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PURPOSE: To construct a quantitative evaluation system for hand tremor during internal limiting membrane (ILM) peeling and investigate changes in hand tremor attributable to the use of the customized passive surgical support robot. METHODS: This analytical and experimental study developed a hand tremor evaluation system that synchronizes three elements: surgical microscope images, an artificial eye module with a force sensor to simulate ILM peeling, and microforceps with an inertial measurement unit. Two surgeons used this system to measure hand tremor during ILM peeling with and without the robot. RESULTS: The horizontal, vertical, and combined vertical and horizontal components of hand tremor were 8.1 ± 6.1, 1.7 ± 1.8, and 8.5 ± 6.2 mG, respectively, in the no robot group. These components decreased to 7.2 ± 6.0, 1.5 ± 1.7, and 7.5 ± 6.1 mG, respectively, in the robot group. In particular, hand tremor was significantly suppressed by 11.9% for the horizontal component using the robot (p = 0.0006). CONCLUSION: The newly constructed system helps to quantitatively evaluate hand tremor during ILM peeling. The customized passive surgical support robot enables to decrease hand tremor during ILM peeling.
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Microvortices are emerging components that impart functionality to microchannels by exploiting inertia effects such as high shear stress, effective fluid diffusion, and large pressure loss. Exploring the dynamic generation of vortices further expands the scope of microfluidic applications, including cell stimulation, fluid mixing, and transport. Despite the crucial role of vortices' development within sub-millisecond timescales, previous studies in microfluidics did not explore the modulation of the Reynolds number (Re) in the range of several hundred. In this study, we modulated high-speed flows (54 < [Formula: see text] < 456) within sub-millisecond timescales using a piezo-driven on-chip membrane pump. By applying this method to microchannels with asymmetric geometries, we successfully controlled the spatiotemporal development of vortices, adjusting their behavior in response to oscillatory flow directions. These different vortices induced different pressure losses, imparting the microchannels with direction-dependent flow resistance, mimicking a diode-like behavior. Through precise control of vortex development, we managed to regulate this direction-dependent resistance, enabling the rectification of oscillatory flow resembling a diode and the ability to switch its rectification direction. This component facilitated bidirectional flow control without the need for mechanical valves. Moreover, we demonstrated its application in microfluidic cell pipetting, enabling the isolation of single cells. Consequently, based on modulating high-speed flow, our approach offers precise control over the spatiotemporal development of vortices in microstructures, thereby introducing innovative microfluidic functionalities.
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Engineered muscle fibers are attracting interest in bio-actuator research as they can contribute to the fabrication of actuators with a high power/size ratio for micro-robots. These fibers require to be stretched during culture for functional regulation as actuators and require a fixation on a rigid substrate for stretching in culture and evaluation of mechanical properties, such as Young's modulus and contraction force. However, the conventional fixation methods for muscle fibers have many restrictions as they are not repeatable and the connection between fixation part and the muscle fibers detaches during culture; therefore, the fixation becomes weak during culture, and direct measurement of the muscle fibers' mechanical properties by a force sensor is difficult. Therefore, we propose a facile and repeatable fixation method for muscle fibers by mixing magnetite nanoparticles at both ends of the muscle fibers to fabricate magnetic ends. The fiber can be easily attached and detached repeatedly by manipulating a magnet that applies a magnetic force larger than 3 mN to the magnetic ends. Thus, the muscle fiber can be stretched fiber during culture for functional regulation, transported between the culture dish and measurement system, and directly connected to the force sensor for measurement with magnetic ends. The muscle fiber connected with magnetic ends have a long lifetime (â¼4 weeks) and the cells inside had the morphology of a skeletal muscle. Moreover, the muscle fiber showed a contraction (specific force of 1.02 mN mm-2) synchronized with electrical stimulation, confirming the muscle fiber fabricated and cultured using our method had similar morphology and function as bio-actuators in previous research. This research demonstrates the advantages of the fixation method using muscle fibers with magnetic ends; the fibers are stretched during culture, and the transportation and force measurement of weak and tiny muscle fibers could be finished in 1 min.
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Contracción Muscular , Fibras Musculares Esqueléticas , Contracción Muscular/fisiología , Músculo Esquelético/fisiología , Fenómenos Mecánicos , Fenómenos MagnéticosRESUMEN
The osmotic stress imposed on microorganisms by hypotonic conditions is perceived to regulate water and solute flux via cell membranes, which are crucial for survival. Some cells that fail to perceive osmotic stress die because this results in the rupture of the cell membrane. The flux through the membrane is characterized by the membrane permeability, which is measured using a stopped-flow apparatus in response to a millisecond-order osmolarity change. However, the obtained data are an ensemble average of each cell response. Additionally, the measurement of permeability, considering cellular viability, contributes to a more accurate evaluation of osmoadaptation. Here, we present a novel on-chip instantaneous extracellular solution exchange method using an air-liquid interface. The presented method provides a concurrent evaluation at the single-cell level in response to a millisecond-order osmotic shock, considering cellular viability by solution exchange. This method utilizes a liquid bridge with a locally formed droplet on the surface of a micropillar fabricated inside a microchannel. We evaluated a solution exchange time of 3.6 ms and applied this method to Synechocystis PCC 6803 under two different osmolarity conditions. The live/dead ratio of 1 M to 0.5 M osmotic down shock condition was 78.8/21.2% while that of 1 M to 0.25 M osmotic down shock condition was 40.0/60.0%. We evaluated the water permeability of two groups: cells that were still live before and after osmotic shock (hereafter named cell type 1), and cells that were live before but were dead 10 minutes after osmotic shock (hereafter named cell type 2). The results indicated that the water permeability of cell type 2 was higher than that of cell type 1. The results obtained using the presented methods confirmed that the effect of osmotic stress can be accurately evaluated using single-cell analysis.
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Agua , Permeabilidad de la Membrana Celular , Presión Osmótica , Membrana Celular/metabolismo , Permeabilidad , Ósmosis , Agua/metabolismoRESUMEN
This paper reports a new concept of a film-shaped micropump array for biomedical perfusion. The detailed concept, design, fabrication process, and performance evaluation using prototypes are described. In this micropump array, an open circuit potential (OCP) is generated by a planar biofuel cell (BFC), which in turn generates electro-osmotic flows (EOFs) in multiple through-holes arranged perpendicular to the micropump plane. The micropump array is thin and wireless, so it can be cut like postage stamps, easily installed in any small location, and can act as a planar micropump in solutions containing the biofuels glucose and oxygen. Perfusion at local sites are difficult with conventional techniques using multiple separate components such as micropumps and energy sources. This micropump array is expected to be applied to the perfusion of biological fluids in small locations near or inside cultured cells, cultured tissues, living organisms, and so on.
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The integration of liquid exchange and microfluidic chips plays a critical role in the biomedical and biophysical fields as it enables the control of the extracellular environment and allows for the simultaneous stimulation and detection of single cells. In this study, we present a novel approach for measuring the transient response of single cells using a system integrated with a microfluidic chip and a probe with a dual pump. The system was composed of a probe with a dual pump system, a microfluidic chip, optical tweezers, an external manipulator, an external piezo actuator, etc. Particularly, we incorporated the probe with the dual pump to allow for high-speed liquid change, and the localized flow control enabled a low disturbance contact force detection of single cells on the chip. Using this system, we measured the transient response of the cell swelling against the osmotic shock with a very fine time resolution. To demonstrate the concept, we first designed the double-barreled pipette, which was assembled with two piezo pumps to achieve a probe with the dual pump system, allowing for simultaneous liquid injection and suction. The microfluidic chip with on-chip probes was fabricated, and the integrated force sensor was calibrated. Second, we characterized the performance of the probe with the dual pump system, and the effect of the analysis position and area of the liquid exchange time was investigated. In addition, we optimized the applied injection voltage to achieve a complete concentration change, and the average liquid exchange time was achieved at approximately 3.33 ms. Finally, we demonstrated that the force sensor was only subjected to minor disturbances during the liquid exchange. This system was utilized to measure the deformation and the reactive force of Synechocystis sp. strain PCC 6803 in osmotic shock, with an average response time of approximately 16.33 ms. This system reveals the transient response of compressed single cells under millisecond osmotic shock which has the potential to characterize the accurate physiological function of ion channels.
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BACKGROUND/AIM: This study describes a rare cell sorter (RCS) method to detect circulating tumor cells (CTCs) and CTC clusters in whole blood without pretreatment. PATIENTS AND METHODS: We collected samples from breast cancer patients at the University of Tsukuba Hospital. A total of 15 whole-blood specimens from patients with breast cancer were collected and analyzed via a microfluidics chip, fluorescence-conjugated antibody staining, and fluorescence microscopy. Of 15 total cases, eight were analyzed by RCS ver3 and seven were analyzed by RCS ver3.5 to reveal potential clinical differences in scanning methods. We then examined the HER2 status on 4 of the 15 patients using our RCS system. RESULTS: RCS efficiently detected all subtypes of CTCs and CTC clusters from the peripheral blood of cancer patients. The concordance rate of HER2 status between tissue and CTCs in 4 tested clinical samples was 100%. CONCLUSION: RCS is a non-invasive method that allows for simultaneous detection of CTCs, cluster presence, and surface marker (e.g., HER2) status. Frequent sampling is, thus, possible and the large amount of data obtained will be clinically useful to predict response to therapy as well as plan adjunct support therapies in cancer patients.
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Neoplasias de la Mama , Células Neoplásicas Circulantes , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Recuento de Células , Femenino , Citometría de Flujo , Humanos , Células Neoplásicas Circulantes/patología , Receptor ErbB-2/metabolismoRESUMEN
BACKGROUND: Magnetosomes (BMPs) are organelles of magnetotactic bacteria (MTB) that are responsible for mineralizing iron to form magnetite. In addition, BMP is an ideal biomaterial that is widely used in bio- and nano-technological applications, such as drug delivery, tumor detection and therapy, and immunodetection. The use of BMPs to create multifunctional nanocomposites would further expand the range of their applications. RESULTS: In this study, we firstly demonstrate that the extracted BMP can remineralize in vitro when it is exposed to AgNO3 solution, the silver ions (Ag+) were transported into the BMP biomembrane (MM) and mineralized into a silver crystal on one crystal plane of Fe3O4. Resulting in the rapid synthesis of an Ag-Fe3O4 hybrid BMP (BMP-Ag). The synergy between the biomembrane, Fe3O4 crystal, and unmineralized iron enabled the remineralization of BMPs at an Ag+ concentration ≥ 1.0 mg mL-1. The BMP-Ag displayed good biocompatibility and antibacterial activity. At a concentration of 2.0 mg/mL, the BMP-Ag and biomembrane removed Ag-Fe3O4 NPs inhibited the growth of gram-negative and gram-positive bacteria. Thus using BMP-Ag as a wound dressing can effectively enhance the contraction of infected wounds. CONCLUSIONS: This study represents the first successful attempt to remineralize organelles ex vivo, realizing the biosynthesis of hybrid BMP and providing an important advancement in the synthesis technology of multifunctional biological nanocomposites.
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Magnetosomas , Óxido Ferrosoférrico/química , Bacterias Gramnegativas , Hierro/química , Magnetosomas/química , Plata/químicaRESUMEN
To provide quantitative feedback on surgical progress to ophthalmologists practicing inner limiting membrane (ILM) peeling, we developed an artificial eye module comprising a quartz crystal resonator (QCR) force sensor and a strain body that serves as a uniform force transmitter beneath a retinal model. Although a sufficiently large initial force must be loaded onto the QCR force sensor assembly to achieve stable contact with the strain body, the highly sensitive and wide dynamic-range property of this sensor enables the eye module to detect the slight forceps contact force. A parallel-plate strain body is used to achieve a uniform force sensitivity over the 4-mm-diameter ILM peeling region. Combining these two components allowed for a measurable force range of 0.22 mN to 29.6 N with a sensitivity error within -11.3 to 4.2% over the ILM peeling area. Using this eye module, we measured the applied force during a simulation involving artificial ILM peeling by an untrained individual and compensated for the long-term drift of the obtained force data using a newly developed algorithm. The compensated force data clearly captured the characteristics of several types of motion sequences observed from video recordings of the eye bottom using an ophthalmological microscope. As a result, we succeeded in extracting feature values that can be potentially related to trainee skill level, such as the mean and standard deviation of the pushing and peeling forces, corresponding, in the case of an untrained operator, to 122.6 ± 95.2 and 20.4 ± 13.2 mN, respectively.
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Among increasing eye diseases, glaucoma may hurt the optic nerves and lead to vision loss, the treatment of which is to reduce intraocular pressure (IOP). In this research, we introduce a new concept of the surgery simulator for Minimally Invasive Glaucoma Surgery (MIGS). The concept is comprised of an anterior eye model and a fluidic circulatory system. The model made of flexible material includes a channel like the Schlemm's canal (SC) and a membrane like the trabecular meshwork (TM) covering the SC. The system can monitor IOP in the model by a pressure sensor. In one of the MIGS procedures, the TM is cleaved to reduce the IOP. Using the simulator, ophthalmologists can practice the procedure and measure the IOP. First, considering the characteristics of human eyes, we defined requirements and target performances for the simulator. Next, we designed and manufactured the prototype. Using the prototype, we measured the IOP change before and after cleaving the TM. Finally, we demonstrated the availability by comparing experimental results and target performances. This simulator is also expected to be used for evaluations and developments of new MIGS instruments and ophthalmic surgery robots in addition to the surgical training of ophthalmologists.
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Glaucoma , Prótesis Visuales , Glaucoma/cirugía , Humanos , Presión Intraocular , Microfluídica , Malla Trabecular/fisiologíaRESUMEN
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.
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Robótica , Campos Magnéticos , Micromanipulación , MicroesferasRESUMEN
We previously proposed a microfluidic bioreactor with glass-Si-glass layers to evaluate the effect of the fluid force on platelet (PLT) production and fabricated a three-dimensional (3D) microchannel by combining grayscale photolithography and deep reactive ion etching. However, a challenge remains in observing the detailed process of PLT production owing to the low visibility of the microfluidic bioreactor. In this paper, we present a transparent microfluidic bioreactor made of cyclo-olefin polymer (COP) with which to observe the process of platelet-like particle (PLP) production under a bright-field, which allows us to obtain image data at a high sampling rate. We succeeded in fabricating the COP microfluidic bioreactor with a 3D microchannel. We investigated the bonding strength of COP-COP layers and confirmed the effectiveness of the microfluidic bioreactor. Results of on-chip PLP production using immortalized megakaryocyte cell lines (imMKCLs) derived from human-induced pluripotent stem cells show that the average total number of produced PLPs per imMKCL was 17.6 PLPs/imMKCL, which is comparable to that of our previous glass-Si-glass microfluidic bioreactor (17.4 PLPs/imMKCL). We succeeded in observing PLP production under a bright-field using the presented microfluidic bioreactor and confirmed that PLP fragmented in a narrow area of proplatelet-like protrusions.
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Collective migration of epithelial cells is a fundamental process in multicellular pattern formation. As they expand their territory, cells are exposed to various physical forces generated by cell-cell interactions and the surrounding microenvironment. While the physical stress applied by neighbouring cells has been well studied, little is known about how the niches that surround cells are spatio-temporally remodelled to regulate collective cell migration and pattern formation. Here, we analysed how the spatio-temporally remodelled extracellular matrix (ECM) alters the resistance force exerted on cells so that the cells can expand their territory. Multiple microfabrication techniques, optical tweezers, as well as mathematical models were employed to prove the simultaneous construction and breakage of ECM during cellular movement, and to show that this modification of the surrounding environment can guide cellular movement. Furthermore, by artificially remodelling the microenvironment, we showed that the directionality of collective cell migration, as well as the three-dimensional branch pattern formation of lung epithelial cells, can be controlled. Our results thus confirm that active remodelling of cellular microenvironment modulates the physical forces exerted on cells by the ECM, which contributes to the directionality of collective cell migration and consequently, pattern formation.
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Movimiento Celular/fisiología , Matriz Extracelular/fisiología , Comunicación Celular , Células Cultivadas , Fibronectinas/fisiología , HumanosRESUMEN
Non-contact manipulation technology has a wide range of applications in the manipulation and fabrication of micro/nanomaterials. However, the manipulation devices are often complex, operated only by professionals, and limited by a single manipulation function. Here, we propose a simple versatile optoelectronic tweezer (OET) system that can be easily controlled for manipulating microparticles with different sizes. In this work, we designed and established an optoelectronic tweezer manipulation system. The OET system could be used to manipulate particles with a wide range of sizes from 2 µm to 150 µm. The system could also manipulate micro-objects of different dimensions like 1D spherical polystyrene microspheres, 2D rod-shaped euglena gracilis, and 3D spiral microspirulina. Optical microscopic patterns for trapping, storing, parallel transporting, and patterning microparticles were designed for versatile manipulation. The sorting, rotation, and assembly of single particles in a given region were experimentally demonstrated. In addition, temperatures measured under different objective lenses indicate that the system does not generate excessive heat to damage bioparticles. The non-contact versatile manipulation reduces operating process and contamination. In future work, the simple optoelectronic tweezers system can be used to control non-contaminated cell interaction and micro-nano manipulation.
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In this research, atomic force microscopy (AFM) with a flat tip cantilever is utilized to measure Young's modulus of a whole yeast cell (Saccharomyces cerevisiae BY4741). The results acquired from AFM are similar to those obtained using a microfluidic chip compression system. The mechanical properties of single yeast cells are important parameters which can be examined using AFM. Conventional studies apply AFM with a sharp cantilever tip to indent the cell and measure the force-indentation curve, from which Young's modulus can be calculated. However, sharp tips introduce problems because the shape variation can lead to a different result and cannot represent the stiffness of the whole cell. It can lead to a lack of broader meaning when evaluating Young's modulus of yeast cells. In this report, we confirm the differences in results obtained when measuring the compression of a poly(dimethylsiloxane) bead using a commercial sharp tip versus a unique flat tip. The flat tip effectively avoids tip-derived errors, so we use this method to compress whole yeast cells and generate a forcedeformation curve. We believe our proposed method is effective for evaluating Young's modulus of whole yeast cells.
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Microscopía de Fuerza Atómica , Saccharomyces cerevisiae , Recuento de Células , Módulo de ElasticidadRESUMEN
The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to ~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.
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Separación Celular/métodos , Espectrometría Raman/métodos , Animales , HumanosRESUMEN
The advent of intelligent image-activated cell sorting (iIACS) has enabled high-throughput intelligent image-based sorting of single live cells from heterogeneous populations. iIACS is an on-chip microfluidic technology that builds on a seamless integration of a high-throughput fluorescence microscope, cell focuser, cell sorter, and deep neural network on a hybrid software-hardware data management architecture, thereby providing the combined merits of optical microscopy, fluorescence-activated cell sorting (FACS), and deep learning. Here we report an iIACS machine that far surpasses the state-of-the-art iIACS machine in system performance in order to expand the range of applications and discoveries enabled by the technology. Specifically, it provides a high throughput of â¼2000 events per second and a high sensitivity of â¼50 molecules of equivalent soluble fluorophores (MESFs), both of which are 20 times superior to those achieved in previous reports. This is made possible by employing (i) an image-sensor-based optomechanical flow imaging method known as virtual-freezing fluorescence imaging and (ii) a real-time intelligent image processor on an 8-PC server equipped with 8 multi-core CPUs and GPUs for intelligent decision-making, in order to significantly boost the imaging performance and computational power of the iIACS machine. We characterize the iIACS machine with fluorescent particles and various cell types and show that the performance of the iIACS machine is close to its achievable design specification. Equipped with the improved capabilities, this new generation of the iIACS technology holds promise for diverse applications in immunology, microbiology, stem cell biology, cancer biology, pathology, and synthetic biology.
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Redes Neurales de la Computación , Programas Informáticos , Algoritmos , Separación Celular , Citometría de FlujoRESUMEN
This paper presents a semi-automatic actuation system which can achieve bio-particles tracking, transportation, and high-precision motion control of robots in a microfluidic chip. This system is mainly applied in magnetically driven robots. An innovative manta ray-like robot was designed to increase stability of robots in a non-contaminated manipulation environment. A multilayer piezo actuator was applied to generate high-frequency vibration to decrease the friction between robots and the glass substrate. We also set up a user-friendly GUI (Graphical User Interface) and realized robot tracking and predetermined trajectory motion through excellent algorithms using Python and C++. In biotechnology, precise transportation of cells is used for the enucleation, microinjection, and investigation of the characteristics of a single cell. Being optimized, the parameters of the robot can effectively reach 10 µm in actuation precision and a maximum actuation speed of 200 mm/s.
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This work describes a hydrogel fluorescence microsensor for prolonged stable temperature measurements. Temperature measurement using microsensors has the potential to provide information about cells, tissues, and the culture environment, with optical measurement using a fluorescent dye being a promising microsensing approach. However, it is challenging to achieve stable measurements over prolonged periods with conventional measurement methods based on the fluorescence intensity of fluorescent dye because the excited fluorescent dye molecules are bleached by the exposure to light. The decrease in fluorescence intensity induced by photobleaching causes measurement errors. In this work, a photobleaching compensation method based on the diffusion of fluorescent dye inside a hydrogel microsensor is proposed. The factors that influence compensation in the hydrogel microsensor system are the interval time between measurements, material, concentration of photo initiator, and the composition of the fluorescence microsensor. These factors were evaluated by comparing a polystyrene fluorescence microsensor and a hydrogel fluorescence microsensor, both with diameters of 20 µm. The hydrogel fluorescence microsensor made from 9% poly (ethylene glycol) diacrylate (PEGDA) 575 and 2% photo initiator showed excellent fluorescence intensity stability after exposure (standard deviation of difference from initial fluorescence after 100 measurement repetitions: within 1%). The effect of microsensor size on the stability of the fluorescence intensity was also evaluated. The hydrogel fluorescence microsensors, with sizes greater than the measurement area determined by the axial resolution of the confocal microscope, showed a small decrease in fluorescence intensity, within 3%, after 900 measurement repetitions. The temperature of deionized water in a microchamber was measured for 5400 s using both a thermopile and the hydrogel fluorescence microsensor. The results showed that the maximum error and standard deviation of error between these two sensors were 0.5 °C and 0.3 °C, respectively, confirming the effectiveness of the proposed method.
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The capability to precisely rotate cells and other micrometer-sized biological samples is invaluable in biomedicine, bioengineering, and biophysics. We propose herein a novel on-chip cell rotation method using acoustic microstreaming generated by oscillating asymmetrical microstructures. When the vibration is applied to a microchip with our custom-designed microstructures, two different modes of highly localized microvortices are generated that are utilized to precisely achieve in-plane and out-of-plane rotational manipulation of microbeads and oocytes. The rotation mechanism is studied and verified using numerical simulations. Experiments of the microbeads are conducted to evaluate the claimed functions and investigate the effects of various parameters, such as the frequency and the driving voltage on the acoustically induced flows. Accordingly, it is shown that the rotational speed and direction can be effectively tuned on demand in single-cell studies. Finally, the rotation of swine oocytes is involved as further applications. By observing the maturation stages of M2 after the exclusion of the first polar body of operated oocytes, the proposed method is proved to be noninvasive. Compared with the conventional approaches, our acoustofluidic cell rotation approach can be simple-to-fabricate and easy-to-operate, thereby allowing rotations irrespective of the physical properties of the specimen under investigation.