Horizontal magnetic tweezers and its applications in single molecule micromanipulation experiments.

Magnetic tweezers (MTs) have become indispensable tools for gaining mechanistic insights into the behavior of DNA-processing enzymes and acquiring detailed, high-resolution data on the mechanical properties of DNA. Currently, MTs have two distinct designs: vertical and horizontal (or transverse) configurations. While the vertical design and its applications have been extensively documented, there is a noticeable gap in comprehensive information pertaining to the design details, experimental procedures, and types of studies conducted with horizontal MTs. This article aims to address this gap by providing a concise overview of the fundamental principles underlying transverse MTs. It will explore the multifaceted applications of this technique as an exceptional instrument for scrutinizing DNA and its interactions with DNA-binding proteins at the single-molecule level.

A vibrating capillary for ultrasound rotation manipulation of zebrafish larvae.

Multifunctional micromanipulation systems have garnered significant attention due to the growing interest in biological and medical research involving model organisms like zebrafish (Danio rerio). Here, we report a novel acoustofluidic rotational micromanipulation system that offers rapid trapping, high-speed rotation, multi-angle imaging, and 3D model reconstruction of zebrafish larvae. An ultrasound-activated oscillatory glass capillary is used to trap and rotate a zebrafish larva. Simulation and experimental results demonstrate that both the vibrating mode and geometric placement of the capillary contribute to the developed polarized vortices along the long axis of the capillary. Given its capacities for easy-to-operate, stable rotation, avoiding overheating, and high-throughput manipulation, our system poses the potential to accelerate zebrafish-directed biomedical research.

Single-Cell Recovery from Tumor Cell Xenotransplanted Zebrafish Embryos for the Study of Metastasis-Initiating Cells.

The study of metastasis-competent cells at the single-cell level represents an opportunity to decipher the molecular mechanisms associated with the metastatic cascade as well as to understand the functional and molecular heterogeneity of these cells. In this context, preclinical in vivo models of cancer metastasis are valuable tools to understand the behavior of cancer cells throughout the process. Here we describe a detailed protocol for the isolation and recovery of individual viable human metastatic cells from zebrafish embryos xenotransplanted with cancer cells for downstream molecular analysis. We cover the critical steps for the dissociation of the xenografted zebrafish embryos to generate a single-cell suspension, and the micromanipulation for their recovery as single cells.

Selective Acoustic Trapping, Translating, Rotating, and Orienting of Organism From Heterogeneous Mixture.

Selective contactless manipulation of organisms with intrinsic mobility from heterogeneous mixture is essential for biomedical engineering and microbiology. Acoustic manipulation, compared to its optical, magnetic, and electrostatic counterparts, provides superior bio-compatibility and additive-free properties. In this study, we present an acoustic manipulation system capable of selectively trapping, translating, rotating, and orienting individual organisms from in-Petri dish organism mixture using a phased transducer array and microscope, by dynamically steering the acoustic field. Specifically, using brine shrimp and zebrafish populations as example, the to-be-manipulated organisms with different sizes or morphologies can be manually designated by the user in microscopic image and interactively localized. Thereafter, the selected organisms can be automatically trapped from the heterogeneous mixture using a multiple focal point-based acoustic field steering method. Finally, the trapped organisms can be translated, rotated, and oriented in regard to the user's distinct manipulation objectives in instant response. In different tasks, closed-loop positioning and real-time motion planning control are performed, highlighting the innovation in terms of automation and accuracy of our manipulation technique. The results demonstrate that our acoustic manipulation system and acoustic field steering method enable selective, stable, precision, real-time, and in-Petri dish manipulation of organisms from heterogeneous mixture.

Conditions for transplantation of primordial germ cells in the yellowtail tetra, Astyanax altiparanae.

Biotechniques, including surrogate propagation derived from primordial germ cell (PGC) transplantation, are valuable tools for the reconstitution of endangered fish species. Although promising, there are no previous studies reporting such approaches using neotropical fish species. The aim of this study was to establish germline chimeras in neotropical fish by using the yellowtail tetra Astyanax altiparanae as a model species of the order Characiformes. Germline chimeras were obtained after transplantation of PGCs cultivated under different conditions: saline medium and supplemented with DMEM, amino acids, vitamins, glutamine, pyruvate, and fetal bovine serum, and subsequently transplanted into A. altiparanae triploids and triploid hybrids from the cross between A. altiparanae (♀) and A. fasciatus (♂). The results indicate ectopic migration in host embryos after transplantation of PGCs cultivated in saline medium. However, PGCs cultivated in supplemented medium migrated to the region of the gonadal ridge in 4.5% of triploid and 19.3% in triploid hybrid. In addition, the higher expression of dnd1, ddx4 and dazl genes was found in PGCs cultivated in supplemented culture medium. This indicates that the culture medium influences the maintenance and development of the cultivated cells. The expression levels of nanos and cxcr4b (related to the differentiation and migration of PGCs) were decreased in PGCs from the supplemented culture medium, supporting the results of ectopic migration. This is the first study to report the transplantation of PGCs to obtain germline chimera in neotropical species. The establishment of micromanipulation procedures in a model neotropical species will open new insights for the conservation of endangered species.

Sulfobetaine-Phosphonate Block Copolymer Coated Iron Oxide Nanoparticles for Genomic Locus Targeting and Magnetic Micromanipulation in the Nucleus of Living Cells.

Exerting forces on biomolecules inside living cells would allow us to probe their dynamic interactions in their native environment. Magnetic iron oxide nanoparticles represent a unique tool capable of pulling on biomolecules with the application of an external magnetic field gradient; however, their use has been restricted to biomolecules accessible from the extracellular medium. Targeting intracellular biomolecules represents an additional challenge due to potential nonspecific interactions with cytoplasmic or nuclear components. We present the synthesis of sulfobetaine-phosphonate block copolymer ligands, which provide magnetic nanoparticles that are stealthy and targetable in living cells. We demonstrate, for the first time, their efficient targeting in the nucleus and their use for magnetic micromanipulation of a specific genomic locus in living cells. We believe that these stable and sensitive magnetic nanoprobes represent a promising tool to manipulate specific biomolecules in living cells and probe the mechanical properties of living matter at the molecular scale.

Manipulation with sound and vibration: A review on the micromanipulation system based on sub-MHz acoustic waves.

Manipulation of micro-objects have been playing an essential role in biochemical analysis or clinical diagnostics. Among the diverse technologies for micromanipulation, acoustic methods show the advantages of good biocompatibility, wide tunability, a label-free and contactless manner. Thus, acoustic micromanipulations have been widely exploited in micro-analysis systems. In this article, we reviewed the acoustic micromanipulation systems that were actuated by sub-MHz acoustic waves. In contrast to the high-frequency range, the acoustic microsystems operating at sub-MHz acoustic frequency are more accessible, whose acoustic sources are at low cost and even available from daily acoustic devices (e.g. buzzers, speakers, piezoelectric plates). The broad availability, with the addition of the advantages of acoustic micromanipulation, make sub-MHz microsystems promising for a variety of biomedical applications. Here, we review recent progresses in sub-MHz acoustic micromanipulation technologies, focusing on their applications in biomedical fields. These technologies are based on the basic acoustic phenomenon, such as cavitation, acoustic radiation force, and acoustic streaming. And categorized by their applications, we introduce these systems for mixing, pumping and droplet generation, separation and enrichment, patterning, rotation, propulsion and actuation. The diverse applications of these systems hold great promise for a wide range of enhancements in biomedicines and attract increasing interest for further investigation.

Surface-Acoustic-Wave (SAW)-Driven Device for Three-Dimensional Cell Manipulation.

Cell orientation is a necessary step in micromanipulation, which significantly affects the outcomes of cell manipulation. Existing cell orientation techniques include the trial-and-error manual approach that suffers from low efficiency, the mechanical contact approaches that have problems of being invasive, and non-contact approaches that are difficult to set up. OBJECTIVE: This paper proposes a system with a surface-acoustic wave (SAWs) to perform noninvasive cell orientation. METHODS: The system employed a SAW chip with a pair of interdigital transducers (IDTs) to rotate embryos around the X and Y axis using acoustic streaming force. Instead of rotating the entire embryos like other methods, the proposed system rotates the cytoplasm alone through the cell chorion. RESULTS: We evaluated the cellular structure recognition algorithm and the rotation control using 100 embryo images and 30 zebrafish embryos. The system successfully recognized all required cellar structures for visual feedback. Furthermore, it rotated all cells into the desired position, including 26 cases completed within 10s with an orientation angle error of less than 4°. All 30 embryos hatched after manipulation. CONCLUSION: The proposed technique can automatically rotate the cytoplasm through the cell chorion noninvasively. SIGNIFICANCE: The system provides a starting point for noninvasive cell manipulation tasks, such as fast intracellular structure scanning and analysis, and microinjection.

Advancements in the future of automating micromanipulation techniques in the IVF laboratory using deep convolutional neural networks.

PURPOSE: To determine if deep learning artificial intelligence algorithms can be used to accurately identify key morphologic landmarks on oocytes and cleavage stage embryo images for micromanipulation procedures such as intracytoplasmic sperm injection (ICSI) or assisted hatching (AH). METHODS: Two convolutional neural network (CNN) models were trained, validated, and tested over three replicates to identify key morphologic landmarks used to guide embryologists when performing micromanipulation procedures. The first model (CNN-ICSI) was trained (n = 13,992), validated (n = 1920), and tested (n = 3900) to identify the optimal location for ICSI through polar body identification. The second model (CNN-AH) was trained (n = 13,908), validated (n = 1908), and tested (n = 3888) to identify the optimal location for AH on the zona pellucida that maximizes distance from healthy blastomeres. RESULTS: The CNN-ICSI model accurately identified the polar body and corresponding optimal ICSI location with 98.9% accuracy (95% CI 98.5-99.2%) with a receiver operator characteristic (ROC) with micro and macro area under the curves (AUC) of 1. The CNN-AH model accurately identified the optimal AH location with 99.41% accuracy (95% CI 99.11-99.62%) with a ROC with micro and macro AUCs of 1. CONCLUSION: Deep CNN models demonstrate powerful potential in accurately identifying key landmarks on oocytes and cleavage stage embryos for micromanipulation. These findings are novel, essential stepping stones in the automation of micromanipulation procedures.

Deconstructing body axis morphogenesis in zebrafish embryos using robot-assisted tissue micromanipulation.

Classic microsurgical techniques, such as those used in the early 1900s by Mangold and Spemann, have been instrumental in advancing our understanding of embryonic development. However, these techniques are highly specialized, leading to issues of inter-operator variability. Here we introduce a user-friendly robotic microsurgery platform that allows precise mechanical manipulation of soft tissues in zebrafish embryos. Using our platform, we reproducibly targeted precise regions of tail explants, and quantified the response in real-time by following notochord and presomitic mesoderm (PSM) morphogenesis and segmentation clock dynamics during vertebrate anteroposterior axis elongation. We find an extension force generated through the posterior notochord that is strong enough to buckle the structure. Our data suggest that this force generates a unidirectional notochord extension towards the tailbud because PSM tissue around the posterior notochord does not let it slide anteriorly. These results complement existing biomechanical models of axis elongation, revealing a critical coupling between the posterior notochord, the tailbud, and the PSM, and show that somite patterning is robust against structural perturbations.

Optoelectronic tweezers: a versatile toolbox for nano-/micro-manipulation.

The rapid development of micromanipulation technologies has opened exciting new opportunities for the actuation, selection and assembly of a variety of non-biological and biological nano/micro-objects for applications ranging from microfabrication, cell analysis, tissue engineering, biochemical sensing, to nano/micro-machines. To date, a variety of precise, flexible and high-throughput manipulation techniques have been developed based on different physical fields. Among them, optoelectronic tweezers (OET) is a state-of-art technique that combines light stimuli with electric field together by leveraging the photoconductive effect of semiconductor materials. Herein, the behavior of micro-objects can be directly controlled by inducing the change of electric fields on demand in an optical manner. Relying on this light-induced electrokinetic effect, OET offers tremendous advantages in micromanipulation such as programmability, flexibility, versatility, high-throughput and ease of integration with other characterization systems, thus showing impressive performance compared to those of many other manipulation techniques. A lot of research on OET have been reported in recent years and the technology has developed rapidly in various fields of science and engineering. This work provides a comprehensive review of the OET technology, including its working mechanisms, experimental setups, applications in non-biological and biological scenarios, technology commercialization and future perspectives.

MiGriBot: A miniature parallel robot with integrated gripping for high-throughput micromanipulation.

Although robotic micromanipulation using microtweezers has been widely explored, the current manipulation throughput hardly exceeds one operation per second. Increasing the manipulation throughput is thus a key factor for the emergence of robotized microassembly industries. This article presents MiGriBot (Millimeter Gripper Robot), a miniaturized parallel robot with a configurable platform and soft joints, designed to perform pick-and-place operations at the microscale. MiGriBot combines in a single robot the benefits of a parallel kinematic architecture with a configurable platform and the use of soft joints at the millimeter scale. The configurable platform of the robot provides an internal degree of freedom that can be used to actuate microtweezers using piezoelectric bending actuators located at the base of the robot, which notably reduces the robot's inertia. The soft joints make it possible to miniaturize the mechanism and to avoid friction. These benefits enable MiGriBot to reach a throughput of 10 pick-and-place cycles per second of micrometer-sized objects, with a precision of 1 micrometer.

Live-cell micromanipulation of a genomic locus reveals interphase chromatin mechanics.

Our understanding of the physical principles organizing the genome in the nucleus is limited by the lack of tools to directly exert and measure forces on interphase chromosomes in vivo and probe their material nature. Here, we introduce an approach to actively manipulate a genomic locus using controlled magnetic forces inside the nucleus of a living human cell. We observed viscoelastic displacements over micrometers within minutes in response to near-piconewton forces, which are consistent with a Rouse polymer model. Our results highlight the fluidity of chromatin, with a moderate contribution of the surrounding material, revealing minor roles for cross-links and topological effects and challenging the view that interphase chromatin is a gel-like material. Our technology opens avenues for future research in areas from chromosome mechanics to genome functions.

Lasers in Live Cell Microscopy.

Due to their unique properties-coherent radiation, diffraction limited focusing, low spectral bandwidth and in many cases short light pulses-lasers play an increasing role in live cell microscopy. Lasers are indispensable tools in 3D microscopy, e.g., confocal, light sheet or total internal reflection microscopy, as well as in super-resolution microscopy using wide-field or confocal methods. Further techniques, e.g., spectral imaging or fluorescence lifetime imaging (FLIM) often depend on the well-defined spectral or temporal properties of lasers. Furthermore, laser microbeams are used increasingly for optical tweezers or micromanipulation of cells. Three exemplary laser applications in live cell biology are outlined. They include fluorescence diagnosis, in particular in combination with Förster Resonance Energy Transfer (FRET), photodynamic therapy as well as laser-assisted optoporation, and demonstrate the potential of lasers in cell biology and-more generally-in biomedicine.

Single-Molecule Micromanipulation and Super-Resolution Imaging Resolve Nanodomains Underlying Chromatin Folding in Mitotic Chromosomes.

The folding of interphase chromatin into highly compact mitotic chromosomes is one of the most recognizable changes during the cell cycle. However, the structural organization underlying this drastic compaction remains elusive. Here, we combine several super resolution methods, including structured illumination microscopy (SIM), binding-activated localization microscopy (BALM), and atomic force microscopy (AFM), to examine the structural details of the DNA within the mitotic chromosome, both in the native state and after up to 30-fold extension using single-molecule micromanipulation. Images of native chromosomes reveal widespread ∼125 nm compact granules (CGs) throughout the metaphase chromosome. However, at maximal extensions, we find exclusively ∼90 nm domains (mitotic nanodomains, MNDs) that are unexpectedly resistant to extensive forces of tens of nanonewtons. The DNA content of the MNDs is estimated to be predominantly ∼80 kb, which is comparable to the size of the inner loops predicted by a recent nested loop model of the mitotic chromosome. With this DNA content, the total volume expected of the human genome assuming closely packed MNDs is nearly identical to what is observed. Thus, altogether, these results suggest that these mechanically stable MNDs, and their higher-order assembly into CGs, are the dominant higher-level structures that underlie the compaction of chromatin from interphase to metaphase.

In vivo acoustic manipulation of microparticles in zebrafish embryos.

In vivo micromanipulation using ultrasound is an exciting technology with promises for cancer research, brain research, vasculature biology, diseases, and treatment development. In the present work, we demonstrate in vivo manipulation of gas-filled microparticles using zebrafish embryos as a vertebrate model system. Micromanipulation methods often are conducted in vitro, and they do not fully reflect the complex environment associated in vivo. Four piezoelectric actuators were positioned orthogonally to each other around an off-centered fluidic channel that allowed for two-dimensional manipulation of intravenously injected microbubbles. Selective manipulation of microbubbles inside a blood vessel with micrometer precision was achieved without interfering with circulating blood cells. Last, we studied the viability of zebrafish embryos subjected to the acoustic field. This successful high-precision, in vivo acoustic manipulation of intravenously injected microbubbles offers potentially promising therapeutic options.

Micromanipulation in Paramecium: From non-mendelian inheritance to the outlook for versatile micromachines.

This review addresses nine areas of knowledge revealed by micromanipulations performed with Paramecium. Microinjection has shown that sexual maturation and senescence of Paramecium caudatum is a programmed process conducted by a specific gene and its product protein. In Paramecium tetraurelia, autogamy was revealed to depend on the number of DNA syntheses rather than the number of cell divisions in clonal aging. The cytoplasmic complementarity test established that microinjection of wild-type cytoplasm can correct genetic defects of mutants. The concept of complementarity together with protein chemistry revealed compounds that control membrane excitability. In non-Mendelian inheritance, noncoding small RNAs made from the parental micronucleus regulate the rearrangement of the progeny's macronuclear DNA. The macronucleus has the potential to be used as a factory for genetic engineering. The development and differentiation of progeny's nuclei in mating pairs are controlled by the parental macronucleus. The chemical reaction processes associated with exocytosis have been revealed by microinjection of various enzymes and antibodies. Using the fusion gene of histone H2B and yellow-fluorescence protein, it was revealed that the fusion gene-mRNA is transferred between cells during mating. Experiments with endosymbiotic bacteria and the host shed light on the conditions needed to establish sustainable symbiotic relationships.

Magnetically Actuated Cell-Robot System: Precise Control, Manipulation, and Multimode Conversion.

Border-nearing microrobots with self-propelling and navigating capabilities have promising applications in micromanipulation and bioengineering, because they can stimulate the surrounding fluid flow for object transportation. However, ensuring the biosafety of microrobots is a concurrent challenge in bioengineering applications. Here, macrophage template-based microrobots (cell robots) that can be controlled individually or in chain-like swarms are proposed, which can transport various objects. The cell robots are constructed using the phagocytic ability of macrophages to load nanomagnetic particles while maintaining their viability. The robots exhibit high position control accuracy and generate a flow field that can be used to transport microspheres and sperm when exposed to an external magnetic field near a wall. The cell robots can also form chain-like swarms to transport a large object (more than 100 times the volume). This new insight into the manipulation of macrophage-based cell robots provides a new concept by converting other biological cells into microrobots for micromanipulation in biomedical applications.

Combination of electronically driven micromanipulation with laser desorption ionization mass spectrometry - The unique tool for analysis of seed coat layers and revealing the mystery of seed dormancy.

Electronically driven micromanipulation (EDM) with microscopic control was used as a novel tool for sample preparation prior to direct (matrix assisted) laser desorption/ionization mass spectrometric ((MA)LDI-MS) analysis of mature pea seed coat composition in defined layers. Microscissors were used for seed coat fragment shape adjustment, microtweezers for sample holding and "microjackhammer" Milling Pro for precise mechanical removing of cell layers in defined depths (2, 5 or 10 µm). These procedures circumvent the application of embedding media or enzymatic digestion of seed coat that would complicate mass spectra interpretation (presence of matrix signals, analyte signals enhancement or attenuation) and represent alternative for 3D metabolites profiling. In addition, microinjector was used to apply a solution on intact or micropeeled seed coat surface in nano-volumes, i.e. MALDI matrix and/or lithium salt, that provide improvement of signal of sugars. Utilization of EDM enabled optimization of matrix composition on a single small fragment of seed coat overcoming thus problems with biological (seed to seed) variability. LDI-MS data were studied by multivariate statistical analysis and significant metabolites in particular layers of seed coats were identified. Normalized intensities of signals (NS) of long-chain hydroxylated fatty acids (HLFA) on intact dormant pea genotype (JI64) seed coats were significantly higher than in their counterparts treated by micropeeling confirming HLFA accumulation in outermost layers (cutin). Fatty acids distribution differences between dormant and non-dormant genotypes were explored in detail. On the other hand, NS of sugar chains and particular polyphenols were significantly higher in micropeeled seed coats of studied dormant and non-dormant genotypes than in intact seed coats. Furthermore, combination of EDM with mass spectrometry imaging (MSI) allowed vertical profiling of metabolites in hilum (a place of former attachment of seed to maternal plant) and comparison of its composition with surrounding tissues. The obtained results contribute to the understanding of relations between seed coat chemical composition and physical seed dormancy.

SonoTweezer: An Acoustically Powered End-Effector for Underwater Micromanipulation.

Recent advances in contactless micromanipulation strategies have revolutionized prospects of robotic manipulators as next-generation tools for minimally invasive surgeries. In particular, acoustically powered phased arrays offer dexterous means of manipulation both in air and water. Inspired by these phased arrays, we present SonoTweezer: a compact, low-power, and lightweight array of immersible ultrasonic transducers capable of trapping and manipulation of sub-mm sized agents underwater. Based on a parametric investigation with numerical pressure field simulations, we design and create a six-transducer configuration, which is small compared to other reported multi-transducer arrays (16-256 elements). Despite the small size of array, SonoTweezer can reach pressure magnitudes of 300 kPa at a low supply voltage of 25 V to the transducers, which is in the same order of absolute pressure as multi-transducer arrays. Subsequently, we exploit the compactness of our array as an end-effector tool for a robotic manipulator to demonstrate long-range actuation of sub-millimeter agents over a hundred times the agent's body length. Furthermore, a phase-modulation over its individual transducers allows our array to locally maneuver its target agents at sub-mm steps. The ability to manipulate agents underwater makes SonoTweezer suitable for clinical applications considering water's similarity to biological media, e.g., vitreous humor and blood plasma. Finally, we show trapping and manipulation of micro-agents under medical ultrasound (US) imaging modality. This application of our actuation strategy combines the usage of US waves for both imaging and micromanipulation.