Minghui Li

For the first time, an improved robotic micropipette aspiration (MA) method considering the effects of the intra-cellular pressure, friction between cell and micropipette, and the micropipette opening size was proposed for improving the measurement accuracy of the cell elasticity properties in this paper. First, an elastic energy model of the aspirated cell was established to analyze the effects of intracellular pressure on MA results. Then a force analysis of MA process was conducted to evaluate the influences of the friction force between the cell surface and micropipette opening as well as the inner diameter of the micropipette opening to MA results. Based on the above work, an improved robotic MA process was developed for reducing the cell elasticity measurement errors through automated release of intracellular pressure, micropipette opening polishing and inner diameter control of the micropipette. The effectiveness of the above work was validated by both finite elemental model (FEM) and MA experiments. The MA results on porcine oocytes demonstrate that our proposed MA method can rectify the 20.11%, 35.08% and 6.80% measure errors of cell Young’s modulus caused by the above three factors, respectively. Our research may provide a more objective evaluation of cell mechanical properties in the related research.

In vivo cell manipulation is crucial for understanding organ functions and dysfunctions at the cellular level. Due to its low optical transparency, the in vivo environment needs to be lighted up first to visualize the targets for manipulation. At present, operators usually carefully blow a micropipette filled with fluorescent dye solution to create a fluorescence field around the micropipette opening, lighting up the in vivo environment. This manual dynamic control of the fluorescence field is usually a low-accuracy, labor-intensive, and high-skill requirement task, becoming even more challenging when the micropipette moves to locate or approach target cells. A large number of in vivo lighting-control tests may improve manual control efficiency of the fluorescent field, but they usually cost many precise animal samples, making them costly and often unaffordable. To enhance the dynamic control efficiency of the fluorescent field while reducing experimental costs, a robotic fluorescent lighting control method is proposed based on an in vivo dynamic fluorescent imaging simulator. First, a dynamic fluorescence imaging model composing an injection model, a diffusion model, and a luminescence model of fluorescent molecules is established to simulate the fluorescence field in vivo. The fluorescent intensity distribution obtained through the model is highly in accordance with experimental results. Based on this model, an adaptive sliding mode controller is employed to achieve the desired fluorescent intensity in the brain tissue. Both the simulation results and the experimental results demonstrate significant advantages of the proposed controller in terms of control accuracy and stability compared to the traditional PID controller and manual operation. the fluorescent intensity at a specified position in the fluorescent field. Simulations based on the established model, along with experiments on brain tissues, demonstrate the controller’s effectiveness. Our system is expected to reduce human involvement and enable high-precision lighting for in vivo cell manipulation in the future.

The inside-out patch clamp technique has been widely applied in brain science and neuroscience research due to its ability to detect extremely weak currents flowing through a single ion channel. The current manual inside-out patch clamp operations are highly expertise-requisite and low efficient. Meanwhile, the existing robotic systems are only applicable for suspended cells due to their new system setups. For the first time, this letter proposed a robotic inside-out patch clamp system for adherent cells based on vesicle rupture control. Firstly, impedance models were established to detect the vesicle rupture state. Then, a force analysis that combines the defocusing imaging model was developed to precisely control the exposure time of the vesicle in the air, which is a key factor in the rupture process of the vesicle. Based on the above works, a robotic inside-out patch clamp process for adherent cells was established. Experimental results demonstrate that the proposed robotic system can detect vesicle rupture state with a 100% success rate, control exposure time with an average error of 0.02$\,\text{s}$ and operate adherent HEK-293 cells with a success rate of 70% at an average operation speed of 61.3$\,$seconds/cell. The success rate of our system is more than three times that of manual operation results, laying a solid foundation for subsequent single ion channel functionality research.

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.

In recent years, the perforated patch clamp technique has been widely applied in cellular electrophysiology research due to its low mechanical disturbance and almost no loss of cellular content in cell membrane perforation process. However, the current passive release process of perforating materials prolongs the perforation process and easily disturbs the gigaseal formation process, significantly lowering the efficiency of perforated patch clamp operation. Addressing this, a robotic perforated patch clamp system was developed based on the active release control of perforating materials in this paper. First, a novel holding module of the patch micropipette integrated with an independently driven transmission channel of the perforating materials was developed for the first time. Then, through release tests of the perforating materials, the appropriate drive mode of transmission channel was determined to be the hydraulic mode with a faster response and higher stability. Further, the concentration gradient field of the perforating materials at the opening of the channel was modeled according to Fick’s law to prevent the false release of them in gigaseal formation process. Furthermore, a cell circuit model was developed to detect perforation degree online for feedback control of the perforating materials release. Experimental results on pyramidal neurons in mouse brain slices demonstrated that, in comparison to the traditional method with passive releases of perforating materials, the proposed system was capable of perforating cell membrane at an almost doubled throughput and with a 57% improvement in the success rate. In comparison to the traditional non-perforated whole-cell patch clamp method, the signal recording duration of neurons operated by our method was doubled due to its fewer negative influences on gigaseal and almost no cellular content loss. Note to Practitioners —The perforated patch clamp technique, utilizes the cell membrane-perforating molecules to drill subnanometer-sized conductive pores in the cell membrane aspirated into a micropipette for the measurement of cellular electrophysiological signals. Unfortunately, the poor controllability of the current passive release of perforating materials in the patch clamp operation easily leads to a long drug diffusion process, and also, disturbs the gigaseal formation between the aspirated cell membrane and micropipette, which is required for the measurement of the extremely weak cellular electrophysiological signals. For the first time, an active release control method of perforating materials was developed in this paper based on the self-developed novel three-channel holding device. With active release control of perforating materials, the proposed method was capable of perforating cell membranes at a doubled speed with a significantly higher success rate, and doubled recording duration in comparison to the traditional perforated patch clamp methods and non-perforated whole-cell patch clamp method, respectively, due to its fewer negative influences on gigaseal and almost no cellular content loss. With the above advantages, our robotic perforated patch clamp method may be applied in cellular electrophysiology research in the future.

Cell transportation is a key step in many biological applications. Currently, most cell transportation research relies on microscopic vision feedback, which limits its application at places when a microscopic view is unavailable. For the first time, this paper proposed a robotic microscopic view-free cell transportation method based on pressure variation model inside a self-made bilayer micropipette. The bilayer micropipette, made by inserting a thin micropipette into a thicker one, picks the target cell with the outsider micropipette and holds it with the inner micropipette in cell transportation. To achieve this, the appropriate fluidic forces to pick and place the cell were determined based on force analysis. Then, the appropriate inner diameter of the inner thin micropipette was determined based on the micropipette aspiration model. Further, the pressure variation inside the bilayer micropipette during holding and releasing process were modeled to conduct a robotic microscopic vision-fee cell transportation. Experimental results demonstrated that our system was capable of transporting zebrafish embryos at an average speed of 25 s/cell with a success rate of 90%, with a transportation efficiency comparable to the related methods with microscopic view feedback. Besides, ignorable mechanical harm to the development competence of the zebrafish embryos was confirmed through culturing experiments. Note to Practitioners —Cell transportation is an important step in many biomedical researches. Currently, cell transportation usually relies on microscopy for picking up, and releasing cells, limiting its application in special environments without microscopic view feedback. In this paper, a robotic microscopic vision-free cell transportation method is proposed for the first time. In this method, cells are transported within a self-made bilayer micropipette. Pressure variations inside the micropipette are utilized to determine cell picking up and release. This robotic transport method demonstrated a high success rate and low harm to cells in experiments. Our research may contribute to the future development of vision-free and fully automated robotic cell culture techniques. Our research may spike inspiration for future research on microscopic vision-free cell transportation in fully automated cell culture system.

Intracellular pressure regulates cell physiological activities and impacts cell micromanipulation results. The measurement of intracellular pressure may reveal the mechanism of these cell physiological activities and improve the micromanipulation accuracy for cells. Current intracellular pressure measurement methods usually rely on specialized and expensive devices or have significant cytotoxicities on cells. In this paper, a simple robotic intracellular measurement method based on an improved balance pressure model is developed on the traditional cell manipulation system setup. First, an improved balance pressure model is proposed to calculate the intracellular pressure. Then, a traditional glass micropipette is utilized as a probe to penetrate the cell membrane to measure intracellular pressure. After cell membrane penetration, the key parameters of the improved balance pressure model including the cell deformation and the moving distance of the gas-liquid interface (GLI) inside the micropipette are measured in time to calculate the intracellular pressure. The experimental results on porcine oocytes demonstrate that the proposed method has an 80% success rate at an average measurement speed of 12 seconds/cell. Further, no intracellular pressure leaking was tested during the measurement process, guaranteeing measurement accuracy. Further, an 87.5% survival rate of operated oocytes was obtained after the measurement, proving limited damage to cell viability. Our method is highly expected to be applied in biological applications requiring in situ measurement of intracellular pressure.

Intracellular pressure, a key physical parameter of the intracellular environment, has been found to regulate multiple cell physiological activities and impact cell micromanipulation results. The intracellular pressure may reveal the mechanism of these cells’ physiological activities or improve the micro-manipulation accuracy for cells. The involvement of specialized and expensive devices and the significant damage to cell viability that the current intracellular pressure measurement methods cause significantly limit their wide applications. This paper proposes a robotic intracellular pressure measurement method using a traditional micropipette electrode system setup. First, the measured resistance of the micropipette inside the culture medium is modeled to analyze its variation trend when the pressure inside the micropipette increases. Then, the concentration of KCl solution filled inside the micropipette electrode that is suitable for intracellular pressure measurement is determined according to the tested electrode resistance–pressure relationship; 1 mol/L KCl solution is our final choice. Further, the measurement resistance of the micropipette electrode inside the cell is modeled to measure the intracellular pressure through the difference in key pressure before and after the release of the intracellular pressure. Based on the above work, a robotic measurement procedure of the intracellular pressure is established based on a traditional micropipette electrode system. The experimental results on porcine oocytes demonstrate that the proposed method can operate on cells at an average speed of 20~40 cells/day with measurement efficiency comparable to the related work. The average repeated error of the relationship between the measured electrode resistance and the pressure inside the micropipette electrode is less than 5%, and no observable intracellular pressure leakage was found during the measurement process, both guaranteeing the measurement accuracy of intracellular pressure. The measured results of the porcine oocytes are in accordance with those reported in related work. Moreover, a 90% survival rate of operated oocytes was obtained after measurement, proving limited damage to cell viability. Our method does not rely on expensive instruments and is conducive to promotion in daily laboratories.

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.