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Micromanipulative techniques, such as subzonal insemination and direct intracytoplasmic sperm injection, are used when standard in vitro fertilization techniques have failed. These new approaches are currently under investigation in a variety of centers across the country. The projected results and the strengths and weaknesses of these techniques are reviewed.
We micromanipulated the nucleus and centrosomes in the zygote of the starfish, Asterina pectinifera, in order to investigate their roles in mitotic apparatus formation and cleavage induction. The zygote cleaved without spindle formation when its nucleus was removed. When one or two centrosomes were transplanted, they formed asters in the recipient cell, which cleaved into three or four blastomeres so that each blastomere might contain one centrosome or aster. When one centrosome was removed, a half-spindle formed in the manipulated cell, which did not cleave until the other centrosome was duplicated. When both centrosomes were removed, no microtubular structures such as the spindle and the aster appeared in the manipulated cell, which failed to cleave. These results indicate that two centrosomes or more in the cell induce cleavage with or without the nucleus and that one centrosome or less does not induce cleavage. It is also concluded that the centrosome(s) together with the nucleus forms a half-spindle or bipolar spindle. However, from the experiments of nucleus transplantation and displacement, spindle formation is found to depend on the distance between chromosomes and centrosomes. The half-spindle formed when the distance from the centrosome to the chromosomes was shorter than 22 microns; on the other hand, when the distance was longer than 22 microns, the nucleus remained apart from the aster, which means that the functional range of the astral microtubule's ability to engage chromosomes was 22 microns from the centrosome.
Natural isolates of Plasmodium falciparum represent a genetically heterogeneous population of parasites. To obtain stable strains of the parasites for long term experiments, a rapid and definitive method of cloning was developed using micropipets and a micromanipulator. Homogeneous parasite clones prepared by this technique were characterized and compared with the parent isolates during 4 years of continuous culture. The process of phenotypic dominance and selection of drug resistance which occur in nature was also simulated in vitro by evaluating population dynamics of two cocultured isolates of P. falciparum.
A simple design for a micromanipulator having fine movements in 3 dimensions is described. An inexpensive and easily executed method of construction for this device is given.
Motorized linear slides used as micromanipulators for biological use suffer from 3 problems: vibration at low speed, poor ergonomic design of the controller and slow coarse positioning. These problems are not hard to solve by minor modifications to the controller and the slides.
A new technique is described which is suitable for long-term recording of the activity of neurones in the brain of an awake, unrestrained cat. By using telescopic electrodes, neurones up to 39 mm from the cranial surface can be reached with a miniature micromanipulator which is small enough to be left in place between recording sessions. The most stable recordings have been obtained with electrodes made from microwire, with which units have been held for up to 8 h.
We have developed an esophageal probe with a precision micromanipulator and a transversely oriented 32 element ultrasonic array which operates at 3.5 MHz. The probe allows us to obtain multiple two-dimensional images of the heart with known angular relationships between them over a series of cardiac cycles. Our ultimate purpose is to acquire images for left ventricular volume estimation with a three-dimensional reconstruction method. Technical details of the probe design are given. In vitro tests have shown that the imaging plane can be angulated within 1.5 degree root mean square error. In vivo results with dogs have demonstrated its ability to obtain multiplanar short axis images of the heart.
Chromatographic particles should not possess only desirable surface chemical properties but also mechanical properties. The latter determine the deformation of the particles under hydrodynamic conditions of packed beds and further the pressure drop. Understanding the mechanical properties of chromatographic particles is essential to successful design and operation of such processing equipment. The mechanical properties of single chromatographic particles made of different formulations and with different surface modifications were characterized by a novel micromanipulation technique. The principle of this technique is to compress single particles between two parallel surfaces. The force being imposed on the particles and their deformation under compression are measured simultaneously. The particles range from 30 to 300 microm, depending on their formulation. 10-30 particles from each sample were compressed up to a particle deformation of 70% in order to get statistically representative data. Particles for hydrophobic interaction chromatography were proved more stable than resins for ion exchange or gel filtration. The difference is remarkable in dry state and is less in wet state. Water seems to "level out" the differences in mechanical stability of resins. The stability seems to be also dependent on the speed of water release out of the resins.
In mitosis, cohesion appears to be present along the entire length of the chromosome, between centromeres and along chromosome arms. By metaphase, sister chromatids appear as two adjacent but visibly distinct rods. Sister chromatids separate from one another in anaphase by releasing all chromosome cohesion. This is different from meiosis I, in which pairs of sister chromatids separate from one another, moving to each spindle pole by releasing cohesion only between sister chromatid arms. Then, in anaphase II, sister chromatids separate by releasing centromere cohesion. Our objective was to find where cohesion is present or absent on chromosomes in mitosis and meiosis and when and how it is released. We determined cohesion directly by pulling on chromosomes with two micromanipulation needles. Thus, we could distinguish for the first time between apparent doubleness as seen in the microscope and physical separability. We found that apparent doubleness can be deceiving: Visibly distinct sister chromatids often cannot be separated. We also demonstrated that cohesion is released gradually in anaphase, with chromosomes looking as if they were unzipped or pulled apart. This implied that tension from spindle forces was required, but we showed directly that no tension was necessary to pull chromatids apart.
Optical tweezers have been little used in experimental studies on filamentous fungi. We have built a simple, compact, easy-to-use, safe and robust optical tweezer system that can be used with brightfield, phase contrast, differential interference contrast and fluorescence optics on a standard research grade light microscope. We have used this optical tweezer system in a range of cell biology applications to trap and micromanipulate whole fungal cells, organelles within cells, and beads. We have demonstrated how optical tweezers can be used to: unambiguously determine whether hyphae are actively homing towards each other; move the Spitzenkörper and change the pattern of hyphal morphogenesis; make piconewton force measurements; mechanically stimulate hyphal tips; and deliver chemicals to localized regions of hyphae. Significant novel experimental findings from our study were that germ tubes generated significantly smaller growth forces than leading hyphae, and that both hyphal types exhibited growth responses to mechanical stimulation with optically trapped polystyrene beads. Germinated spores that had been optically trapped for 25min exhibited no deleterious effects with regard to conidial anastomosis tube growth, homing or fusion.
This is a report of our preliminary experience with assisted hatching by laser with a case of an ongoing twin pregnancy. We used an erbium laser to create a 20 to 30-micrometer opening in the zona pellucida of human embryos to facilitate the embryonic hatching after the embryo transfer. Our report proves that it is possible to obtain a pregnancy using a simple and quick laser method instead of mechanical or chemical procedures within the scope of micromanipulation.
OBJECTIVE: To determine the effects of zona slitting and subsequent embryo transfer performed on different days of in vitro human embryonic development to allow appropriate application of assisted hatching techniques to clinical IVF-ET. DESIGN: A microsurgical technique was used to make a standardized slit in the zona pellucida of bipronucleate human embryos on day 2, 3, or 5 after oocyte retrieval. A mock ET was performed after the procedure and the rate of blastocyst formation and the process of hatching in each embryo was monitored. SETTING: All embryos used were donated for research by patients attending the IVF Unit at the John Radcliffe Hospital, Oxford, United Kingdom. MAIN OUTCOME MEASURES: The effects of the mock ET on the integrity of zona-slit embryos was documented. The embryos were cultured further and parameters such as blastocyst formation, initiation, and completion of hatching were monitored in all three groups of embryos. RESULTS: The mock ET did not have a detrimental effect on embryos as assessed by their morphology and subsequent blastocyst formation rate. The technique used in this study to make an opening in the zona increased the rate and number of blastocysts hatching in all three groups compared with nonmanipulated controls. However, the day on which the slit was made did not significantly alter the outcome. CONCLUSION: A gap in the zona pellucida of human embryos significantly increases the rate of hatching. Micromanipulative techniques such as zona slitting can be suitably used for this purpose without loss of blastomeres through the gap in the zona or trapping of the embryo during the process of hatching.
The introduction of different micromanipulation techniques into reproductive and developmental biology has helped us not only to answer many essential biological questions but it is now evident that these techniques also have wide practical applications. In human-assisted reproduction, the most commonly used approach is the injection of a donor sperm into the oocyte cytoplasm--intracytoplasmic sperm injection. It is, however, speculated that with these techniques it would be also possible to improve the oocyte developmental potential especially in those cases when the quality of the cytoplasm is rather poor and thus its function is compromised. Another important application would be the elimination of mutated mitochondrial DNA (mtDNA) by transferring the nuclear material from an abnormal oocyte into a healthy donor oocyte cytoplast. Some of these techniques were already successfully tested in experimental animals, but it is evident that before their introduction into human medicine many questions must be answered, and we must be sure that these approaches are absolutely or almost absolutely safe. In our contribution, we will be specifically oriented to the nuclear (nuclear material) replacement approaches that could be potentially used to prevent the transmission of mutated mtDNA from mother to offspring. Because these techniques are very delicate, some training with oocytes from other species other than human is strongly recommended.
UV photolysis of caged molecules is a powerful method for studying cellular signaling. However, UV energy is often delivered through the microscope objective which can make certain experiments difficult. We have evaluated the utility of delivering UV pulses directly to the sample through an optical fiber. Visible (635 nm) and UV (337 nm) lasers were coupled into a UV transmitting optical fiber which was micromanipulated over the sample under investigation. Positioning of the fiber, and thus the photolysis beam, was achieved using the visible laser which acted much like a flashlight. By controlling the size of the optical fiber it is also possible to control the area of the sample which is exposed to UV light. After positioning the fiber we demonstrate that the UV beam exiting the optical fiber reliably photolysed NP-EGTA that had been loaded into cells, resulting in an elevation of intracellular calcium. Additionally, caged norepinephrine in the bathing saline was photo-released to activate receptor-operated calcium signaling pathways. Since the delivery of the UV energy is independent of microscope configuration, this approach can be readily incorporated into wide-field fluorescence imaging, confocal microscopy and electrophysiological applications.
Clonal cultures of Histomonas meleagridis, Tetratrichomonas gallinarum and a Blastocystis sp. were established for the first time. Single microbes were successfully isolated from a mixture of micro-organisms obtained from caecal contents of turkeys, using a micromanipulation approach. The cloned parasites were propagated in vitro and maintained through continuous passages multiplying to high numbers. Identification of the protists was done by morphological investigation identifying various forms of each parasite. PCR and partial sequencing of the small subunit rRNA were used to confirm clonality and to determine the relationship of the cloned parasites with known protozoan parasites. The clonal cultures established by this technique will be useful to gain more insight into the biological repertoire of the organisms. In addition, refined infection experiments in different poultry species can now be performed to elucidate the pathological pathways of the respective protozoa.
The inclusion of the gene encoding the Green Fluorescent Protein (GFP) or its derivatives into dicistronic transfer vectors is a useful method to visually identify cells that have incorporated a specific gene of interest. By combining this approach with the use of a micromanipulator, we have developed a protocol for the one-step isolation of cells expressing a specific transgene from a pool of transfected cells. Target fluorescent cells could be identified and isolated even when they occurred at frequencies as low as 1/100,000. The use of Leibowitz L-15 serum- free medium and serum-coated non-charged petri dishes, along with minimal light exposure yielded maximal cell viability and high cloning efficiency (approximately 40%, on average) for a large number of cell lines, both adherent and suspension. Several variations of the basic method are presented, as well as guidelines for the choice of hardware components to implement our cloning workstation.
Complete manipulation by laser light allows precise and gentle treatment of plant cells, subcellular structures, and even individual DNA molecules. Recently, affordable lasers have become available for the construction of microbeams as well as for optical tweezers. This may generate new interest in these tools for plant biologists. Early experiments, reviewed in this journal, showed that laser supported microinjection of material into plant cells or tissues circumvents mechanical problems encountered in microinjection by fragile glass capillaries. Plant protoplasts could be fused with each other when under microscopical observation, and it was no major problem to generate a triple or quadruple fusion product. In the present paper we review experiments where membrane material was prepared from root hair tips and microgravity was simulated in algae. As many plant cells are transparent, it is possible to work inside living, intact cells. New experiments show that it is possible to release by optical micromanipulation, with high spatial resolutions, intracellular calcium from caged compounds and to study calcium oscillations. An example for avian cardiac tissue is given, but the technique is also suitable for plant cell research. As a more technical tool, optical tweezers can be used to spatially fix subcellular structures otherwise moving inside a cell and thus make them available for investigation with a confocal microscope even when the time for image formation is extended (for example at low fluorescence emission). A molecular biological example is the handling of chromosomes and isolated individual DNA molecules by laser microtools. For example, chromosomes can be cut along complex trajectories, not only perpendicular to their long axis. Single DNA molecules are cut by the laser microbeam and, after coupling such a molecule to a polystrene microbead, are handled in complex geometries. Here, the individual DNA molecules are made visible with a conventional fluorescence microscope by fluorescent dyes such as SYBRGreen. The cutting of a single DNA molecule by molecules of the restriction endonuclease EcoRI can be observed directly, i.e. a type of single molecule restriction analysis is possible. Finally, mechanical properties of individual DNA molecules can be observed directly.
A micromanipulation technique has been developed to measure the bursting force of single dry microcapsules coated onto a surface, such as those normally used in carbonless copying paper. For measuring the bursting force of a given microcapsule, a single fine probe with a flat end about 10 microns in diameter was used to squeeze the microcapsule against a flat surface until it burst. The force being imposed on the microcapsule was measured by a transducer connected to the probe. The bursting force and diameter of single dry microcapsules in two samples, different in size and wall thickness, were measured by this technique. The bursting force of the microcapsules in one sample ranged from 50 to 220 microN and the diameter from 1.3 to 7.0 microns, whilst the bursting force in the other was from 20 to 175 microN and the diameter from 0.7 to 3.7 microns. This technique makes it possible to compare the mechanical strength of microcapsules made of different formulations, and to infer information about microcapsule mechanical properties.