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.

Trapping and Detection of Single Viruses in an Optofluidic Chip.

Accurate single virus detection is critical for disease diagnosis and early prevention, especially in view of current pandemics. Numerous detection methods have been proposed with the single virus sensitivity, including the optical approaches and immunoassays. However, few of them hitherto have the capability of both trapping and detection of single viruses in the microchannel. Here, we report an optofluidic potential well array to trap nanoparticles stably in the flow stream. The nanoparticle is bound with single viruses and fluorescence quantum dots through an immunolabeling protocol. Single viruses can be swiftly captured in the microchannel by optical forces and imaged by a camera. The number of viruses in solution and on each particle can be quantified via image processing. Our method can trap and detect single viruses in the 1 mL serum or water in 2 h, paving an avenue for the advanced, fast, and accurate clinical diagnosis, as well as the study of virus infectivity, mutation, drug inhibition, etc.

3D mechanical characterization of single cells and small organisms using acoustic manipulation and force microscopy.

Quantitative micromechanical characterization of single cells and multicellular tissues or organisms is of fundamental importance to the study of cellular growth, morphogenesis, and cell-cell interactions. However, due to limited manipulation capabilities at the microscale, systems used for mechanical characterizations struggle to provide complete three-dimensional coverage of individual specimens. Here, we combine an acoustically driven manipulation device with a micro-force sensor to freely rotate biological samples and quantify mechanical properties at multiple regions of interest within a specimen. The versatility of this tool is demonstrated through the analysis of single Lilium longiflorum pollen grains, in combination with numerical simulations, and individual Caenorhabditis elegans nematodes. It reveals local variations in apparent stiffness for single specimens, providing previously inaccessible information and datasets on mechanical properties that serve as the basis for biophysical modelling and allow deeper insights into the biomechanics of these living systems.

Self-Propelled and Electrobraking Synergetic Liquid Manipulator toward Microsampling and Bioanalysis.

Droplet manipulation is of paramount significance for microfluidics-based biochips, especially for bioanalytical chips. Despite great progresses made on droplet manipulation, the existing bioanalytical methods face challenges in terms of capturing minute doses toward hard-to-obtain samples and analyzing biological samples at low temperatures immediately. To circumvent these limitations, a self-propelled and electric stimuli synergetic droplet manipulator (SES-SDM) was developed by a femtosecond laser microfabrication strategy followed by post-treatment. Combining the inspiration from cactus and Nepenthes pitcher plants, the wedge structure with the microbowl array and silicone oil infusion was endowed cooperatively with the SES-SDM. With the synergy of the ultralow voltage (4.0 V) stimuli, these bioinspired features enable the SES-SDM to transport the droplet spontaneously and controllably, showing the maximum fast motion (15.7 mm/s) and long distance (96.2 mm). Remarkably, the SES-SDM can function at -5 °C without the freezing of the droplets, where the self-propelled motion and electric-responsive pinning can realize the accurate capture and real-time analysis of the microdroplets of the tested samples. More importantly, the SES-SDM can realize real-time diagnosis of excessive heavy metal in water by the cooperation of self-propulsion and electro-brake. This work opens an avenue to design a microsampling (5-20 µL) manipulator toward producing the minute samples for efficient bioanalysis and offers a strategy for microanalysis using the synergistic droplet manipulation.

Tuning the Surface Interactions between Single Cells and an OSTE+ Microwell Array for Enhanced Single Cell Manipulation.

Retrieving single cells of interest from an array of microwells for further off-chip analysis is crucial in numerous biological applications. To this end, several single cell manipulation strategies have been developed, including optical tweezers (OT). OT represent a unique approach for contactless cell retrieval, but their performance is often suboptimal due to nonspecific cell adhesion to the microwell surface. In this study, we focused on improving the surface chemistry of microwell arrays to ensure efficient single cell manipulation using OT. For this purpose, the surface of an off-stoichiometry thiol-ene-epoxy (OSTE+) microwell array was grafted with polyethylene glycol (PEG) molecules with different molecular weights: PEG 360, PEG 500, PEG 2000, and a PEG Mix (an equimolar ratio of PEG 500 and PEG 2000). Contact angle measurements showed that the PEG grafting process resulted in an increased surface energy, which was stable for at least 16 weeks. Next, cell adhesion of two cell types, baker's yeast (Saccharomyces cerevisiae) and human B cells, to surfaces treated with different PEGs was evaluated by registering the presence of cellular motion inside microwells and the efficiency of optical lifting of cells that display motion. Optimal results were obtained for surfaces grafted with PEG 2000 and PEG Mix, reaching an average fraction of cells with motion of over 93% and an average lifting efficiency of over 96% for both cell types. Upon the integration of this microwell array with a polydimethylsiloxane (PDMS) microfluidic channel, PEG Mix resulted in proper washing of non-seeded cells. We further demonstrated the wide applicability of the platform by manipulating non-responding yeast cells to antifungal treatment and B cells expressing surface IgG antibodies. The combination of the optimized microwell surface with continuous microfluidics results in a powerful and versatile platform, allowing high-throughput single cell studies and retrieval of target cells for off-chip analysis.

A protein-coated micro-sucker patch inspired by octopus for adhesion in wet conditions.

In medical robotics, micromanipulation becomes particularly challenging in the presence of blood and secretions. Nature offers many examples of adhesion strategies, which can be divided into two macro-categories: morphological adjustments and chemical adaptations. This paper analyzes how two successful specializations from different marine animals can converge into a single biomedical device usable in moist environments. Taking inspiration from the morphology of the octopus sucker and the chemistry of mussel secretions, we developed a protein-coated octopus-inspired micro-sucker device that retains in moist conditions about half of the adhesion it shows in dry environments. From a robotic perspective, this study emphasizes the advantages of taking inspiration from specialized natural solutions to optimize standard robotic designs.

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.

A new strategy to capture single biological micro particles at the interface between a water film and substrate by ultrasonic tweezers.

Controlled capture of single biological micro particles, with effective capture function, little heat damage to and good stability of captured samples simultaneously, has been a technological challenge in the area of micro manipulation. This paper presents an ultrasonic tweezers based new strategy to meet the challenge. In the strategy, being different from the other ultrasonic methods, the MMP (micro manipulating probe), which vibrates elliptically, is in contact with the substrate. Single yeast cells with a diameter of 3-7 µm and Chlorella vulgaris powders with a diameter of 2-10 µm near the MMP can be sucked onto the MMP's tip. The captured particle can be transferred to a desired location at the interface between the water film and substrate by moving the ultrasonic tweezers. The temperature rise in the capture region is less than 0.1 °C, and the sucking distance can be up to 20 µm. The captured particle is in contact with the MMP's tip, which results in a good stability of the captured particle. The experiments also show that it is possible to use multiple MMPs to individually capture single cells. The finite element analyses indicate that acoustic radiation force generated by the ultrasonic field around the MMP is responsible for the capture. Moreover, the effects of the orthogonal vibration components, tilt angle and length of the MMP on the capture capability are clarified.

Analysis of Lipids in Single Cells and Organelles Using Nanomanipulation-Coupled Mass Spectrometry.

The ability to discriminately analyze the chemical constituents of single cells and organelles is highly sought after and necessary to establish true biomarkers. Some major challenges of individual cell analysis include requirement and expenditure of a large sample of cells as well as extensive extraction and separation techniques. Here, we describe methods to perform individual cell and organelle extractions of both tissues and cells in vitro using nanomanipulation coupled to mass spectrometry. Lipid profiles display heterogeneity from extracted adipocytes and lipid droplets, demonstrating the necessity for single cell analysis. The application of these techniques can be applied to other cell and organelle types for selective and thorough monitoring of disease progression and biomarker discovery.

High-Performance Ultrasonic Tweezers for Manipulation of Motile and Still Single Cells in a Droplet.

The manipulation of motile and still single cells with the simultaneous features of selective trapping, 3-D path free transport, position-controllable release and little heat damage has been a significant challenge. We developed an ultrasonic method for capturing motile and still single cells with the aforementioned features in a droplet. During manipulation, a micromanipulation probe (MMP), which vibrated linearly with a trajectory parallel to a silicon substrate, was immersed in the droplet and was not in contact with the substrate. Motile and still single cells, such as Chattonella marina with a length of 30-50 µm and yeast cells with a diameter of 3-10 µm, at the interface between the droplet and substrate were selectively sucked onto the vibrating MMP and transported via a 3-D route inside the droplet by moving the MMP (or the device). The MMP and captured single cells were in contact, making the release position controllable. The measured temperature rise of the MMP was <0.1°C; thus, it is competitive for the manipulation of biological samples. Finite-element analyses revealed that the contact-type capture was due to acoustic radiation force generated by the ultrasonic field around the vibrating MMP. The dependence of the capture capability and working frequency bandwidth on the working conditions was investigated experimentally.

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.

Translational and rotational manipulation of filamentous cells using optically driven microrobots.

Optical cell manipulation has become increasingly valuable in cell-based assays. In this paper, we demonstrate the translational and rotational manipulation of filamentous cells using multiple cooperative microrobots automatically driven by holographic optical tweezers. The photodamage of the cells due to direct irradiation of the laser beam can be effectively avoided. The proposed method will enable fruitful biomedical applications where precise cell manipulation and less photodamage are required.

A nanostructure platform for live-cell manipulation of membrane curvature.

Membrane curvatures are involved in essential cellular processes, such as endocytosis and exocytosis, in which they are believed to act as microdomains for protein interactions and intracellular signaling. These membrane curvatures appear and disappear dynamically, and their locations are difficult or impossible to predict. In addition, the size of these curvatures is usually below the diffraction limit of visible light, making it impossible to resolve their values using live-cell imaging. Therefore, precise manipulation of membrane curvature is important to understanding how membrane curvature is involved in intracellular processes. Recent studies show that membrane curvatures can be induced by surface topography when cells are in direct contact with engineered substrates. Here, we present detailed procedures for using nanoscale structures to manipulate membrane curvatures and probe curvature-induced phenomena in live cells. We first describe detailed procedures for the design of nanoscale structures and their fabrication using electron-beam (E-beam) lithography. The fabrication process takes 2 d, but the resultant chips can be cleaned and reused repeatedly over the course of 2 years. Then we describe how to use these nanostructures to manipulate local membrane curvatures and probe intracellular protein responses, discussing surface coating, cell plating, and fluorescence imaging in detail. Finally, we describe a procedure to characterize the nanostructure-cell membrane interface using focused ion beam and scanning electron microscopy (FIB-SEM). Nanotopography-based methods can induce stable membrane curvatures with well-defined curvature values and locations in live cells, which enables the generation of a library of curvatures for probing curvature-related intracellular processes.

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.

Advances in Micropipette Aspiration: Applications in Cell Biomechanics, Models, and Extended Studies.

With five decades of sustained application, micropipette aspiration has enabled a wide range of biomechanical studies in the field of cell mechanics. Here, we provide an update on the use of the technique, with a focus on recent developments in the analysis of the experiments, innovative microaspiration-based approaches, and applications in a broad variety of cell types. We first recapitulate experimental variations of the technique. We then discuss analysis models focusing on important limitations of widely used biomechanical models, which underpin the urge to adopt the appropriate ones to avoid misleading conclusions. The possibilities of performing different studies on the same cell are also considered.

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.

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.

Achieving Automated Organelle Biopsy on Small Single Cells Using a Cell Surgery Robotic System.

Single cell surgery such as manipulation or removal of subcellular components or/and organelles from single cells is increasingly used for the study of diseases and their causes in precision medicine. This paper presents a robotic surgery system to achieve automated organelle biopsy of single cells with dimensions of less than 20 µm in diameter. The complexity of spatial detection of the organelle position is reduced by patterning the cells using a microfluidic chip device. A sliding mode nonlinear controller is developed to enable extraction of organelles, such as the mitochondria and the nucleus, from single cells with high precision. An image processing algorithm is also developed to automatically detect the position of the desired organelle. The effectiveness of the proposed robotic surgery system is demonstrated experimentally with automated extraction of mitochondria and nucleus from human acute promyelocytic leukemia cells and human fibroblast cells. Extraction is followed by biological tests to indicate the functionality of biopsied mitochondria as well as the cell viability after removal of mitochondria. The results presented here have revealed that the proposed approach of automated organelle biopsy on single small cells is feasible.

Laryngeal temperature simulations during carbon dioxide laser irradiation delivered by a scanning micromanipulator.

We use scatter-limited phototherapy techniques to calculate the time-dependent temperature profiles of incisions made with a commercial carbon dioxide laser being used to make a 1-mm incision under computer control using the Digital Acublade™ and with incisions made with the same laser under manual control. The goal is to understand the differences in the amount of lateral thermal damage that is likely from the computer-controlled incisions versus the manually controlled incisions. The temperature profiles are calculated from the absorption and scatter of light in a homogeneous material. The resulting temperature profiles are presented as videos showing how the tissue heats up and cools down with the incident laser pulses. The time-dependent thermal distributions indicate that the computer-controlled laser incision could show as little as 210 µm of lateral thermal damage, whereas the manually controlled laser incisions could show as much as 375 µm of lateral thermal damage. The computer-controlled laser incision is able to control laser pulses fast enough that subsequent pulses can ablate away tissue with a significant amount of residual heat from the previous laser pulse. Using the scatter-limited phototherapy techniques, we can see how a computer-controlled laser can make incisions with less thermal damage by ablating away tissue holding a significant amount of heat from the previous pulse before it has time to diffuse through the tissue. This method of heat removal from laser incisions has not been previously described or demonstrated.