RESUMEN

Label-free three-dimensional imaging plays a crucial role in unraveling the complexities of cellular functions and interactions in biomedical research. Conventional single-cell optical tomography techniques offer affordability and the convenience of bypassing laborious cell labelling protocols. However, these methods are encumbered by restricted illumination scanning ranges on abaxial plane, resulting in the loss of intricate cellular imaging details. The ability to fully control cellular rotation across all angles has emerged as an optimal solution for capturing comprehensive structural details of cells. Here, we introduce a label-free, cost-effective, and readily fabricated contactless acoustic-induced vibration system, specifically designed to enable multi-degree-of-freedom rotation of cells, ultimately attaining stable in-situ rotation. Furthermore, by integrating this system with advanced deep learning technologies, we perform 3D reconstruction and morphological analysis on diverse cell types, thus validating groups of high-precision cell identification. Notably, long-term observation of cells reveals distinct features associated with drug-induced apoptosis in both cancerous and normal cells populations. This methodology, based on deep learning-enabled cell 3D reconstruction, charts a novel trajectory for groups of real-time cellular visualization, offering promising advancements in the realms of drug screening and post-single-cell analysis, thereby addressing potential clinical requisites.

RESUMEN

Cytopathology, crucial in disease diagnosis, commonly uses microscopic slides to scrutinize cellular abnormalities. However, processing high volumes of samples often results in numerous negative diagnoses, consuming significant time and resources in healthcare. To address this challenge, a surface acoustic wave-enhanced multi-view acoustofluidic rotation cytometry (MARC) technique is developed for pre-cytopathological screening. MARC enhances cellular morphology analysis through comprehensive and multi-angle observations and amplifies subtle cell differences, particularly in the nuclear-to-cytoplasmic ratio, across various cell types and between cancerous and normal tissue cells. By prioritizing MARC-screened positive cases, this approach can potentially streamline traditional cytopathology, reducing the workload and resources spent on negative diagnoses. This significant advancement enhances overall diagnostic efficiency, offering a transformative vision for cytopathological screening.

RESUMEN

Coordinated cell movement is a cardinal feature in tissue organization that highlights the importance of cells working together as a collective unit. Disruptions to this synchronization can have far-reaching pathological consequences, ranging from developmental disorders to tissue repair impairment. Herein, it is shown that metal oxide nanoparticles (NPs), even at low and non-toxic doses (1 and 10 µg mL-1), can perturb the coordinated epithelial cell rotation (CECR) in micropatterned human epithelial cell clusters via distinct nanoparticle-specific mechanisms. Zinc oxide (ZnO) NPs are found to induce significant levels of intracellular reactive oxygen species (ROS) to promote mitogenic activity. Generation of a new localized force field through changes in the cytoskeleton organization and an increase in cell density leads to the arrest of CECR. Conversely, epithelial cell clusters exposed to titanium dioxide (TiO2) NPs maintain their CECR directionality but display suppressed rotational speed in an autophagy-dependent manner. Thus, these findings reveal that nanoparticles can actively hijack the nano-adaptive responses of epithelial cells to disrupt the fundamental mechanics of cooperation and communication in a collective setting.

RESUMEN

Microfluidic methodologies allow for automatic and high-throughput replicative lifespan (RLS) determination of single budding yeast cells. However, the resulted RLS is highly impacted by the robustness of experimental conditions, especially the microfluidic yeast-trapping structures, which are designed for cell retention, growth, budding, and daughter cell dissection. In this work, four microfluidic yeast-trapping structures, which were commonly used to immobilize mother cells and remove daughter cells for entire lifespan of budding yeast, were systematically investigated by means of finite element modeling (FEM). The results from this analysis led us to propose an optimized design, the yeast rotation (YRot) trap, which is a "leaky bowl"-shaped structure composed of two mirrored microcolumns facing each other. The YRot trap enables stable retention of mother cells in its "bowl" and hydrodynamic rotation of buds into its "leaky orifice" such that matured progenies can be dissected in a coincident direction. We validated the functions of the YRot trap in terms of cell rotation and daughter dissection by both FEM simulations and experiments. With the integration of denser YRot traps in microchannels, the microfluidic platform with stable single-yeast immobilization, long-term cell culturing, and coincident daughter dissection could potentially improve the robustness of experimental conditions for precise RLS determination in yeast aging studies.

Asunto(s)

RESUMEN

Bubbles locating in microfluidic chamber can produce acoustic streaming vortices by applying travelling surface acoustic wave oscillation in an ultrasonic range, which can be used to drive bio-samples to move within the flow field. In this paper, a strategy of bubble array configured in a large number of regularly arranged horseshoe structures is proposed to capture and rotate cells simultaneously. By modifying the geometric parameters of the horseshoe structure and microfluidic setting, high bubble homogeneity and cell trapping percentage was achieved. The simulation and experimental results of the bubble-induced streaming vortices were confirmed to be consistent. Through experiments, we achieved both in-plane and out-of-plane rotation of arrayed HeLa cells trapped by the bubbles. Out-of-plane rotation was used to reconstruct the 3D (three-dimensional) cell morphology, which was demonstrated to be useful in calculating cell geometry related parameters. We believe that this bubble array based cell rotation method is expected to be a promising tool for the investigation of bioengineering, biophysics, medicine, and cell biology.

Asunto(s)

RESUMEN

Single-cell manipulation is considered a key technology in biomedical research. However, the lack of intuitive and effective systems makes this technology less accessible. We propose a new tele-robotic solution for dexterous cell manipulation through optical tweezers. A slave-device consists of a combination of robot-assisted stages and a high-speed multi-trap technique. It allows for the manipulation of more than 15 optical traps in a large workspace with nanometric resolution. A master-device (6+1 degree of freedom (DoF)) is employed to control the 3D position of optical traps in different arrangements for specific purposes. Precision and efficiency studies are carried out with trajectory control tasks. Three state-of-the-art experiments were performed to verify the efficiency of the proposed platform. First, the reliable 3D rotation of a cell is demonstrated. Secondly, a six-DoF teleoperated optical-robot is used to transport a cluster of cells. Finally, a single-cell is dexterously manipulated through an optical-robot with a fork end-effector. Results illustrate the capability to perform complex tasks in efficient and intuitive ways, opening possibilities for new biomedical applications.

RESUMEN

Our group has reported that Melan-A cells and lymphocytes undergo self-rotation in a homogeneous AC electric field, and found that the rotation velocity of these cells is a key indicator to characterize their physical properties. However, the determination of the rotation properties of a cell by human eyes is both gruesome and time consuming, and not always accurate. In this paper, a method is presented to more accurately determine the 3D cell rotation velocity and axis from a 2D image sequence captured by a single camera. Using the optical flow method, we obtained the 2D motion field data from the image sequence and back-project it onto a 3D sphere model, and then the rotation axis and velocity of the cell were calculated. After testing the algorithm on animated image sequences, experiments were also performed on image sequences of real rotating cells. All of these results indicate that this method is accurate, practical, and useful. Furthermore, the method presented there can also be used to determine the 3D rotation velocity of other types of spherical objects that are commonly used in microfluidic applications, such as beads and microparticles.

RESUMEN

It is essential to have three-dimensional orientation of cells under a microscope for biological manipulation. Conventional manual cell manipulation is highly dependent on the operator's experience. It has some problems of low repeatability, low efficiency, and contamination. The current popular robotic method uses an injection micropipette to rotate cells. However, the optimal poking direction of the injection micropipette has not been established. In this paper, a strategy of robotic cell rotation based on optimal poking direction is proposed to move the specific structure of the cell to the desired orientation. First, analysis of the force applied to the cell during rotation was done to find the optimal poking direction, where we had the biggest moment of force. Then, the moving trajectory of the injection micropipette was designed to exert rotation force based on optimal poking direction. Finally, the strategy was applied to oocyte rotation in nuclear transfer. Experimental results show that the average completion time was up to 23.6 s and the success rate was 93.3% when the moving speed of the injection micropipette was 100 µm/s, which demonstrates that our strategy could overcome slippage effectively and with high efficiency.

RESUMEN

The precise rotational manipulation of cells and other micrometer-sized biological samples is critical to many applications in biology, medicine, and agriculture. We describe an acoustic-based, on-chip manipulation method that can achieve tunable cell rotation. In an acoustic field formed by the vibration of a piezoelectric transducer, acoustic streaming was generated using a specially designed, oscillating asymmetrical sidewall shape. We also studied the nature of acoustic streaming generation by numerical simulations, and our simulation results matched well with the experimental results. Trapping and rotation of diatom cells and swine oocytes were coupled using oscillating asymmetrical microstructures with different vibration modes. Finally, we investigated the relationship between the driving voltage and the speed of cell rotation, showing that the rotational rate achieved could be as large as approximately 1800 rpm. Using our device, the rotation rate can be effectively tuned on demand for single-cell studies. Our acoustofluidic cell rotation approach is simple, compact, non-contact, and biocompatible, permitting rotation irrespective of the optical, magnetic, or electrical properties of the specimen under investigation.

RESUMEN

Cell rotation can be achieved by utilizing rotating electric fields through which torques are generated due to phase difference between the dipole moment of cells and the external electric field. While reports of cell rotation under non-rotating electrical fields, such as dielectrophoresis (DEP), are abound, the underlying mechanism is not fully understood. Because of this, contradicting arguments remain regarding if a single cell can rotate under conventional DEP. What's more, the current prevailing DEP theory is not adequate for identifying the cause for such disagreements. In this work we applied our recently developed Volumetric Polarization and Integration (VPI) method to investigate the possible causes for cell rotation under conventional DEP. Three-dimensional (3D) computer models dealing with a cell in a DEP environment were developed to quantify the force and torque imparted on the cell by the external DEP field using COMSOL Multiphysics software. Modeling results suggest that eccentric inclusions with low conductivity inside the cell will generate torques (either in clockwise or counter-clockwise directions) sufficient to cause cell rotation under DEP. For validation of modeling predictions, experiments with rat adipose stem cells containing large lipid droplets were conducted. Good agreement between our modeling and experimental results suggests that the VPI method is powerful in elucidating the underlying mechanisms governing the complicated DEP phenomena.

Asunto(s)

RESUMEN

Single cell manipulation technology has been widely applied in biological fields, such as cell injection/enucleation, cell physiological measurement, and cell imaging. Recently, a biochip platform with a novel configuration of electrodes for cell 3D rotation has been successfully developed by generating rotating electric fields. However, the rotation platform still has two major shortcomings that need to be improved. The primary problem is that there is no on-chip module to facilitate the placement of a single cell into the rotation chamber, which causes very low efficiency in experiment to manually pipette single 10-micron-scale cells into rotation position. Secondly, the cell in the chamber may suffer from unstable rotation, which includes gravity-induced sinking down to the chamber bottom or electric-force-induced on-plane movement. To solve the two problems, in this paper we propose a new microfluidic chip with manipulation capabilities of single cell trap and single cell 3D stable rotation, both on one chip. The new microfluidic chip consists of two parts. The top capture part is based on the least flow resistance principle and is used to capture a single cell and to transport it to the rotation chamber. The bottom rotation part is based on dielectrophoresis (DEP) and is used to 3D rotate the single cell in the rotation chamber with enhanced stability. The two parts are aligned and bonded together to form closed channels for microfluidic handling. Using COMSOL simulation and preliminary experiments, we have verified, in principle, the concept of on-chip single cell traps and 3D stable rotation, and identified key parameters for chip structures, microfluidic handling, and electrode configurations. The work has laid a solid foundation for on-going chip fabrication and experiment validation.