Gene delivery process in a single animal cell after femtosecond laser microinjection

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 μm was formed. The delivery process of extracellular molecules immediately after the hole formation was monitored by a fluorescence staining with fluoresceinisothiocyanate-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.     

The morphology of pulsed laser-induced damage on the surface of a copper film mirror

A copper film mirror was irradiated using a pulsed Nd:YAG laser (1.06 μm,10 ns) through an aperture. After laser irradiation, the damaged spot was analyzed using an optical microscope and periodic damage rings were found at the laser irradiation spot, the average spacing of these rings being about 40–50 μm. The damage ring could be observed in the periphery of the laser spot under lower laser energy densities. As the laser fluence increased, the periodic damage rings grew to cover the whole laser spot. Under higher laser energy density, the whole film was damaged and the rings disappeared. A Fresnel diffraction model is used to explain the appearance of these rings. The laser beam is diffracted by the aperture in the optic path, the laser intensity distribution at the copper film mirror becomes periodic rings and damage ripple pattern forms instantly with the laser ablation. The laser intensity distribution at the mirror surface is calculated using Collins formula, with which the period of the damage rings and other phenomena can be explained.     

High order harmonic generation optimization with an apertured laser beam

We report a systematic study of high order harmonic generation with an infrared laser apertured by an iris, as a function of the aperture size. Measurements were made of harmonic generation efficiency for different gas species, laser energies and focal geometries. Harmonic efficiencies as a function of aperture show a characteristic peaked shape, which is independent of gas species and harmonic order. A one dimensional, time dependent simulation of harmonic generation in a gas cell, taking into account experimentally measured transverse coherence of the laser, closely reproduces the observed behaviours. We show that the aperture diameter which maximizes harmonic yield is the result of a compromise between considerations of focal geometry and ionization (which favour small apertures); and harmonic dipole amplitude and phase (which favour large apertures). Received 31 May 2002 Published online 24 September 2002 RID="a" ID="a"e-mail: kazamias@ensta.fr RID="b" ID="b"UMR 7639 du CNRS     

In situ observation of photo-bleaching in human single living cell excited by a NIR femtosecond laser

The photo-bleaching of single living cells excited by femtosecond laser irradiation was observed in situ to study the nonlinear interaction between ultrafast laser pulses and living human breast MDA-MB-231 cells. We conducted a systematic study of the energy dependence of plasma-mediated photo-disruption of fluorescently labeled subcellular structures in the nucleus of living cells using near-infrared (NIR) femtosecond laser pulses through a numerical aperture objective lens (0.75 NA). The behavior of photo-bleached living cells with fluorescently labeled nuclei was observed for 18 h after femtosecond laser irradiation under a fluorescence microscope. The photo-bleaching of single living cells without cell disruption occurred at between 470 and 630 nJ. To study the photo-disruption of subcellular organelles in single living cells using the nonlinear absorption excited by a NIR femtosecond laser pulse, the process of photo-bleaching without photo-disruption provides key information for clarifying the nonlinear interaction between NIR ultrashort, high-intensity laser light and transparent fluorescently labeled living cells.     

脉冲激光引起铜膜镜面的环形损伤波纹研究

 用波长1.06μm、半高宽10ns的脉冲Nd:YAG激光辐照铜膜镜面，在激光辐照区，用光学显微镜观察到有规律的环形波纹状损伤图案，波纹平均周期约几十μm。通过对光路系统分析，认为样品前的小孔光阑对激光产生了菲涅尔衍射，使得在样品表面光强分布变成周期性环状分布。在极短的相互作用时间内，热扩散很小，损伤图案依赖于光强分布。并依据实验参数，用柯林斯公式对样品表面的光强分布进行了计算，所得光强分布的周期与损伤波纹的周期基本一致。     

Two-Dimensional Multi-Lens Array with Circular Aperture Spherical Lens for Flat-Top Irradiation of Inertial Confinement Fusion Target

A two-dimensional multi-lens array 350 mm in diameter with spherical element lenses has been tested to improve the irradiation uniformity of a high power glass laser for inertial confinement fusion (ICF). A circular shape for the element lens aperture is chosen to eliminate azimuthally asymmetric intensity distribution. An approximate flat-top intensity distribution is obtained with a lens array of 37 lenses. Beam patterns of a coherent laser and partially coherent light (PCL) with the random phase plate (RPP) and the multi-lens array (MLA) are measured in a GEKKO XII glass laser system. Spherical mode structure of the irradiation nonuniformity of 5.6% with MLA is estimated with cos3 θ distribution of absorption coefficient in the target plasma.     

激光细胞微手术的发展和应用

现代生命科学的发展在很大程度上依赖于对活细胞的加工和操纵,比如切除细胞内的某些特定蛋白或细胞器等.激光细胞微手术是一个强有力的工具,近年来在细胞微手术精度控制方面取得很大的进展.综述了激光细胞微手术的最新发展及其在生物学和生物医学工程中的应用,对目前激光细胞微手术中存在的主要问题进行了讨论,对比了普通激光聚焦法、飞秒激...     

Superresolution optical disk with a thermoreversible organic thin film

Recording and retrieving small marks far beyond the optical diffraction limit in a high-speed rotating phase-change optical disk have been investigated by use of a thermoreversible organic thin film as a superresolution mask layer. The organic thin film exhibited significant thermoreversibility and rapid response on laser irradiation. Recorded marks as small as 120 nm in length could be detected by a dynamic disk tester with a laser wavelength of 635 nm and a numerical aperture of 0.6.     

飞秒激光诱导有机薄膜自组装微结构

通过改变辐照激光脉冲数、激光的平均功率、显微镜物镜倍数/数值孔径等研究微皇冠结构形成与加工条件的关系,并借鉴激光熔池形成和液滴溅射的模型对其进行理论分析。当激光脉冲数达到一定数量后,烧蚀区域会出现微皇冠结构。烧蚀区域尺寸的改变是由于形成的液体区域中热毛细作用和化学毛细作用共同所致;微皇冠结构溅射个数的改变是由于形成的气体因多光子吸收体积迅速膨胀所致。     

低强度激光辐照HeLa细胞诱导增殖效应的可见光谱特性

比较研究了可见波段405,514,633和785nm低强度激光辐照HeLa细胞的促增殖效应。实验采用100和1 000J.m-2两种有效能量密度的可见光波辐照细胞,照射后24,48,72h采用MTT法检测细胞活性。结果表明,405,633,785nm激光均能促进HeLa细胞增殖,且呈波长和时间依赖性;633nm激光辐照对细胞促增殖效应最显著;514nm激光辐照对HeLa细胞促增殖效应较不明显。不同光剂量对细胞增殖效应不同,405,633,785nm激光辐照时能量密度为1 000J.m-2的辐照组细胞增殖均较100J.m-2辐照组明显,而514nm激光辐照时1 000J.m-2辐照组与100J.m-2辐照组细胞增殖的差异不明显。     

激光等离子体效应对细胞烧蚀特性研究

研究对比了激光直接辐照、聚焦辐照以及激光等离子体辐照三种辐照方式下,洋葱表皮细胞的烧蚀特征,并基于激光辐照的热力学特性对细胞的温升以及相变过程进行分析。观察发现： 直接辐照对细胞的杀伤效果很不明显;聚焦辐照会引起焦点附近细胞的断裂以及脱水;激光等离子体辐照作用下,细胞会呈现大面积的去除,断裂边缘粗糙,且细胞层有叠加现象。理论分析发现,激光等离子体具有热效应、辐射电离及冲击波效应等,会增加激光脉冲能量到细胞的沉积、以及对细胞冲击剥离等,从而会大大增加细胞的杀伤范围和效率,可用于对细胞进行大面积杀伤。     

Concept study of a 100-PW femtosecond laser based on laser ceramics doped with chromium ions

A new concept of a superpowerful femtosecond laser based on CPA in polycrystalline chromium doped ceramics (Cr:YAG, Cr:YSGG, etc.) pumped by a Nd:glass laser is proposed. In contrast to amplifiers in available petawatt laser systems (neodymium glass, titanium-doped sapphire, and parametric amplifiers with a DKDP crystal), these ceramics combine three key favorable properties: a large gain bandwidth to amplify chirped pulses with less than 20-fs durations, a wide aperture to amplify chirped pulses to the multikilojoule level, and a high conversion efficiency of narrowband Nd:glass laser pulses into chirped pulses. These properties open up the opportunity to create a unique laser with a peak power of 100 PW at a 10-kJ pump power.     

Optical aperture area determination for accurate illuminance and luminous efficacy measurements of LED lamps

The measurement uncertainty of illuminance and, consequently, luminous flux and luminous efficacy of LED lamps can be reduced with a recently introduced method based on the predictable quantum efficient detector (PQED). One of the most critical factors affecting the measurement uncertainty with the PQED method is the determination of the aperture area. This paper describes an upgrade to an optical method for direct determination of aperture area where superposition of equally spaced Gaussian laser beams is used to form a uniform irradiance distribution. In practice, this is accomplished by scanning the aperture in front of an intensity-stabilized laser beam. In the upgraded method, the aperture is attached to the PQED and the whole package is transversely scanned relative to the laser beam. This has the benefit of having identical geometry in the laser scanning of the aperture area and in the actual photometric measurement. Further, the aperture and detector assembly does not have to be dismantled for the aperture calibration. However, due to small acceptance angle of the PQED, differences between the diffraction effects of an overfilling plane wave and of a combination of Gaussian laser beams at the circular aperture need to be taken into account. A numerical calculation method for studying these effects is discussed in this paper. The calculation utilizes the Rayleigh–Sommerfeld diffraction integral, which is applied to the geometry of the PQED and the aperture. Calculation results for various aperture diameters and two different aperture-to-detector distances are presented.     

The Nike electron-beam-pumped KrF laser amplifiers

Nike is a recently completed multikilojoule krypton-fluoride (KrF) laser that has been built to study the physics of direct-drive inertial confinement fusion. The two final amplifiers of the Nike laser are both electron-beam-pumped systems. This paper describes these two amplifiers, with an emphasis on the pulsed power. The smaller of the two has a 20×20 cm aperture, and produces an output laser beam energy in excess of 100 J. This 20 cm Amplifier uses a single 12 kJ Marx generator to inject two 300 kV, 75 kA, 140 ns flat-top electron beams into opposite sides of the laser cell. The larger amplifier in Nike has a 60×60 cm aperture, and amplifies the laser beam up to 5 W. This 60 cm amplifier has two independent electron beam systems. Each system has a 170 kT Marx generator that produces a 670 kV, 540 kA, 240 ns Bat-top electron beam. Both amplifiers are complete, fully integrated into the laser, meet the Nike system requirements, and are used routinely for laser-target experiments     

Biostimulation of HeLa cells by low-intensity visible light

Summary The action of low-intensity red light with λ=633 nm (a He−Ne laser, a filament lamp with light filters a dye-laser pumped by a Cu laser) on the intensity of nucleic-acid synthesis in HeLa cells 1.5 hours after irradiation has been studied. It has been shown that the DNA synthesis is stimulated similarly after irradiation both by the He−Ne laser and by an ordinary source. The RNA synthesis intensity does not alter in both cases. A high-repetition-rate radiation at λ=633 nm acts in the opposite manner: it stimulates the RNA synthesis and the DNA synthesis remains constant. The action spectra of the DNA and RNA synthesis stimulation by continuous light in the range (570÷693) nm are presented.

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Riassunto è stata studiata l'azione della luce rossa a bassa intensità con λ=633nm (un laser a He−Ne, una lampadina a filamento con filtri per la luce, un laser a colorante pompato da un laser a Cu) sull'intensità della sintesi degli acidi nucleici in cellule HeLa un'ora e mezzo dopo irradiazione. Si è mostrato che la sintesi del DNA è stimolata in modo simile dopo irradiazione sia con laser a He−Ne che da una sorgente ordinaria. La sintesi del RNA non è alterata in entrambi i casi. Una radiazione ad alta frequenza di ripetizione a λ=633 nm agisce in maniera opposta: stimola la sintesi del RNA, mentre la sintesi del DNA resta costante. Si forniscono gli spettri d'azione della sintesi di DNA ed RNA mediante luce continua nell'intervallo da 570 a 693 nm.

     

Local stimulation of cultured myocyte cells by femtosecond laser-induced stress wave

When an 800 nm femtosecond laser is tightly focused into cell culture medium a stress wave is generated at the laser focal point. Since the stress wave localizes in a few tens of μm, it is possible to locally stimulate single cells in vitro. In this work, several kinds of cultured mammalian cells, HeLa, PC12, P19CL6, and C2C12, were stimulated by the stress wave and the cell growth after the stress loading with the laser irradiation was investigated. In comparison with the control conditions, cell growth after the laser irradiation was enhanced for the cells of C2C12 and P19CL6, which can differentiate into myocytes, and suppressed for PC12 and HeLa cell lines. These results suggest a possibility of cell growth enhancement due to myogenic cells response to the femtosecond laser-induced stress.     

红外激光对可见光成像系统的硬损伤

采用波长为1315nm的连续波化学氧碘激光对某型摄像机进行了辐照实验,研究了可见光CCD成像系统在响应波段外红外激光辐照下的硬损伤效应。开展了光学系统变光圈尺寸下的激光辐照实验,发现当辐照水平一定的情况下,光圈尺寸越小,光学系统越难发生硬损伤,并解释了该现象的成因;测量到光圈尺寸最大、最小两种状态下的光学系统硬损伤功率阈值分别为几十W、几百W;得到CCD的激光损伤阈值为5.5×104W/cm2;结合相机输出的视频图像与显微镜拍到的被损感光器件实物照片分析了硬损伤机理。     

FIR Laser Efficiency Enhancement by Double Resonance Tuning-Detailed Analysis

In a previous paper by the author (Ref. 1), FIR laser resonators with aperture coupling were shown to have resonance effect at the frequency that could be used to enhance the efficiency of the laser. This paper studies this effect over a wide range of laser parameters such as coupling aperture, cavity length and Fresnel Number. A set of curves are given to help a FIR laser designer optimize the laser.     

Implementation of a diode-pumped Nd:YAG laser with quick-change output couplers for high-beam quality 1064 or 532 nm wavelength generation

A novel diode-pumped Nd:YAG laser system that employs a fixed active laser medium and a pair of quick-change output couplers on a precision linear stage for 1064 or 532 nm wavelength generation is presented. Fixed elements include a rear mirror, an acousto-optical Q-switch, and a diode-pumped solid-state laser (DPSSL). Movable elements for 1064 nm generation include an intra-cavity aperture as a mode selection element (MSE) and an output coupler. Movable elements for 532 nm generation include an intra-cavity frequency conversion with KTP, an intra-cavity aperture as a mode selection element (MSE), and an output coupler. Under stable operating conditions, the 1064 nm configuration produced a beam propagation ratio of 1.18 whereas the 532 nm configuration produced a beam propagation ratio of 1.1, both of which used an intra-cavity MSE with an aperture of 1.2 mm and a length of 5 mm.     

Generation of 5 fs, 0.7 mJ pulses at 1 kHz through cascade filamentation

Two-cycle optical pulses with duration of 5 fs and energy of 0.7 mJ have been generated at 1 kHz by compressing the 38 fs laser pulses from a carrier-envelope phase (CEP) controlled Ti:sapphire laser system through a cascade filamentation compression technique. A simple and effective method is developed to suppress multiple filament formation and stabilize a single filament by inserting a soft aperture with an appropriate diameter into the driving laser beam prior to focusing, resulting in an excellent compressed beam quality. The good beam quality and potentially higher peak power make this ultrashort laser pulse source a significant tool for high-field physics applications.