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Local Ablation of a Single Cell Using Micro/Nano Bubbles
Abstract
We have successfully produced mono-dispersed microgas bubbles less than around 10 μm in diameter in an electrically induced ultrasonic field. The discharged output power and conductive area of the micro-electrode are controlled by glass shell insulation around the copper micro-wire. A small space between the wire and glass tip, a "bubble reservoir," contributes to the stabilization of the electric discharge and directional bubble generation. The directionally dispensed bubbles can be used for processing soft materials such as biological cells. For the present study, the cell membrane has successfully been processed with resolution of a few μm order and without any thermal collateral damage.
... This generates bubbles in front of the injector and shoots the particles in front of the injector into the cell owing to the sudden increase in pressure. (22) As a result, not only will this open a hole, it will also insert the nearby injection solution into the cell, completing the injection without any further action from the operator. ...
This chapter describes the mechanism and structure of the electrically‐induced bubble knife developed by the authors and its wide applications. The novelties are simultaneous local reagent injection and perforation of materials with a wide range of hardness using bubble cavitation and plasma cavitation. Cavitation and plasma discharge were generated by the pulse discharge of a microelectrode having a special tip structure. Injection of soft materials such as animal cells was performed only by bubble cavitation, and pore formation of hard materials such as seeds of plants or metals was achieved by the synergistic effect of bubble cavitation and plasma ablation. The chapter shows the fundamental background of an electrically (and plasma)‐induced bubble knife and injector and their biomedical applications including protein crystallization, which leads to a wide range of biomedical applications.
We have successfully carried out local reagent injection and perforation to materials of variety of hardness by physical and electric stimulation by bubble cavitation and plasma discharge. This synergetic effect was generated by pulse discharge of microelectrode having special tip structure. Injection to soft material such as animal cell was performed only by collapse of directional bubbles, whilst the perforation to hard materials such as plants cells or metals were achieved by synergistic effect of collapse of bubble and plasma ablation. The moment of bubble collapse and simultaneous generation of shock wave was visualized by high-speed camera and Schlieren method. This novel device we proposed has ability to perforate wide range of materials with high dynamic range, and the simultaneous reagent transportation can be applied to wide biomedical applications.
This paper investigated the mechanism of the condensation of protein molecules on air-liquid interface of bubbles. Electrically charged bubbles were confirmed to have clear advantage of producing protein crystal over general bubbles or conventional vapor diffusion method for the first time. The adsorption force between the protein molecular and air-liquid interface of bubble was successfully measured using force sensing optical tweezers. These findings contribute to the effective production of protein crystal and to the protein crystallography.
Minimally-intrusive ablation of cell has been successfully carried out by using the breakdown of electrically-induced bubble. This novel method of fine ablation method provides non-thermal damage on the surface of biological cell as well as minimally invasiveness because this resolution is depend on the width of capillary bubble. This new method of ablation contributes to cell surgery or precise fabrication of soft materials.
We have successfully carried out gene transfer using electrically induced bubble knife. The air-liquid interfaces of the dispensed bubbles have novel characteristics which can carry the various kinds of the materials in different phase and viscosity of solution. This technique has advantages in high efficiency of injection of limited amount of valuable samples such as designed m-RNA. In this paper, we demonstrated m-RNA encoding 3xFLAG-GST into xenopus oocyte to be sure that electrically induced bubble knife has ability to injection chemically unstable and biocompatibility. Furthermore, we injected fluorescent beads whose size was 2.1 μm into BY-2 cell as multiphase injection.
We have succeeded in protein crystallization by electrically-driven mono-dispersed micro-bubble knife. The dispensed directional micro-bubbles whose air-liquid interface tends to combine the ambient molecules due to by their stiction force on the surface of micro-bubbles. These characteristics enable to agglomerate the protein molecule which brings to successful protein crystallization in the solution with low concentration. The rate of protein crystal-nucleation by electrically-driven mono-dispersed micro-bubble was much higher than that of control condition. It was confirmed that induction force of protein crystallization by electrically-driven mono-dispersed micro-bubble increased with the amplification of input power of electrical circuit of electrically-induced micro-bubble knife.
This paper reports a novel micro-fluidic platform for carving protein crystal with electrically driven high-speed mono-dispersed micro-bubble jet. This minimally invasive micro-processing method overcomes the difficulties of processing, holding and positioning fragile material such as protein crystal underwater. The combination of using electrically-induced micro-bubble knife and microfluidic channel provide effective carving of protein crystal by ablation crystal and draining of chips by free vortex flow in microchannel. Three-dimensional positioning of crystal was sufficiently achieved by the configuration of effective micro-fluidic channels. The protein crystal can be carved to the desired shape to fit to X-ray analysis effectively. It seemed that it has potential to contribute to more precise protein analysis
We have succeeded in protein crystallization and processing by electrically-induced bubble knife. The dispensed directional mono-dispersed micro-bubbles whose air-liquid interface tends to combine the ambient molecules due to by their stiction force on the surface of micro-bubbles. Electrically-induced bubble knife enables to generate mono-dispersed micro-bubbles by the electrolysis and local heat, and their generation speed was 30.8 kHz. These characteristics enable to agglomerate the protein molecule which brings to successful protein crystallization in the solution with low concentration. The rate of protein crystal-nucleation by electrically-induced mono-dispersed micro-bubble was much higher than that of control condition. Furthermore, it was confirmed that cavitation induced by electrically-induced bubble knife enabled to curve protein crystal. It seemed that it has potential to contribute to more refined analysis.
This review surveys some capillary flows capable of stretching fluid interfaces down to the micrometric dimension and below. These types of flows have become the basis for attractive techniques to produce monodisperse micro- and nanoparticles with either simple or core-shell structure (micro- and nanocapsules, coaxial nanofibers, and hollow nanofibers). These techniques enable precise control on the particle mean size. We also review the basic physics of the flow and the dimensionless parameters that govern these capillary flows. Some examples of the types of particles produced by a few devices are also presented.
Electrosurgery is broadly used in a wide variety of surgical procedures, yet its underlying mechanisms of interaction are poorly characterized. Fundamentals of electrosurgery have not changed much since the 1930s—cutting is still performed using continuous RF waveforms, leaving a collateral damage zone of hundreds of micrometers in depth. Pulsed waveforms with variable duty cycle are used mostly for tissue coagulation. Recently, we have demonstrated that electrosurgery with microsecond bursts applied via microelectrodes can provide cellular precision in soft tissue dissection. This paper examines dynamics of pulsed electrical discharges in conductive medium, and accompanying phenomena, such as vaporization, cavitation and ionization. It is demonstrated that ionization of the vapor cavity around the electrode is essential for energy delivery beyond the vaporization threshold. It is also shown that the ionization threshold voltage and resistance of the plasma-mediated discharge are much lower in the negative phase of the discharge than in the positive one. Capacitive coupling of the ac waveform to the electrode compensates for this asymmetry by shifting the medium voltage on the electrode, thus increasing the positive and decreasing the negative amplitudes to achieve charge balance in the opposite phases. With planar insulated electrodes having exposed edges of 12.5 μm in width and bursts of 40 μs in duration even tough biological tissues can be dissected with cellular precision. For example, cartilage dissection is achieved with pulse energy of 2.2 mJ per millimeter of length of the blade, and leaves a thermal damage zone of only 5–20 μm in width.
Cavitation bubbles collapsing and rebounding in a pressure gradient ∇p form a "microjet" enveloped by a "vapor jet." This Letter presents unprecedented observations of the vapor jets formed in a uniform gravity-induced ∇p, modulated aboard parabolic flights. The data uncover that the normalized jet volume is independent of the liquid density and viscosity and proportional to ζ ≡ |∇p|R(0)/Δp, where R(0) the maximal bubble radius and Δp is the driving pressure. A derivation inspired by "Kelvin-Blake" considerations confirms this law and reveals its negligible dependence of surface tension. We further conjecture that the jet only pierces the bubble boundary if ζ ≳ 4 × 10(-4).
Electrospun nanofibers composed of biodegradable polymers are attractive candidates for cell culture scaffolds in tissue engineering. Their fine-meshed structures, resembling natural extracellular matrices, effectively interact with cell surfaces and promote cell proliferation. The application of electrospinning, however, is limited to two-dimensional (2D) or single tube-like scaffolds, and the fabrication of arbitrary three-dimensional (3D) scaffolds from electrospun nanofibers is still very difficult due to the fibers’ continuous and entangled form. To address this issue, in this paper, we describe the use of phase-separation-assisted electrospray and electrostatic focusing to perform continuous direct 3D patterning of nanofibrous microcapsules of biodegradable polylactic acid (PLA). These microcapsules exhibit fiber-particle duality because they are composed of nanofibers suitable for cell attachment while also being easy to handle as particles for direct 3D patterning. By varying the flow rate of the polymer solution and the humidity of the electrospray atmosphere during electrospraying, the diameter of the microcapsule and its surface porosity can be controlled. The utility of the direct-patterning process is demonstrated by fabricating high-aspect-ratio microscaffolds and subsequent cell cultures. The nanofibrous and hollow structure of the microcapsules combined with the direct 3D patterning process offers a new approach for fabricating tailor-made scaffolds for regenerative medicine.
Electronic supplementary material
The online version of this article (doi:10.1007/s10544-011-9583-x) contains supplementary material, which is available to authorized users.
To develop a better and more economical instrument for precise, tractionless, "cold" cutting during intraocular surgery. The use of highly localized electric fields rather than laser light as the means of tissue dissection was investigated.
A high electric field at the tip of a fine wire can, like lasers, initiate plasma formation. Micrometer-length plasma streamers are generated when an insulated 25 micron (microm) wire, exposed to physiological medium at one end, is subjected to nanosecond electrical pulses between 1 and 8 kV in magnitude. The explosive evaporation of water in the vicinity of these streamers cuts soft tissue without heat deposition into surrounding material (cold cutting). Streamers of plasma and the dynamics of water evaporation were imaged using an inverted microscope and fast flash photography. Cutting effectiveness was evaluated on both polyacrylamide gels, on different tissues from excised bovine eyes, and in vivo on rabbit retina. Standard histology techniques were used to examine the tissue.
Electric pulses with energies between 150 and 670 microJ produced plasma streamers in saline between 10 and 200 microm in length. Application of electric discharges to dense (10%) polyacrylamide gels resulted in fracturing of the gel without ejection of bulk material. In both dense and softer (6%) gels, layer by layer shaving was possible with pulse energy rather than number of pulses as the determinant of ultimate cutting depth. The instrument made precise partial or full-thickness cuts of retina, iris, lens, and lens capsule without any evidence of thermal damage. Because different tissues require distinct energies for dissection, tissue-selective cutting on complex structures can be performed if the appropriate pulse energies are used; for example, retina can be dissected without damage to the major retinal vessels.
This instrument, called the Pulsed Electron Avalanche Knife (PEAK), can quickly and precisely cut intraocular tissues without traction. The small delivery probe and modest cost make it promising for many ophthalmic applications, including retinal, cataract, and glaucoma surgery. In addition, the instrument may be useful in nonophthalmic procedures such as intravascular surgery and neurosurgery.
Miniaturization is the central theme in modern fabrication technology. Many of the components used in modern products are getting smaller and smaller. In this paper, the recent development of the electron beam lithography technique is reviewed with an emphasis on fabricating devices at the nanometer scale. Because of its very short wavelength and reasonable energy density characteristics, e-beam lithography has the ability to fabricate patterns having nanometer feature sizes. As a result, many nanoscale devices have been successfully fabricated by this technique. Following an introduction of this technique, recent developments in processing, tooling, resist, and pattern controlling are separately examined and discussed. Examples of nanodevices made by several different e-beam lithographic schemes are given, to illustrate the versatility and advancement of the e-beam lithography technique. Finally, future trends in this technique are discussed.
Ti honeycombs with the side of 800 and 400 nm were fabricated by focused ion beam (FIB), though the surfaces of the bottom and wall of the Ti honeycombs were rough, as compared with the surfaces of the bottom and wall of the Si honeycomb. It is demonstrated that the nanoscale Ti components can be fabricated in a short time by FIB.
The electric knife is one of the indispensable surgical devices widely used in surgical operation. However, it has not been remarkably improved since its invention several decades ago. For example, thermal damage of the current radiofrequency electric knife, which is one of the most nonintrusive type of electric knives, is more than several hundred micrometers. A laser device, which was invented approximately 70 years ago, has been developed remarkably, and various research report the interaction between the cells and laser to fulfill the cellular-order ablation [1], [2].
Consecutive images of the fragmentation of capillary waves in an ultrasonic field were obtained using a high-speed video camera through a microscope at a frame rate of 500000 frames per second. The images showed that micro bubbles of uniform diameter from 4 to 15m were generated at a constant periodic rate when a small amount of gas was introduced (via a needle) into a highly viscous liquid whose kinematic viscosity was between 5 and 100 mm. Conditions for stable generation of micro bubbles of uniform diameter were also studied by changing the inner diameter of the needle between 0.08 and 0.34 mm, excitation frequency of around 18.77 and 42.15 kHz, kinematic viscosity of liquid between 5 and 100 mm, surface tension between 20 and 34 mN m, and viscosity of gas between 9.0 and 31.7Pa s. Results revealed that (i) a projection is formed on the oscillatory gas–liquid interface and micro bubbles are released from the tip of the projection; (ii) gas viscosity critically affects the formation of the projection and should be around 20.0Pa s for stable mother bubble oscillation; (iii) conditions for stable generation of micro bubbles are also affected by excitation frequency, surface tension and viscosity of the liquid, and dimensions of the needle; (iv) two controlling parameters for stable generation are the Weber number (, where is the density of the liquid, f is the excitation frequency, is the inner diameter of the needle, and is the surface tension) and the Womersley number (, where is the kinematic viscosity of liquid); and (v) uniform-diameter micro bubbles are generated stably when and . Under the conditions where micro bubbles of uniform diameter were stably generated, the bubble diameter increased almost linearly with increasing gas pressure inside the needle. The gradient of this linear function can be expressed as a function of Wo We, and the normalized outer diameter of the needle, and decreases either with decreasing inner diameter of the needle or with increasing excitation frequency, surface tension and viscosity of the liquid, and outer diameter of the needle.
In this paper, we developed and fabricated a magnetically driven microtool (MMT) and installed it on a microfluidic chip for use in the enucleation of oocytes. The fabricated tool is much smaller than a conventional mechanical micromanipulator used for cell manipulation. We succeeded in driving this MMT in two degrees of freedom-in the X - and Y -directions. The MMT works on the principle of noncontact actuation by magnetic force; therefore, the microfluidic-chip part is fully disposable and inexpensive. The MMT consists of a polymer part with a controllable attitude and a rigid metal (Ni) part with good magnetic properties, which are useful for cutting oocytes. We analytically evaluated that the structure for easy attitude control of the polymer part is a four-leg-type configuration. Based on the novel and original design, the MMT and microfluidic chip were fabricated by photolithography. The MMT could generate a force of 3 mN, which is sufficient to cut an oocyte into half. We successfully demonstrated the cutting of an oocyte on a microfluidic chip by using the MMT.
This paper presents two innovative driving methodologies using a magnetically driven microtool (MMT) for precise cell manipulations and automation systems. First, magnetic analysis has been conducted to show the current MMT problem and proved that static friction makes MMT control difficult. New driving methodologies that reduce the friction on the MMT effectively are introduced, and supported by finite element analysis and experimental results. The positioning accuracy improves 3–10 times and the response speeds become 10 times faster against the driving linear stage than in the conventional drive method. Stage feedback control by PI with a disturbance observer has been also investigated in order to obtain precise positioning accuracy and this was successfully improved by 16 times as compared to the conventional drive. Using this methodology, the enucleation of oocytes is demonstrated to show the effectiveness of the method. The required force to cut a swine oocyte is also estimated by the simplified model to prove that the MMT has sufficient force. Two MMT blades made of nickel were set on the microfluidic chip with a new drive methodology and successfully achieved the enucleation process with high throughput.
An electro-conjugate fluid (ECF) is a type of dielectric and functional fluid that generates a powerful jet flow when subjected to high DC voltage. Although a high voltage is needed to generate the jet flow, the current is quite low at several microamperes, resulting in a total power consumption of several milliwatts. Using this smart fluid, we can develop micro fluid-driven mechanical components without any bulky pumps. Also, it is clarified that the power density of the ECF jet is higher when the electrode pair is miniaturized; therefore, it is suitable for micro actuators. Here, we propose and fabricate three types of soft actuators with an antagonistic configuration: (i) micro artificial muscle cells, (ii) a McKibben-type micro artificial muscle actuator using the ECF effect and (iii) a micro finger actuator with two chambers to bend. The actuators basically consist of a silicone rubber tube covered with a fiber sleeve and a micro pressure source using the ECF effect. Next, we apply and integrate these actuators into a micro robot hand, driven with ECF jets. The driving characteristics of the micro artificial muscle actuator and the integrated micro ECF hand with ECF fingers were fabricated and experimentally investigated. The experimental results show that this ECF jet actuation is effective for driving soft micro hands.
We have successfully produced emulsion droplets on a chip with size control and on-demand droplet generation by using a magnetically driven microtool (MMT). The MMT has a parallel plate structure to be constrained in translational motion. With a lateral motion of MMT in microchannels, the continuous phase can be cut into different size droplets and the dispersed phase flow can inflow into the microchannels by the movement of the MMT to obtain both size-controlled and on-demand droplets actively. The size range of the produced droplet was improved 3 times as large as the previous system by employing a novel hydraulic design of the chip and also leakage of the fluid was successfully prevented when it was chopped by using the MMT.
Laboratory cultures of a single species of bacteria harboring the same genetic background include heterogeneous cell populations, each differing in apparent morphology and physiology, as found in natural environments. To get insights into difference in the genome expression between individual cells, we constructed various types of the cell chip for monitoring the growth and fate of individual bacterial cells. Immobilization of portions of Escherichia coli culture within these cell chips was established after raising the local temperature in the presence of poly-(N-isopropylacrylamide) (PNIPAAm). The newly developed cell-chip system allows the investigation of activity and regulation of green fluorescent protein (GFP)-fused promoter in single live-bacterial cells for prolonged time under controlled culture conditions. Using this single-cell observation system, we succeeded, for the first time, the real-time single-cell assay of promoter activity of the E. coli gcl gene encoding glyoxylate carboligase as a model system, and the kinetics of gcl induction by an effector glyoxylate. Marked heterogeneity was found in the expression level of the gcl promoter. The heterogeneity in gcl promoter activity was, however, confirmed by Flow cytometry of suspension cultures. Our success provides an experimental system for the increased demand of single-cell biology in bacterial studies.
To evaluate a new pulsed-electron avalanche knife design for creating a continuous curvilinear capsulotomy (CCC) and compare the CCC with a mechanical capsulorhexis.
Department of Ophthalmology, Stanford University, Stanford, California, USA.
In this study, CCCs were created in freshly enucleated bovine eyes and in rabbit eyes in vivo. The cutting velocity was adjusted by controlling the burst repetition rate, voltage amplitude, and burst duration. Tissue samples were fixed and processed for histology and scanning electron microscopy (SEM) immediately after surgery.
The study included 50 bovine eyes and 10 rabbit eyes. By adjusting the electrosurgical waveforms, gas-bubble formation was minimized to permit good surgical visualization. The optimum voltage level was determined to be +/-410 V with a burst duration of 20 mus. Burst repetition rate, continuously adjustable from 20 to 200 Hz with footpedal control, allowed the surgeon to vary linear cutting velocity up to 2.0 mm/s. Histology and SEM showed that the pulsed-electron avalanche knife produced sharp-edged capsule cutting without radial nicks or tears.
The probe of the pulsed-electron avalanche knife duplicated the surgical feel of a 25-gauge cystotome and created a histologically smooth capsule cut. It may improve precision and reproducibility of creating a CCC, as well as improve its proper sizing and centration, especially in the face of surgical risk factors, such as weak zonules or poor visibility. FINANCIAL DISCLOSURES: Drs. Palanker and Vankov hold patents to the pulsed electron avalanche knife technology, which are licensed to PEAK Surgical by Stanford University. Drs. Palanker and Chang are consultants to PEAK Surgical. Dr. Vankov is an employee of PEAK Surgical. Neither of the other authors has a financial or proprietary interest in any material or method mentioned.
The PEAK PlasmaBlade is a new electrosurgical device that uses pulsed radiofrequency to generate a plasma-mediated discharge along the exposed rim of an insulated blade, creating an effective cutting edge while the blade stays near body temperature.
Full-thickness incisions were made on the dorsums of pigs with the PlasmaBlade, a conventional electrosurgical device, and a scalpel, and blood loss was quantified. Wounds were harvested at designated time points, tested for wound tensile strength, and examined histologically for scar formation and tissue damage.
Bleeding was reduced significantly (59 percent) in PlasmaBlade incisions compared with scalpel incisions, and acute thermal damage from the PlasmaBlade (66 +/- 5 microm) was significantly less than both cut and coagulation mode electrosurgical incisions (456 +/- 35 microm and 615 +/- 22 microm, respectively). Histologic scoring for injury and wound strength was equivalent between the PlasmaBlade and scalpel incisions. By 6 weeks, the healed PlasmaBlade and scalpel incisions were approximately three times stronger, and scar cosmetic appearance was significantly better compared with electrosurgical incisions.
The PlasmaBlade is a promising new surgical instrument that provides atraumatic, scalpel-like cutting precision and electrosurgical-like hemostasis, resulting in minimal bleeding, tissue injury, and scar formation.
A rapid, viable and safe fabrication method for biomaterials synthesis is reported using high-frequency AC electrospraying. We demonstrate its potential for polymeric nanoparticle fabrication, drug encapsulation in mono-dispersed micron-sized biodegradable polymer shells and the synthesis of 1microm biodegradable fibers with adjustable pore sizes as bioscaffolds for tissue/orthopaedic engineering and wound care therapy. The absence of charge in the ejected drops and fibers facilitates pulmonary drug delivery, polymer encapsulation and minimizes protein/DNA denaturing or compound ionization.
This article describes the process of formation of droplets and bubbles in microfluidic T-junction geometries. At low capillary numbers break-up is not dominated by shear stresses: experimental results support the assertion that the dominant contribution to the dynamics of break-up arises from the pressure drop across the emerging droplet or bubble. This pressure drop results from the high resistance to flow of the continuous (carrier) fluid in the thin films that separate the droplet from the walls of the microchannel when the droplet fills almost the entire cross-section of the channel. A simple scaling relation, based on this assertion, predicts the size of droplets and bubbles produced in the T-junctions over a range of rates of flow of the two immiscible phases, the viscosity of the continuous phase, the interfacial tension, and the geometrical dimensions of the device.
Techniques for stable, rapid and repeatable small-animal immobilization are necessary for high-throughput in vivo genetic/drug screens using cellular and sub-cellular features in multi-cellular organisms. We demonstrate a method for non-invasive and high-throughput on-chip immobilization of physiologically active C. elegans without the use of anesthesia or cooling, but with comparable stability even for the most demanding purposes. We show observation and manipulation of sub-cellular features in immobilized animals using two-photon microscopy and femtosecond-laser microsurgery.
Manual for the enucleation of bovine oocyte and evaluation of quality of embryo
- S Watanabe