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CARS microspectroscopy combined with two-photon fluorescence

Jun 2006
Hank Hogan,

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 changes.

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,” he said.

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:

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