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PubMed1994

Measurements of growth cone adhesion to culture surfaces by micromanipulation.

Neurons were grown on plastic surfaces that were untreated, or treated with polylysine, laminin, or L1 and their growth cones were detached from their culture surface by applying known forces with calibrated glass needles. This detachment force was taken as a measure of the force of adhesion of the growth cone. We find that on all surfaces, lamellipodial growth cones require significantly greater detachment force than filopodial growth cones, but this differences is, in general, due to the greater area of lamellipodial growth cones compared to filopodial growth cones. That is, the stress (force/unit area) required for detachment was similar for growth cones of lamellipodial and filopodial morphology on all surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces, which had a significantly lower stress of detachment than on other surfaces. Surprisingly, the forces required for detachment (760-3,340 mudynes) were three to 15 times greater than the typical resting axonal tension, the force exerted by advancing growth cones, or the forces of retraction previously measured by essentially the same method. Nor did we observe significant differences in detachment force among growth cones of similar morphology on different culture surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces. These data argue against the differential adhesion mechanism for growth cone guidance preferences in culture. Our micromanipulations revealed that the most mechanically resistant regions of growth cone attachment were confined to quite small regions typically located at the ends of filopodia and lamellipodia. Detached growth cones remained connected to the substratum at these regions by highly elastic retraction fibers. The closeness of contact of growth cones to the substratum as revealed by interference reflection microscopy (IRM) did not correlate with our mechanical measurements of adhesion, suggesting that IRM cannot be used as a reliable estimator of growth cone adhesion.

Animals↗

PubMed1969

Chromosome micromanipulation. 3. Spindle fiber tension and the reorientation of mal-oriented chromosomes.

Kinetochore reorientation is the critical process ensuring normal chromosome distribution. Reorientation has been studied in living grasshopper spermatocytes, in which bivalents with both chromosomes oriented to the same pole (unipolar orientation) occur but are unstable: sooner or later one chromosome reorients, the stable, bipolar orientation results, and normal anaphase segregation to opposite poles follows. One possible source of stability in bipolar orientations is the normal spindle forces toward opposite poles, which slightly stretch the bivalent. This tension is lacking in unipolar orientations because all the chromosomal spindle fibers and spindle forces are directed toward one pole. The possible role of tension has been tested directly by micromanipulation of bivalents in unipolar orientation to artificially create the missing tension. Without exception, such bivalents never reorient before the tension is released; a total time "under tension" of over 5 hr has been accumulated in experiments on eight bivalents in eight cells. In control experiments these same bivalents reoriented from a unipolar orientation within 16 min, on the average, in the absence of tension. Controlled reorientation and chromosome segregation can be explained from the results of these and related experiments.

Animals↗

PubMed1999

The usefulness of a piezo-micromanipulator in intracytoplasmic sperm injection in humans.

Intracytoplasmic sperm injection (ICSI) has wide clinical application. In order to achieve good results with this method, it is important to restrict the possibility of oocyte injury as much as possible, and securely inject spermatozoa into the ooplasm. For this purpose, we clinically applied piezo-ICSI, which employs a micromanipulator with piezoelectric elements, to humans, and compared the results with those obtained by conventional ICSI. Conventional ICSI and piezo-ICSI were used in 279 cycles and 335 cycles respectively. Piezo-ICSI showed significantly more favourable results, with a survival rate of 88.1% (conventional ICSI: 81.4, P < 0.001), a fertilization rate of 79.4% (conventional ICSI: 66.4%, P < 0.001), and a pregnancy rate of 23.1% (conventional ICSI: 14.9%, P < 0.05). In piezo-ICSI, the needle used is not sharpened and has a flat tip. However, deformation of the oocyte during insertion of the needle is restrained by vibration of the piezo, and the oolemma is punctured readily and securely by the piezo pulse, at the site where the spermatozoon is injected. Piezo-ICSI is a promising new technique for human ICSI that should improve the survival, fertilization and pregnancy rates after ICSI.

Adult↗

PubMed2000

Human zona pellucida micromanipulation and monozygotic twinning frequency after IVF.

To assess the association of zona pellucida micromanipulation and subsequent development of monozygotic twins, cases of assisted embryo hatching (AH) and intracytoplasmic sperm injection (ICSI) were identified and related to treatment type, implantation and zygosity data. Embryology records from all patients undergoing in-vitro fertilization (IVF) at this centre from January 1995 to March 1998 were reviewed. In this study, 3546 transfer cycles were completed, with clinical pregnancy established in 1911 (54% per transfer) patients undergoing a single IVF cycle. These pregnancies occurred in 1674 (88%) IVF cycles, 120 (6%) donor oocyte cycles (DER), and 117 (6%) frozen embryo transfer (FET) cycles. During the study period, 23 cases of monozygotic (MZ) twins were identified, representing an overall frequency of 1.2%. Chorionicity was determined by transvaginal ultrasound at 7 weeks when the number of embryos transferred was less than the number of fetal heart-beats, or when >1 fetal heartbeat per gestational sac was seen. Zygosity was confirmed by placental evaluation at delivery, and corroborated the antenatal diagnosis in all cases. Among IVF study patients the frequency of MZ twinning was not statistically different between zona manipulated and zona intact subgroups. While this investigation is the largest to date describing the relationship between MZ twins and zona procedures, studies with even greater statistical power are needed to clarify it more precisely, particularly in DER and FET settings. A greater overall frequency of MZ twinning for IVF patients may be a function of the higher number of embryos transferred in IVF, rather than discrete zona manipulations.

Embryo Transfer↗

PubMed1995

Micromanipulation of mouse gametes with laser microbeam and optical tweezers.

Micromanipulation of mouse gametes with a commercially available compact laser microbeam system was studied. Both the normal in-vitro fertilization (IVF) group and the laser zona dissection (LZD) group were tested under normal (2 x 10(6) motile spermatozoa/ml) and low (500,000 motile spermatozoa/ml) insemination conditions. Subzonal insemination (SUZI) was also tried in a small group of gametes and the results were compared with those of the low insemination groups. Fertilization rates and blastocyst formation rates for the IVF and the LZD-treated groups were respectively 53 and 60% and 60 and 78%, which were not significantly different. However, under low insemination conditions, the results were significantly better in the LZD-treated group (58% fertilization rate and 83% blastocyst formation rate) compared to the results of the IVF group (33 and 48%) (P < 0.05). The SUZI-treated group showed the lowest fertilization rate (18%). No significant difference between the LZD and the IVF group was observed with respect to parthenogenetic activation. LZD has a beneficial effect on fertilization rates in cases of reduced sperm quality.

Animals↗

PubMed1989

Biopsy of preimplantation mouse embryos: development of micromanipulated embryos and proliferation of single blastomeres in vitro.

We have developed a technique to sample the preimplantation embryo, which may, in the future, be applied to prenatal diagnosis of genetic disease. Using micromanipulation, we aspirated a single blastomere from 4-cell mouse embryos. This procedure had no effect on in vitro development; 98% of control and 94% of biopsied embryos reached the blastocyst stage after 48 h in culture. Furthermore, after transfer to pseudopregnant recipient mice, the rate of fetal development of biopsied embryos was not significantly different from control embryos, although implantation rate was significantly reduced (mean +/- SD: biopsied 53.1 +/- 4.0, control 81.8 +/- 8.4, p less than 0.001). For the first time we have produced monolayer cell cultures derived from single preimplantation blastomeres. Individual biopsied blastomeres were cultured in vitro on different extracellular matrix components. Significantly greater cell proliferation was obtained in wells coated with fibronectin (FN), laminin (LN), and a complex of laminin and nidogen (LNC) than in a less specific matrix of swine skin gelatin (SSG). Mean (+/- SE) cell nuclei number per well after 6 days in culture was 6.4 +/- 2.1, 11.9 +/- 1.5, 19.8 +/- 2.9, and 20.9 +/- 2.6 in wells coated with SSG, LN, FN, and LNC respectively.

Animals↗

PubMed2002

Effect of partial incision of the zona pellucida by piezo-micromanipulator for in vitro fertilization using frozen-thawed mouse spermatozoa on the developmental rate of embryos transferred at the 2-cell stage.

Cryopreservation of mouse spermatozoa is widely used, although considerable strain differences in fertilization rates using frozen-thawed mouse spermatozoa have been described. The C57BL/6 mouse strain is a very widely used for establishment of transgenic mice, but the fertilization rate associated with the use of cryopreserved C57BL/6 spermatozoa is very low compared with rates for other inbred strains. We have recently solved this difficulty by in vitro fertilization (IVF) in combination with partial zona pellucida dissection (PZD). However, this technique requires culture of fertilized eggs with PZD in vitro up to morula or blastocyst stage before transfer into the uterus because blastomeres are lost after transfer into the oviduct because of the relatively large artificial slit in the zona pellucida. To overcome this problem, we performed a partial zona pellucida incision by using a piezo-micromanipulator (ZIP) for IVF with frozen-thawed mouse spermatozoa. The blunt end of the micropipette touched the surface of the zona pellucida of the oocytes, and piezo pulses were used to incise the zona pellucida while the pipette was moved along by the surface of zona pellucida. The length of the incision was pir/6 microm. When cumulus-free ZIP and PZD oocytes were inseminated with frozen-thawed genetically modified C57BL/6J spermatozoa, the fertilization rates of ZIP and PZD oocytes were 52% and 48%, respectively. After embryo transfer at the 2-cell stage, 18% and 2% of the transferred embryos with ZIP and PZD developed to term, respectively. This difference was significant (P < 0.05). When ZIP and PZD zygotes were cultured to blastocyst stage and subsequently transferred to uterine horns of recipient animals, the difference between ZIP and PZD zygotes for development rate to full term was not significant. Our results indicate that ZIP is an effective alternative technique for IVF using cryopreserved mouse spermatozoa and subsequent embryo transfer.

Animals↗

PubMed1995

Enhancement of fertilization by micromanipulation.

Micromanipulation techniques have been successfully applied to human gametes to assist fertilization when normal function is impaired. The past few years have witnessed the rapid evolution and clinical incorporation of this technology. The unprecedented efficiency of intracytoplasmic sperm injection far surpasses all previous methods. This method has thus become the best treatment option for many forms of male infertility.

Adult↗

PubMed1997

Technical accuracy of a neuronavigation system measured with a high-precision mechanical micromanipulator.

OBJECTIVE: This study was designed to determine and evaluate the different system-inherent sources of erroneous target localization of a light-emitting diode (LED)-based neuronavigation system (StealthStation, Stealth Technologies, Boulder, CO). METHODS: The localization accuracy was estimated by applying a high-precision mechanical micromanipulator to move and exactly locate (+/- 0.1 micron) the pointer at multiple positions in the physical three-dimensional space. The localization error was evaluated by calculating the spatial distance between the (known) LED positions and the LED coordinates measured by the neuronavigator. The results are based on a study of approximately 280,000 independent coordinate measurements. RESULTS: The maximum localization error detected was 0.55 +/- 0.29 mm, with the z direction (distance to the camera array) being the most erroneous coordinate. Minimum localization error was found at a distance of 1400 mm from the central camera (optimal measurement position). Additional error due to 1) mechanical vibrations of the camera tripod (+/- 0.15 mm) and the reference frame (+/- 0.08 mm) and 2) extrapolation of the pointer tip position from the LED coordinates of at least +/- 0.12 mm were detected, leading to a total technical error of 0.55 +/- 0.64 mm. CONCLUSIONS: Based on this technical accuracy analysis, a set of handling recommendations is proposed, leading to an improved localization accuracy. The localization error could be reduced by 0.3 +/- 0.15 mm by correct camera positioning (1400 mm distance) plus 0.15 mm by vibration-eliminating fixation of the camera. Correct handling of the probe during the operation may improve the accuracy by up to 0.1 mm.

Evaluation Studies as Topic↗

PubMed2002

Role of laser-induced plasma formation in pulsed cellular microsurgery and micromanipulation.

We investigate experimentally the physical processes underlying pulsed cellular microsurgery and micromanipulation using nanosecond 532- and 1064-nm laser pulses focused at high numerical aperture. We find that the laser parameters employed for many microirradiation techniques are congruent with those leading to optical breakdown in water. We determine the size and shape of the laser-induced plasma, pressure of the emitted shock wave, and size and energy of the cavitation bubble formed by the expanding plasma. We discuss implications of the results for biophysical microirradiation procedures.

Cells↗

PubMed2005

Micromanipulation of single cells from tissue imprints is an alternative to laser-assisted microdissection.

The characterization and analysis of single cells by molecular biological methods such as the polymerase chain reaction (PCR) is of increasing interest in biomedical research. Different techniques have been developed to obtain single cells from solid tissue. Currently, the most frequently used technique is laser-assisted microdissection (LAM). However, LAM of tissues cannot exclude contamination of the targeted cells by underlying cell fragments. Moreover, this technique can only be performed if a laser microscope is available. Thus, we developed a method to obtain single cells of fresh solid tissue by the simple technique of tissue imprints. After immunostaining of the imprints, single cells were transferred to a reaction tube using a 27-gauge needle guided by a mechanical micromanipulator. Consequently, we used these cells in a single cell PCR.

Base Sequence↗

PubMed2006

Vitreous cryo-sectioning of cells facilitated by a micromanipulator.

Sectioning vitrified cells and tissues for cryo-electron microscopy is more challenging than room-temperature sectioning of plastic-embedded samples. As the sample must be kept very cold (<-130 degrees C) and because there is no liquid upon which the sections can float as they are cut, transferring the sections from the knife edge to a grid is one of the more difficult steps in the process. We employed a micromanipulator to hold and control the cryo-sections as they come off the knife. This allows slower cutting speeds than are typically used in vitreous cryo-sectioning and contributes to better control during cutting, which facilitates repeatable placement of a ribbon of sections onto a grid. The ribbon is kept under tension during the entire cutting process, which may decrease folding and/or compression, features that are inherent to vitreous sections. Furthermore, the added control afforded by this technique makes it easier for multiple ribbons to be placed on a single grid, thereby increasing the number of sections that can be examined and imaged during a microscopy session. It even allows for serial cryo-electron microscopy. As such, this approach is an advance in the cryo-microtomy of vitreous sections.

Cryoelectron Microscopy↗

PubMed1961

Micromanipulation in control and handling of Zygiella x-notata as an experimental animal.

The spider Zygiella x-notata may be brought under direct control analagous to that of the common laboratory animals for an important group of experimental investigations on the nerve, muscle, or secretion of digestive glands. Without anesthetics, chilling, or damage the animal can be fixed for prolonged periods and microinstruments (which include feeding pipettes), positioned by a standard micromanipulator.

Animals↗

PubMed1973

Micromanipulator for yeast genetic studies.

An inexpensive mechanical micromanipulator, designed primarily for separating yeast ascospores, can be assembled from commercially available components and without extensive custom machining.

Genetic Techniques↗

PubMed1982

Production of monozygotic twins by micromanipulation and cervical transfer in the cow.

To develop the practical production of monozygotic twins in cattle, the ability of day 6 to 7 cow embryos to survive after they were split into two halves was tested. Day 6 to 7 embryos were recovered by the cervical method from superovulated Charolais heifers. Only normal embryos were selected at the advanced compaction stage when the cavity appears. Each embryo was cut into two halves and each half was replaced in an empty zona pellucida. After micromanipulation, the half embryos were directly transferred to recipient heifers via the cervix. From 16 normal embryos manipulated, 14 monozygotic half embryo pairs were obtained which were all transferred to 14 recipients (pregnancy rate 64.2 per cent); six recipients carried twins (twinning rate 66.6 per cent). Thus 15 fetuses were recorded from the 14 monozygotic half embryo pairs transferred.

Animals↗

PubMed1984

Production of monozygotic (identical) horse twins by embryo micromanipulation.

The blastomeres of 192- to 8-cell embryos recovered surgically 1-3 days after ovulation from 23 Pony mares were mechanically separated and inserted, in various combinations, into evacuated pig zonae pellucidae to make 27 'half' and 17 'quarter' micromanipulated embryos. These were embedded in agar and cultured in vivo in the ligated oviducts of ewes for 3.5-5 days to allow development to the late morula/early blastocyst stage. Subsequent surgical or non-surgical transfer of 13 'half' and 17 'quarter' embryos to mares resulted in 10 established pregnancies, including 2 monozygotic pairs. Surgical transfer to mares that had not been recently used as donors of embryos was more successful (10/20) than surgical or non-surgical transfer to recently operated mares (0/10).

Animals↗

PubMed1988

Micromanipulation of mammalian embryos.

Micromanipulation of embryos provide a new and valuable biological tool for animal agriculture and medical fields. Current techniques for embryo manipulation are in practice but some of techniques is still way off for application. The present status in new biotechnology applying to animal science and medicine is reviewed and its future is also discussed.

Animals↗

PubMed1991

A micromanipulator for precise control of a brain retractor. Technical note.

A new microsurgical device for delicate brain retraction is described. The device, a micromanipulator with a holder for a brain spatula fixed at the end of a self-retaining retractor arm, allows smooth, steady, fine displacement of the retractor tip ("minimal sliding retraction"), operated by the surgeon's two fingers. This has proved to be a useful and safe method of retraction for critical brain structures such as blood vessels, cranial nerves and aneurysm domes in deep microsurgical fields.

Equipment Design↗