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At least 523 records · Page 29Linked to original sources
PubMed1981

Microdissection and cloning of DNA from a specific region of Drosophila melanogaster polytene chromosomes.

Fragments from section 3 of the salivary gland X chromosome of D. melanogaster were dissected with a micromanipulator. The DNA was extracted, cut and ligated to a lambda vector in a volume of a few nanoliters in an oil chamber monitored through a microscope. From about 10 pg of DNA we obtained 80 recombinant clones, a sample of which were analysed and shown to contain Drosophila DNA which hybridises in situ to the region of section 3 of the X chromosome. With this technique we can isolate clones from any desired region as small as 200 kb from the euchromatic arms of polytene chromosomes.

Bacteriophage lambda↗
PubMed1995

A low drift micropipette holder.

A novel micropipette holder has been designed to overcome the problem of drift encountered when experimenting on small preparations using patch-clamp techniques. In order to increase the stiffness and thermal stability of the system, the holder is designed such that the micropipette is clamped to a rigid quartz rod, and isolated from stresses arising in the connectors. The pressure line, made from stiff tubing, and electrical leads are firmly attached to the table and micromanipulator. This design was able to hold the micropipette tip stable to with 100 nm for more than 1 h, even with changes of 26.7 kPa (200 mmHg) in the pressure line.

Amplifiers, Electronic↗
PubMed1992

Subzonal sperm insertion and the frequency of gamete fusion.

The subzonal sperm insertion technique was applied to assess the potential of motile human spermatozoa to form pronuclei. In 184 mature human oocytes, subzonal sperm insertion was used as the primary mode of insemination in cases with abnormal semen analyses. Oocytes (n = 131) that failed to fertilize in vitro in cases with normal semen profiles were also micromanipulated for secondary insemination. The frequency of sperm fusion, expressed as a percentage, was defined as the total number of male pronuclei formed divided by the total number of spermatozoa inserted subzonally. Our results indicate that 37% of spermatozoa from men with normal semen are able to fuse with the oolemma and decondense within the ooplasm, when placed in the perivitelline space of the oocyte. Excluding the oocytes that appeared morphologically abnormal (presence of cytoplasmic inclusions such as refractile bodies within the ooplasm), the frequency of sperm fusion increased to nearly 60%. Moreover, 14% of subzonally inserted spermatozoa from men with abnormal semen analyses demonstrated an ability to form a pronucleus. The incidence of polyspermy was high, ranging from 30 to 80% in the different groups studied. It is therefore concluded that the human oolemma provides little protection against multiple sperm fusion and that the frequency of gamete fusion is unexpectedly high, even when the spermatozoa are derived from infertile men.

Evaluation Studies as Topic↗
PubMed1996

Combined use of fluorescence microscopy and micromechanical measurement to assess cell and membrane properties.

The combined use of fluorescence microscopy and micromanipulation provides a powerful approach for understanding the mechanochemistry of cell membranes. Fluorescent labeling of erythrocytes has been used to identify particular populations of cells to assess the effects of abnormal deformability on cell survival. It was found that cells deprived of surface area are either eliminated rapidly from the circulation or undergo a reduction in volume to improve cellular deformability. Fluorescence microscopy can also be used to assess the distribution of specific membrane components during mechanical deformation and fragmentation of cell membranes and so lead to more fundamental understanding of the physical association between the membrane bilayer and the underlying membrane cytoskeleton.

Animals↗
PubMed1975

A precision microdissection procedure for undecalcified bone thin sections.

A procedure is described for removing osteons from undecalcified, thin sections of bone. The method involves the use of a microdrilling machine in combination with a micromanipulator. The procedure allows a substantial reduction in the time required for microdissection over previously described methods while permitting the irregular contours of osteons to be followed with precision.

Bone and Bones↗
PubMed1993

Micromachining technology and biomedical engineering.

Medical science and clinical medicine include many microscopic environments. Recent micromachining techniques fit the microscopic environments and are applied to microsurgery, fiberscopic operation, micromanipulation, artificial organs, and drug delivery systems. Microactuators, microsensors, and micro mechanical parts will be prepared for such medical devices and techniques. Virtual reality, stereovision, and fiber imaging support handling of cells and small targets of living body. The paper reports some perspectives of microtechnologies in biomedical engineering.

Angioplasty, Laser↗
PubMed2006

Specific single-cell isolation and genomic amplification of uncultured microorganisms.

We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene sequence analysis and shotgun-cloned for additional genomic analysis. Sequence analysis showed >99% 16S rRNA gene homology to soil crenarchaeotal clone SCA1170 and shotgun fragments had the closest match to a crenarchaeotal BAC clone previously retrieved from a soil sample. The system was validated using Methanothermobacter thermoautotrophicus as single-cell test organism, and the validation setup produced 100% sequence homology to the ten tested regions of the genome of this organism.

Archaea↗
PubMed2005

Computation of adherent cell elasticity for critical cell-bead geometry in magnetic twisting experiments.

Quantification of the cell elastic modulus is a central issue of micromanipulation techniques used to analyze the mechanical properties of living adherent cells. In magnetic twisting cytometry (MTC), magnetic beads of radius R, linked to the cell cytoskeleton through transmembrane receptors, are twisted. The relationships between imposed external torque and measured resulting bead rotation or translation only provide values of the apparent cell stiffness. Thus, specific correcting coefficients have to be considered in order to derive the cell elastic modulus. This issue has been highlighted in previous studies, but general relationships forhandling such corrections are still lacking while they could help to understand and reduce the large dispersion of the reported values of cell elastic modulus. This work establishes generalized abacuses of the correcting coefficients from which the Young's modulus of a cell probed by MTC can be derived. Based on a 3D finite element analysis of an hyperelastic (neo-Hookean) cell, we show that the dimensionless ratio h(u)/2R, where h(u) is the cell height below the bead, is an essential parameter for quantification of the cell elasticity. This result could partly explain the still intriguing question of the large variation of measured elastic moduli with probe size.

Algorithms↗
PubMed2006

Dielectrophoresis tweezers for single cell manipulation.

Positioning single cells is of utmost importance in areas of biomedical research as diverse as in vitro fertilization, cell-cell interaction, cell adhesion, embryology, microbiology, stem cell research, and single cell transfection. Here we describe dielectrophoretic tweezers, a sharp glass tip with electrodes on either side, capable of trapping single cells with electric fields. Mounted on a micromanipulator, dielectrophoresis tweezers can position a single cell in three dimensions, holding the cell against fluid flow of hundreds of microns per second with more than 10 pN of force. We model the electric field produced by the tweezers and the field produced by coaxial microelectrodes. We show that cells are trapped without harm while they divide in the trap. In addition, dielectrophoretic tweezers offer the possibility for trapping, electroporating, and microinjecting a single cell with one probe.

Cell Culture Techniques↗
PubMed1975

Transvitreal chorioretinal biopsy in the rabbit.

Transvitreal chorioretinal biopsy was performed in nine Dutch rabbits by introducing a biopsy instrument through a small eye wall incision, passing the instrument across the eye through the vitreous to the biopsy site, and by trephinating and removing from the eye a 1.6-mm diameter specimen of the posterior eye wall. Biopsy was carried out under stereotaxic control by using a micromanipulator especially designed for ophthalmic surgery. Hemostasis was achieved by circumferential mechanical pressure around the biopsy site. The eye wall defect was sealed by a preplaced explant and repaired by fibrovascular ingrowth from the surrounding choroid and episclera. The normal histologic features of the biopsy specimen were preserved, except for separation of the neurosensory retina from the pigmented epithelium. Complications of the procedure included hemorrhage from the incision site, transvitreal strand formation, and lens abrasion. In no instance, however, did these complications result in significant opacification of the optical media. The biopsy technique has potential use as a clinical procedure in the diagnosis of choroidal tumors and other selected disorders of the choroid and retina and as a technique in investigational animal studies.

Animals↗
PubMed1984

A removable head-mounted microdrive for unit recording in the free-behaving rat.

The microdrive described in this paper has been used for two years for chronic unit recording in the olfactory bulbs of unrestrained rats. It is a removable micromanipulator, suitable for tungsten microelectrodes, designed for stereotaxic exploration of cylindrical areas of nervous tissue 9 mm high and 1.6 mm in dia. The electrode is moved up and down without any rotation and with minimal vibration, with a degree of precision of 5 microns.

Animals↗
PubMed1976

RNA synthesis in puff 2-48BC after experimental induction in Drosophila hydei.

Puffs induced by treatment with vitamin B6 at locus 2-48BC in salivary gland cells of Drosophila hydei were isolated by micromanipulation, and the puff RNA was analyzed electrophoretically. The main RNA species migrated with a mobility of 40S. A similar RNA species was found in this puff after its induction by temperature treatment, except that in this case material with a higher mobility was also found, presumably due to the presence of growing chains. No evidence was found for polyadenylation of the RNA contained within the puff. The RNA profile of nucleoplasm of cells from glands either treated with vitamin B6 or subjected to a temperature shock also displayed a prominent peak with a mobility of 40S which was absent in extracts of nucleoplasm from control glands.

Animals↗
PubMed2004

Chromosome condensation: DNA compaction in real time.

Mitotic chromosomes must be organised into a highly ordered and compacted form to allow proper segregation of DNA during each round of cell division. Two new studies report observations of DNA compaction by eukaryotic and bacterial condensin molecules in real time using magnetic and optical trapping micromanipulation techniques.

Adenosine Triphosphatases↗
PubMed2005

Use of a zona laser treatment system in assisted hatching: optimal laser utilization parameters.

OBJECTIVE: This study aimed to establish the benefit of laser micromanipulation of the zona pellucida as a method of assisted hatching and to determine the optimal technical settings. DESIGN: Prospective pilot study. SETTING: University teaching hospital. ANIMAL(S): Six- to 8-week-old F1 female mice. INTERVENTION(S): Mouse embryos were treated with a laser (Hamilton Thorne Research) using both research and preset clinical settings. The research option allows the user to alter pulse duration, number of pulses, and beam intensity. In this study, laser intensity and duration, as well as the depth and number of disruptions, were altered. MAIN OUTCOME MEASURE(S): Embryos were scored after treatment as either arrested blastocysts, arrested during hatching, hatched, or degenerate. RESULT(S): The treatment where the laser intensity was halved but the number of laser pulses used was doubled was the most beneficial treatment. Zona thinning was not effective. CONCLUSION(S): The study showed that laser-assisted hatching can safely and significantly increase the percentage of embryos reaching the hatched blastocyst stage. A proportionately sized hole in a human embryo may have the same beneficial effect as has been shown in this study.

Animals↗
PubMed2004

Nucleus transfer in mammals: noninvasive approaches for the preparation of cytoplasts.

The production of cloned animals is a difficult and complex procedure that requires two basic steps. First, the cytoplast must be prepared by the enucleation of metaphase II oocytes. Second, the nucleus is transferred either by fusion or by direct microinjection into the cytoplast. The preparation of cytoplasts is a crucial step because they must be able to reprogram the transferred nucleus and to secure the development of reconstructed embryos. Moreover, the classical mechanical enucleation of metaphase II oocytes is rather technically difficult, requiring good equipment and considerable micromanipulation skill. For this reason the simplification of this step is permanently in the centre of interest of those scientists who are involved in the production of clones.

Animals↗
PubMed2004

The role of Emx2 during scapula formation.

The scapula is subdivided into head, collum, and blade. Due to the expression pattern of Emx2 and the absence of the scapula blade in Emx2 knockout mice, it has been suggested that Emx2 is involved in the formation of the scapula. Micromanipulation experiments revealed that ectoderm ablation over the somites does not affect Emx2 expression but inhibits the formation of the scapula blade indicating that Emx2 is not sufficient to induce scapula blade formation. Furthermore, we show that the formation of the scapula head is dependent, scapula blade formation independent of FGFR-1-mediated signaling. Overexpression of Emx2 does not influence scapula blade formation but leads to the development of an additional posterior digit in the anterior border of the limb. Taken together, the data presented implicate that Emx2 expression is necessary but not sufficient for the development of the scapula blade. It is not a marker for scapula development but rather provides positional information along the proximodistal and anterior-posterior limb axes, whereas the specificity of the developing skeletal elements is determined by the concerted interaction of Emx2 with other factors.

Animals↗
PubMed2003

A three-dimensional viscoelastic model for cell deformation with experimental verification.

A three-dimensional viscoelastic finite element model is developed for cell micromanipulation by magnetocytometry. The model provides a robust tool for analysis of detailed strain/stress fields induced in the cell monolayer produced by forcing one microbead attached atop a single cell or cell monolayer on a basal substrate. Both the membrane/cortex and the cytoskeleton are modeled as Maxwell viscoelastic materials, but the structural effect of the membrane/cortex was found to be negligible on the timescales corresponding to magnetocytometry. Numerical predictions are validated against experiments performed on NIH 3T3 fibroblasts and previous experimental work. The system proved to be linear with respect to cytoskeleton mechanical properties and bead forcing. Stress and strain patterns were highly localized, suggesting that the effects of magnetocytometry are confined to a region extending <10 microm from the bead. Modulation of cell height has little effect on the results, provided the monolayer is >5 micro m thick. NIH 3T3 fibroblasts exhibited a viscoelastic timescale of approximately 1 s and a shear modulus of approximately 1000 Pa.

Animals↗
PubMed2003

Evidence that collagen fibrils in tendons are inhomogeneously structured in a tubelike manner.

The standard model for the structure of collagen in tendon is an ascending hierarchy of bundling. Collagen triple helices bundle into microfibrils, microfibrils bundle into subfibrils, and subfibrils bundle into fibrils, the basic structural unit of tendon. This model, developed primarily on the basis of x-ray diffraction results, is necessarily vague about the cross-sectional organization of fibrils and has led to the widespread assumption of laterally homogeneous closepacking. This assumption is inconsistent with data presented here. Using atomic force microscopy and micromanipulation, we observe how collagen fibrils from tendons behave mechanically as tubes. We conclude that the collagen fibril is an inhomogeneous structure composed of a relatively hard shell and a softer, less dense core.

Animals↗