Hank Hogan, email@example.com
When studying cells, Raman spectroscopy offers significant advantages. Because of its
inherent molecular specificity, there is no need for staining or fluorescent tagging,
so no potentially function-altering foreign materials will be introduced into cells.
But one drawback of the technique is that the
Raman cross section, upon which the signal depends, varies across a cell. The signal
from nucleic acid, for example, is weaker than that from cellular proteins or lipids,
according to University of Tokyo chemistry professor Hiro-o Hamaguchi. What’s
more, he said, the spontaneous signal is weak, reducing data acquisition rates.
In response, Hamaguchi and his associate,
Hideaki Kano, have developed a system that combines coherent anti-Stokes Raman scattering
(CARS) microspectroscopy with two-photon excitation fluorescence. The combination
allows the detection of concentration and structural changes in organelles and
saves time in obtaining an overall image of a sample. “We can investigate
detailed dynamic behavior in cellular systems,” Hamagu-chi said.
Coherent anti-Stokes Raman spectroscopy (CARS) signals of a living
yeast cell (red on left) and water (blue on left) show a strong shift difference
at 2840 cm—1, which is due to carbon-hydrogen bond stretching. The signals arise
from the black and white X’s in the image on the right, with the black X marking
the location of a mitochondrian. This signal can be used to spot organelles in the
cell. Researchers swept the spectrum and found this signal using a broadband, supercontinuum
source. Courtesy of Hideaki Kano and the University of Tokyo.
This combination was made possible
by a supercontinuum light source, a white light that covers a large swath of spectrum.
This capability is particularly important in CARS. If the technique is to obtain
full spectral information, there must be a broadband Stokes source. A wider source
frequency range results in a wider measured spectrum.
In CARS, a combination of a narrowband
pump and a broadband Stokes source forces molecules to create a vibrational coherence.
The pump source then puts the molecules into a virtual energy state, from which
they relax into a ground state while emitting an anti-Stokes Raman scattering photon.
The difference in the width of the spectral range between the narrow- and broadband
sources drives multiple vibrational coherences in the system. Being able to cover
more than a single wave number in CARS detection is key, the researchers said, to
distinguishing between concentration changes of a particular molecule and structural
As described in the April 3 issue of
Optics Express, the researchers created their supercontinuum source using
a Coherent femtosecond mode-locked Ti:sapphire oscillator operating at 800 nm
and a nonlinear photonic crystal fiber from Crystal Fibre of Birkerød, Denmark.
As with more traditional fibers, a photonic crystal fiber works by guiding light
through total internal reflection down a core that is surrounded by cladding.
In a traditional fiber, the core has a higher index of refraction than the cladding,
and both are solid. In a photonic crystal fiber, on the other hand, the cladding
is full of holes arranged in a matrix.
The effect, if the holes are small
enough, is to lower the index of refraction of the cladding, which is typically
made of silica. The air-silica material has some unusual properties. “The
photonic crystal fiber in the present study shows negative dispersion in the near-infrared,
or Stokes region,” Kano said.
That, he continued, allows the supercontinuum
to be compressed as it propagates through optics such as a microscope objective.
Given the right fiber length, researchers can deliver the supercontinuum to the
sample with well-compressed ultrashort laser pulses that can be used in two-photon
work. He noted that the photonic crystal fiber’s design, including its dispersion
property, contributes significantly to signal efficiency for multiphoton microscopy
in general. That is important because the researchers employed the source to generate
a two-photon excitation fluorescence signal.
In their setup, they used a narrow
bandpass filter to produce a pump bandwidth of about 20 cm—1. They fed 20 percent
or so of the output from the oscillator, which was diverted before the filter, into
the photonic crystal fiber. Because the fiber was nonlinear, it broadened the narrow
pulse into a supercontinuum.
Kano said that this arrangement was
much simpler than typical supercontinuum sources, which consist of a Ti:sapphire
amplifier, an optical parametric oscillator/amplifier, and two synchronized Ti:sapphire
oscillators. The Japanese supercontinuum source also yielded light across a range
of 2800 cm—1.
The researchers superimposed the pump
and Stokes pulses and sent them through a Nikon microscope into a sample. Using
an opposed objective, they collected the CARS signal and passed that through filters
before dispersing it with an Acton polychromator. They captured the resulting spectrum
using a CCD camera. With the sample mounted on a stage, they scanned across it with
a resolution of better than 0.5 μm in the X- and Y-axes and about 1.5 μm
in the Z, with an exposure time of 100 ms per location.
With this equipment, they studied living
yeast cells with a nucleus labeled by GFP. Using CARS, they spotted mitochondria,
which yielded a strong Raman signal at 2840 cm—1. Using two-photon excitation fluorescence,
they imaged the nucleus and could distinguish between stages of a yeast cell’s
life cycle. Finally, they combined the methods into a single image, providing more
highly detailed information than possible with either of the techniques alone.
Although happy with the source and
its performance, Kano indicated that the setup could be improved, particularly with
regard to the CCD camera. At present, the measurement time is set by the acquisition
time, including readout time, of the camera. “We wish we had a camera with
low noise and high-speed detection. It will reduce the measurement time significantly,”
Even with this constraint, the investigators
reported that they had visualized living cells, lipid vesicles, microcrystals and
several molecular liquids using CARS microspectroscopy. Hamaguchi noted that being
able to obtain three-dimensional images with full spectral information and in short
data-acquisition times makes the technique an excellent tool for bioimaging.
Contact: Hideaki Kano, University
of Tokyo; e-mail: firstname.lastname@example.org.