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At least 73 records · Page 4Linked to original sources

PubMed1997

Molecular studies on single cells harvested by micromanipulation from archival tissue sections previously stained by immunohistochemistry or nonisotopic in situ hybridization.

Molecular analysis of isolated single cells is a powerful tool for analyzing heterogeneity within a population of cells and for clarifying issues of cell origin and clonality. Current techniques are limited by the availability of suitable fresh tissue. To broaden the applicability of molecular techniques at single-cell level, we have developed an approach that uses routinely processed archival tissue. Immunoglobulin heavy chain (IgH) gene rearrangement was analyzed in large tumor cells from four cases of diffuse large cell B-non-Hodgkin's lymphoma and in small reactive T and B lymphocytes from three cases of lymphocytic predominance Hodgkin's disease. One case of Epstein-Barr virus (EBV)-encoded RNA (EBER)-positive angiocentric pulmonary T-cell lymphoma was assayed for the presence of the BamHI-W multiple-copy fragment of the EBV genome. T- and B-lymphoid cells were immunostained with anti-CD3 and CD20, respectively. The tissue sections from the EBER-positive T-cell lymphoma were stained by nonisotopic in situ hybridization. Single cells were mobilized after proteolytic treatment under an inverted microscope using a hydraulic micromanipulator at a magnification of 400 x. Isolated cells were aspirated into a micropipette fixed to a second micromanipulator and transferred into a PCR tube. The IgH complementarity determining region (CDR)3 was successfully amplified in 17 of 52 (33%) small B-lymphocytes from lymphocytic predominance Hodgkin's disease using a previously reported semi-nested PCR method, and the products from each case differed in size as expected of a polyclonal population. None of the 49 small T lymphocytes demonstrated any amplifiable IgH CDR3 products, indicating no significant cellular contamination. The IgH CDR3 sequence analysis of the PCR products indicated a clonal relationship among harvested cells. In the T-cell lymphoma case, the harvested EBER-positive cells were amplifiable for the multiple-copy fragment BamHI-W of the EBV genome. Our study indicates that single-cell analysis can be performed on paraffin-embedded archival tissue after being subjected to immunoperoxidase and in situ hybridization procedures.

Antigens, CD20↗

PubMed1998

Micromanipulation of retinal neurons by optical tweezers.

Micromanipulation by optical tweezers has been tested in cultures of mature isolated retinal cells to determine its potential for use in creating synaptic circuits in vitro. Rod and cone photoreceptors as well as other retinal nerve cell types could be optically trapped with a 980 nm diode laser mounted on an inverted light microscope using a 40x oil immersion objective numerical aperture of 1.3. Manipulation was done under sterile conditions using transparent culture dishes. To form cell groups, one half of a culture dish was made less adhesive by application of a thin layer of silicone elastomer. Unattached cells were trapped and relocated next to cells lying on an adhesive culture substrate. Optical trapping did not affect the ability of neurons to subsequently attach to the culture substrate. Up to 60% of trapped cells survived for 2 or more days. The pattern and rate of process outgrowth for manipulated cells was comparable to unmanipulated cells and by 2 days, cell-cell contacts were observed. Cultures were fixed at 2 and 5 days for electron microscopy. Organelle, nuclear and cytoplasmic structure of manipulated cells was completely normal and in photoreceptors, synaptic vesicles and ribbons were intact. Optical tweezers, therefore, provide a benign technique with which to micromanipulate whole neurons. The procedures also bestow increased precision to the study of cell-cell interactions by allowing the selection of potentially interacting cell types at a single cell level.

Ambystoma↗

PubMed1988

New microspot micromanipulator for carbon dioxide laser surgery in otolaryngology. Early clinical results.

A new micromanipulator with microspot capability has been developed for carbon dioxide laser surgery in otolaryngology. The instrument features new infrared optics, which provide smaller laser spot sizes than those achievable with conventional micromanipulators (300 micron using a 400-mm operating microscope lens). The conventional red helium-neon aiming laser is replaced with a nonlaser fiberoptic image, and a power defocus control on the joystick manipulator eliminates external defocus controls. Less surrounding tissue trauma occurs with this new instrument by using a higher power density with average power settings of 1 to 2 W for cutting and ablation of tissue. Eight patients with benign laryngeal disease and one patient with dysplastic changes of the vocal cord were treated successfully without complications.

Animals↗

PubMed1992

In vitro decondensation of mammalian sperm and subsequent formation of pronuclei-like structures for micromanipulation.

In this study, we describe an efficient protocol for the formation of in vitro developed pronuclei for micromanipulation techniques. Our approach involved incubation of demembranated or permeabilized mammalian sperm in a phosphate buffer supplemented with heparin and beta-mercaptoethanol. Under the prevailing conditions, we achieved a uniform and reliable synchronous decondensation of sperm nuclear DNA. This initial decondensation facilitated the removal of mammalian protamines upon subsequent incubation in an amphibian egg extract. The interchange of protamines for histones to stabilize the DNA structure is recognized as a prerequisite for pronuclear formation. Furthermore, immunocytochemical studies have revealed that pronuclear development is accompanied by the formation of a nuclear lamina with corresponding DNA synthesis. The method described gave a high yield of nuclei during pronuclear formation. Ultimately, our aim is to transfer the in vitro-developed pronuclei into mammalian oocytes by micromanipulation. This novel procedure may prove useful in alleviating severe male factor problems especially in oligozoospermic cases in our in vitro fertilization center.

Animals↗

PubMed1998

[A micromanipulator for intraoperative vibratory hearing assessment with an implantable hearing aid transducer].

First concepts of implantable hearing aids to be coupled to the ossicular chain are available for patients with combined or sensorineural hearing loss (SNHL). To ensure that hearing can be improved intraoperative coupling of a test transducer to the ossicular chain is mandatory for allowing surgical anatomy to be checked and vibratory hearing tests to be performed. To achieve this, the test transducer has to be held and positioned securely in situ for some minutes, avoiding risks for middle or inner ear structures. This is not possible using conventional surgical instruments. Thus, a micromanipulator to hold the test transducer during intraoperative hearing tests was developed. This surgical device allows the surgeon safe, risk-free, and controlled coupling of the test transducer to the ossicular chain with one axial and three rotational degrees of freedom. With the aid of a conventional ear retractor (2x2 prongs), the manipulator is fixed at the patient's ear. In conjunction with a piezoelectric test transducer, the manipulator was used in nine patients during local anesthesia. The test transducer is part of an electronic hearing implant (Tübingen implant) specifically designed for SNHL that may be coupled to a middle ear ossicle or the perilymph of the cochlea. The micromanipulator was easy to handle. It allowed accurate positioning of the test transducer in the ear and the desired coupling of the transducer's probe tip to the ossicular chain during auditory tests. According to the principles of integrated safety, the intraoperative risk of ossicular or inner ear injuries caused, for instance, by the patient's head movement is minimized. The design of the manipulator system is universal, also allowing its use for other electronic hearing implants or minimal invasive surgery after minor modifications.

Ear Ossicles↗

PubMed1980

Mechanical stimulation and micromanipulation with piezoelectric bimorph elements.

Piezoelectric bimorph elements are versatile and inexpensive electromechanical transducers which may be used to construct fast mechanical stimulators and finely controlled micromanipulators. The mechanical stimulators described provide continuously graded displacements ranging from nanometers to about a millimeter, or forces equivalent to 0-7 g. Appropriately designed units can produce small step displacements complete within 100 microseconds. Micromanipulators are described which generate 3-dimensional motion under remote electrical control and which enable positioning within a few tenths of a micrometer. They are sufficiently stable to hold glass microelectrodes for cell penetration or probes for microdissection. The two significant drawbacks of bimorph elements are mechanical resonance and continued movement following displacement, or 'creep", but methods have been developed to compensate for these. A number of methods are available to measure motion of bimorphs with spatial resolution of 10 nm and temporal resolution of 2 microseconds in favorable situations.

Animals↗

PubMed2002

Magnetic tweezers: micromanipulation and force measurement at the molecular level.

Cantilevers and optical tweezers are widely used for micromanipulating cells or biomolecules for measuring their mechanical properties. However, they do not allow easy rotary motion and can sometimes damage the handled material. We present here a system of magnetic tweezers that overcomes those drawbacks while retaining most of the previous dynamometers properties. Electromagnets are coupled to a microscope-based particle tracking system through a digital feedback loop. Magnetic beads are first trapped in a potential well of stiffness approximately 10(-7) N/m. Thus, they can be manipulated in three dimensions at a speed of approximately 10 microm/s and rotated along the optical axis at a frequency of 10 Hz. In addition, our apparatus can work as a dynamometer relying on either usual calibration against the viscous drag or complete calibration using Brownian fluctuations. By stretching a DNA molecule between a magnetic particle and a glass surface, we applied and measured vertical forces ranging from 50 fN to 20 pN. Similarly, nearly horizontal forces up to 5 pN were obtained. From those experiments, we conclude that magnetic tweezers represent a low-cost and biocompatible setup that could become a suitable alternative to the other available micromanipulators.

Biophysical Phenomena↗

PubMed1992

Controlled micromanipulation of human sperm in three dimensions with an infrared laser optical trap: effect on sperm velocity.

Individual human sperm can be micromanipulated in three dimensions using a 1.06 microns Nd:YAG laser trap. Single sperm swimming with velocities in the range of 65 to 85 microns/sec can be trapped with 40 mW of power through 120 seconds without a deleterious effect on velocity. Even though it will be necessary to further evaluate the effects of laser light on specific functions of sperm, our data suggest that decreasing the time of manipulation to a minimum will increase the safety of the micromanipulation procedures. Laser traps may play a role in assisted reproductive technology by facilitating the selective transport of individual sperm.

Analysis of Variance↗

PubMed1989

Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration.

Partial zona dissection (PZD), a method using mechanical force to open the human zona pellucida, and zona drilling, which uses acidic Tyrode's (AT) medium, were compared in 1-day-old oocytes prior to reinsemination. The incidences of monospermy and polyspermy were 13/54 (24%) and 14/54 (26%) following PZD and 6/46 (13%) and 8/46 (17%) following the use of AT medium. This compared favorably with conventional reinsemination: 15/161 (9%) monospermy and 4/161 (3%) polyspermy. Three of the 27 PZD embryos became blastocysts, while none of the AT-exposed embryos developed satisfactorily. Eleven male-factor couples had some of their oocytes randomly treated with PZD prior to insemination; each of the patients had non-micromanipulated control oocytes. Monospermic fertilization and cleavage (23/34; 68%) doubled (P less than 0.05) when PZD was compared with the control oocytes (10/30; 33%). Replacing two PZD and a single control embryo in two patients resulted in twin pregnancies. A third twin pregnancy was established following replacement of only micromanipulated embryos.

Cell Division↗

PubMed1986

Micromanipulated bivalents can trigger mini-spindle formation in Drosophila melanogaster spermatocyte cytoplasm.

Single (individual) bivalents in cultured Drosophila melanogaster primary spermatocytes were detached from the spindle with a micromanipulation needle and placed in the cytoplasm. Such bivalents are prevented from rejoining the spindle by a natural membrane barrier that surrounds the spindle, but they quickly orient as if on a spindle of their own and the half-bivalents separate in anaphase. Serial section electron microscopy shows that a mini-spindle forms around the cytoplasmic bivalent, i.e., the microtubule density in the vicinity of the bivalent is much greater than in other cytoplasmic regions. This microtubule population cannot be accounted for solely by kinetochore nucleation and/or capture of microtubules. Furthermore, the mini-spindles frequently form at odd angles to the main spindle, so that at least one pole has no relationship to the poles of the main spindle. We conclude that a bivalent, or factors that become associated with the bivalent as a result of the manipulation, can either stabilize microtubules or promote their assembly. The bivalent activates latent microtubule organizing centers, or alternatively, polar organizing material has been passively transported from the main spindle to the cytoplasm by the micromanipulation procedure.

Animals↗

PubMed1982

Spindle microtubules and their mechanical associations after micromanipulation in anaphase.

Micromanipulation of living grasshopper spermatocytes in anaphase has been combined with electron microscopy to reveal otherwise obscure features of spindle organization. A chromosome is pushed laterally outside the spindle and stretched, and the cell is fixed with a novel, agar-treated glutaraldehyde solution. Two- and three-dimensional reconstructions from serial sections of seven cells show that kinetochore microtubules of the manipulated chromosome are shifted outside the confusing thicket of spindle microtubules and mechanical associations among microtubules are revealed by bent or shifted microtubules. These are the chief results: (a) The disposition of microtubules invariably is consistent with a skeletal role for spindle microtubules. (b) The kinetochore microtubule bundle is composed of short and long microtubules, with weak but recognizable mechanical associations among them. Some kinetochore microtubules are more tightly linked to one other microtubule within the bundle. (c) Microtubules of the kinetochore microtubule bundle are firmly connected to other spindle microtubules only near the pole, although some nonkinetochore microtubules of uncertain significance enter the bundle nearer to the kinetochore. (d) The kinetochore microtubules of adjacent chromosomes are mechanically linked, which provides an explanation for interdependent chromosome movement in "hinge anaphases." In the region of the spindle open to analysis after chromosome micromanipulation, microtubules may be linked mechanically by embedment in a gel, rather than by dynein or other specific, cross-bridging molecules.

Anaphase↗

PubMed2001

Monozygotic twinning after assisted reproductive techniques: a phenomenon independent of micromanipulation.

A 3 year retrospective analysis was conducted of pregnancies achieved after various assisted reproductive treatment modalities in our infertility practice, to calculate and compare the rates of monozygotic twinning (MZT). A total of 731 pregnancies achieved after various assisted reproduction treatments were reviewed. Gonadotrophin therapy for induction of ovulation and controlled ovarian hyperstimulation (COH) yielded 129 clinical pregnancies. Conventional IVF yielded 139 pregnancies. IVF and intracytoplasmic sperm injection (ICSI) with or without assisted hatching (AH) yielded 463 pregnancies, all during the same time period. The rates of multiple pregnancy (monozygotic and dizygotic) twins and triplets were recorded. MZT was found in 1.5% of ovulation induction or COH pregnancies (2/129). The incidence of MZT after conventional IVF was 0.72% (1/139). After IVF-ICSI/AH, MZT was found in 0.86% (4/463). The overall rate of MZT was 0.95% (7/731). Five cases were dizygotic triplets and two cases were monozygotic twins. We found the rate of MZT after assisted reproduction treatment increased more than two-fold over the background rate in the general population. Dizygotic triplets were found more often than monozygotic twins. The rate of MZT was consistently increased, irrespective of treatment modality or micromanipulation. This may signify that the aetiology of increased MZT after assisted reproduction is the gonadotrophin treatment rather than in-vitro conditions, micromanipulation, or multiple embryo transfer.

Adult↗

PubMed1963

TRANSFORMATION OF BACILLUS SUBTILIS TO MOTILITY AND PROTOTROPHY: MICROMANIPULATIVE ISOLATION OF BACTERIA OF TRANSFORMED PHENOTYPE.

Stocker, B. A. D. (Stanford Medical Center, Palo Alto, Calif.). Transformation of Bacillus subtilis to motility and prototrophy: micromanipulative isolation of bacteria of transformed phenotype. J. Bacteriol. 86:797-804. 1963.-A nonmotile (nonflagellated, fla(-)) try(-) strain of Bacillus subtilis was transformed to fla(+) and to try(+) by wild - type deoxyribonucleic acid (DNA) at comparable rates. Bacteria of fla(+) phenotype were recognized by their motility approximately 3 hr after uptake of DNA, and bacteria of try(+) phenotype at about the same time by their elongation into filaments in a medium lacking tryptophan. Of phenotypically transformed bacteria of each sort isolated by micromanipulation, the majority produced only transformed progeny, a mixture of transformed and untransformed, or a mixture of two kinds of transformant. Some produced only untransformed progeny, or progeny transformed only at a locus linked to that concerned in their phenotypic transformation. In a few clones, some partial heterozygotes were present even ten generations after DNA uptake. In nonmotile clones derived from motile isolates, the unilinear transmission of motility to one to four descendants was detected; it is attributed to persistence of a corresponding number of units of some product of an unincorporated fla(+) gene, probably flagella or cell walls each carrying several flagella. No pedigrees indicating unilinear transmission of an unincorporated fla(+) gene were observed.

Bacillus subtilis↗

PubMedDate not supplied

The impact of the measuring distance on laser-Doppler measurements of the microcirculation in human nasal mucosa. A study of rhinostereometry and micromanipulator-guided laser-Doppler flowmetry.

Fourteen healthy volunteers were examined using rhinostereometry (RSM) and micromanipulator-guided laser-Doppler flowmetry (LDF) on the inferior turbinate and nasal septum. There were significant differences of the flowmetry readings at different distances from the mucosal surface within 0-1.0 mm. Changes in LDF values due to a change in distance between probe and mucosa seem to be an important artefact that will influence the flowmetry, especially if the probe is fixed to the framework of the nose (i.e. the skull) and if the congestive status of the mucosa changes. This can be a problem especially during intranasal challenges with substances that influence congestion. These artefacts can be eliminated by using an RSM-guided micromanipulator that allows the probe to follow changes in congestion.

Adult↗

PubMedDate not supplied

Studies of spontaneous fluctuations in congestion and nasal mucosal microcirculation and the effects of oxymetazoline using rhinostereometry and micromanipulator guided laser Doppler flowmetry.

The mucosa of the inferior turbinate was studied using rhinostereometry and micromanipulator-guided laser Doppler flowmetry in 10 healthy volunteers. First, spontaneous fluctuations were studied measuring congestion and multiple microcirculatory parameters simultaneously every 2 minutes. The subjects were then challenged with oxymetazoline using the same measuring technique studying the effects of the challenge during 12 minutes. There were spontaneous variations in congestion of up to 2.1 mm and variations in perfusion from 38% to 175% of average. There was no correlation between congestion in itself, or change in congestion, to perfusion or any other microcirculatory parameter. After challenge with oxymetazoline there was a rapid decrease in perfusion at 3 minutes after which there were no significant changes. The congestion decreased gradually throughout the procedure. Because congestion reflects the filling of the venous sinusoids and the flowmetry the state of the superficial vessels, we conclude that there are spontaneous short-term fluctuations in the sympathetic tone with independent actions on the different vessels. After challenge with a sympathomimetic drug, there was a decrease in both swelling and flow, but not synchronized. The combination of rhinostereometry and micromanipulator-guided laser Doppler flowmetry is a useful tool to study the dynamics of intranasal challenge reactions.

Depression, Chemical↗

PubMed1991

Micromanipulation in a center for reproductive medicine.

Several variations of micromanipulation of the female gamete (zona drilling, zona cracking, ooplasmic sperm injection, partial zona dissection) have been applied recently to human IVF to overcome severe male factor. Of the first 16 cycles attempted using partial zona dissection, one pregnancy resulted leading to a normal term delivery. Careful removal of the coronal cells, as well as stepwise removal of sucrose postpartial zona dissection, will facilitate this procedure and greatly reduce potential damage to the oocyte by pH, mechanical, or thermal injury. Micromanipulation has become a routine service offered in our program in cases where the likelihood of a poor IVF outcome is either known or suspected, and also serves as a replacement for simple reinsemination in cases of failed fertilization.

Adult↗

PubMed1988

[A floating hydraulic micromanipulator for microelectrode research on brain neurons].

The article describes a simple floating hydraulic micromanipulator of original construction which works in positive-positive pressure and provides maximum stability of the microelectrode tip concerning the neuron in the moving brain. This work is achieved by fixing of the micromanipulator on the hinged suspension bracket and by setting of it on the surface of the object with the help of bearing site. Breathing and vascular brain moving don't influence the neuron activity lead.

Animals↗

PubMed1984

[Compact micromanipulator for the chronic extracellular recording of neuronal activity in restrained and freely moving animals].

A design of a compact micromanipulator giving possibility to introduce the microelectrode into unlimited depth of the brain and to realize the penetration of it into the narrow area of trepanized cranial of the animal several times has been worked out and described. Small size and mass of the design allow to set up several micromanipulators on the cranial of different experimental animals and to record the neuronal activity of the different brain structures simultaneously.

Animals↗