Browsing by Author "Hosokawa, Yoichiroh"

Now showing 1 - 14 of 14

Gene delivery process in a single animal cell after femtosecond laser microinjection
(2009-09-30)
Hosokawa, Yoichiroh
;
Iguchi, Seriya
;
Yasukuni, Ryohei
;
Hiraki, Yuji
;
Shukunami, Chisa
Microinjection of extracellular molecules into a single animal cell was performed by an amplified femtosecond laser irradiation. When a single-shot laser pulse was focused on the plasma membrane of a single fibroblast from the mouse cell line NIH3T3 with a high-numerical aperture objective lens, a transient hole with a diameter of 1 mm was formed. The delivery process of extracellular molecules immediately after the hole formation was monitored by a fluorescence staining with fluoresceini-sothiocyanate-dextran (FITC-dextran). Then the gene expression was confirmed using a DNA plasmid of an enhanced green fluorescent protein (EGFP). The gene expression was observed when the laser pulse was focused first on the cellular membrane and then on the nuclear membrane, while the gene was not expressed when the laser was focused only on the cellular membrane. On the basis of these results, the efficiency of gene delivery by the femtosecond laser microinjection and the subsequent gene expression were clarified. (C) 2009 Elsevier B.V. All rights reserved.
In Situ Dynamic Control of Neurite Growth by Femtosecond Laser Ablation of Substrate Patterns
(2016-01-01)
Okano, Kazunori
;
Liu, Lin-Li
;
Hosokawa, Yoichiroh
;
Masuhara, Hiroshi
;
應用化學系
We are developing a methodology to construct a neuron network on a culture substrate for studying neuronal functions on signal processing and memory. Here we show a method to guide neurite growth on a substrate by using femtosecond laser ablation of polymer layer. The laser was used to control the affinity of the surface to neurons and neurites. We succeeded in neurite growth with directionality.
In situ laser micropatterning of proteins for dynamically arranging living cells
(2013)
Okano, Kazunori
;
Matsui, Ai
;
Maezawa, Yasuyo
;
Hee, Ping-Yu
;
Matsubara, Mie
This study shows the modification of the surface of polymer-layered glass substrates to form biofunctional micropatterns through femtosecond laser ablation in an aqueous solution. Domains of micrometer size on a substrate can be selectively converted from proteinphobic (resistant to protein adsorption) to proteinphilic, allowing patterning of protein features under physiological aqueous conditions. When femtosecond laser pulses (800 nm, 1 kHz, 200-500 nJ per pulse) were focused on and scanned on the substrate, which was glass covered with the proteinphobic polymer 2-methacryloyloxyethylphosphorylcholine (MPC), the surface became proteinphilic. Surface analysis by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) reveals that the laser ablates the MPC polymer. Extracellular matrix (ECM) proteins were bound to the laser-ablated surface by physisorption. Since femtosecond laser ablation is induced under physiological aqueous conditions, this approach can form micropatterns of functional ECM proteins with minimal damage. This method was applied to pattern collagen, laminin, and gelatin on the substrate. Removal of an ECM protein from the substrate followed by replacement with another ECM protein was achieved on demand at a specific location and time by the same laser ablation method. Living cells adhered to the fabricated domains where ECM proteins were arranged. The modification of patterning during cell culture was used to control cell migration and form arrays of different cells.
In situ observation of cell-detachment process initiated by femtosecond laser-induced stress wave
(2010-10-01)
Maezawa, Yasuyo
;
Hosokawa, Yoichiroh
;
Okano, Kazunori
;
Matsubara, Mie
;
Masuhara, Hiroshi
When a stress wave generated by focusing a femtosecond laser is loaded on an animal cell adhered on a substrate, the cell is detached from the substrate. There are two possible mechanisms for the cell detachment: (a) The cell is detached from a scaffold coated on a glass plate, and (b) the cell is detached from the glass plate with the scaffold. In this work, we have studied the cell-detachment mechanism by visualizing the scaffold with a fluorescence probe of quantum dots. When the cell was detached from the substrate, fluorescence from the scaffold simultaneously disappeared from the glass plate, although the scaffold was not irradiated by the laser. This indicates that detachment due to the stress wave is attributed to mechanism (a). On the other hand, when the cell was detached from the substrate by a trypsin treatment, the fluorescence from the scaffold remained, suggesting mechanism (b). By comparing both results, it is considered that physiological damage of the cell membrane during the detachment process by femtosecond laser-induced stress wave is less than that due to the trypsin treatment.
In situ observation of cell-detachment process initiated by femtosecond laser-induced stress wave
(2010-10-01)
Maezawa, Yasuyo
;
Hosokawa, Yoichiroh
;
Okano, Kazunori
;
Matsubara, Mie
;
Masuhara, Hiroshi
When a stress wave generated by focusing a femtosecond laser is loaded on an animal cell adhered on a substrate, the cell is detached from the substrate. There are two possible mechanisms for the cell detachment: (a) The cell is detached from a scaffold coated on a glass plate, and (b) the cell is detached from the glass plate with the scaffold. In this work, we have studied the cell-detachment mechanism by visualizing the scaffold with a fluorescence probe of quantum dots. When the cell was detached from the substrate, fluorescence from the scaffold simultaneously disappeared from the glass plate, although the scaffold was not irradiated by the laser. This indicates that detachment due to the stress wave is attributed to mechanism (a). On the other hand, when the cell was detached from the substrate by a trypsin treatment, the fluorescence from the scaffold remained, suggesting mechanism (b). By comparing both results, it is considered that physiological damage of the cell membrane during the detachment process by femtosecond laser-induced stress wave is less than that due to the trypsin treatment.
Induction of Cell-Cell Connections by Using in situ Laser Lithography on a Perfluoroalkyl-Coated Cultivation Platform
(2011-03-21)
Okano, Kazunori
;
Yu, David
;
Matsui, Ai
;
Maezawa, Yasuyo
;
Hosokawa, Yoichiroh
This article describes a novel laser-directed microfabrication method carried out in aqueous solution for the organization of cell networks on a platform. A femtosecond (fs) laser was applied to a platform culturing PC12, HeLa, or normal human astrocyte (NHA) cells to manipulate them and to facilitate mutual connections. By applying an fs-laser-induced impulsive force, cells were detached from their original location on the plate, and translocated onto microfabricated cell-adhesive domains that were surrounded with a cell-repellent perfluoroalkyl (R(f)) polymer. Then the fs-laser pulse-train was applied to the Rf polymer surface to modify the cell-repellent surface, and to make cell-adhesive channels of several mm in width between each cell-adhesive domain. PC12 cells elongated along the channels and made contact with others cells. HeLa and NHA cells also migrated along the channels and connected to the other cells. Surface analysis by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) confirmed that the Rf polymer was partially decomposed. The method presented here could contribute not only to the study of developing networks of neuronal, glial, and capillary cells, but also to the quantitative analysis of nerve function.
Laser microfabrication and rotation of ship-in-a-bottle optical rotators
(2008-08-04)
Matsuo, Shigeki
;
Kiyama, Satoshi
;
Shichijo, Yoshinori
;
Tomita, Takuro
;
Hashimoto, Shuichi
We have fabricated optical rotators inside a silica substrate and rotated them by a laser trapping technique. The fabrication method used was femtosecond laser-assisted etching, i.e., modification of the host material by irradiation with femtosecond laser pulses along a predesigned pattern, followed by selective chemical etching. The rotators, which consist of the same material as the substrate, can move inside the microcavity but cannot get out. The rotation speed was proportional to the trapping laser power, and the maximum achieved was about 100 rpm. Such rotators will be applicable to micro-total-analysis systems and microfluidics. (c) 2008 American Institute of Physics.
Laser-Assisted Control of Protein Adsorption for Dynamically Arranging Viable Cells
(2013)
Okano, Kazunori
;
Matsui, Ai
;
Maezawa, Yasuyo
;
Matsubara, Mie
;
Hosokawa, Yoichiroh
Polymers and proteins covering glass surface were ablated by femtosecond laser pulses under physiological aqueous condition. The prepared micropatterns of proteins were confirmed by fluorescence imaging and their cell adhesion characteristics was demonstrated.
Micro-channel fabrication by femtosecond laser to arrange neuronal cells on multi-electrode arrays
(2010-11-01)
Hosokawa, Chie
;
Kudoh, Suguru N.
;
Suzuki, Mariko
;
Kiyohara, Ai
;
Hosokawa, Yoichiroh
We propose and demonstrate micro-channel fabrication by femtosecond-laser processing to arrange neuronal cells on multi-electrode arrays (MEAs). When neuronal cells were seeded in the micro-channels on MEAs, they elongated their neurites along the channel and finally formed the network patterned on MEAs. The spontaneous electrical activity of the neuronal networks on MEAs was evaluated by using electrophysiological systems, indicating that patterned network was functionally activated along the channels. The present method is applicable to analyze network dynamics of living neuronal cells patterned artificially.
Micropatterning of perfluoroalkyl self-assembled monolayers for arraying proteins and cells on chips
(2009-06-15)
Kira, Atsushi
;
Okano, Kazunori
;
Hosokawa, Yoichiroh
;
Naito, Akira
;
Fuwa, Koh
Organosilane self-assembled monolayers (SAMs) with perfluoroalkyl groups (R(f)) on glass surfaces were used for arraying proteins and cells on chips. Quartz crystal microbalance measurements confirmed the inhibition of protein adsorption on R(f)-SAM-modified surfaces and showed efficient adsorption on hydroxyl-, carboxyl-, and amino group-modified surfaces. The characteristics of R(f)-modified surfaces were evaluated using solvent contact angle measurement and Fourier transform infrared (FTIR) spectroscopy. The R(f) surface was highly water-and oil-resistant, as inferred from the contact angles of water, oleic acid, and hexadecane. Specific peaks of IR spectra were detected in the region from 1160 to 1360 cm(-1). Etching with dry plasma completely exfoliated the R(f)-SAM, exposing the underlying intact glass surface. Modi. cation conditions were optimized using contact angle and FTIR measurements. After dry plasma processing, the contact angles of all solvents became undetectable, and the IR peaks disappeared. Micrometer scale protein and cell patterns can be fabricated using the proposed method. Protein adsorption on micropatterned R(f)-SAM-modified chips was evaluated using fluorescence analysis; protein adsorption was easily controlled by patterning R(f)-SAM. PC12 and HeLa cells grew well on micropatterned R(f)-SAM-modified chips. Micropatterning of R(f)-SAM by dry plasma treatment with photolithography is useful for the spatial arrangement of proteins and cells. (C) 2009 Elsevier B. V. All rights reserved.
Morphological evaluation of cell differentiation after the isolation of single cells by a femtosecond laser-induced impulsive force
(2011-02-01)
Maezawa, Yasuyo
;
Okano, Kazunori
;
Matsubara, Mie
;
Masuhara, Hiroshi
;
Hosokawa, Yoichiroh
When nerve growth factor (NGF) is interacted with PC12 cells derived from rat pheochromocytoma, they are partially differentiated into neuron-like cells with neurites. In this work, PC12 cells differentiated by NGF were selectively isolated using a localized impulsive force in a mu m-scale area, which was generated by focusing an infrared femtosecond laser into a cell culture medium. In order to evaluate the ability of the isolation method, differentiated and undifferentiated cells were isolated and their morphological changes after the isolation were compared. In both cases, their neurites were once contracted and some of them gradually regenerated day by day. When differentiated cells were isolated, the percentage of differentiated cells with regenerated neurites, 6 h after the isolation, was about 3.3 times higher than that when undifferentiated ones were isolated. This result was compared with a control trypsin experiment. In the comparison, it was indicated that the same degree of cell function was maintained when the present isolation method was used.
SECONDARY CONVERGENCE IN FEMTOSECOND LASER TRAPPING
(2010-06-30)
Jiang, Yuqiang
;
Ma, Cungen
;
Oh, Isamu
;
Hosokawa, Yoichiroh
;
Masuhara, Hiroshi
When femtosecond laser pulses pass through a trapped polystyrene bead, water breakdown is induced even though the energy of laser pulse is much lower compared to the threshold value of breakdown when the femtosecond laser directly irradiates in water. This mechanism is assigned to the secondary convergence of the laser by the trapped bead.
Single femtosecond laser pulse-single crystal formation of glycine at the solution surface
(2013-03-01)
Liu, Tsung-Han
;
Uwada, Takayuki
;
Sugiyama, Teruki
;
Usman, Anwar
;
Hosokawa, Yoichiroh
We demonstrate femtosecond laser-induced crystallization of glycine from its supersaturated solution depending on laser tunable parameters (pulse energy and repetition rate) and focal position, and examine the crystallization probability, crystal morphology, and crystal polymorph. The generation of cavitation bubble through multiphoton absorption of water depends on input laser pulse energy and repetition rate, which strongly determine morphology and number of the obtained crystals. Significant increase in the crystallization probability is observed by irradiating the femtosecond laser pulses to the air/solution interface, and single pulse-induced single crystal formation is successfully achieved. The crystallization mechanism is discussed in view of inhomogeneous mechanical stress induced by cavitation bubble generation and molecular assembly characteristics of the surface. (C) 2012 Elsevier B.V. All rights reserved.
Spatially Precise, Soft Microseeding of Single Protein Crystals by Femtosecond Laser Ablation
(2012-09-01)
Yoshikawa, Hiroshi Y.
;
Hosokawa, Yoichiroh
;
Murai, Ryota
;
Sazaki, Gen
;
Kitatani, Tomoya
We developed a spatially precise, soft microseeding method for the production of single protein crystals that are suitable for X-ray crystallographic studies. We used focused femtosecond laser pulses to produce, via multiphoton absorption processes, seed crystals from small regions (similar to 1 mu m(2)) of crystals. Hen egg-white lysozyme seed crystals, produced in this manner, grew to be single crystals without any deterioration in their crystallinity. We also validated the technique using polycrystals for the membrane protein, acriflavine resistance protein B, for which single crystals are very difficult to obtain. In addition, we found that the shape of a tetragonal lysozyme crystal prepared from the seed could be controlled by altering the time interval between the initiation of crystallization and laser ablation. We also tried to comprehend the mechanism of femtosecond laser-induced microseeding. We visualized the ablated surfaces of the lysozyme crystals by atomic force microscopy and by laser confocal microscopy combined with differential interference microscopy. The results obtained in this study clearly demonstrate that femtosecond laser ablation of protein crystals is based on a photomechanical process, which ejects crystal fragments with little thermal damage. Femtosecond laser ablation is indeed very promising to produce high quality protein seed crystals from polycrystals or cracked crystals that are not suitable for X-ray diffraction studies.