Field:
Chemistry/Biochemistry
Session Topic:
Molecular Imaging
Speaker:
Hideaki Kano/The University of Tokyo
1. Introduction
Since the first development of a microscope in the 17th century, microscopy plays an
important role not only in life sciences but also material sciences. In the 20th century,
various kinds of unique microscopic techniques have been developed such as phase
contrast microscopy, electron microscopy, scanning probe microscopy, and near-field
scanning optical microscopy. Among them, optical microscopy is one of the most
suitable methods in order to observe cells in vivo. A combination of the confocal
technique with fluorescent probes allows us to trace the dynamic behavior of
molecules in a living cell. Although confocal fluorescence microscopy is powerful and
widely used method, we need to introduce fluorescent probes, which may perturb cell
functions. The photobleaching effect is also one of the serious problems in fluorescence
microscopy. On the other hand, vibrational microscopy enables us to obtain direct
molecular images of an unstained sample, because vibrational spectra such as Raman
and infrared spectra embody many inherent and characteristic features that are
specific to a molecule. By analyzing vibrational spectra, we can identify chemical
species and elucidate in details their structure and dynamics. Since biological
materials are made up of molecules, vibrational microspectroscopy should be useful in
life sciences. Although spontaneous Raman microspectroscopy, which is widely used,
provides useful information on molecular composition and structure, spontaneous
Raman process often requires several minutes to obtain one spectrum due to the
small cross section of the Raman scattering. In order to
overcome such a difficulty and boost the signal intensity,
nonlinear Raman microscopy such as coherent
anti-Stokes Raman scattering (CARS) microscopy has
been developed [1-3].
2. CARS microscopy and microspectroscopy
CARS is one of the third-order nonlinear optical
processes. Figure 1(a) shows an energy diagram for the
CARS process. The CARS process requires two laser
sources, namely, a pump (1) and a Stokes laser (2).
When the frequency difference, 1- 2, is matched with a
partiucular vibrational resonance, , a strong and
beam-like CARS signal is launched at the frequency of
21- 2. Since the CARS signal is generated at the
frequency higher than those of the incident laser beams,
the CARS signal is not overwhelmed by the one-photon
fluorescence background. Moreover, CARS microscopy
provides super-resolution, which means that it has
three-dimensional spatial resolution beyond the
diffraction limit due to the nonlinear optical process. In
order to obtain full information on the vibrational
property, we need a spectral profile of the CARS signal. It
Figure 1 Energy diagrams
for a CARS (a) and a
multiplex
CARS
(b)
processes
can be accomplished by a
multiplex CARS process. Figure 1(b) shows an energy diagram for the multiplex
CARS process. In this scheme, we use a broadband Stokes laser. Owing to the wide
range of the frequency difference, multiple vibrational modes can be excited. Thanks
to a recent development of a photonic crystal fiber (PCF), such a broadband laser can
be generated by injecting femtosecond laser pulses into a PCF, giving rise to
supercontinuum. Owing to the ultrabroadband feature of the supercontinuum, we can
obtain not only CARS images but also CARS spectra [4,5].
3. Results and Discussion
Figure 2(a) shows a typical spectral profile
of a single pollen grain of a cherry blossom.
Three strong peaks are clearly observed at
1150, 1520, and 2850 cm-1. The former two
and the latter are assigned to carotenoid
derivatives and lipids, respectively. As shown
in Figs. 2(b-d), the CARS images at the C-C
and the C=C stretching modes are the same
due to the carotenoid derivatives. On the
other hand, the CARS image at the C-H
stretching mode gives the whole shape of the
pollen grain itself.
Figure 3 shows a CARS image of living
yeast cells at different stages of the cell
division cycle. It is clear that various yeast
cells are visualized at the C-H stretching
mode. This signal is found particularly strong
in mitochondria and septum, both of which
are made up of lipid membranes and
polysaccharides, respectively, which are
expected to give strong CARS signals.
Figure 2. (a) CARS spectrum of a single
pollen grain; CARS images of a pollen
grain at the Raman shift of the C-C (b),
C=C (c), and C-H (d) stretching modes.
4. Conclusions
Owing to its inherent molecular specificity,
CARS
microspectroscopy
provides
rich
information
on
molecular
composition,
structure and dynamics in a living cell in vivo.
Combined analysis of CARS with other
nonlinear optical processes enables us to
perform unique multi-nonlinear optical
imaging to gain deep insight into a living
system.
References
[1]
[2]
[3]
[4]
[5]
M. D. Duncan, J. Reintjes, T. J. Manuccia,
Opt. Lett., 7 (1982) 350.
A. Zumbusch, G. R. Holtom, X. S. Xie, Phys.
Rev. Lett., 82 (1999) 4142.
Figure 3. CARS image of living yeast cells
M. Hashimoto, T. Araki, S. Kawata, Opt.
at the Raman shift of the C-H stretching
Lett., 25 (2000) 1768.
mode.
T. W. Kee, M. T. Cicerone, Opt. Lett., 29
(2004) 2701.
H. Kano, H. Hamaguchi, Appl. Phys. Lett., 86 (2005) 121113.