Nikolai Dechev

Development of an Autonomous Biological Cell Manipulator With Single-Cell Electroporation and Visual Servoing Capabilities

Studies of single cells via microscopy and microinjection are a key component in research on gene functions, cancer, stem cells, and reproductive technology. As biomedical experiments become more complex, there is an urgent need to use robotic systems to improve cell manipulation and microinjection processes. Automation of these tasks using machine vision and visual servoing creates significant benefits for biomedical laboratories, including repeatability of experiments, higher throughput, and improved cell viability. This paper presents the development of a new 5-DOF robotic manipulator, designed for manipulating and microinjecting single cells. This biological cell manipulator (BCM) is capable of autonomous scanning of a cell culture followed by autonomous injection of cells using single-cell electroporation (SCE). SCE does not require piercing the cell membrane, thereby keeping the cell membrane fully intact. The BCM features high-precision 3-DOF translational and 2-DOF rotational motion, and a second z-axis allowing top-down placement of a micropipette tip onto the cell membrane for SCE. As a technical demonstration, the autonomous visual servoing and microinjection capabilities of the single-cell manipulator are experimentally shown using sea urchin eggs.

A Scalable

We present a new architecture for a digitally operated 1timesN optical microelectromechanical systems (MEMS) switch supporting a large number of output ports. The switch is based on a novel microassembled rotating micromirror mounted on the top of an optimized micromotor using a rotor-pole-shaping technique. The rotating micromirror consists of a novel electrostatic rotary motor, onto which a 3-D micromirror structure is assembled using a robotic-based microassembly process. The rotating micromirror in the switch can digitally rotate with 480 steps per full revolution allowing for excellent and complete repeatability of the switch. A uniform coupling loss of 1.04 dB across all the output fibers is achieved with a maximum switching time of 24 ms at 70 V signal excitation

1\times N

Studies of individual cells via microscopy and microinjection are a key component in research on gene functions, cancer, stem cells, and reproductive technology. As biomedical experiments become more complex, there is an urgent need for robotic systems to: improve cell manipulation, increase throughput, reduce lost cells, and improve reaction detection. Automation of these tasks using visual servoing creates significant benefits for biomedical laboratories including repeatability of experiments, higher throughput, and a controlled environment capable of operating 24 hours a day. In this work the design and development of a new five degree-of- freedom biomanipulator designed for single-cell microinjection, is described. The biomanipulator employs three degrees of linear motion and two degrees of rotation. This provides the ability to manipulate/micro-inject cells at varying orientations, thereby increasing flexibility in dealing with complex operations such as injecting clustered cells. The capability of the biomanipulator is shown with preliminary experimental results using mouse myeloma cells.

Optical MEMS Switch Architecture Utilizing a Microassembled Rotating Micromirror

This paper presents the design of a novel, portable device for hand rehabilitation. The device provides for CPM (continuous passive motion) and CAM (continuous active motion) hand rehabilitation for patients recovering from damage such as flexor tendon repair and strokes. The device is capable of flexing/extending the MCP (metacarpophalangeal) and PIP (proximal interphalangeal) joints through a range of motion of 0° to 90° for both the joints independently. In this way, typical hand rehabilitation motions such as intrinsic plus, intrinsic minus, and a fist can be achieved without the need of any splints or attachments. The CPM mode is broken into two subgroups. The first mode is the use of preset waypoints for the device to cycle through. The second mode involves motion from a starting position to a final position, but senses the torque from the user during the cycle. Therefore the user can control the ROM by resisting when they are at the end of the desired motion. During the CPM modes the device utilizes a minimum jerk trajectory model under PD control, moving smoothly and accurately between preselected positions. CAM is the final mode where the device will actively resist the movement of the user. The user moves from a start to end position while the device produces a torque to resist the motion. This active resistance motion is a unique ability designed to mimic the benefits of a human therapist. Another unique feature of the device is its ability to independently act on both the MCP and PIP joints. The feedback sensing built into the device makes it capable of offering a wide and flexible range of rehabilitation programs for the hand.

Development of a five degree-of-freedom biomanipulator for autonomous single cell electroporation

Automated robotic bio-micromanipulation can improve the throughput and efficiency of single-cell experiments. Adherent cells, such as fibroblasts, include a wide range of mammalian cells and are usually very thin with highly irregular morphologies. Automated micromanipulation of these cells is a beneficial yet challenging task, where the machine vision sub-task is addressed in this article. The necessary but neglected problem of localizing injection sites on the nucleus and the cytoplasm is defined and a novel two-stage model-based algorithm is proposed. In Stage I, the gradient information associated with the nucleic regions is extracted and used in a mathematical morphology clustering framework to roughly localize the nucleus. Next, this preliminary segmentation information is used to estimate an ellipsoidal model for the nucleic region, which is then used as an attention window in a k-means clustering-based iterative search algorithm for fine localization of the nucleus and nucleic injection site (NIS). In Stage II, a geometrical model is built on each localized nucleus and employed in a new texture-based region-growing technique called Growing Circles Algorithm to localize the cytoplasmic injection site (CIS). The proposed algorithm has been tested on 405 images containing more than 1,000 NIH/3T3 fibroblast cells, and yielded the precision rates of 0.918, 0.943, and 0.866 for the NIS, CIS, and combined NIS–CIS localizations, respectively.

Design of a continuous passive and active motion device for hand rehabilitation

Single cell research has the potential to revolutionize experimental methods in biomedical sciences and contribute to clinical practices. Recent studies suggest analysis of single cells reveals novel features of intracellular processes, cell-to-cell interactions and cell structure. The methods of single cell analysis require mechanical resolution and accuracy that is not possible using conventional techniques. Robotic instruments and novel microdevices can achieve higher throughput and repeatability; however, the development of such instrumentation is a formidable task. A void exists in the state-of-the-art for automated analysis of single cells. With the increase in interest in single cell analyses in stem cell and cancer research the ability to facilitate higher throughput and repeatable procedures is necessary. In this paper, a high-throughput, single cell microarray-based robotic instrument, called the RoboSCell, is described. The proposed instrument employs a partially transparent single cell microarray (SCM) integrated with a robotic biomanipulator for in vitro analyses of live single cells trapped at the array sites. Cells, labeled with immunomagnetic particles, are captured at the array sites by channeling magnetic fields through encapsulated permalloy channels in the SCM. The RoboSCell is capable of systematically scanning the captured cells temporarily immobilized at the array sites and using optical methods to repeatedly measure extracellular and intracellular characteristics over time. The instrument’s capabilities are demonstrated by arraying human T lymphocytes and measuring the uptake dynamics of calcein acetoxymethylester—all in a fully automated fashion.

Machine vision-based localization of nucleic and cytoplasmic injection sites on low-contrast adherent cells

A robotic-based microassembly process has been successfully applied to the construction of a novel micro-mirror design for use in optical switching. This paper is devoted to the description of the microassembly process used to construct the 3D micro-mirror. The microassembly process is based upon the PMKIL (Passive Microgripper, Key and Inter-Lock) assembly system. Details of the assembly process include, the methodology to construct the micro-mirror, the design of the micro-mirror parts, and the design of the tools (microgrippers) that are mounted to the robot to handle the micro-parts. The results of the assembly process are presented, along with examples of prototype 3D micro-mirrors. The entire 3D micromirror consists of a novel electro-static rotary motor, onto which the 3D mirror structure is assembled. The 3D micro-mirror is used as a building element for 1 N optical switching systems and for N×M optical crossconnects.

RoboSCell: an automated single cell arraying and analysis instrument

Implantable biomedical devices play an important role in research for use as stimulators, sensors and biomimetic prosthesis. The use of implantable devices is desired since long-term operation or monitoring is possible, without need for replacement of embedded batteries. This work presents a wireless power transfer (WPT) system that is designed to deliver power efficiently from a stationary primary source to a moving implantable device (secondary circuit) via magnetic resonant coupling. Power is induced in the moving secondary circuit while it translates and rotates within the magnetic field generated by the stationary primary coil cage. In a typical application, a mouse with such a device implanted would move freely inside the cage while continuous power is supplied to the implantable telemetry device. Two different secondary coil configurations are proposed in this work, which are described as the air core and the ferrite core configurations. Finite element analysis (FEA) is done to assist in performance prediction for these configurations. Experimental measurements show that the air core configuration loses magnetic coupling with the primary coil, when the implantable device becomes oriented at 60° or more with respect to the primary field flux direction. For achieving improved coupling for a freely moving mouse within the primary cage, a wrapped ferrite rod could be used in the secondary implant prototype (SIP). This wrapped ferrite rod (WFR) coil provides the coupling required when the SIP moves beyond the 60° and 90° orientations. These two coils are attached to their own tank capacitors and to a separate rectifier. The maximum received power is 60 mW. It is delivered by applying a sinusoidal current through the primary coil of 2.5 A (peak-to-peak). The resonant frequency of the mouse implant device (MID) used in our experiments is 2.135 MHz.

Microassembly of 3D micromirrors as building elements for optical MEMS switching

This study applies 2-D speckle tracking using B-scan ultrasound imaging to estimate the instantaneous and total displacement of the middle flexor digitorum superficialis (FDS) tendon proximal to the wrist. This is achieved by performing the study with human patients, during regular carpal tunnel surgeries. B-Scan images were collected with a 12-MHz transducer placed proximal to the wrist, while a video microscope simultaneously imaged the exposed flexor tendons in the palm as a reference for validation. The accuracy of the proposed speckle-based tracking method is compared using log-compressed Rayleigh (Fisher-Tippet)-, Gaussian (sum of squared differences)- and Laplacian (sum of absolute differences)-based statistics as similarity measures. Overall, tracking was successful and the Rayleigh technique performed better than the Laplacian or Gaussian technique. One goal of this research was to non-invasively monitor FDS tendon displacement in the wrist for the purposes of controlling a prosthetic device. An additional goal was to obtain pre- and post-operative clinical information.

Power transfer via magnetic resonant coupling for implantable mice telemetry device

Gathering behavioral and biological data from small rodents is important for the study of various disease models in biomedical research. Such data acquisition requires a long-term powering method for telemetry electronics and radios, for which a wireless power transfer (WPT) scheme is desirable. This paper investigates a novel WPT system to deliver power from a stationary source (primary coil) to a moving telemetric device (secondary coil) via magnetic resonance coupling. To conduct research with rodents effectively, they must be able to move freely inside their cage. However, the continuously changing orientation of the rodent leads to coupling loss/problems between the primary and secondary coils, presenting a major challenge. We propose novel configurations of the secondary employing ferrite rods placed at specific locations and orientations within the coil. Three-dimensional finite-element analysis using COMSOL software is used to find the magnetic flux density distribution surrounding these secondary configurations. The simulation results show a significant increase of flux through the coil using our ferrite arrangement, with improved coupling at most orientations. Physical prototypes of these secondary coil configurations were constructed and experiments were conducted to test their performance. Measurements show that ferrite rods improved power transfer at most orientations, beyond that of the nominal ferrite-less configuration. The use of angled ferrite rods further improved power transfer, where the medium-ferrite-angled (4MFA) configuration is best. Experiments show the maximum power collected by 4MFA was 113 mW, when parallel to the primary coil, and 28 mW when 60° to the primary coil.