Spatial control of cellular measurements with the laser micropipet

Continued progress in understanding cellular physiology requires new strategies for biochemical measurements in solitary cells, multiple cells, and subcompartments of cells. Large spatial gradients in the concentrations of molecules and presumably the activities of enzymes can occur in cells. Consequently, there is a critical need for measurement techniques for mammalian cells with control over the numbers or regions of cells interrogated. In the present work, we developed a strategy to rapidly load the cytoplasmic contents of either multiple cells or a subregion of a single cell into a capillary. A single, focused pulse from a laser created a mechanical shock wave which disrupted a group of cells or a portion of a cell in the path of the shock wave. Simultaneously, the cytoplasm was loaded into a capillary for electrophoretic separation. The size of the region of cellular disruption (and therefore the volume of cytoplasm collected) was controlled by the amount of energy in the laser pulse. Higher energies could be used to sample groups of cells while much lower energies could be utilized to selectively sample the tip of a neuronal process. The feasibility of performing measurements on subcellular compartments was also demonstrated by targeting reporter molecules to these compartments. A reporter localized to the nucleus was detected on the electropherogram following laser-mediated disruption of the cell and the nucleus. Finally, we demonstrate that this method terminated cellular reactions with sufficient rapidity that cellular membrane repair mechanisms were not activated during cytoplasmic collection. The combined ability to preselect a spatial region of a cell or cells and to rapidly load that region into a capillary will greatly enhance the utility of CE in the biochemical analysis of cells.     

Plasmon-assisted local temperature control to pattern individual semiconductor nanowires and carbon nanotubes

We demonstrate a new versatile strategy to rapidly heat and cool subdiffraction-limited volumes of material with a focused light beam. The local temperature rise is obtained by exploiting the unique optical properties of metallic nanostructures that facilitate efficient light-to-heat conversion through the excitation of surface plasmons (collective electron oscillations). By locally heating nanoscale metallic catalysts, growth of semiconductor nanowires and carbon nanotubes can be initiated and controlled at arbitrarily prespecified locations and down to the single nanostructure level in a room-temperature chamber. This local heating strategy can be orders of magnitude (>10(5)) more energy efficient than conventional chemical vapor deposition (CVD) tools in which an entire chamber/substrate is heated. For these reasons, it has great potential for use in process- and energy-efficient assembly of nanowires into complementary metal-oxide-semiconductor (CMOS) compatible device architectures. In general, the high degree of spatial and temporal control over nanoscale thermal environments afforded by this method inspires new pathways for manipulating a range of important thermally stimulated processes and the development of novel photothermal devices.     

Direct Reprogramming of Human Suspension Cells into Mesodermal Cell Lineages via Combined Magnetic Targeting and Photothermal Stimulation by Magnetic Graphene Oxide Complexes

Suspension cells can provide a source of cells for cellular reprogramming, but they are difficult to transfect by nonviral vectors. An efficient and safe nonviral vector (GO‐Fe₃O₄‐PEI complexes) based on iron oxide nanoparticle (Fe₃O₄)‐decorated graphene oxide (GO) complexed with polyethylenimine (PEI) for the first time is developed for delivering three individual episomal plasmids (pCXLE‐hOCT3/4‐shp53, pCXLE‐hSK, and pCXLE‐hUL) encoding pluripotent‐related factors of Oct3/4, shRNA against p53, Sox2, Klf4, L‐Myc, and Lin28 into human peripheral blood mononuclear cells (PBMCs) simultaneously. The combined treatment of magnetic stirring and near‐infrared (NIR)‐laser irradiation, which can promote contact between the complexes and floating cells and increase the cell membrane permeability, respectively, is used to conduct multiple physical stimulations for suspension PBMCs transfection. The PCR analysis shows that the combinatorial effect of magnetic targeting and photothermal stimulation obviously promoted the transfection efficiency of suspension cells. The transfected cells show positive expression of the pluripotency markers, including Nanog, Oct4, and Sox2, and have potential to differentiate into mesoderm and ectoderm cells. The results demonstrate that the GO‐Fe₃O₄‐PEI complex provides a safe, convenient, and efficient tool for reprogramming PBMCs into partially induced pluripotent stem cells, which are able to rapidly transdifferentiate into mesodermal lineages without full reprogramming.     

Quantum dots as multimodal photoacoustic and photothermal contrast agents

Quantum dots (QDs) have primarily been developed as fluorescent probes with unique optical properties. We herein demonstrate an extension of these QD utilities to photoacoustic (PA) and photothermal (PT) microscopy, using a nanosecond pulse laser excitation (420-900 nm, 8 ns, 10(-3)-10 J/cm(2)). The laser-induced PA, PT and accompanying bubble formation phenomena were studied with an advanced multifunctional microscope, which integrates fluorescence, PA, PT imaging, and PT thermolens modules. It was demonstrated that QDs, in addition to being excellent fluorescent probes, can be used as PA and PT contrast agents and sensitizers, thereby providing an opportunity for multimodal high resolution (300 nm) PA-PT-fluorescent imaging as well as PT therapy. Further improvements for this technology are suggested by increasing the conversion of laser energy in PT, PA, and bubble phenomena in hybrid multilayer QDs that have optimized absorption, thermal, and acoustic properties.     

Gold nanoshell photomodification under a single-nanosecond laser pulse accompanied by color-shifting and bubble formation phenomena

Laser-nanoparticle interaction is crucial for biomedical applications of lasers and nanotechnology to the treatment of cancer or pathogenic microorganisms. We report on the first observation of laser-induced coloring of gold nanoshell solution after a one nanosecond pulse and an unprecedentedly low bubble formation (as the main mechanism of cancer cell killing) threshold at a laser fluence of about 4?mJ?cm(-2), which is safe for normal tissue. Specifically, silica/gold nanoshell (140/15?nm) suspensions were irradiated with a single 4?ns (1064?nm) or 8?ns (900?nm) laser pulse at fluences ranging from 0.1?mJ?cm(-2) to 50?J?cm(-2). Solution red coloring was observed by the naked eye confirmed by blue-shifting of the absorption spectrum maximum from the initial 900?nm for nanoshells to 530?nm for conventional colloidal gold nanospheres. TEM images revealed significant photomodification of nanoparticles including complete fragmentation of gold shells, changes in silica core structure, formation of small 20-30?nm isolated spherical gold nanoparticles, gold nanoshells with central holes, and large and small spherical gold particles attached to a silica core. The time-resolved monitoring of bubble formation phenomena with the photothermal (PT) thermolens technique demonstrated that after application of a single 8?ns pulse at fluences 5-10?mJ?cm(-2) and higher the next pulse did not produce any PT response, indicating a dramatic decrease in absorption because of gold shell modification. We also observed a dependence of the bubble expansion time on the laser energy with unusually very fast PT signal rising (～3.5?ns scale at 0.2?J?cm(-2)). Application of the observed phenomena to medical applications is discussed, including a simple visual color test for laser-nanoparticle interaction.     

Optical and acoustic detection of laser-generated microbubbles in single cells

Acoustically monitored laser-induced optical breakdown (LIOB) has potential as an important tool to diagnose and treat living cells. Laser-induced intracellular microbubbles are readily detectable using high-frequency ultrasound, and LIOB can be controlled to operate within two distinct regimes. In the nondestructive regime, a single, short-lived bubble can be generated within a cell, without affecting its immediate viability. In the destructive regime, the induced photodisruption quickly can kill a targeted cell. To generate and monitor this range of bioeffects in real time, we have developed a system integrating an ultrafast laser source with optical and acoustic microscopy. Experiments were performed on monolayers of Chinese hamster ovary (CHO) cells. A 793 nm, 100 fs laser pulsed at 3.8 kHz was tightly focused within each cell to produce the photodisruption, and a 50 MHz ultrasonic transducer monitored the resultant bubble via continuous pulse-echo recordings. Photodisruption was also observed using bright field microscopy, and cell viability was assessed following laser exposure with a trypan blue assay. By controlling laser pulse fluence and exposure duration, either nondestructive or destructive LIOB could be produced. The intracellular position of the laser focus was also varied to demonstrate that cell viability was affected by the specific location of material breakdown.     

Combination of fiber-guided pulsed erbium and holmium laser radiation for tissue ablation under water

Because of the high absorption of near-infrared laser radiation in biological tissue, erbium lasers and holmium lasers emitting at 3 and 2 μm, respectively, have been proven to have optimal qualities for cutting or welding and coagulating tissue. To combine the advantages of both wavelengths, we realized a multiwavelength laser system by simultaneously guiding erbium and holmium laser radiation by means of a single zirconium fluoride (ZrF(4)) fiber. Laser-induced channel formation in water and poly(acrylamide) gel was investigated by the use of a time-resolved flash-photography setup, while pressure transients were recorded simultaneously with a needle hydrophone. The shapes and depths of vapor channels produced in water and in a submerged gel after single erbium and after combination erbium-holmium radiation delivered by means of a 400-μm ZrF(4) fiber were measured. Transmission measurements were performed to determine the amount of pulse energy available for tissue ablation. The effects of laser wavelength and the delay time between pulses of different wavelengths on the photomechanical and photothermal responses of meniscal tissue were evaluated in vitro by the use of histology. It was observed that the use of a short (200-μs, 100-mJ) holmium laser pulse as a prepulse to generate a vapor bubble through which the ablating erbium laser pulse can be transmitted (delay time, 100 μs) increases the cutting depth in meniscus from 450 to 1120 μm as compared with the depth following a single erbium pulse. The results indicate that a combination of erbium and holmium laser radiation precisely and efficiently cuts tissue under water with 20-50-μm collateral tissue damage.     

Enhanced Photothermal Therapy through the In Situ Activation of a Temperature and Redox Dual‐Sensitive Nanoreservoir of Triptolide

Photothermal therapy (PTT) has attracted tremendous attention due to its noninvasiveness and localized treatment advantages. However, heat shock proteins (HSPs) associated self‐preservation mechanisms bestow cancer cells thermoresistance to protect them from the damage of PTT. To minimize the thermoresistance of cancer cells and improve the efficacy of PTT, an integrated on‐demand nanoplatform composed of a photothermal conversion core (gold nanorod, GNR), a cargo of a HSPs inhibitor (triptolide, TPL), a mesoporous silica based nanoreservoir, and a photothermal and redox di‐responsive polymer shell is developed. The nanoplatform can be enriched in the tumor site, and internalized into cancer cells, releasing the encapsulated TPL under the trigger of intracellular elevated glutathione and near‐infrared laser irradiation. Ultimately, the liberated TPL could diminish thermoresistance of cancer cells by antagonizing the PTT induced heat shock response via multiple mechanisms to maximize the PTT effect for cancer treatment.     

作用于线粒体和细胞核的聚合物纳米材料及其在化疗/光热治疗联合抗癌中的应用

开发本身即具有线粒体靶向能力的亚细胞精准纳米诊疗试剂对于改善癌症治疗效果具有重要意义.本文使用可靶向癌细胞表面过度表达的CD44抗原的透明质酸、胆固醇-聚乙二醇-氨基和可作用于线粒体的花菁类染料IR825-NH2,构建了一种可实现光热治疗的自组装纳米材料(HA-IR825-Chol).相较于游离的IR825-NH2,该结构具有更好的光稳定性、更高的光热转换效率和对癌细胞的识别能力.HA-IR825-Chol可以有效靶向细胞线粒体,并可以在近红外激光照射下诱导线粒体损伤.此外,我们通过疏水作用包裹了化疗试剂10-羟基喜树碱(HCPT)(所形成的药物命名为HAIR825-Chol/HCPT).相关实验结果显示,包裹于纳米材料后HCPT被细胞摄取的效率显著提高,并能够同时分布于线粒体和细胞核中,从而诱导线粒体中细胞色素c的释放和细胞中cleaved caspase-3的上调,最终促进细胞凋亡与死亡.另外,HA-IR825-Chol/HCPT优异的体内肿瘤靶向能力为光化疗联合治疗消除肿瘤提供了必要保证.该工作实现了定位于线粒体的精准亚细胞药物递送,并发展了利用胆固醇提高药物摄取速率和效率的策略,预期将为提高纳米药物抗癌效果提供借鉴.     

Rapidly tuning miniature transversely excited atmospheric-pressure CO2 laser

An experimental study of a rapidly tuning miniature transversely excited atmospheric-pressure CO2 laser is reported. To rapidly shift laser wavelengths over selected transitions in the 9-11 microm wavelength region, we have utilized a high-frequency stepping motor and a diffraction grating. The laser is highly automated with a monolithic microprocessor controlled laser line selection. For the achievement of stable laser output, a system of laser excitation with a voltage of 10 kV, providing effective surface corona preionization and allowing one to work at various gas pressures, is utilized. Laser operation at 59 emission lines of the CO2 molecule rotational transition is obtained and at 51 lines, the pulse energy of laser radiation exceeds 30 mJ. The system can be tuned between two different rotational lines spanning the wavelength range from 9.2 to 10.8 microm within 10 ms.     

Large-Area Alignment of Supramolecular Columns by Photothermal Laser Writing

Controlling the orientation of highly periodic supramolecular structures of small feature size (<5 nm) is the first step for potential applications in optoelectronics, membranes, and template synthesis. A new method, namely, laser photothermal writing, is introduced to direct the orientation of supramolecular columns over a large area. Supramolecular columns consisting of taper-shaped molecules with long aliphatic tail groups are aligned by a thermal gradient, which is induced by exposing a near-infrared laser beam to a graphene photothermal conversion layer. Intriguingly, the orientation of the supramolecular columns can be controlled in a facile manner by varying the laser scanning velocity and power. In contrast to previous methodologies for aligning supramolecular structures, this laser photothermal mechanism allows the directional and continuous alignment of supramolecular structures over an arbitrary large area with the easy control of laser irradiation. Besides, the laser process also enables area-selective orientation of the supramolecular structures for device-oriented nanopatterning.     

Toward the optimization of double-pulse LIBS underwater: effects of experimental parameters on the reproducibility and dynamics of laser-induced cavitation bubble

Double-pulse laser-induced breakdown spectroscopy (LIBS) was recently proposed for the analysis of underwater samples, since it overcomes the drawbacks of rapid plasma quenching and of large continuum emission, typical of single-pulse ablation. Despite the attractiveness of the method, this approach suffers nevertheless from a poor spectroscopic reproducibility, which is partially due to the scarce reproducibility of the cavitation bubble induced by the first laser pulse, since pressure and dimensions of the bubble strongly affect plasma emission. In this work, we investigated the reproducibility and the dynamics of the cavitation bubble induced on a solid target in water, and how they depend on pulse duration, energy, and wavelength, as well as on target composition. Results are discussed in terms of the effects on the laser ablation process produced by the crater formation and by the interaction of the laser pulse with floating particles and gas bubbles. This work, preliminary to the optimization of the spectroscopic signal, provides an insight of the phenomena occurring during laser ablation in water, together with useful information for the choice of the laser source to be used in the apparatus.     

可控碳化废弃聚丙烯制备镍/碳纳米材料用于高效光蒸气转换

利用太阳能实现光蒸气转化是一项极具前景的技术,可应用于海水脱盐和淡水制备等领域.然而,从工业的角度来看,制备低成本、高效率的光热材料仍具有挑战性.本文利用聚离子液体(PIL)和氧化镍(Ni O)作为复合催化剂,实现了聚丙烯(PP)的可控碳化,并制备了镍/碳纳米材料(Ni/CNM).研究结果表明,加入微量的PIL可实现对Ni/CNM形貌和织态结构的调控.Ni/CNM由杯状碳纳米管(CS-CNT)和梨形镍纳米颗粒组成,二者在太阳光吸收上的协同作用使得Ni/CNM具有优异的光热转换性能.此外,Ni/CNM具有较高的比表面积和丰富的微/介/大孔,其构建的三维多孔网络可为水和蒸气的高效传输提供通道.光吸收高、水传输快和热导率低等优势,使Ni/CNM的水蒸发速率高达1.67 kg m^-2h^-1,光-蒸气转换效率高达94.9%,且重复使用10次后性能依然保持稳定.该材料同时适用于染料废水、海水和油/水乳化液等水质的纯化.其中,海水中金属离子的去除效率高达99.99%,染料去除率>99.9%.更重要的是,材料的光蒸气转换性能优于最新报道的碳基光热材料.此工作不仅提出了一种可将废弃聚合物转化为先进的金属/碳杂化物的可持续方法,同时也有助于太阳能利用和海水淡化领域的进一步研究.     

Application of femtosecond-laser induced nanostructures in optical memory

The femtosecond laser induced micro- and nanostructures for the application to the three-dimensional optical data storage are investigated. We have observed the increase of refractive index due to local densification and atomic defect generation, and demonstrated the real time observation of photothermal effect after the femtosecond laser irradiation inside a glass by the transient lens (TrL) method. The TrL signal showed a damped oscillation with about an 800 ps period. The essential feature of the oscillation can be reproduced by the pressure wave creation and propagation to the outward direction from the irradiated region. The simulation based on elastodynamics has shown that a large thermoelastic stress is relaxed by the generation of the pressure wave. In the case of soda-lime glass, the velocity of the pressure wave is almost same as the longitudinal sound velocity at room temperature (5.8 microm/ns). We have also observed the localized photo-reduction of Sm3+ to Sm2+ inside a transparent and colorless Sm(3+)-doped borate glass. Photoluminescence spectra showed that some the Sm3+ ions in the focal spot within the glass sample were reduced to Sm2+ ions after femtosecond laser irradiation. A photo-reduction bit of 200 nm in three-dimensions can be recorded with a femtosecond laser and readout clearly by detecting the fluorescence excited by Ar+ laser (lambda = 488 nm). A photo-reduction bit can be also erased by photo-oxidation with a cw Ar+ laser (lambda = 514.5 nm). Since photo-reduction bits can be spaced 150 nm apart in a layer within glass, a memory capacity of as high as 1 Tbit can be achieved in a glass piece with dimensions of 10 mm x 10 mm x 1 mm. We have also demonstrated the first observation of the polarization-dependent periodic nanostructure formation by the interference between femtosecond laser light and electron acoustic waves. The observed nanostructures are the smallest embedded structures ever created by light. The period of self-organized nanostructures can be controlled from approximately 140 to 320 nm by the pulse energy and the number of irradiated pulses. Furthermore, we have also observed the self-assembled sub-wavelength periodic structures created in silica glass by femtosecond pulses on the plane of the propagation of light.     

Giant Optical Activity in an All‐Dielectric Spiral Nanoflower

Optical activity is an effect of prominent importance in stereochemistry, analytical chemistry, metamaterials, spin photonics, and astrobiology, but is naturally minuscule. Metallic nanostructures are commonly exploited as basic elements for artificially producing large optical activity by virtue of surface plasmon resonance (SPR) on the nanostructures. However, their intrinsic high ohmic loss amplified by the SPR results in low energy efficiency and large photothermal heat generation, severely limiting their performance and practical utility. Giant optical activity by inducing magnetic resonance in an all‐dielectric spiral nanoflower (spiral‐flower‐shaped nanostructure) is demonstrated here. Specifically, a large circular‐intensity difference of ≈35% is theoretically predicted and experimentally demonstrated by optimizing the magnetic quadrupole contribution of the nanoflower to scattered light. The nanoflower overcomes the bottleneck of the traditional metallic platforms and enables the development of diverse chiroptical devices and applications.     

Optical fiber array for the delivery of high peak-power laser pulses for fluid flow measurements

Fiber delivery of 64.7 mJ laser pulses (approximately 6 ns duration) from a Q-switched Nd:YAG laser operating at 532 nm is demonstrated. A custom diffractive optical element was used to shape the laser beam and facilitate coupling into a linear fiber array. This launch arrangement achieves an improvement in launch efficiency compared with a circular fiber bundle evaluated in previous work and the delivery of higher pulse energies is demonstrated. The bundle is capable of delivering light of sufficient pulse energy and, importantly, with suitable focusability, to generate a thin light sheet for the fluid flow measurement technique of particle image velocimetry (PIV). Fiber delivery offers an advantage, in terms of optical access, for the application of PIV to enclosed measurement volumes, such as the cylinder of a combustion engine.     

Nanocomposite tantalum–carbon-based films deposited by femtosecond pulsed laser ablation

Nanostructured coatings of metal (tantalum) containing diamond-like carbon (a-C:Ta) have been prepared by femtosecond pulsed laser deposition (PLD). The films, containing 15 at.% tantalum, have been deposited by ablating sequentially graphite and metallic tantalum in vacuum conditions with an amplified Ti:sapphire laser. The coatings have been investigated by X-ray photoelectron spectroscopy, grazing angle X-ray diffraction, energy filtered transmission electron microscopy, scanning and high resolution transmission electron microscopies. Evidence of metallic α-Ta and β-Ta particles (diameter in the 100 nm range) and smaller quasi-amorphous tantalum clusters embedded in the carbonaceous matrix have been shown. A thin tantalum carbide interface between the carbon matrix and the top surface of the tantalum nodules has also been identified. The ability of femtosecond pulsed laser deposition to synthetize nanocomposite carbon-based films and to control their nanostructure is discussed.     

Micropallet arrays for the separation of single, adherent cells

The selection and collection of single cells from within a heterogeneous population is required to produce genetically engineered cell lines, to develop new stem cell lines, and for single-cell studies. We describe a new platform for the positive selection of single live mammalian cells while the cells remain adherent to their growth surface. Cells were grown on arrays of microfabricated, releasable elements composed of SU-8 polymer termed "cell pallets". The presence of air between the elements restricted the cells to the top surfaces of the pallets. Single pallets situated within large arrays of pallets were released on demand using a single, focused, laser pulse. The laser pulses were low in energy (2-5 muJ) and did not detach nearby, nontargeted pallets. Since the SU-8 pallets and the underlying glass substrate were optically transparent, the cells on the pallets could be visualized by microscopy before and after release. Over 90% of cells remained attached to the pallet during laser-based release. The feasibility of growing the cells from the released pallets into clonal colonies was demonstrated. The pallet array system permits adherent cells to be inspected using conventional microscopy and selected cells released for further analysis. The ability to assess cells while they remain adherent to a surface will broaden the number of attributes that can be utilized for cell separation, for example, cell shape, cytoskeletal properties, and other attributes.     

基于准分子激光微加工的光热微驱动器(英文)

提出了基于光热(PT)微膨胀原理的新型光热微驱动技术.设计了一种能将纵向光热膨胀转化成横向偏转的微驱动器.以AutoCAD设计图为基础,采用KrF准分子激光微加工系统,在单层高密度聚乙烯(HDPE)上加工出长1 500μm、宽250μm、厚40μm的开关式光热微驱动器.从微驱动器的扫描电子显微镜(SEM)图可以看出,微驱动器形状与AutoCAD设计值符合良好.光热微驱动实验采用脉冲频率可调的半导体激光器(4 mW,650 nm)作为驱动源.实验结果表明,在一定的脉冲频率范围(如0～17 Hz)内,光热微驱动器具有良好的静态和动态特性,其横向偏转量最大可达11μm,足以实现微开关功能.这种光热微驱动器可由激光束直接控制,具有原理新颖、结构简洁、体积小、易于加工制作等特点,在微纳米技术领域和微光机电系统(MOEMS)中具有广阔的应用前景.     

A Novel Hierarchical Nanostructure for Enhanced Optical Nonlinearity Based on Scattering Mechanism

Surface modification of nonlinear optical materials (NOMs) is widely applied to fabricate diverse photonic devices, such as frequency combs, modulators, and all‐optical switches. In this work, a double‐layer nanostructure with heterogeneous nanoparticles (NPs) is proposed to achieve enhanced third‐order optical nonlinearity of NOMs. The mechanism of modified optical nonlinearity is elucidated to be the scattering‐induced energy transfer between adjacent NPs layers. Based on the LiNbO₃ platform, as a typical example, double layers of embedded Cu and Ag NPs are synthesized by sequential ion implantation, demonstrating twofold magnitude of near‐infrared enhancement factor and modulation depth in comparison with a single layer of Cu NPs. With the elastic collision model and thermolysis theory being considered, the shift of the localized surface plasmon resonance (LSPR) peak reveals the formation mechanism of the double‐layer nanostructure. Utilizing the enhanced optical nonlinearity of LiNbO₃ as modulators, a Q‐switched mode‐locked waveguide laser at 1 µm is achieved with shorter pulse duration. It suggests potential applications to improve the performance of nonlinear photonic devices by using double‐layer metallic nanostructures.