Comparison of euploid blastocyst expansion with subgroups of single chromosome, multiple chromosome, and segmental aneuploids using an AI platform from donor egg embryos.

PURPOSE: This retrospective observational study compares how different classes of blastocyst genotypes from egg donor cycles differentially blastulate and expand using a standard assay. METHODS: Quantitative measurements of expansion utilized a customized neural network that segments all sequential time-lapse images during the first 10 h of expansion. RESULTS: Analyses were performed using two developmental time perspectives using time-lapse imaging. The first was the time to blastocyst formation (tB), which broadly reflects variations in developmental rate. Euploidy peaked at 100-115 h from fertilization. In contrast, aneuploidy peaks flanked this interval bi-modally. These distributions limit ploidy discrimination based upon traditional standard grading features when assessed in real time. In contrast, from the second perspective of progressive blastocyst expansion that is normalized to each individual blastocyst's tB time, euploidy was significantly increased at expansion values > 20,000µ2 across all tB intervals studied. A Cartesian coordinate plot graphically summarizes information useful to rank order blastocysts within cohorts for transfer. Defined aneuploidy subgroups, distinguished by the number and complexity of chromosomes involved, also showed distributive differences from both euploids and from each other. A small subset of clinically significant trisomies did not show discriminating features separating them from other euploids. CONCLUSION: A standard assay of blastocyst expansion normalized to each individual blastocyst's time of blastocyst formation more usefully discriminates euploidy from aneuploidy than real-time expansion comparisons using absolute developmental time from fertilization.

Deep learning neural network analysis of human blastocyst expansion from time-lapse image files.

RESEARCH QUESTION: Can artificial intelligence (AI) discriminate a blastocyst's cellular area from unedited time-lapse image files using semantic segmentation and a deep learning optimized U-Net architecture for use in selecting single blastocysts for transfer? DESIGN: This platform was retrospectively applied to time-lapse files from 101 sequentially transferred single blastocysts that were prospectively selected for transfer by their highest expansion ranking within cohorts using a 10 h expansion assay rather than standard grading. RESULTS: The AI platform provides expansion curves and raw data files to classify and compare blastocyst phenotypes within both cohorts and populations. Of 35 sequential unbiopsied single blastocyst transfers, 23 (65.7%) resulted in a live birth. Of 66 sequential single euploid blastocyst transfers, also selected for their most robust expansion, 49 (74.2%) resulted in live birth. The AI platform revealed that the averaged expansion rate was significantly (P = 0.007) greater in euploid blastocysts that resulted in live births compared with those resulting in failure to give a live birth. The platform further provides a framework to analyse fragmentation phenotypes that can test new hypotheses for developmental regulation during the preimplantation period. CONCLUSIONS: AI can be used to quantitatively describe blastocyst expansion from unedited time-lapse image files and can be used to quantitatively rank-order blastocysts for transfer. Early clinical results from such single blastocyst selection suggests that live birth rates without biopsy may be comparable to those found using single euploid blastocysts in younger, good responder patients.

Rapid Prototyping of Anomalous Reflective Metasurfaces Using Spray-Coated Liquid Metal.

Reconfigurable intelligent surfaces (RISs) have the potential to improve wireless communication links by dynamically redirecting signals to dead spots. Although a reconfigurable surface is best suited for environments in which the reflected signal must be dynamically steered, there are cases where a static, non-reconfigurable anomalous reflective metasurface can suffice. In this work, spray-coated liquid metal is used to rapidly prototype an anomalous reflective metasurface. Using a pressurized air gun and a plastic thin-film mask, a metasurface consisting of a 6 × 4 array of Galinstan liquid-metal elements is sprayed within minutes. The metasurface produces a reflected wave at an angle of 28° from normal in response to a normal incident 3.5-GHz electromagnetic plane wave. The spray-coated liquid-metal metasurface shows comparable results to an anomalous reflective metasurface with copper elements of the same dimensions, demonstrating that this liquid-metal fabrication process is a viable solution for the rapid prototyping of anomalous reflective metasurfaces.

Electrocapillary Actuation of Liquid Metal in Microchannels.

Controllable deformation of liquid metal by electrocapillary actuation (ECA) is empirically characterized in fluidic channels at the sub-millimeter-length scale. In 100-µm-deep channels of varying widths, the Galinstan liquid metal could move at velocities of more than 40 mm/s. The liquid metal could extend more than 2.5 mm into the channels at an electrocapillary actuation voltage of 3 V DC. The dynamic behavior of the liquid metal as it moves in the microchannels is described. These results are useful for designing microsystems that use liquid metal as a functional material.

Massively parallel manipulation of single cells and microparticles using optical images.

The ability to manipulate biological cells and micrometre-scale particles plays an important role in many biological and colloidal science applications. However, conventional manipulation techniques--including optical tweezers, electrokinetic forces (electrophoresis, dielectrophoresis, travelling-wave dielectrophoresis), magnetic tweezers, acoustic traps and hydrodynamic flows--cannot achieve high resolution and high throughput at the same time. Optical tweezers offer high resolution for trapping single particles, but have a limited manipulation area owing to tight focusing requirements; on the other hand, electrokinetic forces and other mechanisms provide high throughput, but lack the flexibility or the spatial resolution necessary for controlling individual cells. Here we present an optical image-driven dielectrophoresis technique that permits high-resolution patterning of electric fields on a photoconductive surface for manipulating single particles. It requires 100,000 times less optical intensity than optical tweezers. Using an incoherent light source (a light-emitting diode or a halogen lamp) and a digital micromirror spatial light modulator, we have demonstrated parallel manipulation of 15,000 particle traps on a 1.3 x 1.0 mm2 area. With direct optical imaging control, multiple manipulation functions are combined to achieve complex, multi-step manipulation protocols.

Bubbles in microfluidics: an all-purpose tool for micromanipulation.

In recent decades, the integration of microfluidic devices and multiple actuation technologies at the microscale has greatly contributed to the progress of related fields. In particular, microbubbles are playing an increasingly important role in microfluidics because of their unique characteristics that lead to specific responses to different energy sources and gas-liquid interactions. Many effective and functional bubble-based micromanipulation strategies have been developed and improved, enabling various non-invasive, selective, and precise operations at the microscale. This review begins with a brief introduction of the morphological characteristics and formation of microbubbles. The theoretical foundations and working mechanisms of typical micromanipulations based on acoustic, thermodynamic, and chemical microbubbles in fluids are described. We critically review the extensive applications and the frontline advances of bubbles in microfluidics, including microflow patterns, position and orientation control, biomedical applications, and development of bubble-based microrobots. We lastly present an outlook to provide directions for the design and application of microbubble-based micromanipulation tools and attract the attention of relevant researchers to the enormous potential of microbubbles in microfluidics.

Phototransistor-based optoelectronic tweezers for dynamic cell manipulation in cell culture media.

Optoelectronic tweezers (OET), based on light-induced dielectrophoresis, has been shown as a versatile tool for parallel manipulation of micro-particles and cells (P. Y. Chiou, A. T. Ohta and M. C. Wu, Nature, 2005, 436, 370-372). However, the conventional OET device cannot operate in cell culture media or other high-conductivity physiological buffers due to the limited photoconductivity of amorphous silicon. In this paper, we report a new phototransistor-based OET (Ph-OET). Consisting of single-crystalline bipolar junction transistors, the Ph-OET has more than 500x higher photoconductivity than amorphous silicon. Efficient cell trapping of live HeLa and Jurkat cells in Phosphate Buffered Saline (PBS) and Dulbecco's Modified Eagle's Medium (DMEM) has been demonstrated using a digital light projector, with a cell transport speed of 33 microm/sec, indicating a force of 14.5 pN. Optical concentration of cells and real-time control of individually addressable cell arrays have also been realized. Precise control of separation between two cells has also been demonstrated. We envision a new platform for single cell studies using Ph-OET.

Motile and non-motile sperm diagnostic manipulation using optoelectronic tweezers.

Optoelectronic tweezers was used to manipulate human spermatozoa to determine whether their response to OET predicts sperm viability among non-motile sperm. We review the electro-physical basis for how live and dead human spermatozoa respond to OET. The maximal velocity that non-motile spermatozoa could be induced to move by attraction or repulsion to a moving OET field was measured. Viable sperm are attracted to OET fields and can be induced to move at an average maximal velocity of 8.8 ± 4.2 µm s(-1), while non-viable sperm are repelled to OET, and are induced to move at an average maximal velocity of -0.8 ± 1.0 µm s(-1). Manipulation of the sperm using OET does not appear to result in increased DNA fragmentation, making this a potential method by which to identify viable non-motile sperm for assisted reproductive technologies.

A noninvasive, motility independent, sperm sorting method and technology to identify and retrieve individual viable nonmotile sperm for intracytoplasmic sperm injection.

PURPOSE: For intracytoplasmic sperm injection in the absence of sperm motility it can be virtually impossible to distinguish viable from nonviable sperm. A reliable means to identify viable nonmotile sperm is needed and would likely improve the intracytoplasmic sperm injection success rate. Optoelectronic tweezers are a new technology that uses light induced dielectrophoresis fields to distinguish individual live cells from dead cells. We assessed the ability of optoelectronic tweezers to distinguish viable from nonviable individual nonmotile human sperm. MATERIALS AND METHODS: Fresh semen specimens from 6 healthy men were suspended in an isotonic sucrose/dextrose solution and incubated with 0.4% trypan blue dye (Sigma-Aldrich®). Within 15 minutes we randomly selected 5 motile and 50 nonmotile sperm, including 25 trypan negative, followed by 25 trypan positive sperm, under 200× magnification for optoelectronic tweezers assay. We recorded the individual sperm response (attraction or repulsion) to the optoelectronic tweezer field and trypan staining status. RESULTS: From each subject 55 unwashed sperm were individually assayed for a total of 330. All motile sperm were attracted to optoelectronic tweezers. Of 150 trypan negative (viable) sperm 132 (88%) were attracted to the optoelectronic tweezer field with 0.88 sensitivity (95% CI 0.82-0.93) vs that of the trypan blue assay. All 150 trypan positive (nonviable) sperm were repulsed by or neutral to the optoelectronic tweezer field with 1.0 specificity (95% CI 0.98-1.00) vs that of the trypan blue assay. Type I error equaled 0 and overall assay agreement was 94%. CONCLUSIONS: The optoelectronic tweezer assay can distinguish viable from nonviable nonmotile viable sperm with sensitivity comparable to that of the trypan blue assay and equal specificity. Optoelectronic tweezers are a promising means of selecting sperm for intracytoplasmic sperm injection.

NanoPen: dynamic, low-power, and light-actuated patterning of nanoparticles.

We introduce NanoPen, a novel technique for low optical power intensity, flexible, real-time reconfigurable, and large-scale light-actuated patterning of single or multiple nanoparticles, such as metallic spherical nanocrystals, and one-dimensional nanostructures, such as carbon nanotubes. NanoPen is capable of dynamically patterning nanoparticles over an area of thousands of square micrometers with light intensities <10 W/cm(2) (using a commercial projector) within seconds. Various arbitrary nanoparticle patterns and arrays (including a 10 x 10 array covering a 0.025 mm(2) area) are demonstrated using this capability. One application of NanoPen is presented through the creation of surface-enhanced Raman spectroscopy hot-spots by patterning gold nanoparticles of 90 nm diameter with enhancement factors exceeding 10(7) and picomolar concentration sensitivities.

Antifouling coatings for optoelectronic tweezers.

Optoelectronic tweezers enables parallel manipulation of individual single cells using optical addressing and optically induced dielectrophoretic force. This provides a useful platform for performing a variety of biological functions, such as cell manipulation, cell sorting, and cell electroporation. However, in order to obtain more reliable cellular manipulation, especially of adherent mammalian cells, antifouling coatings need to be used to avoid non-specific cell adherence. Two antifouling coatings are discussed here, which can reduce the amount of non-specific adherence by as much as a factor of 30.

Parallel single-cell light-induced electroporation and dielectrophoretic manipulation.

Electroporation is a common technique for the introduction of exogenous molecules across the, otherwise, impermeant cell membrane. Conventional techniques are limited by either low throughput or limited selectivity. Here we present a novel technique whereby we use patterned light to create virtual electrodes which can induce the parallel electroporation of single cells. This technique seamlessly integrates with optoelectronic tweezers to provide a single cell manipulation platform as well. We present evidence of parallel, single cell electroporation using this method through use of fluorescent dyes and dielectrophoretic responses. Additionally, through the use of integrated microfluidic channels, we show that cells remain viable following treatment in the device. Finally, we determine the optimal field dosage to inject propidium iodide into a HeLa cell and maintain cellular viability.

EWOD-driven droplet microfluidic device integrated with optoelectronic tweezers as an automated platform for cellular isolation and analysis.

We report the integration of two technologies: droplet microfluidics using electrowetting-on-dielectric (EWOD) and individual particle manipulation using optoelectronic tweezers (OET)-in one microfluidic device. The integrated device successfully demonstrates a sequence involving both EWOD and OET operations. We encountered various challenges during integration of the two different technologies and present how they are addressed. To show the applicability of the device in cellular biology, live HeLa cells are used in the experiments. The unique advantages of EWOD and OET make their integration a significant step towards a powerful tool for many applications, such as single cell studies involving multiplexed environmental stimuli.

Trap profiles of projector based optoelectronic tweezers (OET) with HeLa cells.

In this paper we present trap profile measurements for HeLa cells in Optoelectronic Tweezers (OET) based on a data projector. The data projector is used as a light source to illuminate amorphous Si creating virtual electrodes which are used to trap particles through dielectrophoresis. We show that although the trap stiffness is typically greater at the edges of the optical spot it is possible to create a trap with constant trap stiffness by reducing the trap's size until it is similar to the object being trapped. We have successfully created a trap for HeLa cells with a constant trap stiffness of 3 x 10(-6) Nm-1 (capable of moving the cell up to 50 microms-1) with a 12 microm diameter trap. We also calculate the depth of the potential well that the cell will experience due to the trap and find that it to be 1.6 x 10(-16)J (4 x 10(4) kBT).

Spray-On Liquid-Metal Electrodes for Graphene Field-Effect Transistors.

Advancements in flexible circuit interconnects are critical for widespread adoption of flexible electronics. Non-toxic liquid-metals offer a viable solution for flexible electrodes due to deformability and low bulk resistivity. However, fabrication processes utilizing liquid-metals suffer from high complexity, low throughput, and significant production cost. Our team utilized an inexpensive spray-on stencil technique to deposit liquid-metal Galinstan electrodes in top-gated graphene field-effect transistors (GFETs). The electrode stencils were patterned using an automated vinyl cutter and positioned directly onto chemical vapor deposition (CVD) graphene transferred to polyethylene terephthalate (PET) substrates. Our spray-on method exhibited a throughput of 28 transistors in under five minutes on the same graphene sample, with a 96% yield for all devices down to a channel length of 50 µm. The fabricated transistors possess hole and electron mobilities of 663.5 cm²/(V·s) and 689.9 cm²/(V·s), respectively, and support a simple and effective method of developing high-yield flexible electronics.

Operational Regimes and Physics Present in Optoelectronic Tweezers.

Optoelectronic tweezers (OET) are a powerful light-based technique for the manipulation of micro- and nanoscopic particles. In addition to an optically patterned dielectrophoresis (DEP) force, other light-induced electrokinetic and thermal effects occur in the OET device. In this paper, we present a comprehensive theoretical and experimental investigation of various fluidic, optical, and electrical effects present during OET operation. These effects include DEP, light-induced ac electroosmosis, electrothermal flow, and buoyancy-driven flow. We present finite-element modeling of these effects to establish the dominant mode for a given set of device parameters and bias conditions. These results are confirmed experimentally and present a comprehensive outline of the operational regimes of the OET device.

Localized Single-Cell Lysis and Manipulation Using Optothermally-Induced Bubbles.

Localized single cells can be lysed precisely and selectively using microbubbles optothermally generated by microsecond laser pulses. The shear stress from the microstreaming surrounding laser-induced microbubbles and direct contact with the surface of expanding bubbles cause the rupture of targeted cell membranes. High-resolution single-cell lysis is demonstrated: cells adjacent to targeted cells are not lysed. It is also shown that only a portion of the cell membrane can be punctured using this method. Both suspension and adherent cell types can be lysed in this system, and cell manipulation can be integrated for cell-cell interaction studies.

Editorial for the Special Issue on Microdevices and Microsystems for Cell Manipulation.

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells.[...].

Cooperative Micromanipulation Using the Independent Actuation of Fifty Microrobots in Parallel.

Micromanipulation for applications in areas such as tissue engineering can require mesoscale structures to be assembled with microscale resolution. One method for achieving such manipulation is the parallel actuation of many microrobots in parallel. However, current microrobot systems lack the independent actuation of many entities in parallel. Here, the independent actuation of fifty opto-thermocapillary flow-addressed bubble (OFB) microrobots in parallel is demonstrated. Individual microrobots and groups of microrobots were moved along linear, circular, and arbitrary 2D trajectories. The independent addressing of many microrobots enables higher-throughput microassembly of micro-objects, and cooperative manipulation using multiple microrobots. Demonstrations of manipulation with multiple OFB microrobots include the transportation of microstructures using a pair or team of microrobots, and the cooperative manipulation of multiple micro-objects. The results presented here represent an order of magnitude increase in the number of independently actuated microrobots in parallel as compared to other magnetically or electrostatically actuated microrobots, and a factor of two increase as compared to previous demonstrations of OFB microrobots.

Vision-assisted micromanipulation using closed-loop actuation of multiple microrobots.

Accurate control and precise positioning of opto-thermocapillary flow-addressed bubble microrobots are necessary for micromanipulation. In addition, micromanipulation using the simultaneous actuation of multiple microrobots requires a robust control system to enable independent motion. This paper demonstrates a hybrid closed-loop vision-assisted control system capable of actuating multiple microrobots simultaneously and positioning them at precise locations relative to micro-objects under manipulation. A vision-assisted grasp-planning application was developed and used to calculate the necessary trajectories of the microrobots to form cages around micro-objects. The location of the microrobots and the micro-objects was detected at the caging locations using a particle-tracking application that used image feedback for precise positioning. The closed-loop image feedback information enabled the position update of the microrobots, allowing them to precisely follow the trajectory and caging locations calculated by the grasp-planning application. Four microrobots were assigned to cage a star-shaped micro-object using the closed-loop control system. Once caged, the micro-object was transported to a location within the workspace and uncaged, demonstrating the micromanipulation task. This microrobotic system is well suited for the micromanipulation of single cells.