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Name
Email
Telephone
Professor INOUYE, Yasushi, Ph.D. +81-6-6879-4243
Associate Prof. VERMA, Prabhat, Ph.D. +81-6-6879-7847
Postdoctoral Fellow TANAKA, Shin-ichi, Ph. D. +81-6-6879-4617

FAX +81-6-6879-7330
Postal Mail Address Department of Frontier Biosciences, Graduate School of Frontier Biosciences, c/o Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 Japan



Researches on Nanobiophotonics:

We carry out research on Nano-biophotonics, a new research field which combines Nanotechnology, Biology and Photonics. Super-resolving capability and ultra high sensitivity for observing/sensing biomolecules and DNA can be achieved by using novel nanotechnologies such as scanning probe techniques, various types of optical spectroscopy and localized surface plasmonics. We are evolving these techniques to create new biological applications.

Up to this point, we have succeeded in;

(1) development of nano-imaging system using near field optical microscope,
(2) construction of high-speed scanning Raman scattering microscope,
(3) observation of intracellular molecular dynamics using surface enhanced Raman scattering,

As nanotechnology and photonics have not been extensively applied yet in biology, we are excited by the possibilities of this developing research field.

1 Near-field optical nano-imaging of biomolecules

Molecules, proteins and organelles composing living cells have nanoscale dimensions. Conventional optical microscopy cannot spatially resolve these small constituents because light cannot be focused into a volume smaller than the wavelength of light due to diffraction. This limitation of spatial resolution is called “diffraction limit”. We have been developing optical imaging techniques with nanometric spatial resolution beyond the diffraction limit since our first proposal in 1994. [1] Our technique uses a metallic probe with a nanosized tip. When irradiated by light, free electrons in the metal collectively oscillate and generate strong optical field in the vicinity of the tip surface. The dimension of the localized field is comparable to the tip, i.e. nanometer scale. Scanning this nano-light spot on the sample surface realizes nanometer-resolution optical imaging. This imaging concept and technique is called near field optical microscopy. Figure 1a shows a fluorescence image of a semiconductor (GeSe) nanoparticle. Here, we achieved 40-nm spatial resolution. [2] In Fig. 1b, a DNA network nano-structure is visualized with a spatial resolution of 15-nm, in which tip-enhanced Raman scattering signal from adenine molecules is detected for molecular vibration imaging. [3] Raman spectroscopy gives information about molecular conformation and species without staining the target molecules by fluorescent dyes. This feature enables us to realize in situ observation of biomolecules in biological environments. Therefore, near field optical microscopy is the only tool that realizes optical imaging with nanometric spatial resolution, and would be a very useful tool in life-science research in the future.
figure1
Figure 1: High resolution images by near field optical microscopy. (a) A fluorescence image of a semiconductor nanoparticle (CdSe quantum dot). (b) A Raman image of a DNA network nanostructure.

[1] Y. Inouye, et al., Opt. Lett. 19, 159 (1994).; N. Hayazawa, et al., Opt. Commun. 183, 333 (2000).; T. Yano, et al., Appl. Phys. Lett. 88, 093125 (2006). etc.
[2] P. Verma, et al., submitted.
[3] T. Ichimura, et al., Phys. Rev. Lett. 92, 220801 (2004).

2 Development of high resolution near field optical microscopy based on metal-molecule interaction

We have previously reported the fundamental difference in spectral shape between Raman spectra enhanced by a metal tip and (unenhanced) normal Raman spectra for the first time in the world. [4] This difference is caused by (1) chemical interaction of the metal tip with sample molecules, in which the molecular orbital is perturbed by the presence of the metal, and (2) mechanical interaction, in which sample molecules are pressurized and deformed by the tip. Figure 2 explains and demonstrates the change in Raman spectrum of adenine. The Raman peak at 723 cm-1, which is assigned to the ring-breathing mode, is shifted to higher frequency when the tip approaches and presses a molecule. Since this effect takes place on molecules directly adjacent to the tip metal, we can realize optical imaging with a spatial resolution of atomic force microscopy by selectively detecting the shifted peak, that is, the signal from the deformed molecules. We are taking efforts to develop novel imaging systems based on the concept of chemical and mechanical interaction.

figure2
Figure 2: Spectral change due to the metal-molecule interaction. (a) Schematic illustration of the metal-molecule interaction under a silver tip. (b) Experimental spectra of the tip enhanced Raman spectrum and normal Raman spectrum.

[4] H. Watanabe, et al., Phys. Rev. B 69, 155418 (2004).; T. Yano, et al., Nano Lett. 6, 6, 1269 (2006). etc.

3 Construction of Raman microscope and observation of cellular dynamicss

The Raman spectrum makes the identification of individual molecule. By measuring the Raman spectrum of an individual molecule, we can investigate biomolecular dynamics in a physiological environment without labeling. However, since the cross section of Raman scattering is extremely low, imaging needs prolonged observation. In order to improve the scanning rate, we developed the slit scanning Raman microscope [5], and succeeded in observing cellular dynamics. Fig. 3 shows the time lapse Raman images of HeLa cells during cell division. The acquisition time of one image was about 3 minutes. Hence, the time resolution was dramatically improved and scanning rate was 100 times faster, compared with conventional methods that scan a focus point in two dimensions. This figure was made by assigning RGB channels to the distributions of cytochrome c (751 cm-1), protein (Amide-I of β-sheet, 1680cm -1), and lipid (CH2 bond, 2852cm -1) in cells during cell division.

figure3
Figure 3: Time lapse Raman images of HeLa cells during cell division

[5] K. Hamada et. al., J. Biomed. Opt. in press.

4 Surface enhanced Raman imaging of live cell using metal nano-particle

We developed a high sensitivity/resolution Raman microscopy by measuring SERS (surface enhanced Raman scattering) signals. SERS is due to the enhancement of electromagnetic field generated by surface plasmon resonance in the vicinity of gold nanoparticles (NPs). We measured Raman spectra from macrophage and HeLa cells in which gold NPs (φ50nm) were introduced via endocytosis, and Raman images were constructed by using these spectra. Consequently, we successfully measured the SERS spectra of biological molecules in the vicinity of gold NPs. This technique will allow us to measure biomolecular dynamics and chemical reactions in endocytosis.

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