Takahiro Kaji

Nondestructive micropatterning of living animal cells using focused femtosecond laser-induced impulsive force

Micropatterning of mouse NIH3T3 fibroblast cells was performed using focused femtosecond laser-induced impulsive force in a culture medium. The cells were detached from an upper substrate by the force and transferred to an underlying substrate with less than spatial resolution of 80μm full width at half maximum. About 80% of the cells were confirmed to be alive at 3h after the patterning. The force exerted to the cell was investigated by high-speed imaging and estimated to be an order of micronewtons. The force origin was not only due to cavitation bubble but also due to shockwave and jet flow.

Nanosecond to submillisecond dynamics in dye-labeled single-stranded DNA, as revealed by ensemble measurements and photon statistics at single-molecule level.

Single-molecule and ensemble time-resolved fluorescence measurements were applied for the investigation of the conformational dynamics of single-stranded DNA, ssDNA, connected with a fluorescein dye by a C6 linker, where the motions both of DNA and the C6 linker affect the geometry of the system. From the ensemble measurement of the fluorescence quenching via photoinduced electron transfer with a guanine base in the DNA sequence, three main conformations were found in aqueous solution: a conformation unaffected by the guanine base in the excited state lifetime of fluorescein, a conformation in which the fluorescence is dynamically quenched in the excited-state lifetime, and a conformation leading to rapid quenching via nonfluorescent complex. The analysis by using the parameters acquired from the ensemble measurements for interphoton time distribution histograms and FCS autocorrelations by the single-molecule measurement revealed that interconversion in these three conformations took place with two characteristic time constants of several hundreds of nanoseconds and tens of microseconds. The advantage of the combination use of the ensemble measurements with the single-molecule detections for rather complex dynamic motions is discussed by integrating the experimental results with those obtained by molecular dynamics simulation.

Controlled Spontaneous Emission of Single Molecules in a Two-Dimensional Photonic Band Gap

We have established a new platform to control the rate of spontaneous emission (SE) of organic molecules in the visible-light region using a combination of a two-dimensional (2D) photonic crystal (PC) slab made of TiO(2) and a single-molecule measurement method. The SE from single molecules of a perylenediimide derivative was effectively inhibited via a radiation field controlled by the 2D PC slab, which has a photonic band gap (PBG) for transverse-electric (TE)-polarized light. The fluorescence lifetimes of the single molecules were extended up to 5.5 times (28.6 ns) by the PBG effect. This result appears to be the first demonstration of drastic lifetime elongation for single molecules due to a PBG effect.

Fabrication of two-dimensional Ta2O5 photonic crystal slabs with ultra-low background emission toward highly sensitive fluorescence spectroscopy

A two-dimensional tantalum pentoxide (Ta2O5) photonic crystal (PC) slab with low-background emission was fabricated and a 12-fold enhancement of fluorescence from the organic dyes of perylene diimide adsorbed on the surface of the PCs was observed. The background emissions of the Ta2O5 substrates with and without the PCs after thermal annealing at 600°C with oxygen gas were comparable to that of a well-cleaned cover glass. This is to date the lowest level of background emissions of two-dimensional PCs using materials with a high refractive index (>2). The results reported here provide new insights into the fabrication of the photonic devices that enable highly sensitive fluorescence microscopy or optical detections.

Bacteriorhodopsin-based bipolar photosensor for biomimetic sensing

Bacteriorhodopsin (bR) is a promising biomaterial for several applications. Optical excitation of bR at an electrode-electrolyte interface generates differential photocurrents while an incident light is turned on and off. This unique functional response is similar to that seen in retinal neurons. The bR-based bipolar photosensor consists of the bR dip-coated thin films patterned on two ITO plates and the electrolyte solution. This bipolar photocell will function as a biomimetic photoreceptor cell. The bipolar structure, due to the photocurrent being generated in alignment with the cathodic direction, makes the excitatory and inhibitory regions possible. This scheme shows our bipolar cell can act as a basic unit of edge detection and forms the artificial visual receptive field.

Orientation of a Dip-Coated Bacteriorhodopsin Thin Film Studied by Second Harmonic Generation Interferometry

We investigated the absolute orientation of bacteriorhodopsin (bR) thin film prepared by the dip-coating technique by means of second harmonic generation (SHG) interferometry. bRs in the bR thin films prepared in this technique tend to have a preference to orient with the cytoplasmic side away from the glass substrate and extracellular side toward the glass substrate. To assist in the analysis, the hyperpolarizability of covalently bound retinal chromophore was also evaluated by a computational calculation.

Non-destructive micro-patterning of protein crystals by focused femtosecond laser

Micro-patterning of proteins has been attracted much attention as a potential technique to realize bio-microdevice. In this work, as a new method to realize non-destructive micro-patterning of proteins, laser transfer printing for a um-sized protein crystal was developed by utilizing focused femtosecond laser. The micro-patterning was performed to transfer the protein crystal which was adhered on a source substrate to a target substrate which was underlaid on the source substrate. An 800-nm femtosecond laser was focused in a water between the source and target substrates on an inverted microscope with a 100x objective lens. When the laser focal point was scanned at the position with distance of a few um far from the source substrate, the protein crystals were detached by a shockwave and cavitation bubble generation at the circumstance of the focal point and transferred to the target substrate forming a line pattern. The line width of the protein crystal was a few tens um with the scanning speed of 90 μm/sec. Furthermore, multi-patterning of several kinds of protein crystals was realized by this method. The pattering resolution is comparable or better than that by another multi-material transfer printing, such as ink jet printing, micro-printing, and laser direct writing.

Terahertz time domain and far-infrared spectroscopies of side-chain electro-optic polymers

We investigated the dielectric properties of side-chain electro-optic polymers in a broad THz frequency region (90 GHz to 7 THz). For this investigation, we used terahertz time domain spectroscopy and the absorption coefficient in a broader frequency region of up to 20 THz that was obtained by far-infrared spectroscopy. The polymers studied were a new methacrylate polymer with a high-hyperpolarizability chromophore as the sidechain, a side-chain copolymer Disperse Red 1 polymethylmethacrylate, and pure polymethylmethacrylate. The dielectric properties in the low THz frequency region (~0.1 THz) provide us with important information about the intrinsic refractive index for ultrahigh-speed electro-optic modulation (~100 GHz), as well as versatile information such as the absorption coefficient and dielectric loss. The THz and far-infrared spectroscopic data in the wide frequency region provide us with the fundamental data for applications of side-chain electro-optic polymers within THz generation and detection.

Photonic-crystal-based platform to control spontaneous emission from single molecules

We have developed a new photonic-crystal-based platform having a two-dimensional photonic band gap (PBG) for the control of spontaneous emission from single molecules and observed fluorescence-lifetime elongation of the single molecules by the PBG effect.

Development of new microscope unit for single molecule spectroscopy under various ambient conditions

This paper introduces techniques we previously developed for single molecule spectroscopy and continues on to describe our studies on dipole orientation imaging of single molecules under various ambient conditions. In these studies, we successfully obtained defocused images of single perylene diimide (PDI) molecules under air, high-vacuum, and pure N2 gas conditions by utilizing the advantages of our new microscope unit. The studies are positioned as one of the important applications of our microscope unit for single molecule spectroscopy. We expect a wide range of applications for this unit for various microscope measurements for many types of materials.