Use Of Micromanipulator Research Articles

A microfluidic device for selective pairing and electrofusion of cell-sized liposomes

Electrofusion is a rapid and straightforward method for merging natural and synthetic cells. The accuracy of the fusion process can be improved through the use of micromanipulation or by implementing microfluidic devices with micro-traps and microelectrodes. However, these approaches have their limitations, including contamination, sample evaporation, inability to exchange solutions, and difficulty in removing unwanted pairs in the traps. In this paper, we present a microfluidic device that utilizes a hydrodynamic pairing system and microelectrodes to achieve accurate electrofusion. Our hydrodynamic pairing system involves two hydrodynamic trapping units that are duplicated in a mirror-symmetric configuration and connected at the two trapping channels. We install alloy-based microelectrodes by vacuum-filling method sandwiching the pairing part. Using the fabricated device, we demonstrate accurate pairing and efficient electrofusion of cell-sized liposomes. Our device would be a valuable tool in controlling biochemical reactions in artificial cells.

Features of CO<sub>2</sub>-laser application in surgical treatment of chronic cicatrical stenoses of the larynx

The aim of the review is to identify current possibilities of carbon dioxide laser application in microsurgical treatment of chronic laryngeal cicatricial stenoses. Currently, laser endolaryngeal surgeries are one of the main standards in surgical treatment of patients with this laryngeal pathology, including when providing high-tech assistance. Various types of lasers and methods of surgical treatment of the larynx are used. The choice of these methods is primarily determined by the opinion of the operating surgeon. The review highlights the principles of laser surgery in general, and when using carbon dioxide laser in particular. The use of micromanipulators makes it possible to combine the work of carbon dioxide laser with the operating microscope, affecting target tissues noninvasively with maximum precision. This technology makes it possible to expand the range of surgical manipulations in endolaryngeal surgeries. The main operating modes of the carbon dioxide laser, applicable for different clinical situations, such as power, impulse, laser power density (PD) are considered. In addition, the combination of a micromanipulator with an operating microscope allows the use of additional techniques aimed at preventing restenosis, such as the use of microflaps, submucosal laryngeal cicatricial tissue vaporization. The possibilities of the recurrence of cicatricial stenosis by different variants of stenting are presented. The risk of complications of this technique is not higher than in other types of larynx laser surgeries, and with proper preventive measures can be minimized. In the review, possible complications, both early and late, measures of their prophylaxis, as well as ways to eliminate their consequences are analyzed in detail.

Micromanipulation of Chromosomes in Insect Spermatocytes.

The micromanipulation of chromosomes has been an essential method for illuminating the mechanism for chromosome congression, the spindle checkpoint, and anaphase chromosome movements, and has been key to understanding what controls chromosome movements during a cell division. A skilled biologist can use a micromanipulator to detach chromosomes from the spindle, to reposition chromosomes within the cell, and to apply forces to chromosomes using a small glass needle with a very fine tip. While perturbations can be made to chromosomes using other methods such as optical trapping and other uses of a laser, to date, no other method allows the repositioning of cellular components on the scale of tens to hundreds of microns with little to no damage to the cell. The selection and preparation of appropriate cells for the micromanipulation of chromosomes, specifically describing the preparation of grasshopper and cricket spermatocyte primary cultures for the use in live-cell imaging and micromanipulation, are described here. In addition, we show the construction of a needle to be used for moving chromosomes within the cell, and the use of a joystick-controlled piezoelectric micromanipulator with a glass needle attached to it to reposition chromosomes within dividing cells. A sample result shows the use of a micromanipulator to detach a chromosome from a spindle in a primary spermatocyte and to reposition that chromosome within the cell.

Use of the supported membrane tube assay system for real-time analysis of membrane fission reactions.

The process of membrane fission is fundamental to diverse cellular processes such as nutrient uptake, synaptic transmission and organelle biogenesis, and it involves the localized application of curvature stress to a tubular membrane intermediate, forcing it to undergo scission. Alternative techniques for creating such substrates necessitate the use of micromanipulators or sophisticated optical traps and require a high level of technical expertise. We present a facile method to generate an array of membrane tubes supported on a passivated glass coverslip, which we refer to as supported membrane tubes (SMrTs). SMrT templates are formed upon hydration of a dry lipid mix in physiological buffer and subsequent flow-induced extrusion of the lipid reservoir into long membrane tubes with variable dimensions. Following surface passivation of coverslips, these templates can be formed from a variety of lipids, with as little as 1-2 nmol of lipid in a matter of 2 h, and can be used in membrane-curvature-sensitive fission assays.

Endoscope Holders in Cranial Neurosurgery: Part I—Technology, Trends, and Implications

Cranial neuroendoscopy is becoming increasingly popular. Endoscopes may be used free-hand or with endoscope holders (EH). The aim of this paper is to summarize the evolution of EH and attempt an overview of currently available devices, features, and usage, identifying weaknesses and focusing on feasibility. A systematic review was conducted on Pubmed, OvidSP, Ebsco, and Google Scholar. Targeted search through cross-references was also implemented, followed by personal contacts, interviews, company websites, and operating manuals. A pilot survey was performed to acquire an impression of the current state of usage. This was conducted by approaching surgeons from several countries via e-mail and online through a neurosurgical exchange forum. A more extensive survey will follow as Part II. EH may be divided into manual and pneumatic. The use of micromanipulators is sometimes implemented. Different characteristics have been described, such as pneumatic, mechanical, friction, micromanipulator, robotic, hybrid, air-locking, and voice-control. Twenty eight surgeons responded. The majority (75%) do not routinely use EH. The rest use EH mainly for intraventricular rather than skull base procedures. Nearly all surgeons (96%) stated using a second surgeon regularly. Reported weaknesses included crude movements, downward drift, loss of depth perception, lack of flexibility, iatrogenic injury, cost, and bulky construction. Although EH have a role in neuroendoscopy, their use seems to be limited and their features regarded as suboptimal. There are no clear indications regarding the procedures that should be performed using EH. Many factors affect the implication of EH, including safety, type of surgery, surgeon's preference, and mechanical properties.

Yeast Replicator: A High-Throughput Multiplexed Microfluidics Platform for Automated Measurements of Single-Cell Aging.

The yeast Saccharomyces cerevisiae is a model organism for replicative aging studies; however, conventional lifespan measurement platforms have several limitations. Here, we present a microfluidics platform that facilitates simultaneous lifespan and gene expression measurements of aging yeast cells. Our multiplexed high-throughput platform offers the capability to perform independent lifespan experiments using different yeast strains or growth media. Using this platform in minimal media environments containing glucose, we measured the full lifespan of individual yeast cells in wild-type and canonical gene deletion backgrounds. Compared to glucose, in galactose we observed a 16.8% decrease in replicative lifespan accompanied by an ∼2-fold increase in single-cell oxidative stress levels reported by PSOD1-mCherry. Using PGAL1-YFP to measure the activity of the bistable galactose network, we saw that OFF and ON cells are similar in their lifespan. Our work shows that aging cells are committed to a single phenotypic state throughout their lifespan.

Early maternal serum ß-human chorionic gonadotropin (ß-hCG) levels and sex-related growth difference of IVF embryos.

Maternal serum ß-human chorionic gonadotropin (ß-hCG) represents the trophoblastic cell mass and is an indirect measurement of embryo development at early implantation stage. Studies in animals and human embryos detected sex-related growth differences (SRGD) in favour of male embryos during the pre-implantation period. The purpose of our study was to correlate SRGD and maternal serum ß-hCG at 16 days after embryo transfer. We retrospectively analysed all (fresh and frozen) non-donor, single embryo transfers (SET), elective and not elective, that were performed between December 2008 and December 2013. We included ß-hCG values from day 16 after oocyte collection of pregnancies resulting in live birth. Neonatal gender was retrieved from patient files. Male and female embryos were further grouped to cleavage and blastocyst stage transfers. Regression analysis for confounding variables included maternal age, maternal body mass index (BMI), use of micromanipulation (ICSI), embryo quality (grade), assisted hatching, day of transfer and fresh or frozen embryo transfer. Seven hundred eighty-six non-donor SETs resulted in live birth. After including only day 16 serum ß-hCG results, 525 SETs were analysed. Neonatal gender was available for 522 cases. Mean maternal serum ß-hCG levels were similar, 347 ± 191 IU/L in the male newborn group and 371 ± 200 IU/L in the female group. The difference between ß-hCG levels remained insignificant after adjusting for confounding variables. Early maternal ß-hCG levels after embryo transfers did not represent SRGD in our study.

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-κB Reporter Transgenic

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as pre-clinical models to test interventions. In these methods, we describe a technique in which lipopolysaccharide (LPS) is injected into the lactating mouse mammary gland via the nipple, effectively modeling mastitis, or inflammation, of the gland. This simulated infection results in increased nuclear factor kappa B (NF-κB) signaling, as visualized through bioluminescent imaging of an NF-κB luciferase reporter mouse. Our ultimate goal in developing these methods was to study the inflammation associated with mastitis in the lactating gland, which often includes redness, swelling, and immune cell infiltration. Therefore, we were keenly aware that incision or any type of wounding of the skin, the nipple, or the gland in order to introduce the LPS could not be utilized in our methods since the approach would likely confound the read-out of inflammation. We also desired a straight-forward method that did not require specially made hand-drawn pipettes or the use of micromanipulators to hold these specialized tools in place. Thus, we determined to use a commercially available insulin syringe and to inject the agent into the mammary duct of an intact nipple. This method was successful and allowed us to study the inflammation associated with LPS injection without any additional effects overlaid by the process of injection. In addition, this method also utilized an NF-κB luciferase reporter transgenic mouse and bioluminescent imaging technology to visually and quantitatively show increased NF-κB signaling within the LPS-injected gland. These methods are of interest to researchers of many disciplines who wish to model disease within the lactating mammary gland, as ultimately, the technique described here could be utilized for injection of a number of substances, and is not limited to only LPS.

Efficient Gene Delivery into Multiple CNS Territories Using <em>In Utero</em> Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos(1,2) that we have successfully adapted(3,4). It is largely based on the success of in ovo electroporation technologies that are commonly used in chick(5). Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described(6). Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells. To date, in utero electroporation has been most widely used for the analysis of neocortical development(1,2,3,4). More recent studies have targeted the embryonic retina(7,8,9) and thalamus(10,11,12). Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3(rd) ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

New cell separation technique for the isolation and analysis of cells from biological mixtures in forensic caseworks

AimTo isolate mucosal cells of the perpetrator in a sexual assault case from a complex mixture of his mucosal cells and the victim’s skin by micromanipulation prior to genomic analysis.MethodsTo capture and analyze mucosal cells we used the micromanipulation with on-chip low volume polymerase chain reaction (LV-PCR). Consensus DNA profiles were generated from 5 replicate experiments.Results and conclusionsWe validated the use of micromanipulation with on-chip LV-PCR for genomic analysis of complex biological mixtures in a fatal rape case. The perpetrator’s mucosal cells were captured from nipple swabs of the victim, and a single-source DNA profile was generated from cell mixtures. These data suggest that micromanipulation with on-chip LV-PCR is an effective forensic tool for the analysis of specific cells from complex samples.

Perinatal outcome of twice-frozen-thawed embryo transfers: a clinical follow-up study

We evaluated our clinical data on refrozen-thawed ETs (92 cycles) and found that human embryos were capable of withstanding two freeze-thaw cycles, resulting in normal live births after transfer at a rate similar to that of primary frozen-thawed embryos. This is the first follow-up study to present perinatal outcomes of children born after embryo re-cryopreservation, and our results should encourage clinicians to explore the possibility of performing the refreezing procedure.

Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production

Global petroleum reserves are shrinking at a fast pace, increasing the demand for alternate fuels. Microalgae have the ability to grow rapidly, and synthesize and accumulate large amounts (approximately 20-50% of dry weight) of neutral lipid stored in cytosolic lipid bodies. A successful and economically viable algae based biofuel industry mainly depends on the selection of appropriate algal strains. The main focus of bioprospecting for microalgae is to identify unique high lipid producing microalgae from different habitats. Indigenous species of microalgae with high lipid yields are especially valuable in the biofuel industry. Isolation, purification and identification of natural microalgal assemblages using conventional techniques is generally time consuming. However, the recent use of micromanipulation as a rapid isolating tool allows for a higher screening throughput. The appropriate media and growth conditions are also important for successful microalgal proliferation. Environmental parameters recorded at the sampling site are necessary to optimize in vitro growth. Identification of species generally requires a combination of morphological and genetic characterization. The selected microalgal strains are grown in upscale systems such as raceway ponds or photobireactors for biomass and lipid production. This paper reviews the recent methodologies adopted for site selection, sampling, strain selection and identification, optimization of cultural conditions for superior lipid yield for biofuel production. Energy generation routes of microalgal lipids and biomass are discussed in detail.

Light propagation in photonic clusters of spherical particles assembled with micromanipulation method

In self-assembled multilayer arrays of micrometer-sized spheres which include small amount of fluorescent particles, unique six-dot-triangular and seven-dot-hexagonal patterns have been known to appear in the fluorescence microscopic image. Although it has been suggested that these patterns correspond to local domain structures, i.e., face centered cubic (fcc) or hexagonal closed packing (hcp), no convincing evidence has been presented. In this study, we fabricated well-controlled photonic clusters by use of micromanipulation and investigated the relation between the propagation patterns and the domain structures. While the minimum fcc and hcp clusters exhibited the triangular and hexagonal patterns, respectively, it was also found that the propagation patterns are dependent on the size of the clusters.

Action potential recording from dielectrophoretically positioned neurons inside micro-wells of a planar microelectrode array

To organise in vitro neural networks at the cellular level and study their electrical patterns, we have fabricated 4 × 4 planar microelectrode arrays using conventional photolithography. The electrode sites of these arrays are located inside micro-wells, for confining the neurons, which are connected with neighbouring wells via micro-trenches capable of guiding the outgrowth of neurites. In order to load a single neuron inside each micro-well, a simple system has been developed that utilises the phenomenon of dielectrophoresis. It operates by moving neurons towards each electrode site of an array using a dielectrophoretic force, checking for the presence of a neuron inside each micro-well using image processing, and stopping the dielectrophoretic force when detecting a neuron inside a micro-well in order to prevent more cells from being trapped. This system provides a fast, effective and inexpensive way to assemble neural grids consisting of contacts between electrodes and single neurons, as the use of micromanipulator guided micropipettes can be avoided. Spontaneous and evoked action potentials from trapped neurons were successfully recorded using a 16-channel acquisition/stimulation unit.

Triggering and Visualizing the Aggregation and Fusion of Lipid Membranes in Microfluidic Chambers

We present a method that makes it possible to trigger, observe, and quantify membrane aggregation and fusion of giant liposomes in microfluidic chambers. Using electroformation from spin-coated films of lipids on transparent indium tin oxide electrodes, we formed two-dimensional networks of closely packed, surface-attached giant liposomes. We investigated the effects of fusogenic agents by simply flowing these molecules into the chambers and analyzing the resulting shape changes of more than 100 liposomes in parallel. We used this setup to quantify membrane fusion by several well-studied mechanisms, including fusion triggered by Ca 2+, polyethylene glycol, and biospecific tethering. Directly observing many liposomes simultaneously proved particularly useful for studying fusion events in the presence of low concentrations of fusogenic agents, when fusion was rare and probabilistic. We applied this microfluidic fusion assay to investigate a novel 30-mer peptide derived from a recently identified human receptor protein, B5, that is important for membrane fusion during the entry of herpes simplex virus into host cells. This peptide triggered fusion of liposomes at an ∼6 times higher probability than control peptides and caused irreversible interactions between adjacent membranes; it was, however, less fusogenic than Ca 2+ at comparable concentrations. Closely packed, surface-attached giant liposomes in microfluidic chambers offer a method to observe membrane aggregation and fusion in parallel without requiring the use of micromanipulators. This technique makes it possible to characterize rapidly novel fusogenic agents under well-defined conditions.

Use of the Quantitative Fluorescent-PCR Assay in the Study of Fetal DNA from Micromanipulated Transcervical Samples

The purpose of this study was to evaluate the validity of the combined use of micromanipulation and quantitative fluorescent (QF)-PCR assay for the identification of fetal elements in transcervical cell (TCC) samples collected in early pregnancy. TCC samples were obtained by intrauterine lavage (IUL) in 113 pregnant women who were between 7 and 12 weeks pregnant before termination of pregnancy. All IUL samples were screened under an inverted microscope, at which time the isolation of fetal cells by micromanipulation was attempted. QF-PCR assay, using 9 small tandem repeat (STR) markers for chromosomes 13, 18, 21, X, and Y, was performed in all specimens to identify fetal cells in TCC samples and the corresponding placental tissue and blood specimens. TCC samples from male fetuses in which either the micromanipulation or QF-PCR analysis were unsuccessful, were studied with fluorescent in situ hybridization (FISH), using probes for X and Y chromosomes. Isolation of supposed fetal material from IUL samples was carried out by means of micromanipulation in 93 cases (82.3%), where discernible chorionic villous filaments or cell clumps of probable trophoblastic origin were present. The QF-PCR analysis was performed in all 93 IUL samples and paternal peaks could be documented in 88 cases (94.6%) thus confirming the presence of fetal cells. Thirteen cases negative to micromanipulation and derived from male fetuses and four male cases not informative with QF-PCR analysis, after micromanipulation, were then tested with FISH assay using probes for sexual chromosomes. In six samples, rare (2-3%) male fetal cells were detected. Considering the combined results obtained from QF-PCR and FISH assays, the overall fetal sexing was correct in 83.2% of cases (94 of 113). This study provides evidence that fetal cells are present in a high proportion of IUL samples. Micromanipulation appears to be an extremely efficient method for the isolation of trophoblastic elements. This study also confirms the potential of IUL as a possible alternative to the traditional prenatal diagnostic procedures for the recovery of fetal cells in precocious stage of gestation, and validates the combination of the isolation of such fetal elements by means of micromanipulation and analysis with the QF-PCR assay for the identification of the most frequent prenatal chromosomal aneuploidies.

Micromanipulator: effectiveness in minimally invasive neurosurgery.

Minimally invasive surgeries by innovative approaches are practiced in all fields. The evolution of microneurosurgery has revolutionized the results in neurosurgery. Use of endoscopes and navigation has made microsurgery less invasive. Another development to make minimally invasive microneurosurgery further lesser invasive is the use of micromanipulator. The use and effectiveness of manually controlled micromanipulator system is presented. The manually controlled micromanipulator system consists of three parts, i.e., a basic micromanipulator, manipulator supporting device and the manual control. The micromanipulator fitted in supporting device is arranged before the start of surgery. The supporting device used is pneumatically driven powered endoscopic holding device (Mitaka Kohki Co., Tokyo) In maximum number of times we used the system for endoscopic assisted cerebrovascular microneurosurgery. In a span of two months we used it in thirty aneurysm clipping surgeries. The endoscope fitted in system has three ranges of motions (forward/backward, upside/down and sideways). We use MACHIDA rigid endoscope with internal diameter of 2.7 mm (smallest diameter endoscope available). Special features of this endoscope are accurate visualization at a deeper plane, stable movements and availability of single focus point for long time. All these features are valuable during pre- and postoperative clipping observation. The aim of development of micromanipulator system was to further reduce invasiveness. A significant improvement in manual dexterity is possible when working through the micromanipulator interface, which dampens human physiological tremor. The physiological tremor would render the manual dexterity unsafe at the end of lever arm of long instruments. Thus, the use endoscope becomes practical. The minimally invasive microneurosurgery can be further made lesser invasive by use of micromanipulator and we are convinced that this will facilitate more accurate and promising results in microneurosurgery.

Morphological identification markers for distinguishing avian from mammalian Giardia species — do they need reconsideration?

The intestinal, parasitic protozoa comprising the genus Giardia are major aetiological agents of diarrhoeal disease in numerous vertebrates, including humans, livestock and domestic pets. In previous investigations, morphological characteristics, in particular caudal flagellar length and asymmetry, have been used to distinguish avian from mammalian Giardia species, with scanning electron microscopy (SEM) often the technique of choice for observation. In this study, however, we observed considerable variation in caudal flagellar length and asymmetry, irrespective of avian or mammalian host origin. In addition, SEM sample preparation and the age of trophozoite cultures caused increases in the number of trophozoites with caudal flagellar length anomalies. Our findings, therefore, suggest that caudal flagellar characteristics are not reliable morphological markers for distinguishing Giardia species. In addition, the novel use of micromanipulation in establishing sub-clonal cultures of Giardia from single trophozoites was a highly accurate and successful microscopic technique.

Use of micromanipulation and "feeder layers" to clone the oyster pathogen Perkinsus marinus.

Genetic and biochemical characterization of microbes often requires the use of clonal cultures. A method to clone the oyster parasite Perkinsus marinus is described. Individual cells are isolated via micromanipulation and maintained above an actively proliferating "feeder layer" of P. marinus on a 0.45-microm membrane. Extracellular products released from the proliferating feeder layer can diffuse across the membrane and bathe the isolated cell, stimulating it to proliferate. The method is relatively simple and should be applicable to most protists that can be cultured in the laboratory.

An improved lamellae drop-off technique for sharp tip preparation in scanning tunneling microscopy

A superior lamellae etching drop-off technique is described for the preparation of W tips without the use of CCl4. Sharp tips with an apex of 25 nm or less can be produced routinely. In the case of Pt/Ir wire, the chemical inertness of the material means that it has to be reduced in diameter before being etched to yield a tip. In order to accomplish this, a capillary diameter reduction technique has also been developed for use in combination with the lamellae etching drop-off technique. The apparatus does not require the use of micromanipulators or micromovers. The shapes of tips with apexes as small as 25 nm can be investigated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). This low cost method of producing sharp tips scanning tunneling microscopy is very effective and it would probably be feasible to develop an automatic tip preparation with a little more technical effort. Since it is possible to produce well shaped W tips almost every time after acquiring a little skill, it is not usually necessary to check their shape by TEM or SEM before use. In contrast, the success rate with Pt/Ir tips is only up to 70%, so that it is advisable to check their shapes by TEM.