Segmenting individual trophectoderm (TE) cells is essential for developing quantitative metrics to assess the developmental potential of human blastocysts. The elongated shape and circular arrangement of TE cells lead to continuously varying orientations across the image, posing challenges for existing cell instance segmentation methods that assume uniformly oriented cells. As a result, most methods segment the TE as a single region, and the development of quantitative, cell-level metrics predictive of live birth potential remains unexplored. In this work, we propose an instance segmentation model that represents elongate, circularly arranged TE cells using elliptical distance maps, with which superior performance in both segmentation accuracy and metric extraction was achieved, compared with state-of-the-art methods. The extracted metrics, including TE cell number, the mean and standard deviation of cell length, width, area, and mean inter-cell distance, serve as effective predictors of a blastocyst’s live birth potential. When used to predict live birth, these metrics achieved a significantly higher area under the receiver operating characteristic curve (AUC = 0.693) than traditional TE morphological grades (AUC = 0.585). The source code is publicly available at https://github.com/robotVisionHang/TESeg.

Deep learning has been widely applied in various domains. Current widely-used optimizers, such as SGD, Adam, and their variants, are designed based on the assumption that the gradient noise generated during model training follows a Gaussian distribution. However, recent empirical studies have found that the gradient noise often does not follow a Gaussian distribution. Instead, the noise exhibits heavy-tailed characteristics consistent with an $\alpha $ -stable distribution, casting doubt on the performance and robustness of optimizers designed under the assumption of Gaussian noise. Inspired by the least mean p-power (LMP) algorithm from the field of adaptive filtering, we propose a novel optimizer called Ape for deep learning. Ape integrates a p-power adjustment mechanism to compress large gradients and amplify small ones, mitigating the impact of heavy-tailed gradient distributions. It also employs an approach for estimating second moments tailored to $\alpha $ -stable distributions. Extensive experiments on benchmark datasets demonstrate Ape's effectiveness in improving both accuracy and training speed compared to existing optimizers. The Ape optimizer showcases the potential of cross-disciplinary approaches in advancing deep learning optimization techniques and lays the groundwork for future innovations in this domain.

Quantifying sperm flagellar beating behavior (e.g., beating amplitude, frequency, and wavelength) plays a crucial role in biological research, clinical diagnostics, and the design of sperm-inspired microrobots. However, existing computational methods struggle to accurately and efficiently analyze the highly dynamic, complex, and fine structures of sperm flagella, especially when portions of the flagellum become invisible due to three-dimensional out-of-focus beating. This paper proposes an automated high-throughput tool for quantitative analysis of sperm flagellar beating. The core innovation is continuous convolution (CConv), which adaptively captures the irregular, time-varying patterns of sperm flagella while ensuring continuity in segmentation outputs, even in the presence of locally invisible regions caused by out-of-focus motion. CConv can be integrated into various neural network architectures as a plug-and-play module. Extensive experiments demonstrate that integrating CConv consistently improves the accuracy and continuity of flagella segmentation across different networks. Furthermore, utilizing a curvature-based approach, we quantified key flagellar beating parameters, including length, amplitude, frequency, and wavelength. Applying the high-throughput tool on 1200 sperm revealed that sperm from fertile donors had significantly higher flagellar beating frequency than sperm from infertile patients. The proposed automated tool unlocks high-throughput, quantitative analysis of sperm flagellar beating, showing the potential for applications in reproductive biology and engineering research. The codes and datasets will be released at https://github.com/Goldfish-Yu/CConv.

Sperm morphology measurement is vital for diagnosing male infertility, which involves quantification of multiple subcellular parts for each sperm. Instance-aware part segmentation networks have been introduced to address this task by automatically identifying individual sperm and segmenting their subcellular parts. However, major limitations of state-of-the-art instance-aware part segmentation networks include: 1) they are time-consuming and computational expensive due to sequential processing and multi-stage frameworks; 2) they perform poorly for densely packed sperm that overlap or cross over one another. To overcome these challenges, this paper proposes 1) integrating instance identification and subcellular part segmentation within a single-stage framework to save inference time and memory usage; 2) dividing a sperm target into simpler components (head and tail) to improve prediction accuracy, followed by a contrastive learning-based matching method to pair the head and tail. Experimental results on our clinically collected human sperm dataset demonstrated that the proposed network not only outperformed state-of-the-art CP-Net (by 3.5% APp vol) but also achieved realtime inference (48.0 frames per second), effectively meeting the clinical requirements for automated parts segmentation of sperm. final part segmentation results. 2) Since the sperm head and tail have simpler shapes, they are detected separately to improve segmentation accuracy. A contrastive learning-based method is then designed to pair head and tail based on similarity of feature embeddings extracted from the proposed instance prediction branch. The proposed method significantly outperformed existing networks, particularly in handling densely packed sperm. The presented method has applicability to analyzing sperm and more broadly other cell types.

Automated morphological measurement of cellular and subcellular structures in live cells is important for evaluating cell functions. Due to their small size and transparent appearance, visualizing cellular and subcellular structures often requires high magnification microscopy and fluorescent staining. However, high magnification microscopy gives a limited field of view, and fluorescent staining alters cell viability and/or activity. Therefore, microscopy image enhancement methods have been developed to predict detailed intracellular structures in live cells. Existing image enhancement networks are mostly CNN-based models lacking global information or Transformer-based models lacking local information. For these purposes, a novel CNN-Transformer combined bilateral U-Net (CTBUnet) is proposed to effectively aggregate both local and global information. Experiments on the collected sperm cell enhancement dataset demonstrate the effectiveness of proposed network for both super-resolution and virtual staining prediction.

An unbiased assessment of sperm morphology and motility is crucial for assessing fertility potential and guiding visual feedback for microrobotic manipulation. Automated analysis and selection of optimal sperm are essential for in vitro fertilization treatments, such as robotic intracytoplasmic sperm injection. However, conventional image processing methods face limitations in analyzing small sperm objects under microscopic imaging. While convolutional neural networks (CNNs) have brought promising advancements in microscopic image analysis, previous CNN methods have struggled to accurately differentiate tiny objects. These methods often require staining or fluorescence techniques to enhance visual contrast between sperm and culture medium, leading to clinical impracticality. To address these limitations, we introduce a novel sperm recognition network named the sperm feature-correlated network (SFCNet), for accurate and efficient segmentation and tracking of minute sperm objects. The SFCNet employs innovative modules, including collateral multi-scale convolution, cross-scale feature map guide, atrous spatial pyramid convolution with pooling, lateral attention, and multi-scale tracking proposal, to preserve essential sperm details despite their small size. Experimental results indicate that the SFCNet surpassed the state-of-the-art models designed for segmenting or tracking small objects, achieving up to a 28.39% higher Sorensen-Dice coefficient in segmentation and a 10.33% higher average precision in tracking. Additionally, the SFCNet excelled in sperm morphometric analysis, achieving errors below 15%. Moreover, the SFCNet also secured top-tier performance in sperm motility analysis, acquiring errors below 13% in seven sperm motility parameters.Note to Practitioners—This study is stimulated by the need to analyze the quality of motile sperms and select the optimal one for in vitro fertilization. Existing methods for detecting sperm fall short as they require a relatively high-magnification microscopic image or the usage of stain or fluorescence to increase sperm visualization, which limits the selection process or even makes the sperm clinically unavailable. To overcome these limitations, the present work proposes a new framework based on deep learning, which includes the design of extracting multi-scale sperm features. Experimental results suggest that the proposed method can perform better than existing methods in real-time analysis of multiple motile sperms’ morphology and motility at 20$\times$objective. In the future, there is a high potential for fertility specialists and healthcare workers to apply the presented framework in fertility treatment with higher accuracy and efficiency.

This paper presents an automated point-of-care semen analysis method that uses smartphone imaging to visually measure sperm concentration and motility of semen samples. The proposed method follows the same visual tracking scheme as laboratory semen analysis systems, aiming to match clinical standards while being suitable for point-of-care use. A boundary-sensitive segmentation network is developed to identify and distinguish sperm from impurities in raw semen. A novel occlusion-aware multi-sperm tracking algorithm is proposed to tackle challenges posed by smartphone imaging and undiluted raw semen samples. For automated motility measurement, an occlusion-awareness module is proposed to robustly track multiple sperm during frequent sperm crossover/occlusion. The module combines the segmented contour and kinematic-based probabilistic modeling to determine the occlusion status of both targets and measurements, facilitating fundamental improvement to feasible joint event enumeration to enable robust data association. The proposed method achieved a high success rate of 95.14% for tracking occluded sperm, with low mean errors for sperm concentration (2.03 million/ml) and motility (1.58%), outperforming existing multi-sperm tracking methods. In clinical tests involving 50 participants, our method exhibited good agreement with clinical standards (Spearman rank correlation coefficients of 0.94 for concentration and 0.89 for motility) even when used by inexperienced users.Note to Practitioners—Semen analysis is the gold standard method for assessing male reproductive capacity. Clinical semen analysis routinely uses professional computer-assisted semen analysis (CASA) systems to examine sperm concentration and motility; however, clinical visits for semen analysis are not always feasible due to the unavailability of such professional systems and the mental stress brought by clinical visits. This work provides a point-of-care semen analysis method. For hardware, a smartphone microscopic imaging modality was developed to enable clear visualization of sperm with the built-in smartphone camera. For software, an occlusion-aware multi-sperm tracking algorithm was proposed to automatically measure sperm concentration and motility. In addition to intensively validating the proposed point-of-care method against clinical CASA systems, this work also analyzed different types of tracking failure and quantified their effects on the automated evaluation of sperm concentration and motility. The techniques pave the way for further improvement in both point-of-care and clinical semen analysis. Overall, this work offers an accessible and reliable tool for automated male fertility evaluation.

Automated positioning systems play a pivotal role in micro-scale cell manipulation. In clinical intracytoplasmic sperm injection (ICSI) for infertility treatment, a motile sperm needs to be immobilized by glass micropipette tapping for subsequent surgical steps. The process requires accurate tracking of the target sperm and precise alignment between the sperm tail and the micropipette. Manual sperm immobilization suffers from inconsistent success rates, and current robotic systems developed for the task fail to comply with the standard clinical setup. Instead of using a motorized micromanipulator as in existing robotic systems, this paper presents an automated and compact three-dimensional (3-D) positioning stage for sperm immobilization that can be seamlessly integrated into standard clinical platforms. To tackle the challenge of accurately tracking the target sperm with degraded detection quality due to the complex 3-D motion of the positioning stage, a multi-stage sperm tracking scheme is designed for detection-to-tracklet association. To prevent physical contact between the sperm head and the micropipette, an adaptive tail-tapping planning strategy based on the sperm head orientation analysis is established. A visual servo controller equipped with a dynamic sperm motion observer is further employed to achieve precise positioning of the target sperm during the immobilization process. Experimental results demonstrated that the proposed system achieved a sperm tracking accuracy of 88.12%, and a sperm positioning accuracy of$2.3~\pm ~1.2~\mu $m. Further experiments revealed the system achieved a success rate of 93.5% and a time cost of 5.5 s for automated sperm immobilization. Note to Practitioners—This work presents an automated positioning system for the robotic immobilization of live human sperm in the intracytoplasmic sperm injection (ICSI) process. Conventional robotic systems developed for sperm immobilization use motorized stages which only provide two degrees of freedom (DOF) and disturb standard clinical setups with bulky sizes and additional equipment. Leveraging the advantages of piezoelectric positioners, a compact 3-D positioning system is developed to perform robotic sperm immobilization in an all-in-one manner. A sperm immobilization tracker is developed to track the target sperm during the immobilization process. The proposed multi-stage data association metric can effectively resist the noisy detection results due to occlusion and out-of-focus blur introduced by the 3-D movement of the positioning stage. Based on the analysis of the sperm head orientation, an adaptive tail-tapping planning strategy is established to avoid the risk of contacting the sperm head where DNA is contained. The developed positioning system can be easily integrated into a standard microscopy operation platform for biological cell manipulation. The proposed tracking scheme is applicable in various microscopy cell analysis scenarios where the detection results are inevitably affected by the degraded image quality.

Traditional sperm morphology analysis is based on tedious manual annotation. Automated morphology analysis of a high number of sperm requires accurate segmentation of each sperm part and quantitative morphology evaluation. State-of-the-art instance-aware part segmentation networks follow a "detect-then-segment" paradigm. However, due to sperm’s slim shape, their segmentation suffers from large context loss and feature distortion due to bounding box cropping and resizing during ROI Align. Moreover, morphology measurement of sperm tail is demanding because of the long and curved shape and its uneven width. This paper presents automated techniques to measure sperm morphology parameters automatically and quantitatively. A novel attention-based instance-aware part segmentation network is designed to reconstruct lost contexts outside bounding boxes and to fix distorted features, by refining preliminary segmented masks through merging features extracted by feature pyramid network. An automated centerline-based tail morphology measurement method is also proposed, in which an outlier filtering method and endpoint detection algorithm are designed to accurately reconstruct tail endpoints. Experimental results demonstrate that the proposed network outperformed the state-of-the-art top-down RP-R-CNN by 9.2% ${\mathbf{AP}}_{vol}^p$, and the proposed automated tail morphology measurement method achieved high measurement accuracies of 95.34%,96.39%,91.20% for length, width and curvature, respectively.

Unbiased measurement of sperm morphometric and motility parameters is essential for assessing fertility potential and guiding visual feedback for microrobotic manipulation. Automated analysis of multiple sperms and selection of an optimal sperm is crucial for in vitro fertilisation treatment such as robotic intracytoplasmic sperm injection. However, conventional image processing methods have limitations in analysing small sperm objects under microscopic imaging. The emergence of convolutional neural networks (CNNs) has offered promising advancements in microscopic image analysis. However, previous CNN methods have struggled to accurately segment tiny objects, requiring staining or fluorescence techniques to enhance visual contrast between sperm and culture medium, leading to clinical impracticality. To address these limitations, we introduce a novel segmentation network named the cross-scale guidance (CSG) network for accurate and efficient segmentation of minute sperm objects. The CSG network employs innovative modules, including collateral multi-scale convolution, cross-scale feature map guide, and multi-scale feature fusion, to preserve essential sperm details despite their small size. Experimental results indicate that the CSG network surpassed the state-of-the-art models designed for small object segmentation, achieving a significant increase up to 18.62% higher mean intersection over union (mIoU). Additionally, the CSG network excelled in sperm morphometric analysis, achieving errors below 20%. Moreover, sperm motility parameters were further derived from the segmentation results for comprehensive sperm fertility analysis.

Automated positioning systems play a pivotal role in micro-scale cell manipulation. In clinical intracytoplasmic sperm injection (ICSI) of in vitro fertilization (IVF) treatment, a motile sperm needs to be immobilized by glass micropipette tapping for subsequent surgical steps. The process requires accurate tracking of the target sperm and precise alignment between the sperm tail and the micropipette. Manual sperm immobilization suffers from inconsistent success rates, and current robotic systems developed for the task fail to comply with the standard clinical setup. Instead of using a motorized micromanipulator as in existing robotic systems, this paper presents an automated, compact three-dimensional positioning stage for sperm immobilization that can be seamlessly integrated into standard clinical platforms. Based on the analysis of the sperm head orientation, an adaptive tail tapping planning strategy is established to avoid the risk of touching the sperm head where DNA is contained. A visual servo controller equipped with a dynamic sperm motion observer is employed to achieve precise tracking and positioning of the target sperm three-dimensionally. Experimental results revealed the system achieved a success rate of 93.5% and a time cost of 5.5 s for automated sperm immobilization.

Obtaining three-dimensional information, especially the z-axis depth information, is crucial for robotic micromanipulation. Due to the unavailability of depth sensors such as lidars in micromanipulation setups, traditional depth acquisition methods such as depth from focus or depth from defocus directly infer depth from microscopic images and suffer from poor resolution. Alternatively, micromanipulation tasks obtain accurate depth information by detecting the contact between an end-effector and an object (e.g., a cell). Despite its high accuracy, only sparse depth data can be obtained due to its low efficiency. This paper aims to address the challenge of acquiring dense depth information during robotic cell micromanipulation. A weakly-supervised depth completion network is proposed to take cell images and sparse depth data obtained by contact detection as input to generate a dense depth map. A two-stage data augmentation method is proposed to augment the sparse depth data, and the depth map is optimized by a network refinement method. The experimental results show that the MAE value of the depth prediction error is less than 0.3 µm, which proves the accuracy and effectiveness of the method. This deep learning network pipeline can be seamlessly integrated with the robotic micromanipulation tasks to provide accurate depth information.

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.

This paper reports, for the first time, automated grading of human blastocysts (day-5 embryos) from multi-focus images. Based on a novel attention module, a convolutional neural network (CNN) was developed to predict the morphological grade of a blastocyst. The attention module integrates high-level features extracted from the blastocyst’s multi-focus images. Experimental results revealed that multi-focus blastocyst images help improve the grading accuracies than a single blastocyst image. Comparisons of the accuracy achieved by the model and the average accuracy of five embryologists demonstrated that the proposed model can outperform embryologists in the morphological grading of blastocysts (88% versus 86% for development stage prediction, 83% versus 79% for inner cell mass grade prediction, 89% versus 82% for trophectoderm grade prediction).Note to Practitioners—This work was motivated by the subjectivity and significant intra-and inter-evaluator variations in manual morphological grading of blastocysts. Existing approaches to automate the grading process mainly use a single blastocyst image although multi-focus images captured at different focal planes reveal more morphological features of a blastocyst than a single blastocyst image. This paper describes a new CNN-based method using multi-focus images to improve the grading accuracy. The accuracy of the proposed method was verified on multi-focus images of human blastocysts captured by a standard time-lapse incubator at fixed focal depths ($-$45$\mu$m,$-$30$\mu$m,$-$15$\mu$m, 0$\mu$m, 15$\mu$m, 30$\mu$m, 45$\mu$m).

Soft tissue cutting is used for incision, separation and removal of tissues or cells. Due to high deformation of soft tissues resulting from their viscosity and elasticity, it is challenging to accurately cut the tissue along a desired path and control the force applied to the tissue for reducing invasiveness, especially at the microscale. This paper presents a robotic biopsy system for cutting and collecting trophectoderm cells from a highly deformable blastocyst. The system, for the first time, enables TE cell junction detection for laser ablation throughout the blastocyst biopsy process by using a convolutional neural network. The overall detection error was 2.13% in every 1,000 cell junctions with position RMSE of$1.63\ \mu \mathrm{m}\pm 0.29\ \mu \mathrm{m}$. A dynamics model was developed to describe the motion of the trophectoderm cells inside a biopsy micropipette. Based on this model, an adaptive control method was developed for trophectoderm cell aspiration and positioning inside the biopsy micropipette. Experimental results revealed that the controller was capable of effectively compensating for the cell positioning error by updating the varying system parameters according to the adaptation law. The success rate was 100%, the cell aggregate positioning accuracy was$\pm 1\ \mu \mathrm{m}$, the average settling time was 2 s, and the largest overshoot was$4.3\ \mu \mathrm{m}$. Compared to manual blastocyst biopsy, the robotic biopsy system shortened the blastocyst's recovery time (35 min vs. 50 min) which indicates lower invasiveness.

Precise rotational positioning of end-effectors under microscopy is crucial for robotic micromanipulation. However, the end-effector is presently limited to a fixed orientation, which is manually set before a given micromanipulation task, lacking accuracy and versatility ofin situreorientation. In this article, we present a unified framework for rotationally positioning the end-effector in three dimensions by establishing a general rotational model, developing a detection method within the limited field of view under microscopy, and designing a three-loop control strategy that adapts to different experimental requirements and model parameters. In experiments, a standard angled micropipette was used as the end-effector to verify the validity of the proposed methods. The performance was evaluated experimentally where the micropipette was robotically rotated to an arbitrarily desired orientation with an average orientation error less than$2^\circ$. In the experiments of sperm manipulation, thein situmicropipette orientation control capability improved the success rate of sperm immobilization and achieved dexterous robotic sperm orientation for facile aspiration.

Cell manipulation is a key technology in many biomedical and clinical applications, in which end-effector alignment is a critical procedure. Presently, end-effector alignment is performed manually and suffers from large misalignment error and inconsistency. Manual alignment often undesirably moves the end-effector (e.g., a glass micropipette) out of the limited field of view under microscopy and risks breaking the fragile end-effector. This paper presents automated end-effector alignment for robotic cell manipulation. A rotational degree of freedom was added to a micromanipulator with translational degrees of freedom. The kinematic model of end-effector’s rotation was established, and the unknown model parameters were calibrated and updated via quadratic optimization. A controller was designed based on the kinematics modeling and parameter optimization to compensate for rotation-induced translation and achieve end-effector alignment. Experimental results demonstrate that the robotic alignment technique achieved an accuracy of 0.6±0.3° and a time cost of 18.5 ± 10.2 s, both significantly less than manual alignment. The developed controller cost significantly less time for micropipette alignment than the PID controller. A glass micropipette was used as the end-effector for human sperm immobilization, a critical procedure in clinical cell surgery. The success rate of sperm immobilization was 97% by robotic micropipette alignment, higher than the success rate of 90% by manual alignment due to the higher accuracy of robotic alignment.

In biology, fluorescent dyes are routinely used for biochemical measurements such as pH and ion concentrations. They, especially when used for detecting a low concentration of ions, suffer from low signal-to-noise ratios (SNR); and increasing the concentration of fluorescent dyes causes more sever cytotoxicity. We invented a new approach that uses a low amount of fluorescent dye-coated magnetic nanoparticles for on-demand, accurately aggregating the nanoparticles and thus fluorescent dyes in a local region inside a cell for intracellular measurement. Experiments proved this approach is capable of achieving a significantly higher SNR and lower cytotoxicity. Different from existing magnetic micromanipulation systems that generate large swarms (several microns and above) or cannot move the generated swarm to an arbitrary position, we developed a five-pole magnetic micromanipulation system and technique for generating a small swarm (e.g., 1 μm; capable of generating a magnetic swarm from 0.52 μm to 52.7 μm with an error <; 7.5 %) and accurately positioning the small swarm (position control accuracy: 0.76 μm). As an example, the system performed intracellular pH mapping using a 1 μm swarm of pH sensitive fluorescent dye-coated magnetic nanoparticles. The swarm had an SNR inside a cell 10 times that by the traditional method, i.e., global dye treatment, with both cases using the same fluorescent dye concentration. Our intracellular measurement results, for the first time, quantitatively revealed the existence of pH gradient and polarized pH distribution in live migrating cells.

Robotic manipulation of deformable objects has been a classic topic in robotics. Compared to synthetic deformable objects such as rubber balls and clothes, biological cells are highly deformable and more prone to damage. This article presents robotic manipulation of deformable cells for orientation control (both out-of-plane and in-plane), which is required in both clinical (e.g., in vitro fertilization) and biomedical (e.g., clone) applications. Compared to manual cell orientation control based on empirical experience, the robotic approach, based on modeling and path planning, effectively rotates a cell, while consistently maintaining minimal cell deformation to avoid cell damage. A force model is established to determine the minimal force applied by the micropipette to rotate a spherical or, more generally, ellipsoidal oocyte. The force information is translated into indentation through a contact mechanics model, and the manipulation path of the micropipette is formed by connecting the indentation positions on the oocyte. An optimal controller is designed to compensate for the variations of mechanical properties across oocytes. The polar body of an oocyte is detected by deep neural networks with robustness to shape and size differences. In experiments, the system achieved an accuracy of 97.6% in polar body detection and an accuracy of 0.7° in oocyte orientation control with maximum oocyte deformation of 2.70 μm throughout the orientation control process.

Robotic manipulation of deformable objects (vs. rigid objects) has been a classic topic in robotics. Compared to deformable synthetic objects such as rubber balls and clothes, biological cells are highly deformable and more prone to damage. This paper presents robotic manipulation of deformable cells for orientation control (both out-of-plane and in-plane), which is required in both clinical (e.g., in vitro fertilization) and biomedical (e.g., clone) applications. Compared to manual cell rotation control based on empirical experience, the robotic approach, based on mathematical modeling and path planning, effectively rotates a cell while consistently maintaining minimal cell deformation to avoid cell damage. A force model is established to determine the minimal force applied by the micropipette to rotate a spherical or more generally, an ellipsoidal mouse oocyte. The force information is translated into indentation through a contact mechanics model, and the manipulation path of the micropipette is formed by connecting the indentation positions on the oocyte. A compensation controller is designed to compensate for the variations of mechanical properties across cells. The polar body of an oocyte is detected by deep neural networks with robustness to shape and size differences. Experimental results demonstrate that the system achieved an accuracy of 97.6% in polar body detection and an accuracy of 0.7° in oocyte orientation control with maximum oocyte deformation of 2.69 μm.

Measuring the motility and morphology parameters of motile cells is important for revealing their functional characteristics. This paper presents automation techniques that, for the first time, enable automated, non-invasive measurement of motility and morphology parameters of individual sperms. Compared to the status quo of qualitative estimation of single sperm's motility and morphology based on embryologists' empirical experience, the automation techniques provide quantitative data in nearly real time. An adapted joint probabilistic data association filter (JPDAF) 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. Experimental results demonstrated an accuracy of 95.6% in sperm motility measurement and errors <;10% in morphology measurement.

Magnetic micromanipulation is an untethered technique and has enabled numerous applications in the scale of millimeters to micrometers from the tissue level to cell level. However, existing systems are not capable of maneuvering a sub-micrometer object for precise force control, preventing the realization of intracellular manipulation or `fantastic voyage' inside a single cell. The magnetic micromanipulation task achieved in this work is sub-micrometer position control and piconewton force control of a sub-micron (0.7 μm) magnetic bead inside a single human bladder cancer cell (RT4). The magnetic bead was 3D positioned in the cell using a generalized predictive controller that effectively tackled the control challenge caused by the slow visual feedback (1 Hz) from high-resolution confocal microscopy. The average positioning error was quantified to be 0.43 μm, which is slightly larger than Brownian motion-imposed constraint (0.31 μm). The system is capable of three-dimensionally applying a maximum force of 60 pN with a resolution of 4 pN. In experiments, a 0.7 μm magnetic bead was controlled to move from an initial position in a cell to target positions on the cell nucleus. Force-displacement data were obtained from multiple locations along the cell nucleus' major and minor axes. The results revealed, for the first time, significantly higher stiffness exists in the cell nucleus' major axis than the minor axis. This stiffness polarity was likely attributed to the aligned stress fibers of actin filament inside the cells.

Manipulation of motile cells such as bacteria and sperm is required in both cell biology and clinical applications. For immobilizing a motile sperm, the sperm head and tail positions must be accurately tracked, interference of proximal sperms on the target sperm must be tackled, and the orientation of the sperm must be properly aligned with the manipulation tool in order not to damage the sperm head where DNA is contained. Manual operation of sperm immobilization has stringent skill requirements, and both manual operation and existing robotic sperm immobilization suffer from inconsistent success rates and incapability of manipulating sperms swimming in all directions. This paper presents a robotic system for fully automated tracking, orientation control, and immobilization of motile sperms. Algorithms were developed for robustly tracking the sperm head and estimating the sperm tail positions under interfering conditions. A new visual servo control strategy was developed to enable the robotic system to actively adjust sperm orientation for immobilizing a sperm swimming in any direction. Experimental results from robotic immobilization of 400 sperms confirmed that the robotic system achieved a consistent success rate of 94.5 %, independent of sperm velocity or swimming direction.

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.

Magnetic micromanipulation has the advantage of untethered control, high precision, and biocompatibility and has recently undergone great advances. The magnetic micromanipulation task to tackle in this paper is to three dimensionally navigate a 5-μm magnetic bead inside a mouse embryo and accurately apply forces to intraembryonic structures to perform mechanical measurements at multiple locations. Existing technologies are not able to achieve these navigation and measurement goals because of poor magnetic force scaling and/or lacking the capability of applying an accurately controlled force. This paper reports a three-dimensional magnetic tweezer system that enables, for the first time, intraembryonic magnetic navigation and force application. A single magnetic bead was introduced into a mouse embryo via robotic microinjection. The magnetic tweezer system accurately controlled the position of the magnetic bead via visually servoed magnetic control. By moving the magnetic bead with known forces inside the embryo, cytoplasm viscosity was measured, which is eight times the viscosity of water. For performing mechanical measurements on the cellular structures inside the mouse embryo, the system should be capable of applying forces up to 120 pN with a resolution of 4 pN. The results revealed that the middle region is significantly more deformable than the side regions of the inner cell mass.

Magnetic micromanipulation has the advantage of untethered control, high precision, and biocompatibility and has recently undergone great advances. The magnetic micromanipulation task to tackle in this work is to three-dimensionally navigate a 5-micrometer magnetic bead inside a mouse embryo and perform mechanical measurements at multiple locations. Existing technologies are not able to achieve these navigation and measurement goals because of poor magnetic force scaling and/or lacking the capability of applying an accurately controlled force. This paper reports a robotic magnetic tweezer system that enables, for the first time, intra- embryonic magnetic navigation and force application. A single magnetic bead was introduced into a mouse embryo via robotic microinjection. The robotic magnetic tweezer system accurately controls the position of the magnetic bead via visually servoed magnetic control. The system is also capable of applying forces up to 120 pN with a resolution of 1.78 pN for performing mechanical measurements on the cellular structures inside the mouse embryo, revealing that the middle region is more deformable than the side regions of the inner cell mass.

Mammalian sperms reorient against fluid flow in the female reproductive tract, known as rheotaxis. Compared to chemotaxis that provides short-distance guidance, rheotaxis provides long-distance guidance for a sperm to find the egg cell. However, only a low number of sperms are capable of rheotaxis and their tail behavior during reorientation is not yet known. We have developed an automated system to manipulate human sperm orientation in fluid flow and quantitatively reveal sperm behavior changes during rheotaxis. The system automatically detects multiple sperms, selects the sperm for analysis, controls fluid flow, and quantifies sperm tail behavior. Sperm head angle is used as feedback to control fluid flow and select reorienting sperms. High accuracy of head angle tracking and automated sperm selection enables the capturing of dynamic sperm turning behavior in a large sample size. Algorithms are developed to track sperm tail skeletons and quantify tail beating amplitude and asymmetry, based on which the first quantitative analysis of sperm tail behavior in rheotaxis is obtained. Experimental results reveal, for the first time, that the sperms that are capable of reorienting against fluid flow beat their tails more asymmetrically than those sperms that are unable to reorient against fluid flow while no significant difference was found in their tail beating amplitudes.