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PubMed2006

Teaching insect retinal physiology with newly designed, inexpensive micromanipulators.

In this article, we detail how to produce two inexpensive micromanipulators that offer high precision (approximately 25 microm) along a single axis of movement. The more expensive of the designs provides improved versatility along multiple axes. Both manipulators offer substantial savings over commercially available micromanipulators with comparable capabilities. Plans and instructions are given such that a novice can produce the manipulators with simple tools. The manipulators are designed to serve undergraduate teaching exercises in physiology. An electroretinogram exercise is suggested in adult house flies (Musca) or flesh flies (Neobellieria). Measuring the intensity-response function and temporal characteristics of visual transduction are discussed. A brief introduction to the field of visual transduction and the physiology of the laboratory exercises is provided as well.

Animals↗
PubMed2007

Treatment of severe male infertility by micromanipulation-assisted fertilization: an update.

In the past 5-10 years the evolution of micromanipulation-assisted fertilization for the treatment of severe male infertility was marked by the introduction of new technical support, refinement of diagnostic methods for the evaluation of sperm developmental potential, and development of new treatment regimens for the newly discovered abnormalities. The new technical support involves the use of non-contact laser technology to assist micromanipulation for fertilization, the evolution of polarized microscopy-based optical systems to non-invasively detect the position of the meiotic spindle in living human oocytes, and the development of high-magnification optical systems for a better morphological selection of spermatozoa to be used for fertilization. Diagnostic approaches were enriched by commercial availability of kits for the analysis of sperm DNA integrity, leading to the definition of sperm nuclear DNA damage as a distinct cause of male infertility, and by the development of tests, based on heterologous ICSI, for detection of sperm failure to activate oocytes. Several treatment options for these conditions have been proposed and are currently being tested in larger-scale trials. Some technical improvement was also achieved in the field of in vitro maturation of germ cells from men with in vivo maturation arrest, but only a modest clinical improvement resulted from their application. As to the risk for the offspring, recent data are rather reassuring. Except for the risk of transmission of genetically based infertility, no straightforward evidence for a health risk derived from these techniques has been provided. Nevertheless, caution is necessary, particularly concerning the eventual increase in genomic-imprinting abnormalities.

DNA Damage↗
PubMed1998

Treatment of severe male infertility by micromanipulation-assisted fertilization: news and views.

Recent progress in cell micromanipulation has made it possible to achieve fertilization even with highly deficient male gametes, by injecting them directly to oocyte cytoplasm. This technique, known as intracytoplasmic sperm injection (ICSI), has proved efficient in most types of sperm pathology. However, ICSI fails when injected spermatozoa are not capable of supporting the process of oocyte activation or when they carry genetic abnormalities incompatible with normal embryonic development. Abnormalities of oocyte activation and the transmission of genetic anomalies to the progeny are also two major concerns relating to the clinical use of ICSI. The use of micromanipulation-assisted fertilization in cases of male germ cell maturation arrest, namely fertilization by round spermatid injection (ROSI), has brought preliminary clinical results, and the improvement of ROSI clinical efficacy remains a major challenge for future research.

Acrosome Reaction↗
PubMed1977

Combined micromanipulation, culture and immunofluorescent techniques for isolation of the coccal organisms comprising the "corn cob" configuration of human dental plaque.

The present study describes methods to 1) selectively isolate Corn Cob Configurations (CCC) from dental plaque by micromanipulation; 2) obtain pure cultures of the coccal constituent; 3) determine by immunofluorescent procedure which organisms originated from the CCC. Using a de Fonbrune micromanipulator, CCC specimens were isolated from supragingival plaque samples. The viability of one specimen thus obtained was established by observing growth on a slide culture. One set of CCC specimens was transferred to broth and incubated aerobically immediately upon collection. Another set was transferred to prereduced transport medium and later plated on blood agar for aerobic and anaerobic culturing. A total of 10 coccal strains were thus isolated. Antisera produced in rabbits against the 10 strains were used to localize these coccal organisms on plaque smears by using the indirect fluorescent antibody technique. Of the 10 antisera tested, 2 produced against streptococcal strains consistently gave a positive immunofluorescent reaction with the coccal component of CCC in the plaque smears; the corresponding streptococci were therefore considered to be CCC forming strains in vivo.

Bacteria↗
PubMed1975

The potentiality of antibody-producing cells. II. Evidence for two antibody molecules of different specificities secreted by micromanipulated bispecific mouse spleen cells.

Bispecific PFC appearing on the 4th day after immunization of mice with sheep erythrocytes conjugated with trinitrophenyl hapten (TNP-SRBC) were identified by their capacity to lyse native SRBC and TNP-conjugated horse erythrocytes (TNP--HoRBC) simultaneously in an open carboxymethylcellulose medium. Individual PFC thus detected, were micromanipulated into two successive media containing the indicators SRBC and TNP--HoRBC. Out of 103 transferred double cells ninety-two (89 per cent) remained double in the second medium and of the ninety-two transferred into the third medium, fifty-four (58 per cent) provoked a double lysis, eighteen (19 per cent) provoked a single lysis and twenty (23 per cent) ceased to lyse. On the other hand, when the second medium contained the indicators and the soluble specific inhibitor (TNP--BSA), out of 156 transferred double PFC, 125 (80 per cent) became single and only nineteen (12 per cent) remained double. Of these cells, 136 were transferred into the third medium (not containing inhibitor) and here again sixty-five (48 per cent) became double, forty-nine (36 per cent) remained single and twenty-two (17 per cent) ceased to provoke any lysis. Other double PFC were micromanipulated from the original revealing medium into two successive media containing only one indicator and the homologous (second medium) or the heterologous third medium) soluble inhibitor (TNP--BSA or soluble SRBC antigen), in order to see whether a soluble inhibitor suppresses only the corresponding specific lysis. Out of 186 double PFC transferred into the media containing the indicator and the corresponding inhibitor 147 (79 per cent) were specifically inhibited, whereas out of 176 double PFC transferred into the third medium (in which about 20 per cent of the cells cease to function) containing one indicator and the unrelated inhibitor, 117 (66-6 per cent) lysed the indicator in spite of the presence of the unrelated inhibitor. Since a specific inhibitor suppresses the lysis of the corresponding indicator, whereas its presence does not interfere with the lysis of the unrelated indicator in the majority of double PFC, it can be concluded that these cells secrete two types of antibody molecules possessing different specificities.

Animals↗
PubMed1993

[Micromanipulation of human gametes within the scope of assisted reproduction].

Poor function of spermatozoa accounts for half of all human infertility. Micromanipulation methods within the framework of in vitro fertilization treatment may enhance fertilization of the egg and may allow the establishment of an ongoing pregnancy in cases of severe male infertility. This review presents different methods of micromanipulation which are routinely applied in male factor infertility and for preimplantation diagnosis.

Embryo Implantation↗
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↗
PubMed2006

Telecontrolled micromanipulator system (NeuRobot) for minimally invasive neurosurgery.

BACKGROUND: To perform less invasive neurosurgery, a telecontrolled micromanipulator system has been developed and applied to clinical situations. Basic experiments for telesurgery have also been conducted. METHOD: A cadaver head was used to carry out surgical simulation of the opening of the sylvian fissure and third ventriculostomy. After obtaining permission from the Ethical Committee of Shinshu University School of Medicine, part of the recurrent meningioma in a 45-year-old man was removed. As basic experiment for telesurgery, surgical simulation was also conducted in a rat brain with the operating console transported to a hospital 40 km distant from the University. FINDINGS: Opening of the sylvian fissure and third ventriculostomy were accurately performed. Tumour removal in a patient with recurrent meningioma was safely achieved. Surgical simulation in the rat brain was accurately and correctly carried out, operated on from a hospital 40 km distant. CONCLUSIONS: The NeuRobot, telecontrolled micromanipulator system, can be used as a tool for less invasive neurosurgery.

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↗
PubMed1985

Use of a uterine-cell monolayer culture system for micromanipulated bovine embryos.

The objective of this study was to evaluate the growth of micromanipulated bovine embryos in two in vitro culture systems. Sixty ova (day 7 from estrus) were collected in Dulbecco's phosphate-buffered saline (PBS), with 2% fetal calf serum, and transferred to a PBS holding medium containing 10% fetal calf serum to prepare for micromanipulation. Forty embryos (morula to expanded blastocyst stages) were selected for embryo splitting using a modified microsurgery procedure. Thirty-nine of these embryos were successfully bisected into demi-embryos (DE) and the halves allotted by post-manipulation quality grades into one of two treatment groups (Trt). DE in Trt A were cultured in Ham's F-10 medium with 10% FCS (HF-10) while the remaining DE halves from each embryo were cocultured in HF-10 on a monolayer of endometrial fibroblasts (8 x 10(4) viable fibroblast cells plated three days prior to culture) in Trt B. Embryo development, recorded at 12-hour intervals, was evaluated by a split-plot analysis of variance. Results indicated that embryo viability decreased (P<0.001) over time in culture. Overall viability was greater (P<0.001) for DE in Trt B than in Trt A, with a significant (P<0.05) Trt x Time interaction, indicating that embryo viability decreased more rapidly across time in HF-10 than in the monolayer coculture system. The percentage of DE developing at 12, 24, 36, 48, 60 and 72 hours in culture was: 44%, 41%, 33%, 28%, 21% and 18% for Trt A and 69%, 69%, 69%, 67%, 62% and 62% for Trt B. Fourteen of the DE in Trt B attached to fibroblast monolayer and initiated trophoblastic outgrowth and four additional DE remained viable for up to 17.5 days in vitro as intact blastocysts. These findings are the first reported that demonstrate that the zona-free bovine DE will develop during in vitro culture. Also, the bovine endometrial fibroblast monolayer system proved to be excellent for both short term (</=12 hours) and long term (up to 72 hours) culture of halved bovine embryos.

Journal Article↗
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↗