The laser microscopy is now an instrument indispensable for life science. Advances in molecular biology, organic chemistry, and materials science have recently enabled us to create several new classes of fluorescent probes for imaging in life science. Through combination with advances in laser microscopy and fluorescent probes, we are now able not only to image protein expression, localization, and activity state, but also to manipulate protein function in the living cell. Here, we first provide a brief introduction to fluorescent probes for cell imaging by laser microscopy and then describe methods for tagging of proteins with fluorescent probes. Finally, we summarize what biological phenomena we can image using fluorescent probes and laser microscopes.

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In the coming age of post-genome science, molecular imaging technology, which is used to investigate the functions and interactions of biomolecules, will play a key role in understanding biological processes. In the context of the trend of “translational research,” many approaches toward transferring the outcomes of basic research employing imaging technology to clinical diagnostics and treatments are being intensively trialed, especially in the United States. In this paper, we review microscope technologies being developed in this rising tide, including recent progress in laser technology.

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With the development of ultrashort pulse laser systems, various nonlinear optical effects have been applied to laser scanning microscopy. These microscopes have unique imaging properties that are not available in conventional optical microscopes. In this article, laser-scanning microscopes that utilize nonlinear optical phenomena, such as second-, third-harmonic generation and coherent anti-Stokes Raman scattering, are introduced. The principle and contrast mechanism of the microscopes, and their biological applications are described.

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One of the major challenges in cell physiology is revealing the molecular mechanism underlying cell functions. This paper reviews applications of near field optics and quantum dots to the cell biology. In the first part (1) imaging of single ion channels with near field illumination and in the following part (2) the process of stress fiber formation examined with quantum dots are introduced.(1) The mechanosensitive channel of large conductance (Msc L) has been examined to explore the molecular basis of the channel gating. We have developed a noble experimental setup; the Msc L is imaged with fluorescent molecular probes for FRET pair, and observed by total internal reflection microscopy.(2) The stress fiber is a long linear structure and orients along the long axis of a cell. We have examined the process of stress fiber formation by employing digitoninpermeabilized semi-intact cells and quantum dots (Qdots).

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Laser speckle microscopy was developed to observe a single living cell without any troublesome labeling preparation. The system consists of an optical microscope, a laser source, and a video camera. The technique has an advantage that ellular conditions can be instantaneously determined by observing speckle fluctuations. In the experiments, living and fixed HeLa cells were compared on the basis of speckle fluctuation and frequency spectra. By applying this technique to cell observations, it is expected to estimate not only cellular activity but also real-time movements of intercellular materials including the cytoplasm without fluorescence observation.

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Femtosecond laser pulses used as a pumping pulse in two-photon excited fluorescence microscopy were adaptively shaped by two separate self-learning schemes. First, the laser pulse was delivered through a singlemode fiber, and its pulse s ape was adaptively optimized before the fiber propagation so that its fluorescent efficiency reaches the highest level. Second, the laser pulse was delivered through a microstructure fiber, and the spectrum shape of the super-continuum generated by the microstructure fiber was adaptively optimized by shaping the input pulse so that the intensity ratio of fluorescence generated from a protein mixture becomes the highest.

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A frequency stabilized single-mode Q-switched Nd: YAG ring cavity laser was developed for compact and high-accuracy spectroscopic sensing systems, such as compact direct-detection Doppler lidars. The average output power was 3.1 W with optical conversion efficiency of 16 % at a pulse repetition frequency of 10 kHz. Injection seeding technique was used for single-mode oscillation, stabilization of the laser frequency was achieved with the feedback control using a Fabry-Perot interferometer as a frequency discriminator. High frequency stability of 0.2 MHz (rms) was realized together with a Fourier limited line width of 4.8 MHz.

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A Bessel beam is suitable for laser micro fabrication because it possesses both a small focal spot on the order of microns and a deep focal depth on the order of centimeters. In the case of laser micro grooving in stainless steel using a Bessel beam, suitable processing conditions were investigated. The results were compared with the results of micro grooving using a focused beam with a convex lens. In the grooving with the Bessel beam, it was possible to increase the groove depth while the groove width was kept almost constant. On the other hand, in the grooving with the focused beam, both the groove depth and the groove width increased when the irradiation energy was increased. A micro groove with an aspect ratio of about 5 and a width of about 2 μm was produced in a 10 μm thick stainless steel foil using a Bessel beam.

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We demonstrate all-optical switching at an extremely low operating energy using silicon photonic crystal nanocavities. The operation is based on the carrier-plasma dispersion effect induced by the two-photon absorption carriers. The switching speed is less than 200ps, which is very fast for a silicon-chip-based optical modulator. In addition to the switching operation using single pulses for modulation, we show the capability of the 5-GHz RZ (Return to Zero) pulse train modulation. Such high speed is achieved because the device is extremely small. The switches can be connected in tandem by input and output waveguides, which paves the way for the realization of integrated optical logic circuits on a single chip, based on photonic crystals.

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[in Japanese]

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