Cell Manipulation Tasks Research Articles

Template-update-based visual tracking for automatic cell manipulation in FluidFM

Visual tracking is an essential process for performing automated cell manipulation tasks. However, Real-time accurate cell tracking in FluidFM is challenging due to probe occlusion during manipulation. This study presents a correlation filtering-based tracking method for real-time, high-precision position tracking of cells and probes. In this method, a target template update strategy is proposed to realize cell tracking under probe occlusion, which abandons the use of pixel values in the occlusion area and reduces the interference of the occlusion area in model learning. Cell tracking and automated manipulation experiments were carried out to validate the proposed method. The Experimental results show that the proposed tracking method can accurately measure the positions of occlusion cells and probe. Furthermore, combined with the precise force control of the FluidFM’s probe, the automated cell Pick & Place experiments was accomplished with high accuracy and robustness.

Surface-Acoustic-Wave (SAW)-Driven Device for Three-Dimensional Cell Manipulation.

This paper proposes a system with a surface-acoustic wave (SAWs) to perform noninvasive cell orientation. The system employed a SAW chip with a pair of interdigital transducers (IDTs) to rotate embryos around the X and Y axis using acoustic streaming force. Instead of rotating the entire embryos like other methods, the proposed system rotates the cytoplasm alone through the cell chorion. We evaluated the cellular structure recognition algorithm and the rotation control using 100 embryo images and 30 zebrafish embryos. The system successfully recognized all required cellar structures for visual feedback. Furthermore, it rotated all cells into the desired position, including 26 cases completed within 10s with an orientation angle error of less than 4°. All 30 embryos hatched after manipulation. The proposed technique can automatically rotate the cytoplasm through the cell chorion noninvasively. The system provides a starting point for noninvasive cell manipulation tasks, such as fast intracellular structure scanning and analysis, and microinjection.

Deep Learning and Computer Vision Strategies for Automated Gene Editing with a Single-Cell Electroporation Platform.

Single-cell delivery platforms like microinjection and nanoprobe electroporation enable unparalleled control over cell manipulation tasks but are generally limited in throughput. Here, we present an automated single-cell electroporation system capable of automatically detecting cells with artificial intelligence (AI) software and delivering exogenous cargoes of different sizes with uniform dosage. We implemented a fully convolutional network (FCN) architecture to precisely locate the nuclei and cytosol of six cell types with various shapes and sizes, using phase contrast microscopy. Nuclear staining or reporter fluorescence was used along with phase contrast images of cells within the same field of view to facilitate the manual annotation process. Furthermore, we leveraged the near-human inference capabilities of the FCN network in detecting stained nuclei to automatically generate ground-truth labels of thousands of cells within seconds, and observed no statistically significant difference in performance compared to training with manual annotations. The average detection sensitivity and precision of the FCN network were 95±1.7% and 90±1.8%, respectively, outperforming a traditional image-processing algorithm (72±7.2% and 72±5.5%) used for comparison. To test the platform, we delivered fluorescent-labeled proteins into adhered cells and measured a delivery efficiency of 90%. As a demonstration, we used the automated single-cell electroporation platform to deliver Cas9-guide RNA (gRNA) complexes into an induced pluripotent stem cell (iPSC) line to knock out a green fluorescent protein-encoding gene in a population of ~200 cells. The results demonstrate that automated single-cell delivery is a useful cell manipulation tool for applications that demand throughput, control, and precision.

Centroid calculation of the blastomere from 3D Z-Stack image data of a 2-cell mouse embryo

During cell surgery, cell manipulation tasks such as placement of cells, extraction of organelles, blastomeres, or other structures from within a cell are essential. To enable these tasks, it is necessary to locate the centroid of such objects within a cell, to be able to position micromanipulators to carry out such tasks. Calculation of the centroid of objects within a cell is necessary to enable vision-based feedback control to micromanipulators for automation of cell tasks. In this paper, we propose a methodology to address the calculation of the centroid of blastomeres within a mouse embryo. This is achieved with z-stack image data from 2D cell images. Z-stack images are captured at uniform intervals over the cell depth, by keeping the embryo fixed and moving the focal plane. Individual (2D) images of the z-stack are then processed. Blastomeres in each image are segmented with existing image processing techniques and converted to mask images. From these masked images, the 2D blastomere image centroids are calculated, allowing the centroid of the blastomere volume in 3D to be computed. The proposed calculation of the blastomere centroid is a critical step in determining the centroid of objects within a cell, to facilitate automation of cell surgery tasks.

Design of a novel magnetic platform for cell manipulation

Cell manipulation tasks, especially in lab-on-a-chip applications for personalized medicine, could greatly benefit from mobile untethered microdevices able to wirelessly navigate in fluidic environments by means of magnetic fields. In this paper, the design, fabrication and testing of a magnetic platform enabling the controlled locomotion and immersion of microrobots placed at the air/liquid interface is proposed and exploited for cell manipulation. The proposed microrobot consists of a polymeric magnetic thin film that acts as cell transporter and a specific coating strategy, devised to enhance a safe cancer cell adhesion to the magnetic film. Experimental results demonstrated an overall cell viability and a fine control of magnetic microrobot locomotion. The proposed technologies are promising in view of future cell manipulation tasks for personalized medicine applications.

Autofocusing and Polar Body Detection in Automated Cell Manipulation.

Autofocusing and feature detection are two essential processes for performing automated biological cell manipulation tasks. In this paper, we have introduced a technique capable of focusing on a holding pipette and a mammalian cell under a bright-field microscope automatically, and a technique that can detect and track the presence and orientation of the polar body of an oocyte that is rotated at the tip of a micropipette. Both algorithms were evaluated by using mouse oocytes. Experimental results show that both algorithms achieve very high success rates: 100% and 96%. As robust and accurate image processing methods, they can be widely applied to perform various automated biological cell manipulations.

Automated Translational and Rotational Control of Biological Cells With a Robot-Aided Optical Tweezers Manipulation System

Research and biomedical applications in cell surgery require transportation and rotation of biological cells. In these cell manipulation tasks, the cell of interest must be translated and oriented properly such that the desired component, such as the polar body or other organelles, can be imaged with optical microscopy. This paper presents a holographic optical tweezers (HOT) based system to carry out automated translational control in the plane, and rotational control about one rotational axes of a suspended cell. Based on the proposed general equations of motion of the cell, held in an optical trap, two controllers, one for cell translational and one for rotational control, are developed to translate and orient the cells to the desired position and orientation in a sequential manner. Experiments are performed to demonstrate the effectiveness of the proposed approach.

Three-Dimensional Rotation of Mouse Embryos

Research and clinical applications, such as microinjection and polar-body biopsy involve 3-D rotation of mammalian oocytes/embryos. In these cell manipulation tasks, the polar body of an embryo/oocyte must be made visible and properly oriented under optical microscopy. Cell rotation in conventional manual operation by skilled professionals is based on trial and error, such as through repeated vacuum aspiration and release. The randomness of this manual procedure, its poor reproducibility, and inconsistency across operators entail a systematic technique for automated, noninvasive, 3-D rotational control of single cells. This paper reports a system that tracks the polar body of mouse embryos in real time and controls multiple motion control devices to conduct automated 3-D rotational control of mouse embryos. Experimental results demonstrated the system's capability for polar-body orientation with a high success rate of 90%, an accuracy of 1.9 °, and an average speed of 22.8 s/cell (versus averagely 40 s/cell in manual operation).

Integrated AC electrokinetic cell separation in a closed-loop device

We integrate electrothermally induced micro-pumps and dielectrophoretic (DEP) traps into micro-circulating fluidic channel loops for yeast cell concentration and separation, two important on-chip cell manipulation tasks, with the same embedded electrodes on-chip. Each fluidic loop design contains well-defined high and low field regions that serve for both fluid transport and cellular manipulation. From a detailed study into the frequency dependent DEP behavior of viable (live) and non-viable (dead) yeast, we demonstrate several operating modes that utilize positive DEP (pDEP) and negative DEP (nDEP) to concentrate both types of cells at either the high or low electric field region and to separate one cell type to a high-field region and one to a low-field region. Because the cells visit the trapping regions repeatedly with the circulating loop design and because of the high shear rates at these stations, our device offers very rapid cell separation and concentration. Two circulating loop designs--one a four-sided square loop, the other a three-sided triangle, with different spatial symmetries and with linear dimensions less than 1 mm, are presented.

A bulk microfabricated multi-axis capacitive cellular force sensor using transverse comb drives

This paper presents design, fabrication and calibration results for a novel 2-DOF capacitive force sensor capable of resolving forces up to 490 µN with a resolution of 0.01 µN in x, and up to 900 µN with a resolution of 0.24 µN in y. A simple fabrication process using deep reactive ion etching (DRIE) on silicon-on-insulator (SOI) wafers forms the 3D high aspect ratio structure. A transverse mode comb drive movement is used to greatly improve device sensitivity. Among other advantages of the developed process is a dice-free release of wafer structures, allowing fragile structures to be individually packaged. Notching or footing effects and bowing effects are well-known problems in DRIE on SOI wafers. Techniques to overcome notching and bowing effects using a PlasmaTherm SLR-770 etcher are presented that do not require hardware modifications. The application of the force sensor is for providing real-time force feedback during individual cell manipulation tasks.