Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

11A/2004G391

Ca distribution in a human HeLa cell examined using XANES peak at the Ca-L
absorption edge
Atsushi ITO*1, Yohei OHNO1, Toshiyuki AKANUMA1, Kunio SHINOHARA2
1
School of Engineering, Tokai Univ., Hiratsuka, Kanagawa 259-1292, Japan
2
JASRI/SPring-8, Sayo-gun, Hyogo 679-5198, Japan
Introduction
XANES of biological specimens provides a unique andsensitive method for elemental imaging using resonanceabsorption peaks with the aid of soft X-ray microscopy.
Ca has been one of major target elements because of its
sharp and prominent XANES peaks at the L absorption
edge[1] and biomedical significance of its distribution intissues and cells. Several studies on the Ca distributions
in calcified tissues have been conducted with scanning Xray microscope at NSLS[1-4], but Ca mapping in a single
mammalian cell has been scarcely reported.
In the present study, we applied contact soft X-ray
microscopy to obtain Ca distribution in a human HeLa
cell by calculating a ratio image between images at thepeak and the bottom energy in XANES at the Ca-L
absorption edge. The results suggested that Ca distributes
predominantly in the nuclear region.
Materials and Methods
Contact X-ray microscopy using an electronic zooming
tube was used to obtain X-ray images of human cancer
HeLa cells treated with critical point drying at the Ca-L
absorption edge at the resolution about 0.5 µm[5]. BL11A beamline was used for this energy region.
XANES of CaCl2 was measured to determine peak andbottom energies for taking X-ray images from 345 eV to365 eV. CaCl2 solution was placed on collodion thin film,
which was followed by air-drying.
Results and Discussion
XANES of CaCl2 at the Ca-L absorption edge wasshown by dotted line in Fig. 1. Characteristic two peaks at351.5 eV and 358.5 eV were observed (arrows “b”, “d”).
0.2

1.5
Nucleus (A)0.19

Absorption

1.45

(a)

(b)

Fig. 2. Soft X-ray image of HeLa cells and Ca distribution.
(a) X-ray image at 349.5 eV; (b) Ca image obtained bytaking the ratio between images at 351.5 eV and 349.5 eV.

To confirm that the dense part is Ca rich region,
absorption spectra of micro-areas with 0.645 µm square
were measured, which were shown by solid lines in Fig. 1.
The locations of two areas, “A” and “B” are from Fig. 2(a). The spectra exhibited a peak at 351.5 eV, but showedslight (position “B”) or no peak (position “A”) at theenergy of 358.5 eV indicated by arrow “d”. Since Ca
content in a cell is expected to be much smaller than thatin calcified tissues, further repeated measurement isrequired to improve the accuracy of the intracellular
localization of Ca.

0.18
Nucleus (B)1.4
ab0.17

dce

0.16

1.35
0.15
CaCl2
1.3345350355360

0.14365

Photon energy (eV)Fig. 1. XANES of nuclear micro-areas in a HeLa cell at theCa-L edge. XANES of CaCl2 is shown with dotted line.

232

To obtain Ca image, the ratio between an image at351.5 eV denoted by arrow “b” and that at 349.5 eV
denoted by arrow “a” was calculated as shown in thepanel (b) of Fig. 2. The denser area has more Ca content.
For identification of Ca distributed area soft X-ray imageof cells at 349.5 eV was shown in Fig. 2 (a). Comparisonof both images indicates that Ca seems to distributearound the nuclear region preferentially compared withcytoplasm.

Users' Report

References[1] C. J. Buckley, Rev. Sci. Instrum., 66, 1318 (1995).
[2] J. M. Kenny et al., J. Microsc., 138, 321-328 (1985).
[3] C. J. Buckley et al., SPIE, 1741, 363-372 (1992).
[4] C. J. Buckley et al., In “X-ray Microscopy IV”, (ed.
by V. V. Aristov and A. I. Erko), pp. 207-212, Moscow:
Bogorodskii Pechatnik Publishing Co., (1994).
[5] K. Shinohara et al., In “X-Ray Microscopy andSpectromicroscopy”, (ed. by J. Thieme et al.), pp. I169~I-173, Heidelberg: Springer, (1998).
* aeito@keyaki.cc.u-tokai.ac.jp