Near-field infrared imaging of molecular changes in cholesteryl oleate
by free electron laser infrared ablation
Tatsuhiro Masaki, Kazuya Goto, and Yasushi Inouye
a)
Department of Applied Physics, Osaka University, Suita, Osaka 565-0871, Japan
Satoshi Kawata
Department of Applied Physics/Handai Frontier Research Center, Osaka University, Suita,
Osaka 565-0871, Japan
͑Received 23 June 2003; accepted 1 October 2003͒
We have applied infrared near-field scanning optical microscopy ͑IR–NSOM͒ to enable evaluation
of detailed molecular changes in cholesteryl oleate, a primary cause of arteriosclerosis. In our
IR–NSOM, a wide wavelength range of 2.9–6.7
m is achieved by use of an optical parametric
amplifier and an apertured cantilever. IR irradiation from a free-electron laser ͑FEL͒ tuned to a 5.75
m wavelength induced molecular structural changes and caused cholesteryl oleate to decompose to
cholesterol and fatty acids in the FEL irradiated areas. The IR–NSOM images at two different
wavelengths, 5.75 and 5.3
m, with a 2
m apertured cantilever probe successfully identified areas
of molecular change in cholesteryl oleate beyond the diffraction limit of IR microspectroscopy.
In-depth molecular structure changes were also evaluated by the IR–NSOM and we demonstrated
that the FEL irradiation induced subsurface molecular structure changes throughout cholesteryl
oleate in the irradiated areas. © 2004 American Institute of Physics. ͓DOI: 10.1063/1.1628380͔
I. INTRODUCTION
Laser treatment with IR lasers allows selective removal
of diseased tissue parts because IR photons corresponding to
the vibrational transition energy of specific molecular bonds
are highly absorbed and the absorbed energy induces mo-
lecular structure change: dissociation of the specific molecu-
lar bonds.
1
It was recently investigated in terms of possibility
of research on treatment of arteriosclerosis and dissociation
of cholesteryl oleate, which accumulates on the vessel wall
and causes arteriosclerosis.
2–4
Awazu et al. used a free elec-
tron laser ͑FEL͒ to perform dissociation of cholesteryl oleate
to cholesterol and fatty acid by 5.75
m IR pulses ͑1.0–7.5
W/cm
2
,15
s, 10 Hz repetition rate͒. They demonstrated
dissociation of cholesteryl oleate by observation of morpho-
logic alteration in optical microscope images, along with ex-
tinction of an IR absorption peak in the Fourier-transform IR
͑FTIR͒ spectrum that conclusively showed dissociation of
the ester bonds in the cholesteryl oleate molecules. However,
they could not evaluate detailed regions where molecular
structure change was induced because the methods they used
were not applicable to evaluation of chemical change in the
microscopic region.
In this article we applied near-field IR micro-
spectroscopy
5–17
to evaluate microscopic molecular changes
in cholesteryl oleate and succeeded in distinguishing the re-
gion where conformation change was induced with super-
resolution ͑i.e., below the diffraction limit͒. A tunable IR
laser, an optical parametric amplifier with a AgGaS
2
crystal
for difference frequency generation, and apertured cantile-
vers fabricated by Si micromachining were used to create
near-field IR microspectroscopy. Near-field IR observation of
cholesteryl oleate was performed at a 5.75
m wavelength
where IR light was highly absorbed due to the ester bonds of
cholesterol. With our method, regions of cholesteryl oleate
with FEL irradiation induced conformation changes were
well distinguished at a spatial resolution of /3. Molecular
structure change inside the FEL irradiation area was also
evaluated from a comparison of transmitted intensity through
FEL irradiated areas and nonirradiated areas.
II. ABLATION OF CHOLESTERYL OLEATE
Cholesteryl oleate is formed by the binding of choles-
terol and fatty acids in the blood vessels and accumulates to
arteriosclerosis. Figure 1 shows the molecular structure of
cholesteryl oleate. We used a 2-
m-thick film of cholesteryl
oleate as a sample. The film was made of 0.02 g cholesteryl
oleate ͑C-9253, C
45
H
78
O
2
; Sigma, St. Louis, MO͒ dissolved
in 5 ml carbon tetrachloride and coated on the BaF
2
sub-
strate. After evaporation of the solvent, a microcrystalline
cholesteryl oleate film was formed. The IR absorption spec-
trum of the film was measured by a commercial FTIR ͑spec-
tral resolution: 1 cm
Ϫ1
͒, and is shown in Fig. 2. The spec-
trum shows an IR absorption peak at a wavelength of 5.75
m, which appears from ester bonds of cholesteryl oleate.
The produced film was ablated by IR laser irradiation. The
FEL at Science University of Tokyo ͑FEL–SUT͒ was used as
an IR laser source.
18
The wavelength used for ablation was
5.75
m, tuned to the absorption peak of ester bonds in
cholesterol. A pulse train from the FEL–SUT was formed by
macropulses of 1
s duration at a repetition rate of 3 Hz,
each of which includes 2856 micropulses with 1–2 ps dura-
tion. IR pulses from the FEL–SUT irradiated the sample
after being focused with a Cassegrain objective ͓numerical
aperature ͑NA͒: 0.58, magnification: 15͔ in an IR micro-
a͒
Electronic mail: ya-inoue@ap.eng.osaka-u.ac.jp
JOURNAL OF APPLIED PHYSICS VOLUME 95, NUMBER 1 1 JANUARY 2004
3340021-8979/2004/95(1)/334/5/$22.00 © 2004 American Institute of Physics