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.     

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.     

Effect of low level laser irradiation on the proliferation of myoblasts—the skeletal muscle precursor cells: an experimental in vitro study

The aim of this paper is to study the effect of low-level laser irradiation (LLLI) on proliferation of myoblasts in culture. Myoblasts derived from rat skeletal muscle were irradiated by He-Ne laser with different doses. Compared with nonirradiated control group, the number of myoblasts increased when the cells in normal culture conditions were exposed to the laser of specific energy density. The amount of cells with proliferating cell nuclear antigen (PCNA) positive expression and the 5-bromo-2′-deoxyuridine (BrdU) incorporation rate after laser irradiation were also higher than that of the control group, suggesting that LLLL at certain doses can effectively enhance myoblasts growth activity in vitro. This study firstly demonstrated that stimulating myoblasts to enter into proliferative stage from initial resting state was an important mechanism of regeneration and repair of injured skeletal muscle promoted by LLLI in clinical treatment.     

Femtosecond laser-induced stimulation of a single neuron in a neuronal network

We demonstrated the stimulation of neurons at a single-cell level in cultured neuronal network by a focused femtosecond laser. When the femtosecond laser was focused on a neuron loaded with a fluorescent calcium indicator, the fluorescence intensity immediately increased at the laser spot, suggesting that intracellular Ca²⁺ increases in the neuronal cell due to the femtosecond laser irradiation. The probability of Ca²⁺ elevation at the laser spot depended on the average laser power, irradiation time, and position of the focal point along the optical axis, indicating that the femtosecond laser activates neurons because of multiphoton absorption. Moreover, after laser irradiation of a single neuron cultured on multielectrode arrays, the evoked electrical activity of the neurons was demonstrated by electrophysiological systems, which concluded that the focused femtosecond laser could achieve stimulating a single neuron in a neuronal network with high spatial and temporal resolution.     

飞秒激光诱导Li2O-Nb2O5-SiO2玻璃析晶拉曼谱研究

通过使用波长 800nm、重复频率 250kHz的飞秒激光脉冲聚焦进Li2O-Nb2O5-SiO2组分的玻璃内部,空间选择性诱导出铌酸锂微晶.为了进一步地研究辐照区域晶体的生长机制,使用显微拉曼光谱仪分析了玻璃样品辐照区域不同位置的结构变化.研究表明,在飞秒激光辐照一段时间后的聚焦区域,由于非线性效应和热累积效应形成了一个高温辐射梯度场,在激光辐照中心区域超过玻璃的析晶温度而促使玻璃熔融析晶.     

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。     

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.     

Femtosecond laser microstructuring: an enabling tool for optofluidic lab‐on‐chips

This paper provides an overview of the rather new field concerning the applications of femtosecond laser microstructuring of glass to optofluidics. Femtosecond lasers have recently emerged as a powerful microfabrication tool due to their unique characteristics. On the one hand, they enable to induce a permanent refractive index increase, in a micrometer‐sized volume of the material, allowing single‐step, three‐dimensional fabrication of optical waveguides. On the other hand, femtosecond‐laser irradiation of fused silica followed by chemical etching enables the manufacturing of directly buried microfluidic channels. This opens the intriguing possibility of using a single laser system for the fabrication and three‐dimensional integration of optofluidic devices. This paper will review the state of the art of femtosecond laser fabrication of optical waveguides and microfluidic channels, as well as their integration for high sensitivity detection of biomolecules and for cell manipulation.     

High repetition rate femtosecond laser irradiation-induced elements redistribution in Ag-doped glass

A 250 kHz femtosecond laser was used to induce a one-step precipitation of Ag nanoparticles and the simultaneous element redistribution in Ag ion doped glass. After femtosecond laser exposure, a ring-shape region was formed. Besides, as a result of an optical spherical aberration effect, a nonuniform laser intensity distribution along the incident direction caused most of the formed Ag nanoparticles to precipitate in the lower part of cross section of this structure. By an EPMA analysis, the relative concentration of the Ag element was both high in the center and in the boundary of the ring-shape region. These Ag nanoparticles could potentially increase the refractive index in their precipitation regions. We proposed that the induced ion redistribution and the precipitation of Ag nanoparticles should be due to the heat accumulation effect by the high repetition rate femtosecond laser irradiation.     

Ultra fast melting process in femtosecond laser crystallization of thin a-Si layer

In this paper, we investigated the mechanism of crystallization induced by femtosecond laser irradiation for an amorphous Si (a-Si) thin layer on a crystalline Si (c-Si) substrate. The fundamental, SHG, THG wavelength of a Ti:Sapphire laser was used for the crystallization process. To investigate the processed areas we performed Laser Scanning Microscopy (LSM), Transmission Electron Microscopy (TEM) and Imaging Pump-Probe measurements. Except for 267 nm femtosecond laser irradiation, the crystallization occurred well. The threshold fluences for the crystallization using 800 nm and 400 nm femtosecond laser irradiations were 100 mJ/cm² and 30 mJ/cm², respectively. TEM observation revealed that the crystallization occurred by epitaxial growth from the boundary surface between the a-Si layer and c-Si substrate. The melting depths estimated by Imaging Pump-Probe measurements became shallower when the shorter wavelength was used.     

Femtosecond laser-induced inverted microstructures inside glasses by tuning refractive index of objective's immersion liquid

We report the formation of inverted microstructures inside glasses after femtosecond laser irradiation by tuning the refractive index contrast between the immersion liquid and the glass sample. By using water as well as 1-bromonaphthalene as immersion liquids, microstructures with similar shape but opposite directions are induced after femtosecond laser irradiation. Interestingly, the elemental distribution in the induced structures is also inverted. The simulation of laser intensity distribution along the laser propagation direction indicates that the interfacial spherical aberration effect is responsible for the inversion of microstructures and elemental distribution.     

Cover Picture: Ann. Phys. 3'2013

Biological modulation using a femtosecond‐pulsed laser: When a femtosecond‐pulsed laser (yellow beam) is used to irradiate the cytosol of the cell (upper left), it generates free electrons (blue particles) in the focused area. Laser‐induced free electrons subsequently produce reactive oxygen species (ROS, red particles), which are amplified by inter‐mitochondrial networks. Amplified ROS signals can stimulate endoplasmic reticulum to release calcium ions (green particles). Femtosecond‐pulsed laser stimulation can modify blood vessel permeability (lower right). After short‐term laser irradiation, small chemicals (e.g., drugs) and proteins to which the intact blood vessel is impermeable can now flow out while red blood cells stay within the lumen. The cascade mechanism, which consists of low‐density plasma, ROS and calcium, is responsible for femtosecond‐pulsed laser‐induced biological modulation in both excitable and non‐excitable cells. J. Yoon et al., pp. 205‐214 in this issue     

Realignment process of actin stress fibers in single living cells studied by focused femtosecond laser irradiation

Three-dimensional dissection of a single actin stress fiber in a living cell was performed based on multi-photon absorption of a focused femtosecond laser pulse. The realignment process of an actin stress fiber was investigated after its direct cutting by a single-shot femtosecond laser pulse irradiation by high-speed transmission and fluorescence imaging methods. It was confirmed that mechanical force led by the femtosecond laser cutting propagates to entire cell through the cytockelton in a 100 μs time scale. The cut actin stress fiber was realigned in the time scale of a few tens of minutes. The dynamic analysis of the realignment induced by single-shot femtosecond laser gives new information on cell activity.     

Single crystal formation of amino acid with high temporal controllability by combining femtosecond and continuous wave laser trapping

We investigated laser trapping crystallization of glycine by using femtosecond (fs) laser as a trapping light source. Impulsively exerted fs laser pulses crystallized glycine more effectively than that induced by continuous wave (CW) laser trapping. Highly efficient crystallization and crystal growth behavior indicates fs laser irradiation increased the concentration not only at the focal spot, but also around the laser focus. Furthermore, we found that irradiation of fs pulses to CW laser-induced locally high supersaturation region enables immediate crystallization. Spatiotemporally controlled triggering of a single crystal formation with sub-second time resolution has achieved by integrating fs and CW laser trapping techniques.     

Biocompatibility of nanoactuators: stem cell growth on laser-generated nickel–titanium shape memory alloy nanoparticles

Nanoactuators made from nanoparticulate NiTi shape memory alloy show potential in the mechanical stimulation of bone tissue formation from stem cells. We demonstrate the fabrication of Ni, Ti, and NiTi shape memory alloy nanoparticles and their biocompatibility to human adipose-derived stem cells. The stoichiometry and phase transformation property of the bulk alloy is preserved during attrition by femtosecond laser ablation in liquid, giving access to colloidal nanoactuators. No adverse effect on cell growth and attachment is observed in proliferation assay and environmental electron scanning microscopy, making this material attractive for mechanical stimulation of stem cells.     

Femtosecond laser induced periodic large-scale surface structures on metals

The periodic ripple structures on wolfram and titanium surfaces are induced experimentally by linear polarized femtosecond laser pulses at small incident angles. The structural features show a material difference in the s- and p-polarized laser irradiation. The interspace between the ripples increases significantly for p-polarized laser irradiation when it exceeds a threshold angle, and the ripples' periodicities are larger than the wavelength of the incident p-polarized femtosecond laser; however, no significant change in the period of the ripples is observed with increasing incident angle for s-polarized laser irradiation. To explain these phenomena we propose a resonant absorption mechanism, by which the experimental observations can be interpreted.     

Photoinduced multiple microchannels inside silicon produced by a femtosecond laser

Photoinduced multiple microchannels in the interior of silicon produced by an 800-nm femtosecond laser were observed. The multiple microchannels were aligned spontaneously with a period along the propagation direction of the laser beam, which could be attributed to the interface spherical aberration induced due to refractive-index mismatch. We also observed that the depth of the photoinduced microchannels increased with the increase of the laser power. The power dependence of the depth of photoinduced microchannels in silicon was different from that in transparent materials, which probably arose from the competition between self-focusing due to the nonlinear Kerr effect and self-defocusing related to the thermal accumulation in the process of laser irradiation.     

Repetition rate dependency of low-density plasma effects during femtosecond-laser-based surgery of biological tissue

Femtosecond laser based nanosurgery of biological tissue is usually done in two different regimes. Depending on the application, low kHz repetition rates above the optical breakdown threshold or high MHz repetition rates in the low-density plasma regime are used. In contrast to the well understood optical breakdown, mechanisms leading to dissection below this threshold are not well known due to the complexity of chemical effects with high numbers of interacting molecules. Furthermore, the laser repetition rate may influence their efficiency. In this paper, we present our study on low-density plasma effects in biological tissue depending on repetition rate by static exposure of porcine corneal stroma to femtosecond pulses. We observed a continuous increase of the laser-induced damage with decreasing repetition rate over two orders of magnitude at constant numbers of applied laser pulses or constant laser pulse energies. Therefore, low repetition rates in the kHz regime are advantageous to minimize the total delivered energy to biological tissue during femtosecond laser irradiation. However, due to frequent excessive damage in this regime directly above the threshold, MHz repetition rates are preferable to create nanometer-sized cuts in the low-density plasma regime.     

Study of the microstructural transformations of borate glass and barium metaborate crystals induced by femtosecond laser

This paper describes the microstructural transformations of borate glass and barium metaborate crystals induced by femtosecond laser. Such structural transformations were verified by Raman spectroscopy. The borate glass is transformed into low temperature (LT) phase of barium metaborate (BaB_2O_4) crystals after being irradiated for 10 min by a femtosecond laser. In addition, after 20 min of irradiation, high temperature (HT) phase of BaB_2O_4 crystals is also produced. Further studies demonstrate that LT phase BaB_2O_4 crystals are formed in the HT phase BaB_2O_4 crystals after femtosecond laser irradiation for 10 s.     

光学相干层析成象技术的医学诊断应用

利用超短脉冲飞秒激光建立了OCT实验装置,通过对动物肾脏组织样品的层析成象,OCT图象与传统组织学图谱能找到很好的对应关系;对动物脑梗塞模型组织的扫描成象,得到了脑梗塞侧和正常侧的OCT图象,实验中,大脑病变组织和正常组织能够被正确区分。