CP-Net: Instance-aware part segmentation network for biological cell parsing.

Instance segmentation of biological cells is important in medical image analysis for identifying and segmenting individual cells, and quantitative measurement of subcellular structures requires further cell-level subcellular part segmentation. Subcellular structure measurements are critical for cell phenotyping and quality analysis. For these purposes, instance-aware part segmentation network is first introduced to distinguish individual cells and segment subcellular structures for each detected cell. This approach is demonstrated on human sperm cells since the World Health Organization has established quantitative standards for sperm quality assessment. Specifically, a novel Cell Parsing Net (CP-Net) is proposed for accurate instance-level cell parsing. An attention-based feature fusion module is designed to alleviate contour misalignments for cells with an irregular shape by using instance masks as spatial cues instead of as strict constraints to differentiate various instances. A coarse-to-fine segmentation module is developed to effectively segment tiny subcellular structures within a cell through hierarchical segmentation from whole to part instead of directly segmenting each cell part. Moreover, a sperm parsing dataset is built including 320 annotated sperm images with five semantic subcellular part labels. Extensive experiments on the collected dataset demonstrate that the proposed CP-Net outperforms state-of-the-art instance-aware part segmentation networks.

Correction: A spiral microfluidic device for rapid sorting, trapping, and long-term live imaging of Caenorhabditis elegans embryos.

[This corrects the article DOI: 10.1038/s41378-023-00485-4.].

Modeling and Compensation of Positioning Error in Micromanipulation.

In order to improve the positioning accuracy of the micromanipulation system, a comprehensive error model is first established to take into account the microscope nonlinear imaging distortion, camera installation error, and the mechanical displacement error of the motorized stage. A novel error compensation method is then proposed with distortion compensation coefficients obtained by the Levenberg-Marquardt optimization algorithm combined with the deduced nonlinear imaging model. The compensation coefficients for camera installation error and mechanical displacement error are derived from the rigid-body translation technique and image stitching algorithm. To validate the error compensation model, single shot and cumulative error tests were designed. The experimental results show that after the error compensation, the displacement errors were controlled within 0.25 µm when moving in a single direction and within 0.02 µm per 1000 µm when moving in multiple directions.

A spiral microfluidic device for rapid sorting, trapping, and long-term live imaging of Caenorhabditis elegans embryos.

Caenorhabditis elegans embryos have been widely used to study cellular processes and developmental regulation at early stages. However, most existing microfluidic devices focus on the studies of larval or adult worms rather than embryos. To accurately study the real-time dynamics of embryonic development under different conditions, many technical barriers must be overcome; these can include single-embryo sorting and immobilization, precise control of the experimental environment, and long-term live imaging of embryos. This paper reports a spiral microfluidic device for effective sorting, trapping, and long-term live imaging of single C. elegans embryos under precisely controlled experimental conditions. The device successfully sorts embryos from a mixed population of C. elegans at different developmental stages via Dean vortices generated inside a spiral microchannel and traps the sorted embryos at single-cell resolution through hydrodynamic traps on the sidewall of the spiral channel for long-term imaging. Through the well-controlled microenvironment inside the microfluidic device, the response of the trapped C. elegans embryos to mechanical and chemical stimulation can be quantitatively measured. The experimental results show that a gentle hydrodynamic force would induce faster growth of embryos, and embryos developmentally arrested in the high-salinity solution could be rescued by the M9 buffer. The microfluidic device provides new avenues for easy, rapid, high-content screening of C. elegans embryos.

A Novel Monopolar Cross-Scale Nanopositioning Stage Based on Dual Piezoelectric Stick-Slip Driving Principle.

The precise characterization and measurement of new nanomaterials and nano devices require in situ SEM nanorobotic instrumentation systems, which put forward further technical requirements on nanopositioning techniques of compact structure, cross-scale, nanometer accuracy, high vacuum and non-magnetic environment compatibility, etc. In this work, a novel cross-scale nanopositioning stage was proposed, which combined the advantages of piezoelectric stick-slip positioner and piezoelectric scanner techniques and adopted the idea of macro/micro positioning. A new structure design of a single flexible hinge shared by a small and large PZT was proposed to effectively reduce the size of the positioning stage and achieve millimeter stroke and nanometer motion positioning accuracy. Then, the cross-scale motion generation mechanism of the dual piezoelectric stick-slip drive was studied, the system-level dynamics model of the proposed positioning stages was constructed, and the mechanism design was optimized. Further, a prototype was manufactured and a series of experiments were carried out to test the performance of the stage. The results show that the proposed positioning stage has a maximum motion range of 20 mm and minimum step length of 70 nm under the small piezoceramic ceramic macro-motion stepping mode, and a maximum scanning range of 4.9 µm and motion resolution of 16 nm under the large piezoceramic ceramic micro-motion scanning mode. Moreover, the proposed stage has a compact structure size of 30 × 17 × 8 mm3, with a maximum motion speed of 10 mm/s and maximum load of 2 kg. The experimental results confirm the feasibility of the proposed stage, and nanometer positioning resolution, high accuracy, high speed, and a large travel range were achieved, which demonstrates that the proposed stage has significant performance and potential for many in situ SEM nanorobotic instrument systems.

Automated Denudation of Oocytes.

Denudation is a technique for removal of the cumulus cell mass from oocytes in clinical intracytoplasmic sperm injection (ICSI). Manual oocyte denudation requires long training hours and stringent skills, but still suffers from low yield rate and denudation efficiency due to human fatigue and skill variations across operators. To address these limitations, this paper reports a robotic system for automated oocyte denudation. In this system, several key techniques are proposed, including a vision-based contact detection method for measuring the relative z position between the micropipette tip and the dish substrate, recognition of oocytes and the surrounding cumulus cells, automated calibration algorithm for eliminating the misalignment angle, and automated control of the flow rate based on the model of oocyte dynamics during micropipette aspiration and deposition. Experiments on mouse oocytes demonstrated that the robotic denudation system achieved a high yield rate of 97.0 ± 2.8% and denudation efficiency of 95.0 ± 0.8%. Additionally, oocytes denuded by the robotic system showed comparable fertilization rate and developmental competence compared with manual denudation. Our robotic denudation system represents one step towards the automation and standardization of ICSI procedures.

Microrobotic Swarms for Intracellular Measurement with Enhanced Signal-to-Noise Ratio.

In cell biology, fluorescent dyes are routinely used for biochemical measurements. The traditional global dye treatment method suffers from low signal-to-noise ratios (SNR), especially when used for detecting a low concentration of ions, and increasing the concentration of fluorescent dyes causes more severe cytotoxicity. Here, we report a robotic technique that controls how a low amount of fluorescent-dye-coated magnetic nanoparticles accurately forms a swarm and increases the fluorescent dye concentration in a local region inside a cell for intracellular measurement. Different from existing magnetic micromanipulation systems that generate large swarms (several microns and above) or that cannot move the generated swarm to an arbitrary position, our system is capable of generating a small swarm (e.g., 1 µm) and accurately positioning the swarm inside a single cell (position control accuracy: 0.76 µm). In experiments, the generated swarm inside the cell showed an SNR 10 times higher than the traditional global dye treatment method. The high-SNR robotic swarm enabled intracellular measurements that had not been possible to achieve with traditional global dye treatment. The robotic swarm technique revealed an apparent pH gradient in a migrating cell and was used to measure the intracellular apparent pH in a single oocyte of living C. elegans. With the position control capability, the swarm was also applied to measure calcium changes at the perinuclear region of a cell before and after mechanical stimulation. The results showed a significant calcium increase after mechanical stimulation, and the calcium increase was regulated by the mechanically sensitive ion channel, PIEZO1.

A Novel Stick-Slip Nanopositioning Stage Integrated with a Flexure Hinge-Based Friction Force Adjusting Structure.

The piezoelectrically-actuated stick-slip nanopositioning stage (PASSNS) has been applied extensively, and many designs of PASSNSs have been developed. The friction force between the stick-slip surfaces plays a critical role in successful movement of the stage, which influences the load capacity, dynamic performance, and positioning accuracy of the PASSNS. Toward solving the influence problems of friction force, this paper presents a novel stick-slip nanopositioning stage where the flexure hinge-based friction force adjusting unit was employed. Numerical analysis was conducted to estimate the static performance of the stage, a dynamic model was established, and simulation analysis was performed to study the dynamic performance of the stage. Further, a prototype was manufactured and a series of experiments were carried out to test the performance of the stage. The results show that the maximum forward and backward movement speeds of the stage are 1 and 0.7 mm/s, respectively, and the minimum forward and backward step displacements are approximately 11 and 12 nm, respectively. Compared to the step displacement under no working load, the forward and backward step displacements only increase by 6% and 8% with a working load of 20 g, respectively. And the load capacity of the PASSNS in the vertical direction is about 72 g. The experimental results confirm the feasibility of the proposed stage, and high accuracy, high speed, and good robustness to varying loads were achieved. These results demonstrate the great potential of the developed stage in many nanopositioning applications.

Location detection of key areas in medical images based on Haar-like fusion contour feature learning.

BACKGROUND: Key area location is an important content of medical image processing and an important detail of auxiliary medical diagnosis. OBJECTIVE: In this paper, a prior knowledge fusion method based on Haar-like feature and contour feature is proposed to locate and detect key areas in medical images. METHOD: For the image to be processed, six Haar-like features and five contour features are extracted respectively. The improvement of Haar-like feature extraction template better adapts to the complexity of regional structure of medical images. The design of the contour feature extraction process fully reflects the consideration of feature invariance. The two features, together with prior knowledge, are fed into their respective decision makers and final fusers as the basis for determining and locating key regions. RESULTS: The experimental results show that the proposed method has excellent performance in locating key regions of medical images on MRI. When the capacity of the database increases from 10 to 200, the accuracy of locating the key areas of the image to be processed still reaches more than 90%. CONCLUSION: The proposed method realizes the accurate location of the key areas of medical images, which is of great significance for the auxiliary medical diagnosis.

Robotic Immobilization of Motile Sperm for Clinical Intracytoplasmic Sperm Injection.

OBJECTIVE: In clinical intracytoplasmic sperm injection (ICSI), a motile sperm must be immobilized before insertion into an oocyte. This paper aims to develop a robotic system for automated tracking, orientation control, and immobilization of motile sperms for clinical ICSI applications. METHODS: We adapt the probabilistic data association filter by adding sperm head orientation into state variables for robustly tracking the sperm head and estimating sperm tail positions under interfering conditions. The robotic system also utilizes a motorized rotational microscopy stage and a new visual servo control strategy that predicts and compensates for sperm movements to actively adjust sperm orientation for immobilizing a sperm swimming in any direction. RESULTS: The system robustly tracked sperm head with a tracking success rate of 96.0% and estimated sperm tail position with an accuracy of 1.08 µm under clinical conditions where the occlusion of the target sperm and interference from other sperms occur. Experimental results from robotic immobilization of 400 sperms confirmed that the system achieved a consistent immobilization success rate of 94.5%, independent of sperm velocity or swimming direction. CONCLUSION: Our adapted tracking algorithm effectively distinguishes the target sperm from interfering sperms. Predicting and compensating for sperm movements significantly reduce the positioning error during sperm orientation control. These features make the robotic system suitable for automated sperm immobilization. SIGNIFICANCE: The robotic system eliminates stringent skill requirements in manual sperm immobilization. It is capable of manipulating sperms swimming in an arbitrary direction with a high success rate.

Injectable Silk-Vaterite Composite Hydrogels with Tunable Sustained Drug Release Capacity.

Improving the efficiency of chemotherapy remains a key challenge in drug delivery. Many drug carriers have been designed to achieve multifunctional factors as part of their performance, including controlled release, dispersibility in aqueous environments, and targeting to cancer sites. However, it is difficult to optimize multiple properties simultaneously for a single carrier system. Here, synergistic carriers composed of vaterite microspheres and silk nanofiber hydrogels were developed to improve the dispersibility of vaterite spheres and the control of drug delivery without compromising the injectability or sensitivity to pH. The vaterite microspheres were dispersed homogeneously and remained stable in the silk nanofiber hydrogels. Doxorubicin (DOX) was effectively loaded on the vaterite spheres and silk nanofibers, forming synergistic silk-vaterite hydrogel delivery systems. The sustained delivery of DOX was tuned and controlled by vaterite stability and the DOX content loaded on the spheres and nanofibers. The cytotoxicity was regulated via the controlled delivery of DOX, suggesting the possibility of optimizing chemotherapeutic strategies. These silk-vaterite delivery hydrogels suggest a useful strategy for designing novel delivery systems for improved delivery and therapeutic benefits.

A Paper-Based Piezoelectric Accelerometer.

This paper presents the design and testing of a one-axis piezoelectric accelerometer made from cellulose paper and piezoelectric zinc oxide nanowires (ZnO NWs) hydrothermally grown on paper. The accelerometer adopts a cantilever-based configuration with two parallel cantilever beams attached with a paper proof mass. A piece of U-shaped, ZnO-NW-coated paper is attached on top of the parallel beams, serving as the strain sensing element for acceleration measurement. The electric charges produced from the ZnO-NW-coated paper are converted into a voltage output using a custom-made charge amplifier circuit. The device fabrication only involves cutting of paper and hydrothermal growth of ZnO NWs, and does not require the access to expensive and sophisticated equipment. The performance of the devices with different weight growth percentages of the ZnO NWs was characterized.

Effect of Cell Inner Pressure on Deposition Volume in Microinjection.

Microinjection is a widely used technique for introducing exogenous materials into cells. Many applications of microinjection, such as gene editing and drug testing, rely on the accurate control of the deposition volume. However, the deposition volume in microinjection is presently calibrated in an open medium without considering the cell inner pressure effect, which we experimentally show in this paper that it can induce an error as large as 30% between the actual deposition volume and the set volume. In this work, the relationship between the cell inner pressure and the deposition volume was analytically modeled and experimentally validated. On the basis of the developed model, the cell inner pressure of a given cell type can be well estimated from the injection pressure and the resulting deposition volume. The quantitated cell inner pressure is then used to reduce the error between the set volume and the actual deposition volume. Experiments conducted on human bladder cancer cells (T24 and RT4) showed that T24 cells have a higher inner pressure than RT4 cells (405 ± 45 Pa vs 341 ± 34 Pa), and after compensating for the cell inner pressure, the error between the intended set volume and the actual deposition volume into a cell became less than 3%.

Automated Non-Invasive Measurement of Single Sperm's Motility and Morphology.

Measuring cell motility and morphology is important for revealing their functional characteristics. This paper presents automation techniques that enable automated, non-invasive measurement of motility and morphology parameters of single sperm. Compared to the status quo of qualitative estimation of single sperm's motility and morphology manually, the automation techniques provide quantitative data for embryologists to select a single sperm for intracytoplasmic sperm injection. An adapted joint probabilistic data association filter was used for multi-sperm tracking and tackled challenges of identifying sperms that intersect or have small spatial distances. Since the standard differential interference contrast (DIC) imaging method has side illumination effect which causes inherent inhomogeneous image intensity and poses difficulties for accurate sperm morphology measurement, we integrated total variation norm into the quadratic cost function method, which together effectively removed inhomogeneous image intensity and retained sperm's subcellular structures after DIC image reconstruction. In order to relocate the same sperm of interest identified under low magnification after switching to high magnification, coordinate transformation was conducted to handle the changes in the field of view caused by magnification switch. The sperm's position after magnification switch was accurately predicted by accounting for the sperm's swimming motion during magnification switch. Experimental results demonstrated an accuracy of 95.6% in sperm motility measurement and an error <10% in morphology measurement.

A Flexure-Guided Piezo Drill for Penetrating the Zona Pellucida of Mammalian Oocytes.

Mammalian oocytes such as mouse oocytes have a highly elastic outer membrane, zona pellucida (ZP) that cannot be penetrated without significantly deforming the oocyte, even with a sharp micropipette. Piezo drill devices leverage lateral and axial vibration of the micropipette to accomplish ZP penetration with greatly reduced oocyte deformation. However, existing piezo drills all rely on a large lateral micropipette vibration amplitude ( 20 ) and a small axial vibration amplitude (0.1 ). The very large lateral vibration amplitude has been deemed to be necessary for ZP penetration although it also induces larger oocyte deformation and more oocyte damage. This paper reports on a new piezo drill device that uses a flexure guidance mechanism and a systematically designed pulse train with an appropriate base frequency. Both simulation and experimental results demonstrate that a small lateral vibration amplitude (e.g., 2 ) and an axial vibration amplitude as large as 1.2 were achieved. Besides achieving 100% effectiveness in the penetration of mouse oocytes (n = 45), the new piezo device during ZP penetration induced a small oocyte deformation of 3.4 versus larger than 10 using existing piezo drill devices.

Microfluidic Assessment of Frying Oil Degradation.

Monitoring the quality of frying oil is important for the health of consumers. This paper reports a microfluidic technique for rapidly quantifying the degradation of frying oil. The microfluidic device generates monodispersed water-in-oil droplets and exploits viscosity and interfacial tension changes of frying oil samples over their frying/degradation process. The measured parameters were correlated to the total polar material percentage that is widely used in the food industry. The results reveal that the steady-state length of droplets can be used for unambiguously assessing frying oil quality degradation.

Human sperm rheotaxis: a passive physical process.

A long-standing question in natural reproduction is how mammalian sperm navigate inside female reproductive tract and finally reach the egg cell, or oocyte. Recently, fluid flow was proposed as a long-range guidance cue for sperm navigation. Coitus induces fluid flow from oviduct to uterus, and sperm align themselves against the flow direction and swim upstream, a phenomenon termed rheotaxis. Whether sperm rheotaxis is a passive process dominated by fluid mechanics, or sperm actively sense and adapt to fluid flow remains controversial. Here we report the first quantitative study of sperm flagellar motion during human sperm rheotaxis and provide direct evidence indicating that sperm rheotaxis is a passive process. Experimental results show that there is no significant difference in flagellar beating amplitude and asymmetry between rheotaxis-turning sperm and those sperm swimming freely in the absence of fluid flow. Additionally, fluorescence image tracking shows no Ca(2+) influx during sperm rheotaxis turning, further suggesting there is no active signal transduction during human sperm rheotaxis.

Recent advances in nanorobotic manipulation inside scanning electron microscopes.

A scanning electron microscope (SEM) provides real-time imaging with nanometer resolution and a large scanning area, which enables the development and integration of robotic nanomanipulation systems inside a vacuum chamber to realize simultaneous imaging and direct interactions with nanoscaled samples. Emerging techniques for nanorobotic manipulation during SEM imaging enable the characterization of nanomaterials and nanostructures and the prototyping/assembly of nanodevices. This paper presents a comprehensive survey of recent advances in nanorobotic manipulation, including the development of nanomanipulation platforms, tools, changeable toolboxes, sensing units, control strategies, electron beam-induced deposition approaches, automation techniques, and nanomanipulation-enabled applications and discoveries. The limitations of the existing technologies and prospects for new technologies are also discussed.

Suspended, Shrinkage-Free, Electrospun PLGA Nanofibrous Scaffold for Skin Tissue Engineering.

Electrospinning is a technique for creating continuous nanofibrous networks that can architecturally be similar to the structure of extracellular matrix (ECM). However, the shrinkage of electrospun mats is unfavorable for the triggering of cell adhesion and further growth. In this work, electrospun PLGA nanofiber assemblies are utilized to create a scaffold. Aided by a polypropylene auxiliary supporter, the scaffold is able to maintain long-term integrity without dimensional shrinkage. This scaffold is also able to suspend in cell culture medium; hence, keratinocyte cells seeded on the scaffold are exposed to air as required in skin tissue engineering. Experiments also show that human skin keratinocytes can proliferate on the scaffold and infiltrate into the scaffold.

Robotic adherent cell injection for characterizing cell-cell communication.

Compared to robotic injection of suspended cells (e.g., embryos and oocytes), fewer attempts were made to automate the injection of adherent cells (e.g., cancer cells and cardiomyocytes) due to their smaller size, highly irregular morphology, small thickness (a few micrometers thick), and large variations in thickness across cells. This paper presents a robotic system for automated microinjection of adherent cells. The system is embedded with several new capabilities: automatically locating micropipette tips; robustly detecting the contact of micropipette tip with cell culturing surface and directly with cell membrane; and precisely compensating for accumulative positioning errors. These new capabilities make it practical to perform adherent cell microinjection truly via computer mouse clicking in front of a computer monitor, on hundreds and thousands of cells per experiment (versus a few to tens of cells as state of the art). System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4000 cells. This paper also reports the use of the new robotic system to perform cell-cell communication studies using large sample sizes. The gap junction function in a cardiac muscle cell line (HL-1 cells), for the first time, was quantified with the system.