. The
diaphragm was apparent in these sections.
The present study demonstrates that PXCT may beconsidered a powerful tool for visualization of internal
structures of embryos, and for the detection of novel
abnormalities during the embryonic period, without theneed for histological analysis. The non-invasive andnon-destructive properties of the technique are important for analysis of scarce specimens, such as humanembryos. The present study is the first step toward elucidating the latent abnormalities that result in spontaneous abortion in externally normal embryos [8].

REFERENCES[1] K. Shiota, Congenital Anomalies 31, 67 (1991).
[2] R. O’Rahilly and F. Müller, Developmental stages in humanembryos: including a revision of Streeter’s” horizons” and asurvey of the Carnegie Collection, (Carnegie Institution ofWashington Publishing. 1987).
[3] H. Nishimura, K. Takano, T. Tanimura and M. Yasuda,Teratology 1, 281 (1968).
[4] K. Shiota, S. Yamada, T. Nakatsu-Komatsu, C. Uwabe,
K. Kose, Y. Matsuda, T. Haishi, S. Mizuta and T. Matsuda,
Am J Med Genet A 143A, 3121 (2007).
[5] T. Takakuwa, Antat Rec (2017), in press.
[6] A. Yoneyama, S. Yamada and T. Takeda, Fine Biomedical
Imaging Using X-Ray Phase-Sensitive Technique, eds. G. D.
Gargiulo and A. McEwan, Advanced Biomedical Engineering
107 (2011).
[7] A. Hirose, T. Nakashima, S. Yamada, C. Uwabe, K. Kose andT. Takakuwa, Anat Rec 295, 51 (2012).
[8] T. Kanahashi, S. Yamada, M. Tanaka, A. Hirose, C. Uwabe,
K. Kose, A. Yoneyama, T. Takeda and T. Takakuwa, Anat
Rec 299, 8 (2016).

BEAMLINE
BL-14C

T. Takakuwa (Kyoto Univ.)

HIGHLIGHTS

67