Cell extraction automation in single cell surgery using the displacement method.

Micromanipulation is the precise in vitro handling and study of individual biological cells, where the smallest error can be disastrous. One such example is the extraction of cellular material from multicellular organisms, such as cells from early stage embryos. In this paper, we propose automation methods for the extraction and retrieval of individual cells from a multicellular organism in vitro using the displacement method. Computer-controlled syringe pumps and micromanipulators combined with custom computer vision algorithms are used for automated cell extraction and retrieval. Automation feasibility is demonstrated through automated controlled extraction of one or two blastomeres from cleavage-stage embryos. Preliminary proof of concept blastomere extraction experiments involving mouse embryos obtained success rates ranging from 72% to 88% for the different extraction tasks: displacement, detection, and retrieval. These automated blastomere extraction experiments demonstrate that automated cell extraction is indeed feasible, but the process may still be improved. To the best of these authors' knowledge, this paper is the first to report the automation of single cell extraction from multicellular organisms using the displacement method, and especially for automated blastomere extraction from cleavage-stage embryos. These methods provide a set of tools for moving towards fully automated single cell surgery procedures.

Effects of dielectrophoresis on thrombogenesis in human whole blood.

Thrombogenesis (blood clot formation) is a major barrier to the development of biomedical devices that interface with blood. Although state-of-the-art chemically and pharmacologically mediated clot mitigation strategies are effective, some limitations of such approaches include depletion of active agents, or adverse reactions in patients. Increased clotting protein adsorption and platelet adhesion, which occur when artificial surfaces are exposed to blood result in enhanced clot formation on artificial surfaces. It is hypothesized that repelling proteins and platelets using dielectrophoresis (DEP), a contact-free particle manipulation technique, will reduce clot formation in biomedical devices. In this paper, the effect of DEP on thrombogenesis in human blood is investigated. Undiluted whole blood from human donors is pumped through microchannels at a physiological shear rate (400 s-1 ). Experiments are performed by applying 0 V, 0.5 Vrms , 2 Vrms , and 3 Vrms to electrodes in the channel. Clot formation is observed to decrease in experiments in which DEP electrodes are active (average of 6% coverage @ 0V reduced to 0.08% coverage @ 3 Vrms ). Repulsion is more effective at higher voltages. DEP causes a quantifiable reduction in microscopic and macroscopic clot formation in PDMS microchannels.

Design of optimal electrode geometries for dielectrophoresis using fitness based on simplified particle trajectories.

Dielectrophoretic (DEP) forces applied to microscopic particles are highly dependent on the gradient of the electric field experienced by the particles. These DEP forces can be used to selectively capture and remove cells from fluid flows within a micro-channel above the DEP electrodes. Modification of the geometry of the electrodes that generate the electric field is the main approach available to increase the electric field gradient over a wide area, and hence increase the applied dielectrophoretic force. Optimized DEP forces increase attraction or repulsion of target cells from the electrode surface, enhancing the efficacy of electrodes for cell sorting applications. In this paper, we present a design approach, using genetic optimization techniques, to develop novel electrode geometries that effectively capture target particles. The performance of candidate electrode designs is evaluated by calculating simplified particle trajectories.

Simulation of a high-speed superimposed fiber Bragg gratings interrogation system.

This paper presents a simulation of high-speed nonuniform random sampling in a superimposed fiber Bragg gratings (SFBGs) interrogation system. The simulated Gauss SFBGs are used to generate a nonuniform sensing pulse train during each scanning cycle. Six different conditions that can cause nonuniform sampling are simulated, and a random sine-wave driving method to improve the driving speed is proposed. An 11.8 kHz dynamic strain is measured by generating an additive nonuniform randomly distributed 12 kHz optical sensing pulse train from a mean 2 kHz sinusoidal periodically changing scanning frequency and three SFBGs. Four conditions that can improve the sampling results are also simulated.

A universal piezo-driven ultrasonic cell microinjection system.

Over the past decade, the rapid development of biotechnologies such as gene injection, in-vitro fertilization, intracytoplasmic sperm injection (ICSI) and drug development have led to great demand for highly automated, high precision equipment for microinjection. Recently a new cell injection technology using piezo-driven pipettes with a very small mercury column was proposed and successfully applied in ICSI to a variety of mammal species. Although this technique significantly improves the survival rates of the ICSI process, shortcomings due to the toxicity of mercury and damage to the cell membrane due to large lateral tip oscillations of the injector pipette may limit its application. In this paper, a new cell injection system for automatic batch injection of suspended cells is developed. A new design of the piezo-driven cell injector is proposed for automated suspended cell injection. This new piezo-driven cell injector design relocates the piezo oscillation actuator to the injector pipette which eliminates the vibration effect on other parts of the micromanipulator. A small piezo stack is sufficient to perform the cell injection process. Harmful lateral tip oscillations of the injector pipette are reduced substantially without the use of a mercury column. Furthermore, ultrasonic vibration micro-dissection (UVM) theory is utilized to analyze the piezo-driven cell injection process, and the source of the lateral oscillations of the injector pipette is investigated. From preliminary experiments of cell injection of a large number of zebrafish embryos (n = 200), the injector pipette can easily pierce through the cell membrane at a low injection speed and almost no deformation of the cell wall, and with a high success rate(96%) and survival rate(80.7%) This new injection approach shows good potential for precision injection with less damage to the injected cells.

Automated Embryo Manipulation and Rotation via Robotic nDEP-Tweezers.

Embryo manipulation is a fundamental task in assisted reproductive technology (ART). Nevertheless, conventional pick-place techniques often require proper alignment to avoid causing damage to the embryo and further, the tools have limited capability to orient the embryo being handled. OBJECTIVE: This paper presents a novel and non-invasive technique that can easily manipulate mouse embryos on a polyvinyl chloride (PVC) Petri dish. METHODS: An inverted microchip with quadrupole electrodes was attached to a micromanipulator to become a robotic dielectrophoresis (DEP) tweezers, and a motorized platform provided additional mobility to the embryos lying on a Petri dish. Vision-based algorithms were developed to evaluate relevant information of the embryos from the image, and to provide feedback signals for precise position and orientation control of the embryo. RESULTS: A series of experiments was conducted to examine the system performance, and the embryo can be successfully manipulated to a specified location with the desired orientation for subsequent processing. CONCLUSION: This system offers a non-contact, low cost, and flexible method for rapid cell handling. SIGNIFICANCE: As the DEP tweezers can grasp the embryo without the need for precise alignment, the overall time required to process a large number of embryos can be shortened.

Automatic System for the Blastocyst Embryo Manipulation and Rotation.

Cell manipulation plays a vital role in the success rate and efficiency of the cell microsurgical operations, including biopsy of cell internal organelles such as the embryo biopsy, in which the embryo is manipulated and reoriented safely to a predefined desired position and orientation. In this paper, a simplified approach for the blastocyst embryo reorientation is proposed. It utilizes conventional tools and techniques currently in use in manual approaches in research labs and In Vitro Fertilization clinics, and controls the process using a vision feedback system. An experimental setup is developed to verify the dynamic behavior of the proposed approach, in which a stationary holding micropipette is used to hold the embryo, which is then rotated in two coordinate directions through friction contact with a moving substrate, in our case a glass microscope slide. The embryo rotates on the holding micropipette tip, due to the relatively low friction of this contact. A computer vision algorithm is used to estimate the embryo orientation coordinates, and use this information as a feedback signal to a simple proportional controller to control the embryo rotation angle. Experimental results demonstrate that the system is capable of cell rotation in two independent coordinates, suitable for embryo microsurgical task execution.

Out-of-Plane Rotation Control of Biological Cells With a Robot-Tweezers Manipulation System for Orientation-Based Cell Surgery.

In many cell surgery applications, cell must be oriented properly such that the microsurgery tool can access the target components with minimum damage to the cell. In this paper, a scheme for out of image plane orientation control of suspended biological cells using robotic controlled optical tweezers is presented for orientation-based cell surgery. Based on our previous work on planar cell rotation using optical tweezers, the dynamic model of cell out-of-plane orientation control is formulated by using the T-matrix approach. Vision-based algorithms are developed to extract the cell out of image plane orientation angles, based on 2-D image slices obtained under an optical microscope. A robust feedback controller is then proposed to achieve cell out-of-plane rotation. Experiments of automated out of image plane rotational control for cell nucleus extraction surgery are performed to demonstrate the effectiveness of the proposed approach. This approach advances robot-aided single cell manipulation and produces impactful benefits to cell surgery applications such as nucleus transplantation and organelle biopsy in precision medicine.

Achieving Automated Organelle Biopsy on Small Single Cells Using a Cell Surgery Robotic System.

Single cell surgery such as manipulation or removal of subcellular components or/and organelles from single cells is increasingly used for the study of diseases and their causes in precision medicine. This paper presents a robotic surgery system to achieve automated organelle biopsy of single cells with dimensions of less than 20 µm in diameter. The complexity of spatial detection of the organelle position is reduced by patterning the cells using a microfluidic chip device. A sliding mode nonlinear controller is developed to enable extraction of organelles, such as the mitochondria and the nucleus, from single cells with high precision. An image processing algorithm is also developed to automatically detect the position of the desired organelle. The effectiveness of the proposed robotic surgery system is demonstrated experimentally with automated extraction of mitochondria and nucleus from human acute promyelocytic leukemia cells and human fibroblast cells. Extraction is followed by biological tests to indicate the functionality of biopsied mitochondria as well as the cell viability after removal of mitochondria. The results presented here have revealed that the proposed approach of automated organelle biopsy on single small cells is feasible.

Automation and Optimization of Multipulse Laser Zona Drilling of Mouse Embryos During Embryo Biopsy.

Laser zona drilling (LZD) is a required step in many embryonic surgical procedures, for example, assisted hatching and preimplantation genetic diagnosis. LZD involves the ablation of the zona pellucida (ZP) using a laser while minimizing potentially harmful thermal effects on critical internal cell structures. OBJECTIVE: Develop a method for the automation and optimization of multipulse LZD, applied to cleavage-stage embryos. METHODS: A two-stage optimization is used. The first stage uses computer vision algorithms to identify embryonic structures and determines the optimal ablation zone farthest away from critical structures such as blastomeres. The second stage combines a genetic algorithm with a previously reported thermal analysis of LZD to optimize the combination of laser pulse locations and pulse durations. The goal is to minimize the peak temperature experienced by the blastomeres while creating the desired opening in the ZP. RESULTS: A proof of concept of the proposed LZD automation and optimization method is demonstrated through experiments on mouse embryos with positive results, as adequately sized openings are created. CONCLUSION: Automation of LZD is feasible and is a viable step toward the automation of embryo biopsy procedures. SIGNIFICANCE: LZD is a common but delicate procedure performed by human operators using subjective methods to gauge proper LZD procedure. Automation of LZD removes human error to increase the success rate of LZD. Although the proposed methods are developed for cleavage-stage embryos, the same methods may be applied to most types LZD procedures, embryos at different developmental stages, or nonembryonic cells.

Localized Electroporation With Dielectrophoretic Field Flow Fractionation: Toward Removal of Circulating Tumour Cells From Human Blood.

This paper presents the design and experimental performance of a microelectrode-based device to selectively lyse cells in a flow in a microfluidic channel. Localized cell lysis is achieved by utilizing "irreversible electroporation," in which cells are exposed to high magnitude electric pulses. Localized cell lysis in a flow has research applications and may allow for the removal of harmful cells, such as circulating tumor cells from blood. Due to the dependence of this technique on the magnitude of the applied electric field, lethal electric field regions can be localized in the channel by the calibration of the applied voltage. Dielectrophoresis field flow fractionation is used to levitate target cancer cells in the lethal region of the device microchannel. Experiments are performed to demonstrate the localized lysis of MCF7 cancer cells in a mixture of blood cells. Due to their larger size, these circulating tumor cell analogues levitate to a greater height in the channel than erythrocytes. MCF7 lysis is observed to increase from 4.6% in control experiments to 57.3% in active experiments. Leukocyte viability was unaffected in active experiments. These results demonstrate the feasibility of localizing cell lysis in a microfluidic flow environment.

Dynamic modeling and torque estimation of FES-assisted arm-free standing for paraplegics.

This paper presents an application of recent findings in the field of redundant robotic systems' control, toward investigating the feasibility of functional electrical stimulation (FES) assisted arm-free standing for paraplegics. Twelve degrees-of-freedom (DOF) forward and inverse dynamic models of quiet standing have been developed. These models were used to investigate the minimum number of DOF that would need to be actuated in order to generate stable quiet standing in paraplegics despite internal and external disturbances. The results presented herein suggest that the proposed nonlinear dynamic model could achieve guaranteed asymptotic stability with only six active DOF, assuming that the remaining six DOF are passive, i.e., there is no active or passive torques applied to those DOF. The stability analyses were performed using a proportional and derivative (PD) controller coupled with gravity compensation. The results of this analysis suggest that if only six particular DOF are actively controlled in a paraplegic subject, this individual should be able to achieve stable quiet standing despite disturbances. This result has both clinical and system-design implications for the development of a device that will facilitate FES-assisted arm-free quiet standing. The clinical implication is, if a paraplegic patient can exert voluntary control over specified six DOF in the lower limbs, that patient, after intensive physiotherapy, will have the potential to perform quiet standing unassisted. The system-design implication is that FES-assisted arm-free standing for paraplegics is theoretically plausible if one would actively control only six out of 12 DOF in the lower limbs. The proposed solution does not require the locking of joints in the lower limbs (commonly applied in the field) or voluntary control of the upper body to compensate for the internal and external disturbances. Another important finding of this study is the existence of six different combinations of six active DOF able to facilitate stable quiet standing. This dynamic redundancy of the biological bipedal stance allows the selection of an ideal subset of six DOF in designing a neuroprosthesis for standing. This further implies that a considerably less complex FES system than previously anticipated needs to be developed for FES-assisted standing.

Multi-Pulse laser ablation modeling with applications to automated zona removal.

Laser zona drilling (LZD), the ablation of a portion of the zona pellucida (ZP) in embryos with the use of a laser, is a required step in many embryonic surgical procedures such as assisted hatching and preimplantation genetic diagnosis. The objective of LZD is to remove specific locations of the ZP while minimizing potential harmful thermal effects to important structures of the embryo, namely the blastomeres. Current thermal analyzes of lasers used in LZD only encompass the use of a single pulse, whereas LZD is typically performed using multiple pulses. In this paper we analyze the effect of multipulse LZD and introduce a linear approximation method for multi-pulse LZD. Furthermore, we describe a novel method of measuring the thermal effect of a single laser pulse using the thermosensitive fluorescent dye Rhodamine B and a high speed camera.

Optimal combination of minimum degrees of freedom to be actuated in the lower limbs to facilitate arm-free paraplegic standing.

Arm-free paraplegic standing via functional electrical stimulation (FES) has drawn much attention in the biomechanical field as it might allow a paraplegic to stand and simultaneously use both arms to perform daily activities. However, current FES systems for standing require that the individual actively regulates balance using one or both arms, thus limiting the practical use of these systems. The purpose of the present study was to show that actuating only six out of 12 degrees of freedom (12-DOFs) in the lower limbs to allow paraplegics to stand freely is theoretically feasible with respect to multibody stability and physiological torque limitations of the lower limb DOF. Specifically, the goal was to determine the optimal combination of the minimum DOF that can be realistically actuated using FES while ensuring stability and able-bodied kinematics during perturbed arm-free standing. The human body was represented by a three-dimensional dynamics model with 12-DOFs in the lower limbs. Nakamura's method (Nakamura, Y., and Ghodoussi, U., 1989, "Dynamics Computation of Closed-Link Robot Mechanisms With Nonredundant and Redundant Actuators," IEEE Trans. Rob. Autom., 5(3), pp. 294-302) was applied to estimate the joint torques of the system using experimental motion data from four healthy subjects. The torques were estimated by applying our previous finding that only 6 (6-DOFs) out of 12-DOFs in the lower limbs need to be actuated to facilitate stable standing. Furthermore, it was shown that six cases of 6-DOFs exist, which facilitate stable standing. In order to characterize each of these cases in terms of the torque generation patterns and to identify a potential optimal 6-DOF combination, the joint torques during perturbations in eight different directions were estimated for all six cases of 6-DOFs. The results suggest that the actuation of both ankle flexionextension, both knee flexionextension, one hip flexionextension, and one hip abductionadduction DOF will result in the minimum torque requirements to regulate balance during perturbed standing. To facilitate unsupported FES-assisted standing, it is sufficient to actuate only 6-DOFs. An optimal combination of 6-DOFs exists, for which this system can generate able-bodied kinematics while requiring lower limb joint torques that are producible using contemporary FES technology. These findings suggest that FES-assisted arm-free standing of paraplegics is theoretically feasible, even when limited by the fact that muscles actuating specific DOFs are often denervated or difficult to access.