Cell Injection System Research Articles

Development, modeling, and control of a syringe-based picoliter-scale microinjection system

Microinjection, which is widely employed in modern biology and medicine research, is a crucial method for transferring biomaterial in a single cell. Despite substantial research into microinjection automation technology, how to achieve high precision, which is crucial for quantitative analysis, has gotten scant attention. This paper offers a dual-loop control syringe-based picoliter-scale injection system, with the inner being a pneumatic servo loop with a baroreceptor providing pressure feedback and the outside being a position servo loop with a microscope providing visual feedback. Specifically, significant crawling phenomenon are found throughout the characteristic test trial, rendering the gas-liquid interface uncontrollable. To mathematically characterize this phenomenon, a theoretical model is developed that accounts for static friction, capillary force, capillary cone angle, and fluid viscous force. The genetic algorithm is used to identify difficult-to-measure and calibrate parameters. Based on the established model, an integral sliding-mode controller (ISMC) with saturation function and a PID controller for the inside loop control were established, both of which have a fast convergence rate and do not overshoot. Finally, experimentation is used to validate the designed cell injection system, demonstrating that the suggested system can regulate the smallest droplet in the tip of the needle to within 0.25 pL.

An Inchworm and Stick-Slip Dual Mode Piezoelectric Linear Actuator for Cell Injection

Cell injection system for biomedical engineering demands that actuators have large stroke, high resolution and speed for cell positioning and injection. Single mode piezoelectric actuators could meet some of these requirements but hardly all. Stick-slip actuator is easy to achieve high-speed operation, but it is prone to setback; The speed characteristics of the inchworm actuator are lower than those of the stick-slip actuator, but the step is more stable and the setback is lower. This study proposes an inchworm and stick-slip dual mode piezoelectric actuator (ISSPA) for cell injection. It integrates two working modes into one actuator and both modes drive the same slider. The stick-slip mode works at high frequency for fast positioning to the cell and the inchworm mode drives the needle to puncture the cell membrane and accurately locates to the specified location. The structure and working principle of the ISSPA are provided. A prototype of the proposed ISSPA is fabricated and experiments are conducted. When the driving voltage of the piezoelectric stack is 100 V and the frequency is 500 Hz,the maximum speed of the ISSPA is 2.287 mm/s in the stick-slip mode. When the driving voltage of the piezoelectric stack is 60 V, the frequency is 3 Hz and the load is 0.5 kg,the minimum step displacement is 57 nm in inchworm mode. Demonstration of cell injection is carried out by using the ISSPA to positioning and puncturing a zebrafish embryo. Fast positioning with the stick-slip mode and accurate puncture with the inchworm mode are achieved with the ISSPA alone, taking 12 seconds for the whole injection process. It could be expected to apply the ISSPA to cells with wide range of diameter, as the frequency of the stick-slick mode and the voltage of the inchworm mode could be well regulated.

Formal Verification of Robotic Cell Injection systems up to 4-DOF using HOL Light

Abstract Cell injection is an approach used for the delivery of small sample substances into a biological cell and is widely used in drug development, gene injection, intracytoplasmic sperm injection and in-vitro fertilization. Robotic cell injection systems provide the automation of the process as opposed to the manual and semi-automated cell injection systems, which require expert operators and involve time consuming processes and also have lower success rates. The automation of the cell injection process is obtained by controlling the orientation and movement of its various components, like injection manipulator, microscope etc., and planning the motion of the injection pipette by controlling the force of the injection. The conventional techniques to analyze the cell injection process include paper-and-pencil proof and computer simulation methods. However, both these techniques suffer from their inherent limitations, such as, proneness to human error for the former and the approximation of the mathematical expressions involved in the numerical algorithms for the latter. Formal methods have the capability to overcome these limitations and can provide an accurate analysis of these cell injection systems. Model checking, i.e., a state-based formal method, has been recently used for analyzing these systems. However, it involves the discretization of the differential equations capturing the continuous dynamics of the system and thus compromises on the completeness of the analysis of these safety-critical systems. In this paper, we propose a higher-order-logic theorem proving (a deductive-reasoning based formal method) based framework for analyzing the dynamical behavior of the robotic cell injection systems upto 4-DOF. The proposed analysis, based on the HOL Light theorem prover, enabled us to identify some discrepancies in the simulation and model checking based analysis of the same robotic cell injection system.

Oocyte activation, oolemma piercing, and real-time viability confirmation in human oocytes using electrophysiological techniques.

To discuss the recent applications of electrophysiological principles to the optimization and automation of the IVF laboratory. There is growing evidence showing improvement of live birth rates following oocyte electro-activation. Novel applications using electrophysiological techniques are now employed to determine oocyte penetration and viability in real-time. In this short review, we summarize the recent advances in the integration of electrophysiological techniques into the assisted reproductive technology laboratories. We describe the potential clinical applications and their advantages such as creation of reliable automated cell injection systems and novel manual intracytoplasmic sperm injection (ICSI) training platforms. We also discuss theoretical adverse effects and ways to mitigate them.

High Fidelity Force Feedback Facilitates Manual Injection in Biological Samples

Micro-teleoperated interaction with biological cells is of special interest. The low fidelity of previous systems aimed at such small scale tasks prompted the design of a novel manual bilateral cell injection system. This systems employed the coupling of a null-displacement active force sensor with a haptic device having negligible effective inertia. This combination yielded a bilateral interaction system that was unconditionally stable even when the scaling gains were high. To demonstrate the capability of this system, two experiments were performed. A hard trout egg was delicately punctured and a small dye amount was injected in an embryo within a zebra fish egg without causing other forms of damage. The results demonstrate that the system let an operator dextrously interact with reduced reliance on visual feedback.

Low-Invasive Cell Injection based on Rotational Microrobot.

The advancement of cell injections has created a need for accurate, efficient, and low-invasive injections. However, the conventional approaches to reduce cell damage during penetration, mainly optimization of micropipette tips and vision-based automatic injections, have almost reached the limit. Here, described are the design and implementation of a robotic-aided rotatory microinjection system to reduce cell deformation and penetration force during the injection. The homocentric rotation technology integrates an ultraprecise manipulation system with multiple degrees of freedom, which tremendously diminishes the damage to the cell. Through systematic tests on zebrafish embryo microinjection, compared with traditional straightforward cell injection techniques, the rotary cell injection approach is able to reduce the penetration deformation and force of the specimen up to 30%. This work breaks the performance limit of current microinjection techniques, and paves a new way for the development of low-invasive cell injection systems. It is envisioned that this rotary microrobot system can excavate enormous applications in the field of biomedicine, such as artificial fertilization and gene therapy.

A Survey of Force-Assisted Robotic Cell Microinjection Technologies

Cell injection plays an important role in genetics, transgenics, molecular biology, drug discovery, reproductive study, and other biomedical fields. Compared with manual cell microinjection and robotic cell microinjection with sole position feedback, force-assisted robotic cell microinjection can improve the success rate and survival rate of cell injection. In this paper, the state-of-the-art research on microinjection of both adherent cells and suspended cells with microforce sensing techniques is reviewed. The significance of force sensors in the robotic cell injection system is also discussed. Five types of prevalent force sensing methods and their applications in cell microinjection are reviewed. The challenges and promising solutions in automating the cell microinjection process are addressed. Note to Practitioners —Microinjection process is a complex task to perform. As the advance progress of high-performance microforce sensors, force-assisted robotic cell injection has been a hot topic in recent years. This paper presents the state-of-the-art survey of recent developments on microforce sensing for robotic cell microinjection to address the research challenges. Based on microforce sensing and control, microinjection of both adherent and suspended cells can benefit from the force-assisted robotic cell injection process. The main challenges and promising solutions in terms of micromanipulator design, injection control design, cell holder design, penetration scheme design, injecting pipette maintenance, injection volume, cost reduction, and microforce sensor calibration issues have been discussed. The related research trends are summarized.

Design and development of a piezo-driven microinjection system with force feedback

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Design and Precision Position/Force Control of a Piezo-Driven Microinjection System

This paper presents the design and development of a piezo-driven cell injection system with the fusion of force and position control. By integrating a polyvinylidene fluoride force sensor for detecting the penetration force of biological cells and a strain-gage sensor for measuring the micropipette relative position in real time, the entire cell micromanipulation system features low cost, convenient installation, and easy maintenance. The challenge of transition jerk between the force and position controller switching is reduced by introducing a weight coefficient method. An adaptive sliding mode control with parameter estimation scheme is implemented to compensate for the hysteresis nonlinearity and disturbance of the piezoelectric actuator. The injection force is controlled by realizing an incremental proportional-integral-derivative controller. The effectiveness of the developed cell injection system is experimentally verified by penetrating zebrafish embryos. The experimental results lay a foundation for the further biological manipulation research, such as the automatic batch injection of zebrafish embryos.

Towards Probabilistic Formal Modeling of Robotic Cell Injection Systems

Cell injection is a technique in the domain of biological cell micro-manipulation for the delivery of small volumes of samples into the suspended or adherent cells. It has been widely applied in various areas, such as gene injection, in-vitro fertilization (IVF), intracytoplasmic sperm injection (ISCI) and drug development. However, the existing manual and semi-automated cell injection systems require lengthy training and suffer from high probability of contamination and low success rate. In the recently introduced fully automated cell injection systems, the injection force plays a vital role in the success of the process since even a tiny excessive force can destroy the membrane or tissue of the biological cell. Traditionally, the force control algorithms are analyzed using simulation, which is inherently non-exhaustive and incomplete in terms of detecting system failures. Moreover, the uncertainties in the system are generally ignored in the analysis. To overcome these limitations, we present a formal analysis methodology based on probabilistic model checking to analyze a robotic cell injection system utilizing the impedance force control algorithm. The proposed methodology, developed using the PRISM model checker, allowed to find a discrepancy in the algorithm, which was not found by any of the previous analysis using the traditional methods.

細胞への自動インジェクション装置の開発と展望

細胞への自動インジェクション装置の開発と展望

PD-based trajectory tracking control in automatic cell injection system

Trajectory tracking technology has been the focus of industrial manipulatory applications for many years, and its research has been found in micromanipulation in bioengineering recently. In this paper, a hybrid vision and force control method is applied to the automatic cell injection. The three-dimensional cell injection process involves the trajectory tracking in free space and the force control in contact space. A PD plus feedforward compensation control method is applied to the trajectory tracking in 3D space. Further, a PD-based robust controller is introduced into trajectory tracking while the systemic uncertainty of the cell injection is additionally considered. Both of the two control methods are theoretically proved to be exponential convergent. Finally, the effectiveness of the proposed method is verified as compared with other control methods by its application to trajectory tracking problem.

Haptic Microrobotic Cell Injection System

Microrobotic cell injection is the subject of increasing research interest. At present, an operator relies completely on visual information and can be subject to low success rates, poor repeatability, and extended training times. This paper focuses on increasing operator performance during cell injection in two ways. First, our completed haptic cell injection system aims to increase the operator's performance during real-time cell injection. Haptic bilateralism is investigated and a mapping framework provides an intuitive method for manoeuvring the micropipette in a manner similar to handheld needle insertion. Volumetric virtual fixtures are then introduced to haptically assist the operator to penetrate the cell at the desired location. The performance of the volumetric virtual fixtures is also discussed. Second, the haptically enabled cell injection system is replicated as a virtual environment facilitating virtual offline operator training. Virtual operator training utilizes the same mapping framework and haptic virtual fixtures as the physical system allowing the operator to train offline and then directly transfer their skills to real-time cell injection.

Minimally invasive injectable short nanofibers of poly(glycerol sebacate) for cardiac tissue engineering

Myocardial tissue lacks the ability to appreciably regenerate itself following myocardial infarction (MI) which ultimately results in heart failure. Current therapies can only retard the progression of disease and hence tissue engineering strategies are required to facilitate the engineering of a suitable biomaterial to repair MI. The aim of this study was to investigate the in vitro properties of an injectable biomaterial for the regeneration of infarcted myocardium. Fabrication of core/shell fibers was by co-axial electrospinning, with poly(glycerol sebacate) (PGS) as core material and poly-l-lactic acid (PLLA) as shell material. The PLLA was removed by treatment of the PGS/PLLA core/shell fibers with DCM:hexane (2:1) to obtain PGS short fibers. These PGS short fibers offer the advantage of providing a minimally invasive injectable technique for the regeneration of infarcted myocardium. The scaffolds were characterized by SEM, FTIR and contact angle and cell–scaffold interactions using cardiomyocytes. The results showed that the cardiac marker proteins actinin, troponin, myosin heavy chain and connexin 43 were expressed more on short PGS fibers compared to PLLA nanofibers. We hypothesized that the injection of cells along with short PGS fibers would increase cell transplant retention and survival within the infarct, compared to the standard cell injection system.

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.

Visual-Based Impedance Control of Out-of-Plane Cell Injection Systems

In this paper, a vision-based impedance control algorithm is proposed to regulate the cell injection force, based on dynamic modeling conducted on a laboratory test-bed cell injection system. The injection force is initially calibrated to derive the relationship between the force and the cell deformation utilizing a cell membrane point-load model. To increase the success rate of injection, the injector is positioned out of the focal plane of the camera, used to obtain visual feedback for the injection process. In this out-of-plane injection process, the total cell membrane deformation is estimated, based on the X-Y coordinate frame deformation of the cell, as measured with a microscope, and the known angle between the injector and the X-Y plane. Further, a relationship between the injection force and the injector displacement of the cell membrane, as observed with the camera, is derived. Based on this visual force estimation scheme, an impedance control algorithm is developed. Experimental results of the proposed injection method are given which validate the approach.

Robotic Cell Injection System With Position and Force Control: Toward Automatic Batch Biomanipulation

Biological cell injection is laborious work that requires lengthy training and suffers from a low success rate. In this paper, a robotic cell-injection system for automatic injection of batch-suspended cells is proposed. To facilitate the process, these suspended cells are held and fixed to a cell array by a specially designed cell-holding device, and injected one by one through an ldquoout-of-planerdquo cell-injection process. A micropipette equipped with a polyvinylidene fluoride microforce sensor to measure real-time injection force is integrated in the proposed system. Through calibration, an empirical relationship between the cell-injection force and the desired injector pipette trajectory is obtained in advance. Then, after decoupling the out-of-plane cell injection into a position control in the <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">X</i> - <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Y</i> horizontal plane and an impedance control in the <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Z</i> -axis, a position and force control algorithm is developed to control the injection pipette. The depth motion of the injector pipette, which cannot be observed by microscope, is indirectly controlled via the impedance control, and the desired force is determined from the online <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">X</i> - <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Y</i> position control and cell calibration results. Finally, experimental results demonstrate the effectiveness of the proposed approach.

Application of machine vision for automated cell injection

This paper presents image processing algorithms recognising cell structures for an automated cell injection system. For suspended zebrafish embryos, the convex hull and convexity defects of the cytoplasm contour are used. For adherent endothelial cells, the surface and shadow information of the nucleoli is used. Overall, 550 zebrafish embryos were 100% correctly recognised in online injection experiments. Compared to manual injection, the automated injection speed and reproducibility increase by up to 1.5-fold and 67% respectively. In the endothelial cell case, 436 nucleoli were 92% correctly recognised, paving the way for an automated adherent cell injection system to be developed.

Vision-Servo System for Automated Cell Injection

Recent developments in nuclear reprogramming and intracytoplasmic sperm injection reflect an increasing need for more advanced and automatic micromanipulation technologies. In this paper, we present an automatic cell injection system, which is capable of visually monitoring the injecting process and controlling the microactuators. Traditionally, cell injection was manually operated, and it was laborious, time consuming, of low accuracy, and prone to contamination due to the handling requirements. An automatic and efficient strategy is required to eliminate these drawbacks. In this paper, a system is developed where the injection process is monitored and controlled automatically via integration of a vision system to an injector manipulation system. The cell is located, and the pipette is positioned and driven by the algorithm to achieve effective penetration. The precision achieved is physically proven to be within a good tolerance range.

Effects of Flow Rate on Sensitivity and Affinity in Flow Injection Biosensor Systems Studied by 55-MHz Wireless Quartz Crystal Microbalance

In this paper, we developed a 55-MHz wireless-electrodeless quartz crystal microbalance (QCM) and systematically studied the effects of flow rate on the sensitivity to the detection of proteins and on the affinity between biomolecules evaluated by the flow injection system. Brownian motion of proteins in liquid suggests a low probability of meeting, and the convection effect plays an important role in the sensitivity and the affinity in the flow cell injection system. The wireless quartz crystal was isolated in the QCM cell, and flow rates between 50 and 1000 microL/min were used for monitoring binding reactions between human immunoglobulin G and Staphylococcus aureus protein A. The sensitivity was significantly increased as the flow rate increased, while the affinity value remained unchanged. However, the affinity value was affected by the reaction time for a large-concentration analyte, indicating the need of a high-sensitivity biosensor system for accurate evaluation of affinity. The electrode effect on the QCM sensitivity was also theoretically investigated, showing that the electrode significantly deteriorates the QCM sensitivity and makes the Sauerbrey equation invalid.