Cell rotation has great significance for cell manipulation, which is applied to intracytoplasmic sperm injection, preimplantation genetic screening and diagnosis, somatic cell nuclear transfer, etc. In this letter, an automatic cell rotation method is proposed based on real-time detection and tracking of the polar body. Image segmentation and visual tracking are combined to detect the polar body accurately and robustly during cell rotation. Both out-of-plane and in-plane rotation are realized by utilizing a pair of standard glass micropipettes. In experiments, two kinds of oocytes, which are quite different in appearance, are used to validate the system. For mouse oocytes, the robotic system achieved a 98% success rate of out-of-plane rotation and a 93% success rate of in-plane rotation. For porcine oocytes, the robotic system achieved an 80% success rate of out-of-plane rotation and a 93% success rate of in-plane rotation. The average time of cell rotation is approximately 9.1s for mouse oocytes and 12.8s for porcine oocytes. The experiment results demonstrate that the proposed method is effective, stable and high speed for automatic cell rotation in 3D.

Objective: The invisibility of domestic oocyte nucleus in bright field currently forces operators to blindly aspirate nucleus out in oocyte enucleation, usually causing large cytoplasm losses and poor developmental competences of cloned embryos. Although fluorescent labeling of nucleus allows for nucleus localization, the involved photobleaching problems and barriers to the execution of enucleation process limit its online-application in oocyte enucleation. This paper reports a novel label-free oocyte enucleation method for precise removal of the nucleus with less cytoplasm loss. Methods: The relative positions between the injection pipette and nucleus for complete removal of nucleus with less cytoplasm loss were determined through a finite element modeling of nucleus aspiration. To position injection pipette to the above positions relative to nucleus, the appropriate oocyte orientation and trajectory of injection pipette inside oocyte were derived according to the offline-calibrated 3-D distribution of nucleus and the simulated dynamic drift of nucleus that occurs as injection pipette is maneuvered inside oocyte. Finally, a robotic label-free precise enucleation procedure was established. Results: The experimental results on more than 1000 porcine oocytes proved that this system is capable of reducing cytoplasm loss by 60% at the same level of enucleation success rate and almost doubling the cleavage rate of clone embryos in comparison to blind aspiration method. Conclusions: The results prove that our method significantly improves the developmental competence of cloned embryos in comparison to manual enucleation method. Significance: Our method is expected to improve the extremely low success rate of animal cloning in the future.

The formation of the gigaseal, a giga Omega-scale seal between the micropipette electrode and cell surface, is the keystone to measure the extremely weak electric signals of cell ion channels in patch clamp technique. To determine a point on the cell surface facilitating gigaseal formation, 3-D cell morphology information is required. However, the current 3-D cell morphology measurement methods relying on special devices or easily causing clogging issues of the micropipette electrode due to contact on cells are usually not applicable in the traditional patch clamp system. Addressing this, a noninvasive 3-D cell morphology measurement method was developed in this article for robotic patch clamp with a higher success rate of gigaseal formation. First, the measured bath impedance of the micropipette electrode was modeled and then utilized to measure the cell surface height noninvasively. Using the measured cell surface heights at key positions, the 3-D cell morphology was fitted to determine a contact position on the cell surface to facilitate gigaseal formation. Finally, a robotic whole-patch clamp process was conducted at the determined contact position. Experimental results demonstrated that 100% and 90% success rates of noninvasive 3-D cell morphology measurements were achieved on the cultured human embryonic kindey (HEK)-293 cells and pyramidal neurons in mouse brain slices, respectively, with an average measurement error of cell surface height less than $0.15~\mu \text{m}$ . At the determined contact position, significant improvements in success rates of gigaseal formation and whole-cell patch clamp operation were achieved in comparison to the results at the positions recommended in related work. Our research may spike inspiration to improve the success rates of gigaseal formation and patch clamp operation based on 3-D cell morphologies.

Cell aspiration is a common technique in cell manipulation for cell transfer or intracellular property measurement. In this paper, we present a robotic micromanipulation system for cell aspiration and positioning by a micropipette. Considering the relative motion of the object and the fluid, we first establish an overall dynamic model of microbead motion inside and outside the micropipette based on computational fluid dynamics (CFD). Then we design an adaptive sliding mode controller (ASMC) for microbead aspiration outside the micropipette and positioning inside the pipette based on the dynamic model. The controller is proven to achieve asymptotic stability by Lyapunov techniques. Simulation and experimental results demonstrate the effectiveness of the fluid model and the performance of the designed control system.

Polar body biopsy has been widely applied in preimplantation genetic diagnosis for assisted reproductive technology. The key step in the polar body biopsy is picking up the polar body from the oocyte/embryo using a micropipette. Unfortunately, the almost transparent appearance of the polar body as well as its dynamic drift when the micropipette approaches it inside the cell makes it a challenging task to pick it up with less cytoplasm loss for the cell. The unnecessary cytoplasm loss in the picking up process of the polar body easily causes damage to the development competence of the cell and may lead to disturbances to the biopsy results of the polar body. This paper proposes a precise robotic picking up method of polar bodies with less cytoplasm loss for biopsy purposes. First, a defocus imaging method is proposed to locate the polar body with an almost transparent appearance. Then, the dynamic drift of the polar body with the micropipette moving inside the cell is modeled online based on force analysis to determine an appropriate trajectory for the micropipette to approach the polar body. Further, an Active Disturbance Rejection Controller (ADRC) is designed to move the micropipette along the desired trajectory to approach the polar body and then aspirate it into the micropipette. The experimental results on porcine oocytes demonstrate that our system is capable of localizing the polar body with a success rate of 95 $\%$ and an average error of 1.12 $\pm$ 0.14 $\mu$ m. Moving along the determined trajectory, the micropipette is capable of approaching the edge of the polar body with an average error of 1.84 $\pm$ 0.31 $\mu$ m (n $=$ 20), which is only 11 $\%$ of the results obtained without dynamic drift estimation of the polar body. With this advantage, our system picks up the polar body with a close 60 $\%$ improvement in success rate (95 $\%$ vs 60 $\%$ ) and only half of the average cytoplasm loss (5 $\%$ vs 10 $\%$ ) in comparison to operation results without dynamic drift estimation. Note to Practitioners —Picking up of polar body from the oocyte/embryo using a micropipette is a vital operation in the polar body biopsy. Precisely picking up the polar body with less cytoplasm is vital to maintaining the developmental competence of the embryo/oocyte and reducing disturbances to biopsy result. This article presented a precise robotic picking up process of polar body. This process introduced defocus imaging method for polar body localization, dynamic drift estimation of polar body, and micropipette trajectory design and motion control by Active Disturbance Rejection Controller (ADRC). Experimental results have demonstrated the efficiency of the proposed robotic picking up process. Application of this process may provide an economical and practical method to carry polar body biopsy for practitioners.