Regulation of biological processes is often based on physical interactions between cells and their microenvironment. To unravel how and where interactions occur, micromanipulation methods can be used that offer high-precision control over the duration, position and magnitude of interactions. However, lacking an in vivo system, micromanipulation has generally been done with cells in vitro, which may not reflect the complex in vivo situation inside multicellular organisms. Here using optical tweezers we demonstrate micromanipulation throughout the transparent zebrafish embryo. We show that different cells, as well as injected nanoparticles and bacteria can be trapped and that adhesion properties and membrane deformation of endothelium and macrophages can be analysed. This non-invasive micromanipulation inside a whole-organism gives direct insights into cell interactions that are not accessible using existing approaches. Potential applications include screening of nanoparticle-cell interactions for cancer therapy or tissue invasion studies in cancer and infection biology.

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

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

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.

Poor semen quality is increasingly the reason for human infertility. For couples who cannot be helped by conventional in vitro fertilization (IVF) treatment, micromanipulation methods and special sperm preparation techniques in in vitro fertilization hold considerable promise to enhance fertilization of the egg. "Partial zona dissection" (PZD n = 59) or "subzonal insemination" (SUZI n = 36) were performed in 88 IVF cycles obtaining a fertilization rate between 12.2% and 34.8% depending on the micromanipulation technique applied and a pregnancy rate of 19.2% per embryo transfer. So far ten ongoing pregnancies have occurred and five healthy children have been born. In certain cases of male factor infertility micromanipulation and special sperm preparation techniques therefore represent the only possibility of conceiving.

The structure of mitotic chromosomes in cultured newt lung cells was investigated by a quantitative study of their deformability, using micropipettes. Metaphase chromosomes are highly extensible objects that return to their native shape after being stretched up to 10 times their normal length. Larger deformations of 10 to 100 times irreversibly and progressively transform the chromosomes into a "thin filament," parts of which display a helical organization. Chromosomes break for elongations of the order of 100 times, at which time the applied force is around 100 nanonewtons. We have also observed that as mitosis proceeds from nuclear envelope breakdown to metaphase, the native chromosomes progressively become more flexible. (The elastic Young modulus drops from 5,000 +/- 1,000 to 1,000 +/- 200 Pa.) These observations and measurements are in agreement with a helix-hierarchy model of chromosome structure. Knowing the Young modulus allows us to estimate that the force exerted by the spindle on a newt chromosome at anaphase is roughly one nanonewton.

With technologies rapidly evolving, many research institutions are now opting to invest in costly, high-quality, specialized microscopes which are shared by many researchers. As a consequence, the user may not have the ability to adapt a microscope to their specific needs and limitations in experimental design are introduced. A flexible work-horse microscopy system is a valuable tool in any laboratory to meet the diverse needs of a research team and promote innovation in experimental design. We have developed the Flexiscope; a multi-functional, adaptable, efficient and high-performance microscopy/electrophysiology system for everyday applications in a neurobiology laboratory. The core optical components are relatively constant in the three configurations described here: an upright configuration, an inverted configuration and an upright/electrophysiology configuration. We have provided a comprehensive description of the Flexiscope. We show that this method is capable of oblique infrared illumination imaging, multi-channel fluorescent imaging and automated three-dimensional scanning of larger specimens. Image quality is conserved across the three configurations of the microscope, and conversion between configurations is possible quickly and easily, while the motion control system can be repurposed to allow sub-micrometre computer-controlled micromanipulation. The Flexiscope provides similar performance and usability to commercially available systems. However, as it can be easily reconfigured for multiple roles, it can remove the need to purchase multiple microscopes, giving significant cost savings. The modular reconfigurable nature allows the user to customize the system to their specific needs and adapt/upgrade the system as challenges arise, without requiring specialized technical skills.

We examined the effects of endoplasmic reticulum (ER) stress inhibitor treatment during the micromanipulation of porcine somatic cell nuclear transfer (SCNT) on the in vitro development of SCNT embryos. ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxycholic acid (TUDCA; 100 μM) were added to the micromanipulation medium and holding medium. The expression of X-box binding protein 1 (Xbp1), ER-stress-associated genes, and apoptotic genes in SCNT embryos was confirmed at the one-cell and blastocyst stages. Levels of Xbp1 splicing and expression of ER-stress-associated genes in SCNT embryos at the one-cell stage decreased significantly with TUDCA treatment (p<0.05). The expression of ER-stress-associated genes also decreased slightly with the addition of both salubrinal and TUDCA (Sal+TUD). The expression levels of caspase-3 and Bcl2-associated Xprotein (Bax) mRNA were also significantly lower in the TUDCA and Sal+TUD treatments (p<0.05). At the blastocyst stage, there were no differences in levels of Xbp1 splicing, and transcription of ER-stress-associated genes and apoptosis genes between control and treatment groups. However, the blastocyst formation rate (20.2%) and mean blastocyst cell number (63.0±7.2) were significantly higher (p<0.05) for embryos in the TUDCA treatment compared with those for control (12.6% and 41.7±3.1, respectively). These results indicate that the addition of ER-stress inhibitors, especially TUDCA, during micromanipulation can inhibit cellular damage and enhance in vitro development of SCNT embryos by reducing stress levels in the ER.

Meiosis produces four haploid cells after two successive divisions in sexually reproducing organisms. A critical event during meiosis is construction of the synaptonemal complex (SC), a large, protein-based bridge that physically links homologous chromosomes. The SC facilitates meiotic recombination, chromosome compaction, and the eventual separation of homologous chromosomes at metaphase I. We present experiments directly measuring physical properties of captured mammalian meiotic prophase I chromosomes. Mouse meiotic chromosomes are about ten-fold stiffer than somatic mitotic chromosomes, even for genetic mutants lacking SYCP1, the central element of the SC. Meiotic chromosomes dissolve when treated with nucleases, but only weaken when treated with proteases, suggesting that the SC is not rigidly connected, and that meiotic prophase I chromosomes are a gel meshwork of chromatin, similar to mitotic chromosomes. These results are consistent with a liquid- or liquid-crystal SC, but with SC-chromatin stiff enough to mechanically drive crossover interference.

The mechanical properties of tissues are pivotal for morphogenesis and disease progression. Recent approaches have enabled measurements of the spatial distributions of viscoelastic properties among embryonic and pathological model systems and facilitated the generation of important hypotheses such as durotaxis and tissue-scale phase transition. There likely are many unexpected aspects of embryo biomechanics we have yet to discover which will change our views of mechanisms that govern development and disease. One area in the blind spot of even the most recent approaches to measuring tissue stiffness is the potentially anisotropic nature of that parameter. Here, we report a magnetic micromanipulation device that generates a uniform magnetic field gradient within a large workspace and permits measurement of the variation of tissue stiffness along three orthogonal axes. By applying the device to the organ-stage mouse embryo, we identify spatially heterogenous and directionally anisotropic stiffness within the mandibular arch. Those properties correspond to the domain of expression and the angular distribution of fibronectin and have potential implications for mechanisms that orient collective cell movements and shape tissues during development. Assessment of anisotropic properties extends the repertoire of current methods and will enable the generation and testing of hypotheses.

For nearly a century, conventional microbiological methods have been standard practice for detecting and identifying pathogens in food. Nevertheless, the microbiological safety of food has improved and various rapid methods have been developed to overcome the limitations of conventional methods. Alternative methods are expected to detect low cell numbers, since the presence in food of even a single cell of a pathogenic organism may be infectious. With respect to low population levels, the performance of a detection method is assessed by producing serial dilutions of a pure bacterial suspension to inoculate representative food matrices with highly diluted bacterial cells (fewer than 10 CFU/ml). The accuracy of data obtained by multiple dilution techniques is not certain and does not exclude some colonies arising from clumps of cells. Micromanipulation techniques to capture and isolate single cells from environmental samples were introduced more than 40 years ago. The main limitation of the current micromanipulation technique is still the low recovery rate for the growth of a single cell in culture medium. In this study, we describe a new single cell isolation method and demonstrate that it can be used successfully to grow various types of microorganism from picked individual cells. Tests with Gram-positive and Gram-negative organisms, including cocci, rods, aerobes, anaerobes, yeasts and molds showed growth recovery rates from 60% to 100% after micromanipulation. We also highlight the use of our method to evaluate and challenge the detection limits of standard detection methods in food samples contaminated by a single cell of Salmonella enterica.

We investigated the effects of endoplasmic reticulum (ER) stress inhibitor and antioxidant treatments during the micromanipulation of somatic cell nuclear transfer (SCNT) on in vitro development of SCNT embryos. Tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor and vitamin C (Vit. C), an antioxidant, were treated by alone or in combination, then, the level of X-box binding protein 1 (Xbp1) splicing and the expressions of ER stress-associated genes, oxidative stress-related genes, and apoptotic genes were confirmed in the 1-cell and blastocyst stages. In the 1-cell stage, the levels of Xbp1 splicing were significantly decreased in TUDCA and Vit. C treatment groups compared to the control (p<0.05). In addition, the expression levels of most ER stress-associated genes and oxidative stress-related genes were significantly lower in all treatment groups than the control (p<0.05), and the transcript levels of apoptotic genes were also significantly lower in all treatment groups than the control (p<0.05). In the blastocyst stage, decreased expression of ER stress-, oxidative stress-, and apoptosis-related genes were observed only in some treatments. However, the blastocyst formation rates in TUDCA and Vit. C treatment groups (24.8% and 22.0%, respectively) and mean blastocyst cell number in all treatment groups (59.7±4.3 to 63.5±3.3) were significantly higher (p<0.05) than those of control. The results showed that the TUDCA or Vit. C treatment during micromanipulation inhibited both ER and oxidative stresses in the early stage of SCNT embryos, thereby reducing cell damage and promoting in vitro development.

Micromanipulation is a powerful technique to measure the mechanical properties of microparticles including microcapsules. For microparticles with a homogenous structure, their apparent Young's modulus can be determined from the force versus displacement data fitted by the classical Hertz model. Microcapsules can consist of a liquid core surrounded by a solid shell. Two Young's modulus values can be defined, i.e., the one is that determined using the Hertz model and another is the intrinsic Young's modulus of the shell material, which can be calculated from finite element analysis (FEA). In this study, the two Young's modulus values of microplastic-free plant-based microcapsules with a core of perfume oil (hexyl salicylate) were calculated using the aforementioned approaches. The apparent Young's modulus value of the whole microcapsules determined by the classical Hertz model was found to be EA = 0.095 ± 0.014 GPa by treating each individual microcapsule as a homogeneous solid spherical particle. The previously obtained simulation results from FEA were utilised to fit the micromanipulation data of individual core-shell microcapsules, enabling to determine their unique shell thickness to radius ratio (h/r)FEA = 0.132 ± 0.009 and the intrinsic Young's modulus of their shell (EFEA = 1.02 ± 0.13 GPa). Moreover, a novel theoretical relationship between the two Young's modulus values has been derived. It is found that the ratio of the two Young's module values (EA/EFEA) is only a function on the ratio of the shell thickness to radius (h/r) of the individual microcapsule, which can be fitted by a third-degree polynomial function of h/r. Such relationship has proven applicable to a broad spectrum of microcapsules (i.e., non-synthetic, synthetic, and double coated shells) regardless of their shell chemistry.

Clonal composition and T cell receptor (TCR) repertoire of CD4(+) and CD8(+) T cells infiltrating actively demyelinating multiple sclerosis (MS) lesions were determined with unprecedented resolution at the level of single cells. Individual CD4(+) or CD8(+) T cells were isolated from frozen sections of lesional tissue by micromanipulation and subjected to single target amplification of TCR-beta gene rearrangements. This strategy allows the assignment of a TCR variable region (V region) sequence to the particular T cell from which it was amplified. Sequence analysis revealed that in both cases investigated, the majority of CD8(+) T cells belonged to few clones. One of these clones accounted for 35% of CD8(+) T cells in case 1. V region sequence comparison revealed signs of selection for common peptide specificities for some of the CD8(+) T cells in case 1. In both cases, the CD4(+) T cell population was more heterogeneous. Most CD4(+) and CD8(+) clones were represented in perivascular infiltrates as well as among parenchymal T cells. In case 2, two of the CD8(+) clones identified in brain tissue were also detected in peripheral blood. Investigation of the antigenic specificities of expanded clones may help to elucidate their functional properties.

In this study, we developed an online graphical and intuitive interface connected to a server aiming to facilitate professional access worldwide to those facing problems with bovine blastocysts classification. The interface Blasto3Q, where 3Q refers to the three qualities of the blastocyst grading, contains a description of 24 variables that were extracted from the image of the blastocyst and analyzed by three Artificial Neural Networks (ANNs) that classify the same loaded image. The same embryo (i.e., the biological specimen) was submitted to digital image capture by the control group (inverted microscope with 40× magnification) and the experimental group (stereomicroscope with maximum of magnification plus 4× zoom from the cell phone camera). The images obtained from the control and experimental groups were uploaded on Blasto3Q. Each image from both sources was evaluated for segmentation and submitted (only if it could be properly or partially segmented) for automatic quality grade classification by the three ANNs of the Blasto3Q program. Adjustments on the software program through the use of scaling algorithm software were performed to ensure the proper search and segmentation of the embryo in the raw images when they were captured by the smartphone, since this source produced small embryo images compared with those from the inverted microscope. With this new program, 77.8% of the images from smartphones were successfully segmented and from those, 85.7% were evaluated by the Blasto3Q in agreement with the control group.

The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs). The objective of this study was to identify and to analyse the promoter region of rabbit POU5F1 gene; furthermore to examine its expression pattern in preimplantation stage rabbit embryos.

Morphologically classifying embryos is important for numerous laboratory techniques, which range from basic methods to methods for assisted reproduction. However, the standard method currently used for classification is subjective and depends on an embryologist's prior training. Thus, our work was aimed at developing software to classify morphological quality for blastocysts based on digital images.

We aim to visualize the external physical damages and distinct external phenotypic effects between mechanical and laser-assisted immobilized human spermatozoa using scanning electronic microscopy (SEM). Human spermatozoa were immobilized mechanically or with laser assistance for SEM examination and the membrane integrities were checked on both types of immobilized spermatozoa. We found evidence of external damages at SEM level on mechanically kinked sperm, but not on laser-assisted immobilized sperm. Although no external damage was found on laser-assist immobilized sperm, there were two distinct types of morphological changes when spermatozoa were stricken by infra-red laser. Coiled tails were immediately formed when Laser pulse was applied to the sperm end piece area, whereas laser applied to the sperm principal piece area resulted in a sharp bend of sperm tails. Sperm immobilized by laser did not exhibit any morphological change if the laser did not hit within the on-screen central target zone or if the laser hit the sperm mid piece or head. Our modified membrane integrity assay revealed that the external membrane of more than half of the laser-assisted immobilized sperm remained intact. In conclusion, mechanical immobilization produced membrane damages whilst laser-assisted immobilization did not result in any external membrane damages besides morphological changes at SEM level.

Cholesteric liquid crystal (CLC) has attracted intensive attention due to its ability to form a periodic helical structure with broad tunability. CLC gratings in open systems are especially promising in sensing and micromanipulation. However, there is still much to learn about the inherent mechanism of such gratings. We investigate the light-driven rotation and pitch-tuning behaviors of CLC gratings in semi-free films which are formed by spin-coating the CLC mixtures onto planarly photoaligned substrates. The doped azobenzene chiral molecular switch supplies great flexibility to realize the continuous grating rotation. The maximum continuous rotational angle reaches 987.8°. Moreover, dependencies of light-driven rotation and pitch tuning on the dopant concentration and exposure are studied. The model of director configuration in the semi-free film is constructed. Precise beam steering and synchronous micromanipulation are also demonstrated. Our work may provide new opportunities for the CLC grating in applications of beam steering, micromanipulation, and sensing.

Highly precise micromanipulation tools that can manipulate and interrogate cell organelles and components must be developed to support the rapid development of new cell-based medical therapies, thereby facilitating in-depth understanding of cell dynamics, cell component functions, and disease mechanisms. This paper presents a literature review on micro/nanomanipulation tools and their control methods for single-cell surgery. Micromanipulation methods specifically based on laser, microneedle, and untethered micro/nanotools are presented in detail. The limitations of these techniques are also discussed. The biological significance and clinical applications of single-cell surgery are also addressed in this paper.