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.

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.

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.

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.

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.

Single Cell Mass Spectrometry With a Robotic Micromanipulation System for Cell Metabolite Analysis.

OBJECTIVE: The increasing demand for unraveling cellular heterogeneity has boosted single cell metabolomics studies. However, current analytical methods are usually labor-intensive and hampered by lack of accuracy and efficiency. METHODS: we developed a first-ever automated single cell mass spectrometry system (named SCMS) that facilitated the metabolic profiling of single cells. In particular, extremely small droplets of sub nano-liter were generated to extract the single cells, and the underlying mechanism was verified theoretically and experimentally. This was crucial to minimize the dilution of the trace cellular contents and enhance the analytical sensitivity. Based on the precise 3D positioning of the pipette tip, we established a visual servoing robotic micromanipulation platform on which single cells were sequentially extracted, aspirated, and ionized, followed by the mass spectrometry analyses. RESULTS: With the SCMS system, inter-operator variability was eliminated and working efficiency was improved. The performance of the SCMS system was validated by the experiments on bladder cancer cells. MS and MS2 analyses of single cells enable us to identify several cellular metabolites and the underlying inter-cell heterogeneity. CONCLUSION: In contrast to traditional methods, the SCMS system functions without human intervention and realizes a robust single cell metabolic analysis. SIGNIFICANCE: the SCMS system upgrades the way how single cell metabolites were analyzed, and has the potential to be a powerful tool for single cell metabolomics studies.

Magnetic Nanoparticles as a Tool for Remote DNA Manipulations at a Single-Molecule Level.

Remote control of cells and single molecules by magnetic nanoparticles in nonheating external magnetic fields is a perspective approach for many applications such as cancer treatment and enzyme activity regulation. However, the possibility and mechanisms of direct effects of small individual magnetic nanoparticles on such processes in magneto-mechanical experiments still remain unclear. In this work, we have shown remote-controlled mechanical dissociation of short DNA duplexes (18-60 bp) under the influence of nonheating low-frequency alternating magnetic fields using individual 11 nm magnetic nanoparticles. The developed technique allows (1) simultaneous manipulation of millions of individual DNA molecules and (2) evaluation of energies of intermolecular interactions in short DNA duplexes or in other molecules. Finally, we have shown that DNA duplexes dissociation is mediated by mechanical stress and produced by the movement of magnetic nanoparticles in magnetic fields, but not by local overheating. The presented technique opens a new avenue for high-precision manipulation of DNA and generation of biosensors for quantification of energies of intermolecular interaction.

Micromanipulation of single cells and fingerprints for forensic identification.

Crime scene samples often include biological stains, handled items, or worn clothes and may contain cells from various donors. Applying routine sample collection methods by using a portion of a biological stain or swabbing the entire suspected touched area of the evidence followed by DNA extraction often leads to DNA mixtures. Some mixtures can be addressed with sophisticated interpretation protocols and probabilistic genotyping software resulting in DNA profiles of their contributors. However, many samples remain unresolved, providing no investigative information. Samples with many contributors are often the most challenging samples in forensic biology. Examples include gang rape situations or where the perpetrator's DNA is present in traces among the overwhelming amounts of the victim's DNA. If this is the only available evidence in a case, it is of paramount importance to generate usable information. An alternative approach, to address biological mixtures, could be the collection of individual cells directly from the evidence and testing them separately. This method could prevent cells from being inadvertently blended during the extraction process, thus resulting in DNA mixtures. In this study, multiple tools coupled with adhesive microcarriers to collect single cells were evaluated. These were tested on epithelial (buccal) and sperm cells, as well as on touched items. Single cells were successfully collected but fingerprints were swabbed in their entirety to account for the extracellular DNA of these samples and the poor DNA quality of shed skin flakes. Furthermore, micromanipulation devices, such as the P.A.L.M.® and the Axio Zoom.V16 operated manually or with a robotic arm aureka®, were compared for their effectiveness in collecting cells. The P.A.L.M.® was suitable for single cell isolation when smeared on membrane slides. Manual or robotic manipulations, by utilizing the Axio Zoom.V16, have wider applications as they can be used to isolate cells from various substrates such as glass or membrane slides, tapes, or directly from the evidence. Manipulations using the Axio Zoom.V16, either with the robotic arm aureka® or manually, generated similar outcomes which were significantly better than the outcomes by using the P.A.L.M.®. Robotic manipulations using the aureka® produced more consistent results, but operating the aureka® required training and often needed re-calibrations. This made the process of cell manipulations slower than when manually operated. Our preferred method was the manual manipulations as it was fast, cost effective, required little training, but relied on a steady hand of the technician.

Automated Embryo Manipulation and Rotation via Robotic nDEP-Tweezers.

Embryo manipulation is a fundamental task in assisted reproductive technology (ART). Nevertheless, conventional pick-place techniques often require proper alignment to avoid causing damage to the embryo and further, the tools have limited capability to orient the embryo being handled. OBJECTIVE: This paper presents a novel and non-invasive technique that can easily manipulate mouse embryos on a polyvinyl chloride (PVC) Petri dish. METHODS: An inverted microchip with quadrupole electrodes was attached to a micromanipulator to become a robotic dielectrophoresis (DEP) tweezers, and a motorized platform provided additional mobility to the embryos lying on a Petri dish. Vision-based algorithms were developed to evaluate relevant information of the embryos from the image, and to provide feedback signals for precise position and orientation control of the embryo. RESULTS: A series of experiments was conducted to examine the system performance, and the embryo can be successfully manipulated to a specified location with the desired orientation for subsequent processing. CONCLUSION: This system offers a non-contact, low cost, and flexible method for rapid cell handling. SIGNIFICANCE: As the DEP tweezers can grasp the embryo without the need for precise alignment, the overall time required to process a large number of embryos can be shortened.

Puncture of the Equine Embryonic Capsule and Its Repair In Vivo and In Vitro.

Vitrification of embryos >300 µm in diameter requires puncture of the glycoprotein capsule, although the size of the hole compatible with embryo survival is unknown. Forty-five day-7 or -8 embryos were punctured using a 30-µm glass biopsy pipette mounted on a micromanipulator (n = 20) or manually with either an acupuncture needle (∼100-µm diameter -hole; n = 10) or a microneedle with a <1 µm tip to produce a ∼30-µm diameter hole (n = 15) before transferring to recipient mares; further 12 embryos were punctured with either the acupuncture needle or microneedle before being cultured in vitro for 48 hrs (n = 3 per puncture group) or transferred to recipient mares and recovered 48 hrs later (n = 3 per puncture group). No pregnancies resulted from the 10 embryos punctured with the acupuncture needle, whereas 15 of 20 (75%) and 10 of 15 (67%) punctured on the micromanipulator or manually with the microneedle resulted pregnancies. Neither acupunctured nor microneedle-punctured embryos repaired their capsules in vitro. The acupunctured embryos also failed to repair their capsule after 48 hrs in vivo and subsequent uterine flushing yielded numerous capsular vesicles. The microneedle-punctured embryos did repair their capsule in vivo. Puncture with the microneedle opens the way for development of a manual method to vitrify equine embryos.

The acoustic radiation force of a focused ultrasound beam on a suspended eukaryotic cell.

Although ultrasound tools for manipulating and permeabilizing suspended cells have been available for nearly a century, accurate prediction of the distribution of acoustic radiation force (ARF) continues to be a challenge. We therefore developed an analytical model of the acoustic radiation force (ARF) generated by a focused Gaussian ultrasound beam incident on a eukaryotic cell immersed in an ideal fluid. The model had three layers corresponding to the nucleus, cytoplasm, and membrane, of a eukaryotic cell. We derived an exact expression for the ARF in relation to the geometrical and acoustic parameters of the model cell components. The mechanics of the cell membrane and nucleus, the relative width of the Gaussian beam, the size, position and aspect ratio of the cell had significant influence on the ARF. The model provides a theoretical basis for improved acoustic control of cell trapping, cell sorting, cell assembly, and drug delivery.

Magnetic Measurement and Stimulation of Cellular and Intracellular Structures.

From single-pole magnetic tweezers to robotic magnetic-field generation systems, the development of magnetic micromanipulation systems, using electromagnets or permanent magnets, has enabled a multitude of applications for cellular and intracellular measurement and stimulation. Controlled by different configurations of magnetic-field generation systems, magnetic particles have been actuated by an external magnetic field to exert forces/torques and perform mechanical measurements on the cell membrane, cytoplasm, cytoskeleton, nucleus, intracellular motors, etc. The particles have also been controlled to generate aggregations to trigger cell signaling pathways and produce heat to cause cancer cell apoptosis for hyperthermia treatment. Magnetic micromanipulation has become an important tool in the repertoire of toolsets for cell measurement and stimulation and will continue to be used widely for further explorations of cellular/intracellular structures and their functions. Existing review papers in the literature focus on fabrication and position control of magnetic particles/structures (often termed micronanorobots) and the synthesis and functionalization of magnetic particles. Differently, this paper reviews the principles and systems of magnetic micromanipulation specifically for cellular and intracellular measurement and stimulation. Discoveries enabled by magnetic measurement and stimulation of cellular and intracellular structures are also summarized. This paper ends with discussions on future opportunities and challenges of magnetic micromanipulation in the exploration of cellular biophysics, mechanotransduction, and disease therapeutics.

Automatic System for the Blastocyst Embryo Manipulation and Rotation.

Cell manipulation plays a vital role in the success rate and efficiency of the cell microsurgical operations, including biopsy of cell internal organelles such as the embryo biopsy, in which the embryo is manipulated and reoriented safely to a predefined desired position and orientation. In this paper, a simplified approach for the blastocyst embryo reorientation is proposed. It utilizes conventional tools and techniques currently in use in manual approaches in research labs and In Vitro Fertilization clinics, and controls the process using a vision feedback system. An experimental setup is developed to verify the dynamic behavior of the proposed approach, in which a stationary holding micropipette is used to hold the embryo, which is then rotated in two coordinate directions through friction contact with a moving substrate, in our case a glass microscope slide. The embryo rotates on the holding micropipette tip, due to the relatively low friction of this contact. A computer vision algorithm is used to estimate the embryo orientation coordinates, and use this information as a feedback signal to a simple proportional controller to control the embryo rotation angle. Experimental results demonstrate that the system is capable of cell rotation in two independent coordinates, suitable for embryo microsurgical task execution.

The optoelectronic microrobot: A versatile toolbox for micromanipulation.

Microrobotics extends the reach of human-controlled machines to submillimeter dimensions. We introduce a microrobot that relies on optoelectronic tweezers (OET) that is straightforward to manufacture, can take nearly any desirable shape or form, and can be programmed to carry out sophisticated, multiaxis operations. One particularly useful program is a serial combination of "load," "transport," and "deliver," which can be applied to manipulate a wide range of micrometer-dimension payloads. Importantly, microrobots programmed in this manner are much gentler on fragile mammalian cells than conventional OET techniques. The microrobotic system described here was demonstrated to be useful for single-cell isolation, clonal expansion, RNA sequencing, manipulation within enclosed systems, controlling cell-cell interactions, and isolating precious microtissues from heterogeneous mixtures. We propose that the optoelectronic microrobotic system, which can be implemented using a microscope and consumer-grade optical projector, will be useful for a wide range of applications in the life sciences and beyond.

MEMS impedance flow cytometry designs for effective manipulation of micro entities in health care applications.

Efficient manipulation of micro biological cells has always been a very important task in healthcare sector for which a Micro Electro Mechanical System (MEMS) based impedance flow cytometry has been proven to be a promising technique. This technique utilise the advantage of dielectrophoresis (DEP) force which is generated by non-uniform electric field in a microfluidic channel using an appropriate external AC supply at certain frequency range. The DEP forces generated in micro-channel depend upon various biological and physical parameters of cell and suspending medium. Apart from that design parameters of microfluidic channel and dimension of electrodes used for generating DEP action also plays major role in micro cell/bead manipulation. This article give remarks on the operating parameters which affects the cell manipulation and interrogates the currently accepted various electrode orientations in microfluidic MEMS flow cytometer technologies for effective manipulation of micro entities like healthy human cells (T-lymphocytes, B- lymphocytes, Monocytes, Leukocytes erythrocytes and human kidney cells HEK293), animal cells (neuroblastoma N115 and sheep red blood cells), cancer cells (MCF-7, MDA-435 and CD34+), yeast cells (saccharomyces cerevisiae, listeria innocua and E. coli) and micro particles (polystyrene beads) based on their dielectric properties using DEP action. Article focuses on the key electrode orientations for generation of non-uniform electric field in microfluidic flow cytometer like tapered electrodes, trapezoidal electrode arrays, Interdigitated electrodes, curved microelectrode and 3D electrode orientations and give remarks on their advantages and limitations. The cell manipulation with current MEMS impedance flow cytometry orientations targeting possibilities of implementation of the lab-on-chip devices has been discussed.

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment.

Blood-borne metastasis accounts for most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Small, Clickable, and Monovalent Magnetofluorescent Nanoparticles Enable Mechanogenetic Regulation of Receptors in a Crowded Live-Cell Microenvironment.

Multifunctional magnetic nanoparticles have shown great promise as next-generation imaging and perturbation probes for deciphering molecular and cellular processes. As a consequence of multicomponent integration into a single nanosystem, pre-existing nanoprobes are typically large and show limited access to biological targets present in a crowded microenvironment. Here, we apply organic-phase surface PEGylation, click chemistry, and charge-based valency discrimination principles to develop compact, modular, and monovalent magnetofluorescent nanoparticles (MFNs). We show that MFNs exhibit highly efficient labeling to target receptors present in cells with a dense and thick glycocalyx layer. We use these MFNs to interrogate the E-cadherin-mediated adherens junction formation and F-actin polymerization in a three-dimensional space, demonstrating the utility as modular and versatile mechanogenetic probes in the most demanding single-cell perturbation applications.

Micropipette Aspiration of Single Cells for Both Mechanical and Electrical Characterization.

Cellular physical properties have been identified to reflect cell states. Existing techniques are able to characterize either mechanical or electrical properties of a cell. This paper presents a micropipette aspiration technique that enables the characterization of both mechanical (instantaneous elastic modulus, equilibrium elastic modulus, and viscosity), and electrical (specific membrane capacitance) properties of the same single cell. Two bladder cancer cell lines (RT4 and T24) with different metastatic potential were used to evaluate the technique. The results showed that high-grade bladder cancer cells (T24, grade III) possess lower viscosity, lower elastic modulus, and larger SMC than the low-grade cancer cells (RT4, grade I). The Naive Bayes classifier was utilized to assess the classification accuracy using single-physical and multi-physical parameters. The classification results confirmed that the use of multi-biophysical parameters resulted in higher accuracy (97.5%), sensitivity (100%), and specificity (95.2%) than the use of a single-physical parameter for distinguishing T24 and RT4 cells.

Out-of-Plane Rotation Control of Biological Cells With a Robot-Tweezers Manipulation System for Orientation-Based Cell Surgery.

In many cell surgery applications, cell must be oriented properly such that the microsurgery tool can access the target components with minimum damage to the cell. In this paper, a scheme for out of image plane orientation control of suspended biological cells using robotic controlled optical tweezers is presented for orientation-based cell surgery. Based on our previous work on planar cell rotation using optical tweezers, the dynamic model of cell out-of-plane orientation control is formulated by using the T-matrix approach. Vision-based algorithms are developed to extract the cell out of image plane orientation angles, based on 2-D image slices obtained under an optical microscope. A robust feedback controller is then proposed to achieve cell out-of-plane rotation. Experiments of automated out of image plane rotational control for cell nucleus extraction surgery are performed to demonstrate the effectiveness of the proposed approach. This approach advances robot-aided single cell manipulation and produces impactful benefits to cell surgery applications such as nucleus transplantation and organelle biopsy in precision medicine.

Combined OCT distance and FBG force sensing cannulation needle for retinal vein cannulation: in vivo animal validation.

PURPOSE: Retinal vein cannulation is an experimental procedure during which a clot-dissolving drug is injected into an obstructed retinal vein. However, due to the fragility and minute size of retinal veins, such procedure is considered too risky to perform manually. With the aid of surgical robots, key limiting factors such as: unwanted eye rotations, hand tremor and instrument immobilization can be tackled. However, local instrument anatomy distance and force estimation remain unresolved issues. A reliable, real-time local interaction estimation between instrument tip and the retina could be a solution. This paper reports on the development of a combined force and distance sensing cannulation needle, and its experimental validation during in vivo animal trials. METHODS: Two prototypes are reported, relying on force and distance measurements based on FBG and OCT A-scan fibres, respectively. Both instruments provide an 80 [Formula: see text] needle tip and have outer shaft diameters of 0.6 and 2.3 mm, respectively. RESULTS: Both prototypes were characterized and experimentally validated ex vivo. Then, paired with a previously developed surgical robot, in vivo experimental validation was performed. The first prototype successfully demonstrated the feasibility of using a combined force and distance sensing instrument in an in vivo setting. CONCLUSION: The results demonstrate the feasibility of deploying a combined sensing instrument in an in vivo setting. The performed study provides a foundation for further work on real-time local modelling of the surgical scene. This paper provides initial insights; however, additional processing remains necessary.