Vol. 129 (2016)

ACTA PHYSICA POLONICA A

No. 2

Proceedings of the 50th Zakopane School of Physics, Zakopane, Poland, May 18–23, 2015

Biomedical Application of Soft X-ray Microscopy
with Special Reference to Spectromicroscopy
A. Itoa,∗ , T. Inoueb , M. Kadoc , T. Ohigashid , S. Tonee and K. ShinoharaaaSchool of Engineering, Tokai University, Hiratsuka 259-1292, Japanb
Kao Corporation, Odawara, Kanagawa 250-0002, Japanc
Japan Atomic Energy Agency, Kizugawa 619-0215, Japand
UVSOR Facility, Institute for Molecular Science, Okazaki 444-8585, Japane
Department of Biochemistry, Kawasaki Medical School, Kurashiki 701-0192, Japan
Soft X-ray microscopy has great advantages over other types of microscopy on the observation of hydrated
specimens at high spatial resolution below 100 nm, and on the elemental and molecular mapping using absorption characteristics. These advantages are particularly suitable for biomedical applications. A possible problem
associated with observation of specimens at high spatial resolution in a hydrated environment is described and astrategy to overcome the problem is presented. The latter part of the present report deals with spectromicroscopyto obtain molecular distribution in biological specimens. DNA and protein distributions in isolated nuclei are
examined following to the progression of apoptosis at high resolution, and the oxidation product is visualized inhuman hair in relation to calcium content.
DOI: 10.12693/APhysPolA.129.260
PACS: 87.59.–e

1. Introduction
The potential advantages of soft X-ray microscopy
with X-ray energies below several keV [1] in biology overconventional microscopy such as light and electron microscopy can be summarized as follows: (1) High spatialresolution imaging in the natural physiological state, particularly using X-rays with photon energies in the “waterwindow” region between K absorption edges of carbon(284 eV) and oxygen (544 eV), (2) capability of computed tomography (CT) imaging to observe thick specimens, and (3) mapping of elements and molecules using
spectromicroscopy for characteristic elements or specificmolecules.
Comparing with hard X-ray microscopy which is being actively developed due to its large penetration depth
covering tissues, soft X-ray microscopy has remarkablefeatures including achievement of higher spatial resolution at absorption contrast better than 50 nm or evendown to 10 nm. The absorption contrast gives direct information about electron density and is easy to interpret
without any image reconstruction processes definitely required for phase contrast imaging. Thus soft X-ray microscopy would be suitable for high resolution observation of a whole cell or intracellular molecular assembly ina thickness of a few µm.
On the observation of elemental and molecular map,
soft X-ray spectromicroscopy has still several advantages
over electron microscopy. Electron-probe microanalysis∗ corresponding author; e-mail:aeito@keyaki.cc.u-tokai.ac.jp(EPMA) or electron energy loss spectroscopy (EELS) forsuch purposes causes much more radiation damage on
the specimens due to the presence of optically forbidden
transition and the production of secondary electrons. Energy resolution in chemical mapping is also advantageous
for soft X-ray spectromicroscopy. The energy resolutionof EELS is around 20 eV, while in the case of the soft Xray spectromicroscopy the energy resolution below 1 eVis achievable depending on a monochromator.
2. Observation of hydrated specimens
In order to achieve high spatial resolution in water environment, it is necessary to avoid image blurring caused
by Brownian motion. We estimated the degree of blurring for freely diffusible particles as a function of imaging
time [2]. This estimate is an extreme case and may occasionally be overestimated, but we could know the upper
limit for the required imaging time. According to ourcalculation, if we need 50 nm in spatial resolution for example, imaging should be completed within 0.5 ms. In
addition, we have to consider X-ray dose to achieve a required spatial resolution. Sayre et al. proposed a method
to estimate radiation dose as a function of required spatial resolution [2]. We applied their method to a model
nucleoprotein, a complex of DNA and its binding protein
histone, with a thickness of 1 µm, which is surroundedby medium of water containing protein with a ratio of
9 to 1. Under this condition, we calculated photon fluence in the order of 1013 photons/cm2 to achieve 100 nm
spatial resolution. Incidentally, in the same situation theabsorbed dose required for electron microscope was estimated about 102 times larger than X-ray microscope [3].
To meet these requirements, laser plasma X-rays produced with highly intense laser pulse have been applied(260)

Biomedical Application of Soft X-ray Microscopy. . .
to capture X-ray image of hydrated specimens in a very
short time. A thin-foiled gold target has been irradiatedwith an intense pulsed laser with 20 J energy in 600 ps
pulse duration to produce laser plasma. The photon fluxof the laser-plasma X-ray source in the water window(2.3–4.4 nm) has been measured to be 1.4 × 1015 photons/pulse/sr and it makes possible to capture a soft Xray image of live hydrated biological cells with a single
pulse exposure.

261

using the difference in X-ray absorption near edge structure (XANES) profiles between DNA and protein at theC K absorption edge [6]. The same methodology was successfully applied to DNA distribution in a bull sperm [7].
We proposed that the use of XANES at the N K absorption edge would be better for the mapping of DNA due
to the presence of a unique π∗ peak [8]. Using newly
built STXM installed at UVSOR in Japan [9], we haveobtained the N K edge spectra and examined DNA andprotein distribution in cell nuclei during the progressionof apoptosis. Apoptosis is well known as a programmed
cell death and is characterized by remarkable morphological changes in a cell nucleus. Tone et al. has extensively
studied the progression of the morphological changes bylabelling DNA with fluorescent dye for isolated nuclei using optical microscope, and proposed that the changes ina nucleus proceeded via three stages [10]. We intended
to follow the process with a spatial resolution around
100 nm.

Fig. 1. Soft X-ray images of live hydrated Leydig cellsof mouse testis taken with laser plasma X-rays.

Figure 1 shows a recent observation of live mammaliancells, which was achieved below 100 nm spatial resolution
at the single exposure with time duration of 600 ps using
a contact microscopy. In Fig. 1, intracellular structures
such as chromatin, mitochondria and actin filaments
could be clearly identified [4]. Interestingly dense whiteparticle-like structures were identified as oil droplets relating to steroid hormone. Alternative to it, a rapid freezing method frequently used to maintain hydrated structure could be suitable for CT imaging. Such effort has
been continued at a beamline dedicated for soft X-ray
microscopy, e.g. NCXT at ALS [5].
3. Spectromicroscopy for biomedical specimens
X-ray spectromicroscopy is a powerful imaging tool
to obtain molecular distribution in biological specimens.
The scanning transmission X-ray microscope (STXM)
has been frequently used for this purpose. The illustrated
system is inserted in Fig. 2, i.e., specimen is scanned on a
monochromatic X-rays focused by a zone plate and transmitted X-rays are detected. The pioneering work by Adeet al. was conducted for DNA mapping in a chromosome

Fig. 2. Relative distribution of DNA and protein indried apoptotic nucleus in the “stage 1”. (a) Horizontal
line profile of DNA distribution in part (b), (b) DNA
distribution, (c) protein distribution, (d) horizontal line
profile of protein distribution in part (c). Insert figure
shows typical optical layout of STXM. Scale bar corresponds to 1 µm.

Although the research is in progress, the preliminary
results for the relative distributions of DNA and nuclear
protein such as histone in dried nucleus at stage 1 areshown in Fig. 2. DNA and protein were found to be preferentially located in the peripheral region leading to theformation of a ring structure. The horizontal line profiles
across the nucleus (part (a) and (d) in Fig. 2) indicated262

A. Ito et al.

Fig. 3. Chemical structures of sulfur containing aminoacids and their XANES at the S K absorption edge. (a)Chemical structures of cystine and its oxidation product, cysteic acid. (b) XANES of cystine and cysteic
acid. Imaging energies (a) to (d) indicated by arrows
were used to obtain maps of cystine and cysteic acid bya subtraction method.

that DNA content was nearly constant with slight increase in the peripheral region, while protein was moreaccumulated in the peripheral region, which suggests thatDNA may be present without histone in the inner region.
The meaning of this observation is left for future research.
Molecular images of all stages in the process of apoptosis are referred to the proceedings of the International
Conference on X-Ray Microscopy in 2014 [11].
Another application is an imaging of oxidation stateof cystine in human hair, which was conducted at relatively high X-ray energy region near hard X-rays including the S K edge (2.47 keV) and the Ca K edge
(4.04 keV). Human hair always undergoes various outeroxidative stimuli such as bleach treatment and exposure
to ultraviolet radiation. In order to provide the efficient
protection of hair against these oxidative stresses, the information about the distribution of oxidative damage isdefinitely useful. Human hair is primarily composed ofkeratin protein characterized by high content of sulfurcontaining amino acid, cystine. Exposure to oxidative
stress induces cysteic acid, an oxidative form of cystine(Fig. 3a). Since these two forms of amino acids havedifferent XANES peaks at the S K absorption edge asshown in Fig. 3b [12], we could obtain the distributionof cysteic acid (oxidation map) by the subtraction of images taken at the energy (c) and (d) [13]. For a cystine
map, the subtraction between energy (a) and (b) was performed. The images were taken by a contact X-ray microscopy with a high spatial resolution two-dimensional
detector, an electronic zooming tube at a spatial resolution of about 0.5 µm over the energies used at the Photon
Factory, Japan.
Figure 4 indicates the influence of bleach treatment at
a beauty parlor on the oxidation in female hair. Hair
without any bleach treatment has little oxidation as seenfrom the cysteic acid map, while with increasing times of
bleach treatment the oxidation was evidently increased
over the whole area of the hair. The right column pictures show Ca distribution. Accumulation of calcium

Fig. 4. Distributions of oxidation product and Ca in
human hair with bleach treatment at a beauty parlor.
Dried cross-sectionally sliced thin section with a thickness of about 20 µm of hair was observed by an X-ray
contact microscopy. Cystine and cysteic acid maps wereshown for hair specimens treated with different degreeof bleaching. Ca maps are displayed for comparison.
Scale bar corresponds to 20 µm.

has been reported to have a close correlation to oxidative damage [14], and recent interesting observations suggested the possible correlation between Ca content inhair and the incidence of breast cancer [15]. Based onthese preceding observations we measured the localization of Ca to compare with an oxidation map at highspatial resolution. The calcium map was obtained at theCa K edge using XANES profiles of Ca compounds. Figure 4 shows distributions of cystine, cysteic acid and Ca
to compare bleached hair with normal unbleached hair.
Cystine maps reflect the thickness of hair specimens, thatis, the specimen with no treatment has largest thickness
compared with other specimens. Considering the thickness of specimens, increasing bleach treatment increased
Ca content as well as oxidation in the regions of cuticle,
the peripheral part of hair, and cortex, the inner part ofhair, which was in accord with the previous reports except for the central structure called medulla. Ca content
in medulla is not likely to depend on the degree of oxidation. For example, Ca content was significant even in untreated hair. The Ca distributions were also confirmed by
X-ray fluorescence mapping using scanning microbeam
X-rays at the Photon Factory (data not shown). Theexperiments using hair specimens with bleach treatment
in our laboratory give similar results [16]. These findings indicate that in determining the Ca contents in hair
the detailed distribution of calcium inside hair should betaken into account.
The present results demonstrated the potential of thesoft X-ray spectromicroscopy to provide a high spatial
resolution map of a specific molecule which cannot be ob-Biomedical Application of Soft X-ray Microscopy. . .
tained by other imaging technique. However, note thatthese experiments have been done with dry specimens.
The method should be extended to the observation inhydrated specimens using rapid freezing method for theadvancement of soft X-ray spectromicroscopy in biomedical applications.
Acknowledgments
The work was performed at the Photon Factory under the application number 2008G536, 2010G542, and2012G626 and also a part of this work was conductedin UVSOR, supported by Nanotechnology Platform Program (Molecule and Material Synthesis) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This work was partly supported by the Japan Society for the Promotion of Science(JSPS) KAKENHI, Grant-in-Aid for Scientific Research(C) (25390128).
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