纳米尺度激光紧聚焦光穿孔技术

论述了一种新型的细胞转导方法——纳米尺度激光紧聚焦光穿孔技术.按照光穿孔可能的物理机制,分4类讨论了现在主流的光穿孔技术:连续激光光穿孔,其中最前沿的技术是紫外二极管激光光穿孔;纳秒、皮秒激光脉冲和经放大的千赫兹飞秒激光脉冲光穿孔,其中应用范围最广的是Nd:YAG激光器;飞秒激光振荡器发出的兆赫兹脉冲序列光穿孔,该技术在多尺度、跨学科及在体研究中扮演重要角色;最新出现的利用双脉冲微射流进行光穿孔,这是光穿孔技术中仅有的一种将外源物主动引入细胞内的方法.可以预见:当激光紧聚焦技术结合了双光子荧光显微镜、微芯片实验室、膜片钳技术等各种单细胞分析技术后,必将在细胞间通信、干细胞谱系提交、癌症转移、激光基因疗法、胚胎发育学以及神经功能再生等学科领域中产生重大影响.     

Adaptive subwavelength control of nano-optical fields

Adaptive shaping of the phase and amplitude of femtosecond laser pulses has been developed into an efficient tool for the directed manipulation of interference phenomena, thus providing coherent control over various quantum-mechanical systems. Temporal resolution in the femtosecond or even attosecond range has been demonstrated, but spatial resolution is limited by diffraction to approximately half the wavelength of the light field (that is, several hundred nanometres). Theory has indicated that the spatial limitation to coherent control can be overcome with the illumination of nanostructures: the spatial near-field distribution was shown to depend on the linear chirp of an irradiating laser pulse. An extension of this idea to adaptive control, combining multiparameter pulse shaping with a learning algorithm, demonstrated the generation of user-specified optical near-field distributions in an optimal and flexible fashion. Shaping of the polarization of the laser pulse provides a particularly efficient and versatile nano-optical manipulation method. Here we demonstrate the feasibility of this concept experimentally, by tailoring the optical near field in the vicinity of silver nanostructures through adaptive polarization shaping of femtosecond laser pulses and then probing the lateral field distribution by two-photon photoemission electron microscopy. In this combination of adaptive control and nano-optics, we achieve subwavelength dynamic localization of electromagnetic intensity on the nanometre scale and thus overcome the spatial restrictions of conventional optics. This experimental realization of theoretical suggestions opens a number of perspectives in coherent control, nano-optics, nonlinear spectroscopy, and other research fields in which optical investigations are carried out with spatial or temporal resolution.     

大鼠背根神经元中Ca2+的激光共聚焦显微成像与定量分析

成功实现体外培养背根神经元(Dorsal Root Ganglion,DRG),利用差速贴壁法进行提纯,通过细胞免疫荧光技术鉴定所培养的细胞纯度,发现其纯度高达90%,完全适合运用于细胞分子机理研究.在此基础上,采用激光共聚焦显微成像实验观察DRG内的Ca2+荧光信号,并对Ca2+浓度进行了定量分析.研究结果有助于深入探究和定量分析神经元中Ca2+参与特定的细胞生理功能.     

Brain-wide neuronal dynamics during motor adaptation in zebrafish

A fundamental question in neuroscience is how entire neural circuits generate behaviour and adapt it to changes in sensory feedback. Here we use two-photon calcium imaging to record the activity of large populations of neurons at the cellular level, throughout the brain of larval zebrafish expressing a genetically encoded calcium sensor, while the paralysed animals interact fictively with a virtual environment and rapidly adapt their motor output to changes in visual feedback. We decompose the network dynamics involved in adaptive locomotion into four types of neuronal response properties, and provide anatomical maps of the corresponding sites. A subset of these signals occurred during behavioural adjustments and are candidates for the functional elements that drive motor learning. Lesions to the inferior olive indicate a specific functional role for olivocerebellar circuitry in adaptive locomotion. This study enables the analysis of brain-wide dynamics at single-cell resolution during behaviour.     

飞秒脉冲激光产生及捕获微气泡的实验研究

采用高速光学摄像及高频超声成像技术对飞秒脉冲激光在介质水中发生光学诱导击穿的过程进行实验研究.结果表明,光学诱导击穿产生后,在高能激光自聚焦的焦点处产生一系列微气泡,最后只有一个微气泡可以被激光束稳定地捕获,并且这个微气泡可以在超声作用下进行非接触式三维操控.进一步分析了沿激光束产生和捕获的微气泡的时空特性以及激光束捕获微气泡的力学特性.实验结果为应用微气泡进行分子水平的靶向性治疗提供了全新的技术手段.     

Editor’s note

The past two decades have witnessed great progress in development of ultrashort laser pulse in laser science, often at an unexpected speed. It has found various applications, both theoretical and practical, in the frontiers of science.     

利用双温模型理论分析飞秒激光烧蚀金过程

为了了解飞秒激光脉冲烧蚀金过程中的电子温度和晶格温度变化。利用显式有限差分法对飞秒激光脉冲烧蚀金的过程中电子和晶格的温度场进行一维数值计算。理论研究了不同激光脉宽和电声耦合系数对金属表层电子和晶格温度的影响,同时还研究了双脉冲激光烧蚀金过程中电子和晶格温度的变化。得出激光脉冲越短,加工热效应越小,多脉冲具有热累积效应。因此,为了实现飞秒激光冷加工,应该采用单个超短脉冲降低热效应。     

基于纳飞秒双脉冲激光的蓝宝石蚀除研究

蓝宝石作为一种典型的宽带隙半导体材料具有良好的理化特性,在机械电子、航空航天、微电子等领域具有广阔的应用前景.本文提出采用纳飞秒双束激光进行蓝宝石加工的方法,利用飞秒脉冲峰值功率高的特性,通过多光子电离与碰撞电离,激发蓝宝石表面自由电子从而提高了蓝宝石对纳秒激光的吸收率,同时利用纳秒激光能量相对较大,技术成熟的优势,实现对蓝宝石材料的高质高效加工.为了揭示纳飞秒双束激光与蓝宝石材料的能量耦合机制,采用Fokker-Plank方程、Drude模型、双温模型对飞秒激光诱发的自由电子的演化过程及自由电子与入射激光之间的相互作用进行研究,并通过有限元仿真模拟了纳飞秒双激光与蓝宝石材料相互作用的能量耦合过程与蚀除过程,分析了纳飞秒双脉冲激光作用下,不同脉冲延时下等离子浓度的时空演化规律及其对入射激光吸收系数及反射率等表面光学特性的影响规律,揭示了纳飞秒双激光高效高质加工蓝宝石材料的机理.该研究对实现蓝宝石等宽带隙半导体等脆硬材料的高质高效加工具有重要意义.     

Modulation of synchronous calcium oscillations in hippocampal neurons by photostimulation of astrocytes with femtosecond laser

A large body of evidence indicates that astrocytes play an important role in a range of brain functions through calcium (Ca²⁺) signaling. Experimentally evoking Ca²⁺ signaling is a useful technique for investigating the functions of astrocytes. However, conventional stimulation methods typically have poor spatio-temporal precision, and some are invasive. Our group has developed a technique to overcome these problems, in which astrocytes are photostimulated with a femtosecond laser. In the current study, we applied this method to a hippocampal neural network to explore astrocytic functions in detail. The results revealed that applying photostimulation to astrocytes in a cultured hippocampal astrocyte-neuron network caused the following changes: (i) Synchronous Ca²⁺ oscillations in neurons were induced; (ii) spontaneous Ca²⁺ synchrony instantaneously emerged; and (iii) high-frequency spontaneous Ca²⁺ synchrony was regulated. Thus, astrocytic Ca²⁺ signaling evoked by photostimulation was found to modulate synchronous Ca²⁺ oscillations in hippocampal neurons. We propose that photostimulation with a femtosecond laser will serve as a powerful tool in investigating astrocytic functions at the network level.     

钛宝石飞秒激光系统及其应用

目前最为成熟的飞秒激光系统是基于啁啾脉冲放大技术（CPA）的太瓦级钛宝石飞秒激光系统。本文以东莞理工学院光电子技术实验室的钛宝石飞秒激光系统为例,主要介绍飞秒激光系统的组成和一些主流应用,为飞秒激光系统的搭建与商业化提供了可参考的路线和方向。     

Experience with moving visual stimuli drives the early development of cortical direction selectivity

The onset of vision occurs when neural circuits in the visual cortex are immature, lacking both the full complement of connections and the response selectivity that defines functional maturity. Direction-selective responses are particularly vulnerable to the effects of early visual deprivation, but it remains unclear how stimulus-driven neural activity guides the emergence of cortical direction selectivity. Here we report observations from a motion training protocol that allowed us to monitor the impact of experience on the development of direction-selective responses in visually naive ferrets. Using intrinsic signal imaging techniques, we found that training with a single axis of motion induced the rapid emergence of direction columns that were confined to cortical regions preferentially activated by the training stimulus. Using two-photon calcium imaging techniques, we found that single neurons in visually naive animals exhibited weak directional biases and lacked the strong local coherence in the spatial organization of direction preference that was evident in mature animals. Training with a moving stimulus, but not with a flashed stimulus, strengthened the direction-selective responses of individual neurons and preferentially reversed the direction biases of neurons that deviated from their neighbours. Both effects contributed to an increase in local coherence. We conclude that early experience with moving visual stimuli drives the rapid emergence of direction-selective responses in the visual cortex.     

超衍射相位板设计

对超衍射理论进行了研究和分析,通过利用优化算法设计了对应于横向、纵向,特别是三维超衍射的3种二元相位元件;模拟结果表明在飞秒激光微加工系统中,考虑双光子效应,这种相位元件的超衍射性能尤为理想.讨论了光源的光谱范围对相位元件超衍射效果的影响,结果表明当光谱较窄时,超衍射性能几乎与使用单波长光源时一样.所设计的超衍射相位元件可以应用于飞秒激光加工系统.     

高阶孤子飞秒激光脉冲的成形机制

根据实验上普遍观察到的10fs左右激光脉冲的脉冲分裂现象，以高阶孤子特征分析了飞秒激光器中克尔透镜锁模脉冲的类孤子成形机制，以至进一步了解克尔透镜锁模脉冲成形机制。     

Significant effects of branches and bromine substitution of near-infrared two-photon photosensitizers on the generation of singlet oxygen

This letter reports the influence of branches and bromine substitution of the photosensitizers on one-and two-photon absorption generation of singlet oxygen.Near-infrared femtosecond Ti:squassier laser was utilized to determine two-photon properties of the photosensitizers tuning wavelength from 700 to 880 nm at intervals of 20 nm.One-and two-photon optical and photophysical properties of the photosensitizers show significant dependence on the branches and substituted bromine atoms.     

飞秒脉冲在透明介质中的三维光存储及读出对比度研究

将波长为800nm，脉冲宽度为150fs的近红外激光脉冲，聚焦到聚甲基丙烯酸甲酯(Ploymethyl Metacrtlate，简记PMMA)和熔融石英中，实现了三维逐位式光数据存储．分别记录了5，10，15和20层数据位点，并利用相位对比光学显微原理，对各层数据并行读出，从而分析了各层数据位点读出对比度的变化．结果表明，各层数据位点的折射率对比度由内至外依次增加．记录层数越多，内部层的对比度下降越明显．由于飞秒激光脉冲与透明介质相互作用中熔融石英比PMMA内部产生的残余应力大，因此，在数据位点参数相同的情况下，利用聚甲基丙烯甲酯(PMMA)材料记录的层数更多．     

Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy

Molecular vibrations have oscillation periods that reflect the molecular structure, and are hence being used as a spectroscopic fingerprint for detection and identification. At present, all nonlinear spectroscopy schemes use two or more laser beams to measure such vibrations. The availability of ultrashort (femtosecond) optical pulses with durations shorter than typical molecular vibration periods has enabled the coherent excitation of molecular vibrations using a single pulse. Here we perform single-pulse vibrational spectroscopy on several molecules in the liquid phase, where both the excitation and the readout processes are performed by the same pulse. The main difficulty with single-pulse spectroscopy is that all vibrational levels with energies within the pulse bandwidth are excited. We achieve high spectral resolution, nearly two orders of magnitude better than the pulse bandwidth, by using quantum coherent control techniques. By appropriately modulating the spectral phase of the pulse we are able to exploit the quantum interference between multiple paths to selectively populate a given vibrational level, and to probe this population using the same pulse. This scheme, using a single broadband laser source, is particularly attractive for nonlinear microscopy applications, as we demonstrate by constructing a coherent anti-Stokes Raman (CARS) microscope operating with a single laser beam.     

Functional mapping of single spines in cortical neurons in vivo

The individual functional properties and spatial arrangement of afferent synaptic inputs on dendrites have a critical role in the processing of information by neurons in the mammalian brain. Although recent work has identified visually-evoked local dendritic calcium signals in the rodent visual cortex, sensory-evoked signalling on the level of dendritic spines, corresponding to individual afferent excitatory synapses, remains unexplored. Here we used a new variant of high-resolution two-photon imaging to detect sensory-evoked calcium transients in single dendritic spines of mouse cortical neurons in vivo. Calcium signals evoked by sound stimulation required the activation of NMDA (N-methyl-D-aspartate) receptors. Active spines are widely distributed on basal and apical dendrites and pure-tone stimulation at different frequencies revealed both narrowly and widely tuned spines. Notably, spines tuned for different frequencies were highly interspersed on the same dendrites: even neighbouring spines were mostly tuned to different frequencies. Thus, our results demonstrate that NMDA-receptor-dependent single-spine synaptic inputs to the same dendrite are highly heterogeneous. Furthermore, our study opens the way for in vivo mapping of functionally defined afferent sensory inputs with single-synapse resolution.     

飞秒激光在蓝宝石晶体表面加工微细结构的实验研究

由于蓝宝石晶体具有很高的硬度和耐磨蚀性,很难进行机械和化学腐蚀加工。笔者利用波长780 nm、频率1 kHz和脉冲宽度164 fs的飞秒脉冲激光在蓝宝石晶体表面进行了微细结构加工的实验研究。采用飞秒激光静态照射蓝宝石晶体表面,通过飞秒激光烧蚀孔的直径和脉冲能量的关系,计算了飞秒激光烧蚀蓝宝石晶体的两种烧蚀状态下的烧蚀阈值和有效烧蚀半径。通过直线扫描实验,在不同实验条件下在蓝宝石晶体表面加工微槽,获得微槽的宽度和深度与飞秒激光主要参数之间的关系。研究结果表明,微槽的加工表面可通过增加扫描次数而得到明显的提高,且扫描次数的增加对微槽的宽度和深度基本无影响。利用聚焦的飞秒激光束沿着轨迹扫描,在蓝宝石晶体表面加工出比较清洁的微小结构,可以为实现微结构的精密加工提供指导。     

阶跃相位整形的飞秒激光脉冲时间光强分布

利用飞秒超快脉冲相位整形方法,计算并研究了阶跃相位整形后激光脉冲的时域光强分布特点。研究结果表明,通过改变调制位置和调制深度,高斯激光脉冲可以整形形成强度可调的双脉冲结构,且子脉冲的中心时间和脉宽可以采用调制参数来调节。同时,研究发现阶跃位置的有效可调范围和激光的线宽密切相关。     

超短脉冲激光及其应用

评述了国际上超短脉冲激光技术的最新研究进展以及超短脉冲激光在超高速光通讯、海量信息存储、光合作用研究、化学反应过程研究等领域广泛的应用状况