Acoustic characterization of microbubble dynamics in laser-induced optical breakdown

A real-time acoustic technique to characterize microbubbles produced by laser-induced optical breakdown (LIOB) in water was developed. Femtosecond laser pulses are focused just inside the surface of a small liquid tank. A tightly focused, high frequency, single-element ultrasonic transducer is positioned so its focus coincides axially and laterally with this laser focus. When optical breakdown occurs, a bubble forms and a pressure wave is emitted (i.e., acoustic emission). In addition to this acoustic signal, the microbubble is actively probed with pulse-echo measurements from the same transducer. After the bubble forms, received pulse-echo signals have an extra pulse, describing the bubble location and providing a measure of axial bubble size. Wavefield plots of successive recordings illustrate the generation, growth, and collapse of cavitation bubbles due to optical breakdown. These same plots also can be used to quantify LIOB thresholds.     

Bubble-based acoustic radiation force elasticity imaging

Acoustic radiation force is applied to bubbles generated by laser-induced optical breakdown (LIOB) to study viscoelastic properties of the surrounding medium. In this investigation, femtosecond laser pulses are focused in the volume of gelatin phantoms of different concentrations to form bubbles. A two-element confocal ultrasonic transducer generates acoustic radiation force on individual bubbles while monitoring their displacement within a viscoelastic medium. Tone burst pushes of varying duration have been applied by the outer element at 1.5 MHz. The inner element receives pulse-echo recordings at 7.44 MHz before, during, and after the excitation bursts, and crosscorrelation processing is performed offline to monitor bubble position. Maximum bubble displacements are inversely related to the Young's moduli for different gel phantoms, with a maximum bubble displacement of over 200 microm in a gel phantom with a Young's modulus of 1.7 kPa. Bubble displacements scale with the applied acoustic radiation force and displacements can be normalized to correct for differences in bubble size. Exponential time constants for bubble displacement curves are independent of bubble radius and follow a decreasing trend with the Young's modulus of the surrounding medium. These results demonstrate the potential for bubble-based acoustic radiation force methods to measure tissue viscoelastic properties.     

脉冲激光击穿水介质特性的实验研究

针对激光击穿水介质过程中的微观及宏观特性研究,利用调QNd：YAG激光聚焦击穿水介质形成激光声源,采用高速摄像机、高频测量水听器对激光击穿水介质过程中的等离子腔体闪光、空泡脉动、近／远场声波特性等综合效应进行了实验测量。实验表明：激光空泡的特征与水动力空化空泡相似;激光声信号强度在光击穿条件下与入射激光能量具有一定的线性关系;声脉冲高频段占声能的主要部分。研究结果可为水下激光加工、激光医学、激光声的研究提供一定的理论和实验支持。     

Optical micromanipulations inside yeast cells

We present a combination of nonlinear microscopy and optical trapping applied to three-dimensional imaging and manipulation of intracellular structures in living cells. We use Titanium-sapphire laser pulses for nonlinear microscopy of the nuclear envelope and the microtubules marked with green fluorescent protein in fission yeast. The same laser source is also used to trap small lipid granules naturally present in the cell. The trapped granule is used as a handle to exert a pushing force on the cell nucleus. The granule is moved in a raster-scanning fashion to cover the area of the nucleus and hence displace the nucleus away from its normal position in the center of the cell. Such indirect manipulations of an organelle (e.g., nucleus) can be useful when direct trapping of the chosen organelle is disadvantageous or inefficient. We show that nonlinear microscopy and optical manipulation can be performed without substantial damage or heating of the cell. We present this method as an important tool in cell biology for manipulation of specific structures, as an alternative to genetic and biochemical methods. This technique can be applied to several fundamental problems in cell biology, including the mechanism of nuclear positioning and the spatial coordination of nuclear and cell division.     

In situ analysis of living embryonic stem cells by coherent anti-stokes Raman microscopy

Embryonic stem cells (ESC), derived from preimplantation embryos, are defined by their ability to both self-renew and differentiate into all of the cells and tissues of a mature animal. Efforts to develop methods for in vitro culture of ESC for research or eventual therapeutic applications are hampered by the lack of rapid, nondestructive assays for distinguishing ESC from other (differentiated) cells within a growing culture. Coherent anti-Stokes Raman scattering (CARS) microscopy is shown here to be a sensitive and nondestructive method for identifying mouse ESC based on selective observation of specific molecular vibrations believed to be spectroscopic markers indicating the differentiated vs undifferentiated states of such cells. The nonlinear nature of CARS also permits imaging with subcellular resolution, potentially offering a means by which chemical changes accompanying the early stages of differentiation may be associated with certain intracellular compartments (e.g., nucleus, cytoplasm, membranes). A novel exposure/collection configuration is described, which yields high collection efficiency and low interference from nonresonant background components.     

Transient bubble domain configuration in garnet materials observed using high speed photography

The dynamic domain configurations of bubbles in garnet materials have been studied using a sampling optical microscope capable of single exposure photographs with a 10 nsec exposure time. The microscope is an integral part of a sampling system so that the transient shape of the bubble is recorded at various times after a field pulse or, for bubbles in field access devices, during a clock cycle. A triggerable flowing nitrogen gas laser pumping a low Q Rodamine 6G Dye laser is used as an illumination source giving light pulses of ∼1.5 KW for 10 nsec. This light is sufficient to expose Kodak 4 × 16 mm movie film. Standard pulse generators (HP 214A) are used to make free bubble radial velocity measurements. A modified generator allows free bubble collapse measurement to be made. For bubbles propagating at operating frequency within field access devices, a standard bubble exerciser is used, synchronized to the sampling system. In this case, special samples with an internal mirror and epi-mode illumination are used. Illustrative results of bubble domain shapes in a chevron propagating structure and a 90° chevron expander detector are included.     

Detection of cytochrome C in a single cell using an optical nanobiosensor

In this work, the intracellular measurement of cytochrome c using an optical nanobiosensor is demonstrated. The nanobiosensor is a unique fiberoptics-based tool which allows the minimally invasive analysis of intracellular components. Cytochrome c is a very important protein to the process which produces cellular energy. In addition, cytochrome c is well-known as the protein involved in apoptosis, or programmed cell death. delta-Aminolevulinic acid (5-ALA) was used to induce apoptosis in MCF-7 human breast carcinoma cells. 5-ALA, a photodynamic therapy (PDT) drug in cells was activated by a HeNe laser beam. After the PDT photoactivation, the release of cytochrome c from the mitochondria to the cytoplasm in a MCF-7 cell was monitored by the optical nanobiosensor inserted inside the single cell and followed by an enzyme-linked immunosorbent assay (ELISA) outside the cell. The combination of the nanobiosensor with the ELISA immunoassay improved the detection sensitivity of the nanobiosensor due to enzymatic amplification. Our results lead to the investigation of an apoptotic pathway at the single cell level.     

Visualization in cryogenic environment: Application to two-phase studies

This paper reviews recent technical developments devoted to the study of cryogenic two-phase fluids. These techniques span from simple flow visualization to quantitative measurements of light scattering. It is shown that simple flow pattern configurations are obtained using classical optical tools (CCD cameras, endoscopes), even in most severe environments (high vacuum, high magnetic field). Quantitative measurements include laser velocimetry, particle sizing, and light scattering analysis. In the case of magnetically compensated gravity boiling oxygen, optical access is used to control the poistioning of a bubble subject to buoyancy forces in an experimental cell. Flow visualization on a two-phase superfluid helium pipe-flow, performed as a support of LHC cooldown studies, leads to flow pattern characterization. Visualization includes stratified and atomized flows. Thanks to the low refractive index contrast between the liquid and its vapor, quantitative results on droplet densities can be obtained even in a multiple scattering regime.     

Vortex breakdown generated by off-axis bifurcation in a cylinder with rotating covers

Summary Vortex breakdown of bubble type is studied for the flow in a cylinder with rotating top and bottom covers. For large ratios of the angular velocities of the covers, we observe numerically that the vortex breakdown bubble in the steady regime may occur through the creation of an off-axis vortex ring. This scenario does not occur in existing bifurcation theory based on a simple degeneracy in the flow field. We extend the theory to cover a non-simple degeneracy, and derive the associated bifurcation diagrams. We show that the vortex breakdown scenario involving a vortex ring can be explained from this theory, and that the numerically generated bifurcation diagrams are consistent with the theory.     

Nondestructive subharmonic imaging

Ultrasound contrast agent microbubbles are intravascular agents that can be used to estimate blood perfusion. Blood perfusion may be estimated by destroying the bubbles in a vascular bed and observing the refresh of contrast agents back into the vascular bed. Contrast agents can be readily destroyed by traditional imaging techniques. The design of a nondestructive imaging technique is necessary for the accurate quantification of contrast agent refresh. In this work, subharmonic imaging is investigated as a method for nondestructive imaging with the contrast agent microbubble MP1950 (Mallinckrodt, Inc., St. Louis, MO). Optical observation during insonation, in conjunction with a modified Rayleigh-Plesset (R-P) analysis, provides insight into the mechanisms of and parameters required for subharmonic frequency generation. Subharmonic imaging with a transmission frequency that is the same as the resonant frequency of the bubble is shown to require a minimum pressure of insonation that is greater than the experimentally-observed bubble destruction threshold. Subharmonic imaging with a transmission frequency that is twice the resonant frequency of the bubble produces a subharmonic frequency response while minimizing bubble instability. Optimization is performed using optical experimental analysis and R-P analysis     

Impact of release dynamics of laser-irradiated polymer micropallets on the viability of selected adherent cells

We use time-resolved interferometry, fluorescence assays and computational fluid dynamics (CFD) simulations to examine the viability of confluent adherent cell monolayers to selection via laser microbeam release of photoresist polymer micropallets. We demonstrate the importance of laser microbeam pulse energy and focal volume position relative to the glass–pallet interface in governing the threshold energies for pallet release as well as the pallet release dynamics. Measurements using time-resolved interferometry show that increases in laser pulse energy result in increasing pallet release velocities that can approach 10 m s⁻¹ through aqueous media. CFD simulations reveal that the pallet motion results in cellular exposure to transient hydrodynamic shear stress amplitudes that can exceed 100 kPa on microsecond timescales, and which produces reduced cell viability. Moreover, CFD simulation results show that the maximum shear stress on the pallet surface varies spatially, with the largest shear stresses occurring on the pallet periphery. Cell viability of confluent cell monolayers on the pallet surface confirms that the use of larger pulse energies results in increased rates of necrosis for those cells situated away from the pallet centre, while cells situated at the pallet centre remain viable. Nevertheless, experiments that examine the viability of these cell monolayers following pallet release show that proper choices for laser microbeam pulse energy and focal volume position lead to the routine achievement of cell viability in excess of 90 per cent. These laser microbeam parameters result in maximum pallet release velocities below 6 m s⁻¹ and cellular exposure of transient hydrodynamic shear stresses below 20 kPa. Collectively, these results provide a mechanistic understanding that relates pallet release dynamics and associated transient shear stresses with subsequent cellular viability. This provides a quantitative, mechanistic basis for determining optimal operating conditions for laser microbeam-based pallet release systems for the isolation and selection of adherent cells.     

Rapidly prototyped three-dimensional nanofluidic channel networks in glass substrates

Microfluidic and nanofluidic technologies have long sought a fast, reliable method to overcome the creative limitations of planar fabrication methods, the resolution limits of lithography, and the materials limitations for fast prototyping. In the present work, we demonstrate direct 3D machining of submicrometer diameter, subsurface fluidic channels in glass, via optical breakdown near critical intensity, using a femtosecond pulsed laser. No postexposure etching or bonding is required; the channel network (or almost any arbitrary-shaped cavity below the surface) is produced directly from "art-to-part". The key to this approach is to use very low energy, highly focused, pulses in the presence of liquid. Microbubbles that result from laser energy deposition gently expand and extrude machining debris from the channels. These bubbles are in a highly damped, low Reynolds number regime, implying that surface spalling due to bubble collapse is unimportant. We demonstrate rapid prototyping of three-dimensional "jumpers", mixers, and other key components of complex 3D microscale analysis systems in glass substrates.     

Cell death induced by the application of alternating magnetic fields to nanoparticle-loaded dendritic cells

In this work, the capability of primary, monocyte-derived dendritic cells (DCs) to uptake iron oxide magnetic nanoparticles (MNPs) is assessed and a strategy to induce selective cell death in these MNP-loaded DCs using external alternating magnetic fields (AMFs) is reported. No significant decrease in the cell viability of MNP-loaded DCs, compared to the control samples, was observed after five days of culture. The number of MNPs incorporated into the cytoplasm was measured by magnetometry, which confirmed that 1-5 pg of the particles were uploaded per cell. The intracellular distribution of these MNPs, assessed by transmission electron microscopy, was found to be primarily inside the endosomic structures. These cells were then subjected to an AMF for 30 min and the viability of the blank DCs (i.e. without MNPs), which were used as control samples, remained essentially unaffected. However, a remarkable decrease of viability from approximately 90% to 2-5% of DCs previously loaded with MNPs was observed after the same 30 min exposure to an AMF. The same results were obtained using MNPs having either positive (NH(2)(+)) or negative (COOH(-)) surface functional groups. In spite of the massive cell death induced by application of AMF to MNP-loaded DCs, the number of incorporated magnetic particles did not raise the temperature of the cell culture. Clear morphological changes at the cell structure after magnetic field application were observed using scanning electron microscopy. Therefore, local damage produced by the MNPs could be the main mechanism for the selective cell death of MNP-loaded DCs under an AMF. Based on the ability of these cells to evade the reticuloendothelial system, these complexes combined with an AMF should be considered as a potentially powerful tool for tumour therapy.     

Complex permittivity measurement as a new noninvasive tool for monitoring in vitro tissue engineering and cell signature through the detection of cell proliferation, differentiation, and pretissue formation

In in vitro tissue engineering, microporous scaffolds are commonly used to promote cell proliferation and differentiation in three-dimensional structures. Classic measurement methods are particularly time consuming, difficult to handle, and destructive. In this study, a new nondestructive method based on complex permittivity measurement (CPM) is proposed to monitor and track the osteoblast and macrophage differentiation through their morphological variation upon cell attachment and proliferation inside the microporous scaffolds. CPM is performed using a vector network analyzer and a dielectric probe under sterile conditions in a laminar-flow hood. A suitable effective medium approximation (EMA) is applied to fit the data in order to extract the parameters of the different constituents. Our data show that the EMA depolarization factor can be monitored to assess the variation of cell morphology characterizing cell attachment. Discrimination between two batches of scaffolds seeded, respectively, with 2 million and 1 million osteoblast cells is possible; the ratio of their CPM-derived cell volume fractions is in agreement with the ratio of their cell seeding numbers. In addition, cell proliferation inside scaffolds seeded with osteoblasts cultured in alpha minimum essential medium and inside scaffolds seeded with osteoblasts cultured in alpha minimum essential medium supplemented to induce the formation of extracellular matrix is monitored via CPM over several days. CPM-determined cell volume fraction is compared to DNA assay cell counts. Extracellular matrix formation and cell presence was confirmed by scanning electron microscopy. A set of three signature parameters (epsilon'mem, epsilon'cyt, kappa'cyt) characteristic of cell line is extracted from CPM. Distinct signatures are recorded for osteoblasts and macrophages, thus confirming the ability of CPM to discriminate between different cell types. This study demonstrates the potential of CPM as a diagnostic tool to monitor quickly and noninvasively cell growth and differentiation inside microporous scaffolds. Our findings suggest that the use of CPM could be extended to many biomedical applications, such as drug detection and automation of tissue and bacterial cultures in bioreactors.     

Photothermal nanoblade for large cargo delivery into mammalian cells

It is difficult to achieve controlled cutting of elastic, mechanically fragile, and rapidly resealing mammalian cell membranes. Here, we report a photothermal nanoblade that utilizes a metallic nanostructure to harvest short laser pulse energy and convert it into a highly localized explosive vapor bubble, which rapidly punctures a lightly contacting cell membrane via high-speed fluidic flows and induced transient shear stress. The cavitation bubble pattern is controlled by the metallic structure configuration and laser pulse duration and energy. Integration of the metallic nanostructure with a micropipet, the nanoblade generates a micrometer-sized membrane access port for delivering highly concentrated cargo (5 × 10(8) live bacteria/mL) with high efficiency (46%) and cell viability (>90%) into mammalian cells. Additional biologic and inanimate cargo over 3-orders of magnitude in size including DNA, RNA, 200 nm polystyrene beads, to 2 μm bacteria have also been delivered into multiple mammalian cell types. Overall, the photothermal nanoblade is a new approach for delivering difficult cargo into mammalian cells.     

Optical breakdown in fused silica and argon gas: application to Nd:YAG laser limiter

A gas cell filed with argon gas under pressure is placed in a tightly focused laser beam to provide a limiter for laser pulses above a certain peak power, corresponding to the optical breakdown threshold for the creation of a laser-induced plasma. Measurements of the threshold intensity as a function of argon gas pressure are given for a laser wavelength of 1.064 microm (Nd:YAG) and a pulse length of 6.4 ns. Threshold intensities for optical breakdown in fused silica were measured with the same optical system, enabling a relative comparison of breakdown thresholds, of interest for protecting fused-silica optical components in fiber-optic delivery systems for laser material processing applications. The threshold intensity was measured to 220 GW/cm2 in Ar at 1.0 x 10(5) N/m2 (1 atm), 80 GW/cm2 in Ar at 8.0 x 10(5) N/m2 (7.9 atm), and 55 GW/cm2 in fused silica. Even though the threshold in argon is higher than that in fused silica, the limiter will protect the optical components if the laser beam is focused to a tighter spot in the gas cell than at the input end of the fiber.     

Scaling considerations for sub-90 nm split-gate flash

A systematic methodology is presented to scale split-gate (SG) flash memory cells in the sub-90 nm regime within the presently known scaling constraints of flash memory. The numerical device simulation results show that the high performance sub-90 nm NOR-type SG cells can be achieved by a suitable channel and source-drain engineering. An asymmetric channel doping profile along with ultra-shallow source-drain junctions was used to achieve the target drain programming voltage (V_sp) for an efficient cell programming while keeping the cell breakdown voltage, BV > V_sp, with tolerable leakage currents. The study shows that with properly optimised technology parameters, 65 nm SG-NOR flash memory can be achieved with an adequate cell read current, a tolerable programmed cell leakage current at the read condition and efficient write and erase times.     

Towards ready-to-use 3-D scaffolds for regenerative medicine: adhesion-based cryopreservation of human mesenchymal stem cells attached and spread within alginate–gelatin cryogel scaffolds

Cultivation and proliferation of stem cells in three-dimensional (3-D) scaffolds is a promising strategy for regenerative medicine. Mesenchymal stem cells with their potential to differentiate in various cell types, cryopreserved adhesion-based in fabricated scaffolds of biocompatible materials can serve as ready-to-use transplantation units for tissue repair, where pores allow a direct contact of graft cells and recipient tissue without further preparation. A successful cryopreservation of adherent cells depends on attachment and spreading processes that start directly after cell seeding. Here, we analyzed different cultivation times (0.5, 2, 24 h) prior to adhesion-based cryopreservation of human mesenchymal stem cells within alginate–gelatin cryogel scaffolds and its influence on cell viability, recovery and functionality at recovery times (0, 24, 48 h) in comparison to non-frozen control. Analysis with confocal laser scanning microscopy and scanning electron microscopy indicated that 2 h cultivation time enhanced cryopreservation success: cell number, visual cell contacts, membrane integrity, motility, as well as spreading were comparable to control. In contrast, cell number by short cultivation time (0.5 h) reduced dramatically after thawing and expanded cultivation time (24 h) decreased cell viability. Our results provide necessary information to enhance the production and to store ready-to-use transplantation units for application in bone, cartilage or skin regenerative therapy.     

Optothermophysical properties of demineralized human dental enamel determined using photothermally generated diffuse photon density and thermal-wave fields

Noninvasive dental diagnostics is a growing discipline since it has been established that early detection and quantification of tooth mineral loss can reverse caries lesions in their incipient state. A theoretical coupled diffuse photon density and thermal-wave model was developed and applied to photothermal radiometric frequency responses, fitted to experimental data using a multiparameter simplex downhill minimization algorithm for the extraction of optothermophysical properties from artificially demineralized human enamel. The aim of this study was to evaluate the reliability and robustness of the advanced fitting algorithm. The results showed a select group of optical and thermal transport parameters and thicknesses were reliably extracted from the computational fitting algorithm. Theoretically derived thicknesses were accurately predicted, within about 20% error, while the estimated error in the optical and thermal property evaluation was within the values determined from early studies using destructive analyses. The high fidelity of the theoretical model illustrates its efficacy, reliability, and applicability toward the nondestructive characterization of depthwise inhomogeneous sound enamel and complex enamel caries lesions.     

The use of rat, rabbit or human bone marrow derived cells for cytocompatibility evaluation of metallic elements

Rat, rabbit and human bone marrow cells were cultured according to the method previously reported for cells of rat origin [1] and were exposed, or not (control), to corrosion products of a Co–Cr orthopaedic alloy as well as to metal salts containing Co2+, Cr3+ and Cr6+. Cells were cultured for 21 days and analysed for the following biochemical parameters: intracellular MTT reduction (i.e. cell viability/proliferation), alkaline phosphatase (ALP) activity and protein production. Morphological observations included both histochemistry (detection of ALP-positive cells, calcium and phosphate deposits) and scanning electron microscopy (SEM). Control cultures of rat and rabbit cells showed higher proliferation rates than human cells at the start of culture, but they all reached similar values on day 21. Protein production was parallel to cell proliferation. In contrast, ALP activity of rat cultures was much stronger than rabbit or human cultures. All cell types were able to develop the osteogenic phenotype in vitro.Co–Cr extract caused inhibitory effects on cell viability, on ALP activity and, to a lower extent, on protein production of all rat, rabbit and human cell cultures. Compared to rat and rabbit cultures, human cultures were the most sensitive to metal ions exposure.