Femtosecond laser modification of living neuronal network

An all-wet femtosecond laser microprocessing technique was utilized for patterning and cutting functional network of living neuronal cells on a multi-electrode dish (MED). The neuronal cells cultured on a source substrate were transferred onto an electrode in a MED probe in solution by utilizing a femtosecond laser-induced impulsive force and a pattern of neuronal cells were formed on the MED probe. The cellular activity of the detached neurons was supported that neurites could be regenerated around the electrodes. As another processing method, the neurons stretching between electrodes were selectively cut by the direct femtosecond laser irradiation and the spontaneous electrical activity of the neuronal network was evaluated. While the spontaneous action potentials of neurons were synchronized before the cutting, the synchronization disappeared after the cutting, indicating that the neuronal network is locally disconnected by the laser cutting. The present method is applicable to artificial reconstruction of living neuronal network.     

Formation of periodic surface nanostructures on Ti:Al2O3 crystals using femtosecond laser pulses

Periodic surface nanostructures are observed on Ti³⁺:Al₂O₃ single crystals that have been irradiated by a single focused beam from a femtosecond pulsed laser (wavelength: 800 nm; pulse duration: 130 and 152 fs). Atomic force microscopy images of single-ablated zones and modified structures created by fixing and translating samples through the focal region of a linearly polarized laser beam reveal self-organized periodic surface nanostructures (ripples) with a subwavelength spacing, which are oriented perpendicular to the electric-field vector of the laser beam. The period of the subwavelength ripples obtained by linearly polarized laser irradiation varies from ∼λ/5 to 2λ/5 (λ: incident laser wavelength) depending on the laser pulse energy. This phenomenon can be explained by assuming that the incident light field interferes with the electric field of electron plasma waves propagating inside the material; this interference periodically modulates the electron plasma density and modifies the surface ablation. In addition, for the first time, we observe screw-shaped nanostructures in the focal spot of circularly polarized beam irradiation. The morphology of these nanostructures appears to reflect the circular polarization of the laser light.     

Crystallization of amorphous Ge2Sb2Te5 films induced by a single femtosecond laser pulse

Crystallization is achieved in amorphous Ge₂Sb₂Te₅ films upon irradiation with a single femtosecond laser pulse. Transmission electron microscopy images evidence the morphology of the crystallized spot which depends on the fluence of the femtosecond laser pulse. Fine crystalline grains are induced at low fluence, and the coarse crystalline grains are obtained at high fluence. At the damage fluence, ablation of the films occurs.     

Nonlinear optical effects of near-IR femtosecond laser radiation on the morphology and structure of a nerve cell in the field of an optical trap

Changes in the morphology and structure of a neuron cell in the process of manipulating it with an femtosecond laser tweezers operating at a wavelength of 800 nm are examined. The changes in the morphology and structure are caused by the nonlinear optical absorption and multiphoton excitation of cell biomacromolecules by femtosecond pulses of light. The cell nucleus is demonstrated to be destroyed by focused femtosecond laser radiation (FLR). Changes in the state of the cytoplasmic nucleoproteins and the hydrophobicity of the plasmatic membrane under the action of FLR focused inside the cell are observed. By the example of the simultaneous displacement of the neuron with a cw laser and cutting of a neuron with FLR, the operation of holographic optical manipulator and a scalpel based on the use of femtosecond and cw lasers is considered. The possibility of the simultaneous microsurgical operation with several optical foci of FLR is demonstrated.     

Generation of calcium waves in living cells induced by 1 kHz femtosecond laser protuberance microsurgery

We have demonstrated that intracellular calcium waves in a living olfactory ensheathing cell (OEC) can be induced by femtosecond laser surgery on cellular protuberance. In this paper, calcium wave generation mechanisms are further investigated using different culture mediums and protuberance diameters. The protuberances of living OECs are cut by home-made 1 kHz femtosecond laser surgery system with 130 fs pulsewidth and 800 nm wavelength, and the average power of 200 μW is chosen for stable and effective cell surgery. Whether the cells are cultured in mediums with Ca²⁺ or not, intracellular calcium waves can be induced after cell surgery. The generation of calcium waves is independent on the dimension of protuberance diameter. Based on these results, we analyze generation mechanisms of calcium wave and conclude that shockwave-induced mechanical force and laser-induced cytoskeleton depolymerization are two key factors.     

Ablation depth control with 40 nm resolution on ITO thin films using a square,flat top beam shaped femtosecond NIR laser

We reported on the ablation depth control with a resolution of 40 nm on indium tin oxide (ITO) thin film using a square beam shaped femtosecond (190 fs) laser (λ_p=1030 nm). A slit is used to make the square, flat top beam shaped from the Gaussian spatial profile of the femtosecond laser. An ablation depth of 40 nm was obtained using the single pulse irradiation at a peak intensity of 2.8 TW/cm². The morphologies of the ablated area were characterized using an optical microscope, atomic force microscope (AFM), and energy dispersive X-ray spectroscopy (EDS). Ablations with square and rectangular types with various sizes were demonstrated on ITO thin film using slits with varying x–y axes. The stereo structure of the ablation with the depth resolution of approximately 40 nm was also fabricated successfully using the irradiation of single pulses with different shaped sizes of femtosecond laser.     

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.     

Effect of Doping on the Properties of Hydrogenated Amorphous Silicon Irradiated with Femtosecond Laser Pulses

A comparative analysis of the effect of femtosecond laser irradiation on the structure and conductivity of undoped and boron-doped hydrogenated amorphous silicon (a-Si: H) is performed. It is demonstrated that the process of nanocrystal formation in the amorphous matrix under femtosecond laser irradiation is initiated at lower laser energy densities in undoped a-Si: H samples. The differences in conductivity between undoped and doped a-Si: H samples vanish almost completely after irradiation with an energy density of 150–160 mJ/cm².     

Structural changes induced on strontium barium niobate glass by femtosecond laser irradiation

Localized modification of the optical properties of erbium doped strontium barium niobate (SBN) glass has been performed using femtosecond laser irradiation. The samples, with composition SrO–BaO–Nb₂O₅–B₂O₅ and doped with 5%mol of Er³⁺, were fabricated using a melt-quenching method. The samples were irradiated with different number of pulses per spot (1–50 pulses) at two different laser fluences (2.6 and 5.6 J/cm²) by using an fs laser amplifier operating at 800 nm and generating pulses with a duration of 120 fs. Micro-luminescent microscopy, using an Ar⁺ laser as excitation source, has been used to analyze the modifications of the luminescent properties of the sample upon fs laser exposure. The emissions of the Er³⁺: ⁴I_11/2→⁴I_15/2 and ⁴I_13/2→⁴I_15/2 transitions allow appreciating the structural modifications caused by femtosecond laser exposure. The lifetimes of the levels involved in these transitions were measured inside and outside the laser irradiated region. These measurements have been compared with those obtained in bulk glass ceramic sample, which is obtained from the glass precursor by a thermal treatment in order to estimate the optimal conditions to produce nanocrystals in a localized region by ultrafast laser irradiation.     

Neuron participation in a synchrony-encoding assembly

Background

Caenorhabditis elegans actively crawls down thermal gradients until it reaches the temperature of its prior cultivation, exhibiting what is called cryophilic movement. Implicit in the worm's performance of cryophilic movement is the ability to detect thermal gradients, and implicit in regulating the performance of cryophilic movement is the ability to compare the current temperature of its surroundings with a stored memory of its cultivation temperature. Several lines of evidence link the AFD sensory neuron to thermotactic behavior, but its precise role is unclear. A current model contends that AFD is part of a thermophilic mechanism for biasing the worm's movement up gradients that counterbalances the cryophilic mechanism for biasing its movement down gradients.

Results

We used tightly-focused femtosecond laser pulses to dissect the AFD neuronal cell bodies and the AFD sensory dendrites in C. elegans to investigate their contribution to cryophilic movement. We establish that femtosecond laser ablation can exhibit submicrometer precision, severing individual sensory dendrites without causing collateral damage. We show that severing the dendrites of sensory neurons in young adult worms permanently abolishes their sensory contribution without functional regeneration. We show that the AFD neuron regulates a mechanism for generating cryophilic bias, but we find no evidence that AFD laser surgery reduces a putative ability to generate thermophilic bias. In addition, although disruption of the AIY interneuron causes worms to exhibit cryophilic bias at all temperatures, we find no evidence that laser killing the AIZ interneuron causes thermophilic bias at any temperature.

Conclusion

We conclude that laser surgical analysis of the neural circuit for thermotaxis does not support a model in which AFD opposes cryophilic bias by generating thermophilic bias. Our data supports a model in which the AFD neuron gates a mechanism for generating cryophilic bias.     

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.     

Controllable photoinduced optical attenuation in a single-mode optical fiber by irradiation of a femtosecond pulse laser

Novel optical attenuation fibers were fabricated by the irradiation of a focused infrared femtosecond pulsed laser onto the core of a silica glass single-mode optical fiber. Optical attenuation at a wavelength of 1.55 microm proportionally increased with increasing numbers of irradiation points and was controllable under laser irradiation conditions. The single-mode property of the waveguide and the mode-field diameter of the optical fiber were maintained after irradiation of the femtosecond laser. It is suggested that the attenuation results from optical scattering at photoinduced spots formed inside the fiber core.     

Distinct roles of presynaptic dopamine receptors in the differential modulation of the intrinsic synapses of medium-spiny neurons in the nucleus accumbens

Background

In both schizophrenia and addiction, pathological changes in dopamine release appear to induce alterations in the circuitry of the nucleus accumbens that affect coordinated thought and motivation. Dopamine acts principally on medium-spiny GABA neurons, which comprise 95% of accumbens neurons and give rise to the majority of inhibitory synapses in the nucleus. To examine dopamine action at single medium-spiny neuron synapses, we imaged Ca²⁺ levels in their presynaptic varicosities in the acute brain slice using two-photon microscopy.

Results

Presynaptic Ca²⁺ rises were differentially modulated by dopamine. The D1/D5 selective agonist SKF81297 was exclusively facilitatory. The D2/D3 selective agonist quinpirole was predominantly inhibitory, but in some instances it was facilitatory. Studies using D2 and D3 receptor knockout mice revealed that quinpirole inhibition was either D2 or D3 receptor-mediated, while facilitation was mainly D3 receptor-mediated. Subsets of varicosities responded to both D1 and D2 agonists, showing that there was significant co-expression of these receptor families in single medium-spiny neurons. Neighboring presynaptic varicosities showed strikingly heterogeneous responses to DA agonists, suggesting that DA receptors may be differentially trafficked to individual varicosities on the same medium-spiny neuron axon.

Conclusion

Dopamine receptors are present on the presynaptic varicosities of medium-spiny neurons, where they potently control GABAergic synaptic transmission. While there is significant coexpression of D1 and D2 family dopamine receptors in individual neurons, at the subcellular level, these receptors appear to be heterogeneously distributed, potentially explaining the considerable controversy regarding dopamine action in the striatum, and in particular the degree of dopamine receptor segregation on these neurons. Assuming that post-receptor signaling is restricted to the microdomains of medium-spiny neuron varicosities, the heterogeneous distribution of dopamine receptors on individual varicosities is likely to encode patterns in striatal information processing.     

Characterization of channel waveguides in Pr:YLiF<Subscript>4</Subscript> crystals fabricated by direct femtosecond laser writing

Single tracks and pairs of tracks are written in the volume of Pr-doped LiYF₄-crystals using tightly focused femtosecond laser radiation (λ=1045 nm, τ _p=400–500 fs, f=0.1–1 MHz). Waveguiding between the tracks is demonstrated and optimized by varying the distance between the tracks and the laser writing conditions. The stress-induced guiding mechanism is explained based on TEM, interference microscopy, near-field and far-field measurements. It is shown that the single-crystalline material is getting poly-crystalline under femtosecond laser irradiation. By measuring the lifetime of the ³P₁→³H₅ transition and the emission spectrum at excitation with λ=444 nm, no influence on these properties of the guided light is observed. This possibly enables the realization of a channel waveguide laser in the visible spectral range.     

Removal of doped poly(methylmetacrylate) from tungsten and titanium substrates by femto- and nanosecond laser cleaning

The influence of different laser pulse lengths on the removal of a polymer layer from metal substrates was investigated. As model systems, doped poly(methylmetacrylate) (PMMA) on titanium and tungsten substrates were selected.The ablation threshold and irradiation spot morphology of titanium and tungsten were compared for femtosecond (fs) and nanosecond (ns) laser irradiation and different pulse numbers. Nanosecond laser treatment resulted in a non-homogeneous surface morphology for both titanium and tungsten substrates. Femtosecond irradiation of tungsten revealed a homogeneous ablation spot with little changes in the surface morphology. For titanium, the formation of columnar structures within the irradiation spot was observed.Two different dopant concentrations were used for PMMA to achieve an equal linear absorption coefficient for the femto- and nanosecond laser wavelengths of 790 and 1064 nm. The best results were achieved for the removal of doped PMMA by femtosecond laser irradiation, where only a minimal modification of the metal surface was detected. In the case of nanosecond laser exposure, a pronounced change of the structure was observed, suggesting that damage-free cleaning of the selected metal may only be possible using femtosecond laser pulses. Different experimental parameters, such as laser fluence, pulse repetition rate and sample speed were also investigated to optimize the cleaning quality of doped PMMA from tungsten substrates with femtosecond laser pulses.     

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 pulse irradiation of Sb-rich AgInSbTe films: Scanning electron microscopy and atomic force microscopy investigations

The single-layer and multilayer Sb-rich AgInSbTe films were irradiated by a single femtosecond laser pulse with the duration of 120 fs. The morphological feature resulting from the laser irradiation have been investigated by scanning electron microscopy and atom force microscopy. For the single-layer film, the center of the irradiated spot is a dark depression and the border is a bright protrusion; however, for the multilayer film, the center morphology changes from a depression to a protrusion as the energy increases. The crystallization threshold fluence of the single-layer and the multilayer films is 46.36 mJ/cm², 63.74 mJ/cm², respectively. PACS 79.20.Ds; 78.55.Qr; 78.66.Jg; 68.37.Ef; 68.37.Ps     

Femtosecond laser induced microripple on PDMS surface

We find the femtosecond laser induced microripple beside the focused femtosecond laser spot and along the movement direction of the laser spot on polydimethylsiloxane （PDMS） surface. The microripple may be due to the melting of PDMS induced by femtosecond laser pulses and the subsequent cool-down solidification of the melting PDMS along with the movement of the femtosecond laser spot. This result will be helpful to understand the interaction between the femtosecond laser and the polymer.     

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.     

Femtosecond laser processing of indium-tin-oxide thin films

Micro- and nano-scale crystalline indium-tin-oxide (c-ITO) patterns fabricated from amorphous ITO (a-ITO) thin films on a glass substrate using a (low NA 0.26) femtosecond laser pulse that is not tightly focused are demonstrated. Different types of c-ITO patterns are obtained by controlling the laser pulse energies and pulse repetition rate of a femtosecond laser beam at a wavelength of 1064 nm: periodic micro c-ITO dots with diameters of ~1.4 μm, two parallel c-ITO patterns with/without periodic-like glass nanostructures at a laser scanning path and nano-scale c-ITO line patterns with a line width ~900 nm, i.e. ~1/8 of the focused beam׳s diameter (7 μm at 1/e²).