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

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

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.

Microinjection and Micromanipulation: A Historical Perspective.

Microinjection/micromanipulation is more than 100 years old. It is a technique that is instrumental in biomedical research and healthcare. Its longevity lies in its preciseness in mechanical retrieval, or delivery of biological materials, which in some cases is simply necessary or more effective than other retrieval/delivery means. Microinjection is favored for its straightforwardness in transferring contents from micromolecules to macromolecules and from organelles to cells. Microinjection/micromanipulation has been practiced over the century like an art form. Variations in handlings and instruments can be tolerated to a surprising degree with satisfactory outcomes. Throughout the century, microinjection developed as an indispensable tool along with the evolution of biomedical fields: from transgenics to gene targeting, from animal cloning to human infertility treatment, from nuclease-guided genetic engineering to RNA-guided genome editing (Fig. 1). The birth of the CRISPRology rejuvenated microinjection. For microinjection/micromanipulation, the second century has already begun with the early arrival of computerized instrumentation and lately of the high-throughput nanomanipulators potentially operable by artificial intelligence. As we yin-yang both systemic and precision approaches in research and medicine, microinjection will no doubt continue to find its unique place in the future.

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.

Acoustic radiation force and torque on an absorbing compressible particle in an inviscid fluid.

Exact formulas of the acoustic radiation force and torque exerted by an arbitrary time-harmonic wave on an absorbing compressible particle that is suspended in an inviscid fluid are presented. It is considered that the particle diameter is much smaller than the incident wavelength, i.e., the so-called Rayleigh scattering limit. Moreover, the particle absorption assumed here is due to the attenuation of compressional waves only. Shear waves inside and outside the particle are neglected, since the inner and outer viscous boundary layer of the particle are supposed to be much smaller than the particle radius. The obtained radiation force formulas are used to establish the trapping conditions of a particle by a single-beam acoustical tweezer based on a spherically focused ultrasound transducer. In this case, it is shown that the particle absorption has a pivotal role in single-beam trapping at the transducer focal region. Furthermore, it is found that only the first-order Bessel vortex beam can generate the radiation torque on a small particle. In addition, numerical evaluation of the radiation force and torque exerted on a benzene and an olive oil droplet suspended in water are presented and discussed.

On-demand weighing of single dry biological particles over a 5-order-of-magnitude dynamic range.

We report a simple and highly versatile system to select and weigh individual dry biological particles. The system is composed of a microtweezer to pick and place individual particles and a cantilever-based resonator to weigh them. The system can weigh entities that vary from a red blood cell (~10(-11) g) to the eye-brain complex of an insect (~10(-6) g), covering a 5-order-of-magnitude mass range. Due to its versatility and ease of use, this weighing method is highly compatible with established laboratory practices. The system can provide complementary mass information for a wide variety of individual particles imaged using scanning electron microscopy and determine comparative weights of individual biological entities that are attached to microparticles as well as weigh fractions of individual biological entities that have been subjected to focused ion beam milling.

Induction and coverage times for crude oil droplets spreading on air bubbles.

The interactions between crude oil droplets and air bubbles were studied by the droplet-bubble micromanipulator technique. Eight crude oils were investigated, and some aspects of the involved mechanisms were discussed. The induction time was measured for air bubbles approaching crude oil droplets in different aqueous phases. Distinct differences were observed in the presence and absence of salts, which showed the importance of long-ranged electrostatic repulsive forces on thin-film stability. The results also suggested that adsorption of dissolved hydrocarbons at air bubble surfaces may increase the potential energy barrier in the thin liquid film. Furthermore, the time needed for crude oil droplets to spread over the air bubble surfaces (referred to as coverage time) was determined for the crude oils. The results showed that the spreading velocity decreased with increasing viscosity of the crude oil. The detailed understanding of this type of interaction is considered to be a precursor for improving the oil removal efficiency during the flotation process.

Hydrogel-based microactuators with remote-controlled locomotion and fast Pb2+-response for micromanipulation.

Hydrogel-based microactuators that enable remote-controlled locomotion and fast Pb(2+)-response for micromanipulation in Pb(2+)-polluted microenvironment have been fabricated from quadruple-component double emulsions. The microactuators are Pb(2+)-responsive poly(N-isopropylacrylamide-co-benzo-18-crown-6-acrylamide) microgels, each with an eccentric magnetic core for magnetic manipulation and a hollow cavity for fast Pb(2+)-response. Micromanipulation of the microactuators is demonstrated by using them for preventing Pb(2+)-leakage from microchannel. The microactuators can be remotely and precisely transported to the Pb(2+)-leaking site under magnetic guide, and then clog the microchannel with Pb(2+)-responsive volume swelling to prevent flowing out of Pb(2+)-contaminated solution. The proposed microactuator structure provides a potential and novel model for developing multifunctional actuators and sensors, biomimetic soft microrobots, microelectro-mechanical systems and drug delivery systems.

Photothermal genetic engineering.

Optical methods for manipulation of cellular function have enabled deconstruction of genetic and neural circuits in vitro and in vivo. Plasmonic gold nanomaterials provide an alternative platform for external optical manipulation of genetic circuits. The tunable absorption of gold nanoparticles in the infrared spectral region and straightforward surface functionalization has led to applications in intracellular delivery and photorelease of short RNAs, recently enabling bidirectional photothermal modulation of specific genes via RNA interference (RNAi). We discuss recent advances in optical gene circuit engineering and plasmonic nanomaterials, as well as future research opportunities and challenges in photothermal gene manipulation.

Use of a micromanipulator system (NeuRobot) in endoscopic neurosurgery.

NeuRobot, a micromanipulator system with a rigid neuroendoscope and three micromanipulators, was developed for less invasive and telecontrolled neurosurgery. This system can be used to perform sophisticated surgical procedures through a small, 10-mm-diameter, window. The present study was performed to evaluate the feasibility of using NeuRobot in neuroendoscopy. Four different intraventricular neurosurgical procedures were simulated in three fixed cadaver heads using NeuRobot: (1) fenestration of the floor of the third ventricle; (2) fenestration of the septum pellucidum; (3) biopsy of the thalamus; and (4) biopsy of the choroid plexus of the lateral ventricle. Each procedure required less than 2 min, and all procedures were performed accurately. After these surgical simulations, a third ventriculostomy was carried out safely and adequately in a patient with obstructive hydrocephalus due to a midbrain venous angioma. Our results confirmed that NeuRobot is applicable to lesions in which conventional endoscopic neurosurgery is indicated. Furthermore, NeuRobot can perform more complex surgical procedures than a conventional neuroendoscope because of its maneuverability and stability. NeuRobot will become a useful neurosurgical tool for dealing with lesions that are difficult to treat by conventional neuroendoscopic surgery.

Roughness and pH changes of enamel surface induced by soft drinks in vitro-applications of stylus profilometry, focus variation 3D scanning microscopy and micro pH sensor.

This study aimed to evaluate enamel surface roughness (Ra) and pH before and after erosion by soft drinks. Enamel was exposed to a soft drink (cola, orange juice or green tea) for 1, 5 or 60 min; Ra was measured using contact-stylus surface profilometry (SSP) and non-contact focus variation 3D microscope (FVM). Surface pH was measured using a micro pH sensor. Data were analyzed at significance level of alpha=0.05. There was a significant correlation in Ra between SSP and FVM. FVM images showed no changes in the surface morphology after various periods of exposure to green tea. Unlike cola and orange juice, exposure to green tea did not significantly affect Ra or pH. A significant correlation was observed between surface pH and Ra change after exposure to the drinks. Optical surface analysis and micro pH sensor may be useful tools for non-damaging, quantitative assessment of soft drinks erosion on enamel.

Comparison of the debridement efficacy of the EndoVac irrigation system and conventional needle root canal irrigation in vivo.

INTRODUCTION: The purpose of this study was to compare the debridement efficacy of EndoVac irrigation versus conventional needle irrigation in vivo. METHODS: Seven adult patients with a total of 22 matched pairs of single-canaled vital teeth with fully formed apices were recruited. Canals were instrumented to a master apical file size #40/.04 taper. One tooth from each matched pair was irrigated by using the EndoVac system. The other tooth was irrigated by conventional needle irrigation. Five additional teeth were used as positive controls. A #10 K-file was inserted into the control canals to determine working length (WL), with no other instrumentation or irrigation performed to confirm the presence of debris. The teeth were extracted, fixed, and decalcified. Six histologic slides each 6 µm thick were made from sections at 1 and 3 mm from WL and stained. The slide with the most debris was photographed at each level for each tooth. A Wilcoxon signed rank test was used to compare the percentage of debris remaining in the canals between the 2 irrigation techniques. RESULTS: The median amount of debris remaining at 1 mm was 0.05% for the EndoVac group and 0.12% for the conventional irrigation group (P < .05). The median amount of debris remaining at 3 mm was 0.09% for the EndoVac group and 0.07% for the conventional needle irrigation group (P > .05). CONCLUSIONS: EndoVac irrigation resulted in significantly less debris at 1 mm from WL compared with conventional needle irrigation. There was no significant difference at the 3-mm level.

Monitoring of laser micromanipulated optically trapped cells by digital holographic microscopy.

For a precise manipulation of particles and cells with laser light as well as for the understanding and the control of the underlying processes it is important to visualize and quantify the response of the specimens. Thus, we investigated if digital holographic microscopy (DHM) can be used in combination with microfluidics to observe optically trapped living cells in a minimally invasive fashion during laser micromanipulation. The obtained results demonstrate that DHM multi-focus phase contrast provides label-free quantitative monitoring of optical manipulation with a temporal resolution of a few milliseconds.

The thickness of the intestinal mucous layer in the colon of rats fed various sources of non-digestible carbohydrates is positively correlated with the pool of SCFA but negatively correlated with the proportion of butyric acid in digesta.

The present experiment aimed to study the influence of six sources of non-digestible carbohydrates (NDC) on the mucous layer in the colon of rats. The NDC sources used were as follows: cellulose (C); pectin (P); inulin; resistant starch (RS); barley hulls. The diets contained 108-140 g NDC/kg DM. A fibre-free (FF) diet served as a control. The diets were fed to forty-eight rats for 34-41 d. The thickness of the total mucous layer in the colon was increased (P < 0.05) in rats fed C, P and RS when compared with rats fed a FF diet. In the colon, positive correlations were observed between the total thickness of the mucous layer and the area of neutral mucins, the pool of SCFA and the pool of acetic acid, while it was negatively correlated with the proportion of butyrate. The total thickness of the mucous layer was not correlated with the MUC gene transcription. The transcription of the gene MUC2 was negatively correlated (P = 0.04), whereas the transcription of MUC3 was positively correlated (P = 0.05) with the butyrate pool in the caecum. No correlations between the MUC2 or MUC3 transcription and SCFA were found in the colon. Hence, the regulation of the MUC genes differs between the compartments of the hindgut and, within compartments, the MUC genes may be regulated differently. In conclusion, a diet providing a large pool of SCFA with a low proportion of butyrate in the colon stimulates the formation of a thick mucous layer, which probably benefits intestinal health.

Elastic and viscoelastic characterization of microcapsules for drug delivery using a force-feedback MEMS microgripper.

This paper reports a monolithic, force-feedback MEMS (microelectomechanical systems) microgripper and its application to micro-scale compression testing of swollen hydrogel microcapsules at wet state during manipulation. The single-chip microgripper integrates an electrothermal microactuator and two capacitive force sensors, one for contact detection (force resolution: 38.5 nN) and the other for gripping force measurements (force resolution: 19.9 nN). With the capability of resolving gripping forces down to 19.9 nN and material deformations with a 20.5 nm resolution, the system quantified Young's modulus values and viscoelastic parameters of alginate microcapsules (15-25 microm), demonstrating an easy-to-operate, accurate compression testing technique for characterizing soft, micrometer-sized biomaterials.

Magnetic resonance propulsion, control and tracking at 24 Hz of an untethered device in the carotid artery of a living animal: an important step in the development of medical micro- and nanorobots.

Our recent demonstration of a ferromagnetic bead being navigated automatically inside the carotid artery of a living animal at an average speed of 10 cm/s using a clinical MRI system may be considered as a significant step in the field of medical micro- and nanorobotics. This is particularly true when we consider that an appropriate tracking method was embedded in the closed-loop control process allowing the blood vessels to be considered as navigational routes, providing maximum access for conducting operations inside the human body. But more importantly, this demonstration not only validates preliminary theoretical models but provides us with initial insights about the strategies and approaches that are likely to be used to navigate under computer control, micro- and nanodevices including nanorobots from the largest to the smallest diameter blood vessels that could be used to reach targets inside the human body. Here, based on these initial experimental data obtained in vivo, such strategies and methods are briefly described with some initial design concepts of medical interventional micro- and nanorobots.

Membrane dielectric responses of bufalin-induced apoptosis in HL-60 cells detected by an electrorotation chip.

A non-invasive electrorotation (ROT) technique was used to monitor the apoptosis-induced changes in HL-60 cells. The specific membrane capacitance of the cells fell from 15.6 +/- 0.9 mF/cm(2) to 6.4 +/- 0.6 mF/cm(2) after 48 h treatment with 10 nM bufalin, a component of bufadienolides in traditional Chinese medicine, Chan Su. However, the average membrane conductance remained almost constant during the first 24 h of treatment and then increased afterwards. Apoptosis was verified by a DNA fragmentation assay and scanning electron microscopy. The results demonstrate that the ROT technique gives a quantitative analysis of the toxic damage by chemicals to cells and can be exploited in the testing and development of new pharmaceuticals and active cell agents.

Method of propulsion of a ferromagnetic core in the cardiovascular system through magnetic gradients generated by an MRI system.

This paper reports the use of a magnetic resonance imaging (MRI) system to propel a ferromagnetic core. The concept was studied for future development of microdevices designed to perform minimally invasive interventions in remote sites accessible through the human cardiovascular system. A mathematical model is described taking into account various parameters such as the size of blood vessels, the velocities and viscous properties of blood, the magnetic properties of the materials, the characteristics of MRI gradient coils, as well as the ratio between the diameter of a spherical core and the diameter of the blood vessels. The concept of magnetic propulsion by MRI is validated experimentally by measuring the flow velocities that magnetized spheres (carbon steel 1010/1020) can withstand inside cylindrical tubes under the different magnetic forces created with a Siemens Magnetom Vision 1.5 T MRI system. The differences between the velocities predicted by the theoretical model and the experiments are approximately 10%. The results indicate that with the technology available today for gradient coils used in clinical MRI systems, it is possible to generate sufficient gradients to propel a ferromagnetic sphere in the larger sections of the arterial system. In other words, the results show that in the larger blood vessels where the diameter of the microdevices could be as large as a couple a millimeters, the few tens of mT/m of gradients required for displacement against the relatively high blood flow rate is well within the limits of clinical MRI systems. On the other hand, although propulsion of a ferromagnetic core with diameter of approximately 600 microm may be possible with existing clinical MRI systems, gradient amplitudes of several T/m would be required to propel a much smaller ferromagnetic core in small vessels such as capillaries and additional gradient coils would be required to upgrade existing MRI systems for operations at such a scale.

Manipulation and extraction of genomic DNA from cell lysate by functionalized magnetic particles for lab on a chip applications.

A novel approach for extracting living cells' genomic DNA materials utilizing functionalized magnetic particles (MPs) is reported in this investigation. This strategy is amenable to handle bio-samples in a miniaturized environment and it offers a possibility to separate and purify DNA from other cell lysate mixtures "on-chip", which is known to be a bottle-neck step in an integrated micro-total-analysis-system (muTAS). "Species-specific" genomic DNA of interest is captured by the MPs based on the hybridization interaction between the biotinylated probes modified MPs and a complementary region of the targeted genome. The genome DNA anchored on the particles can be separated from the rest of cellular mixtures by a simple buffer washing upon the exertion of external magnetic force. Surface modifications of MPs and hybridization conditions affecting the genome capturing efficiency are investigated. Extraction of genomic DNA from E. coli is demonstrated in a silicon/glass-based micro-reactor patterned with a platinum heater and sensors. On-chip extraction and manipulation of genomic DNAs illustrated in this study is a step forward toward a total integrated bioanalytical microsystem for crude cells/sample analysis.

The solution to the streptavidin-biotin paradox: the influence of history on the strength of single molecular bonds.

In the past few years, many studies have attempted to measure the strength of a single molecular bond. In general, these experiments consisted in pulling on the bond and measuring the force necessary to dissociate the molecules. However, seemingly contradictory experimental results led to draw the intriguing conclusion that the strength of the bond could depend on the experiment even if the pulling conditions are similar: this paradox was first observed on the widely used streptavidin-biotin bond. Here, by doing supplementary measurements and by reanalyzing the controversial experimental results using Kramers' theory, we show that they can be conciliated. This allows us to show that the strength of a bond is very sensitive to the history of its formation, which is the key to the paradox.