P1423AG019

BL-5

Visualization of boron distributions in cancer cells
dosed with a boron delivery drug
Takanori Wakitaa, Kazuyo Igawab, Miyu Kanedac, Naoshi Ikedac,Hiroaki Teratod, Yuji Muraokaa,c, Takayoshi Yokoyaa,ca

Research Institute for Interdisciplinary Science, Okayama University
bNeutron Therapy Research Center, Okayama University
cGraduate School of Natural Science and Technology, Okayama University
dAdvanced Science Research Center, Okayama University
Keywords: Photoemission electron microscopy, local X-ray absorption spectroscopy, elemental distributions

Boron Neutron Capture Therapy (BNCT) has attracted attention in recent years as a cancer treatment
method that aims to destroy only cancer cells without damaging normal cells. It has achieved remarkable
results in the treatments of cephalon and cervix cancers. [1]. In this method, cancer cells are destroyed by analpha-ray and a lithium ion emitted through a nuclear reaction between a boron nuclear and a thermal neutronray. In order to increase the probability of selectively destroying only cancer cells while causing as little
damage to normal cells as possible, it is essential to increase the accumulation of boron compounds on thetarget cancer cells, which will contribute to maximize the effect of BNCT with thermal neutrons of the lowest
possible intensity. Visualization of intracellular boron distribution using microscopic techniques provides
important clues for the development of boron compounds that can efficiently accumulate in cancer cells.
Electron probe micro analyzer (EPMA) has been widely used in visualization of microscopic elemental
distributions on various material surfaces as one of the most popular techniques, by means of combinationof a scanning electron microscope and an X-ray fluorescence analyzer [2]. X-ray fluorescence is produced
via relaxation of core-holes excited, in this case, by an electron beam, which is therefore element specific innature. For light elements such as boron (B), however, Auger transition rates are more than 99.9% in theircore-hole relaxation processes, which means that detection of trace light elements by X-ray fluorescence has
difficulty. In contrast, X-ray absorption spectroscopy (XAS) with total electron yield (TEY) is suitable todetect trace light elements because TEY is proportional to the amount of secondary electrons generated
mainly by Auger electrons [3]. The intensity of photoemission electron microscope (PEEM) is also
proportional to the TEY intensity when it is used with X-ray as a light source and without both an energy
filter and an energy analyzer [4]. One can obtain a local XAS spectrum by plotting intensities in a certain
area on a series of PEEM images as a function of X-ray energies. In addition, an elemental distribution image
can be obtained as a difference image of two PEEM images measured at X-ray energies of (1) a pre-edgeand (2) a peak position above a certain absorption edge.
In this study, we have performed PEEM and TEY measurements at BL5 in HiSOR for (1) a micro-droplet
of solidified L-boronophenylalanine (L-BPA), which is used as a boron delivery drug in clinical BNCTs, ona Si substrate and (2) cancer cells dosed with L-BPA, in order to investigate microscopic chemical states oftrace B atoms in them from fine structures in local- and wide-area-XAS spectra near B K-edge and tovisualize B distributions on their surfaces. For this kind of measurements with PEEM, we have developed anew auto-measurement system where we can obtain a serial PEEM images with excitation x-ray energies
for a certain energy range with a fixed energy step. The experimental station of BL5 is equipped with aPEEM III (Elmitec GmbH) and with a manipulator connected to a digital amperemeter for a TEYmeasurement. As a light source, the second-order X-rays from the monochromator were used because they
have higher intensity and energy resolution around B K-edge than the first-order X-ray from themonochromator with the same entrance and exit slit widths. All the measurements were performed at roomtemperature.
In order to elucidate the XAS signals from L-BPA, first we have performed PEEM and TEY measurements
for a micro-droplet of solidified L-BPA on a Si substrate. Figure 1 (a) shows an optical microscope image ofa micro-droplet on a Si substrate. The size of the micro-droplet is about 100 µm. Figure 1 (b) shows a PEEM
image of the same micro-droplet measured with Hg lamp showing 2 dimensional local work function
distribution for it. The field of view is 150 µm. The blue curve in figure 1 (c) shows plots of averaged
intensities per a pixel in blue square in figure 1 (b) as a function of X-ray energy around B K-edge, showing
a local XAS spectrum which has a sharp peak structure at 193.6 eV and a small bump at 191.6 eV. These
clear structures are not observed in an XAS spectrum measured by TEY (black curve in figure 1 (c)) which
provides an averaged information of the sample surface from a wide area illuminated by X-ray (the size ofX-ray is about 1 x 5 mm2 on the sample surface). These results demonstrate the effectiveness of PEEM
measurements for detecting signals from small amounts of substances on a sample surface.
Figure 2 (a) shows an optical microscope image of a colony of cancer cells dosed with L-BPA on a silicon
substrate. Figure 2 (b) shows a PEEM image of the same colony measured with X-ray of 195 eV. The fieldof view is 150 µm. The red curve in figure 2 (c) shows plots of averaged intensities per a pixel in red squarein figure 2 (b) as a function of X-ray energy around B K-edge. For the blue curve in figure 2 (c), 30 PEEM
images are summed for each X-ray energy, providing better S/N ratio of the blue curve than the one of thered curve. The small structures in this local-XAS spectrum for the cancer cell does not resemble the ones infigure 1 (c) for micro-droplet of L-BPA. This suggests that the L-BPA may be decomposed in the cancer cell.
Further measurements to obtain more better S/N ratio in a local-XAS spectrum for the cancer cells are inprogress.

FIGURE 1. (a) An optical microscope image of a micro-droplet on a Si substrate. The size of the micro-droplet is about 100µm. (b) A PEEM image of the same micro-droplet measured with Hg lamp. The field of view is 150 µm. The blue curve in (c)shows plots of averaged intensities per a pixel in blue square in figure 1 (b) as a function of X-ray energy around B K-edge.
The black curve shows an XAS spectrum measured by TEY.

FIGURE 2. (a) An optical microscope image of a colony of cancer cells dosed with L-BPA on a silicon substrate. (b) A PEEM
image of the same colony measured with X-ray of 195 eV. The field of view is 150 µm. The red curve in (c) shows plots ofaveraged intensities per a pixel in red square in figure 2 (b) as a function of X-ray energy around B K-edge. For the blue

curve in figure 2 (c), 30 PEEM images are summed for each X-ray energy.

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