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At least 739 records · Page 41Linked to original sources
PubMed1991

An inexpensive inverted microscope for patch-clamp and other electrophysiological studies at the cellular level.

The popularization of the patch-clamp technique has increased the demand for inverted light microscopes that allow the optimal or almost free movement of patch-clamp pipettes and their support drives. However, commercially available models of inverted microscopes have not been specifically designed for this line of research and, as a consequence, patch-clamp pipette movements are restricted by the small space available between the sample, and the light source and its modulating attachments. This paper provides the details for the construction of a relatively inexpensive inverted microscope that meets the specifications required for patch-clamp and other electrophysiological investigations at the cellular level. The microscope allows the free positioning of the conventional probes for patch-clamp, microelectrode amplifiers, and other micromanipulator probes and attachments. The construction of the microscope is simple and, therefore, since it is relatively inexpensive, the microscope may be easily upgraded in many ways for special purposes, including special optical effects. Finally, although the instrument was developed for patch-clamp and classical electrophysiological studies, it may be used in other types of investigations where freedom of microtool movement is imperative, such as in microsurgery applications.

Electrophysiology↗
PubMed1992

A fast perfusion system for single cell physiology optimized for microscopes with water immersion objectives.

A perfusion system was constructed which allows the fast application of different solutes underneath a water immersion objective. The perfusion system is mounted into the immersion objective by milling a slot into the frontal metal plate of the lens holder. It consists of a five-channel pipette fixed to the objective and solution reservoirs gated by computer controlled magnetic valves. Up to five different solutions can be applied to the specimen under study. The solution between objective and specimen is completely exchanged after 1-2 s as determined from fluorescence measurements. This arrangement is optimized for [Ca2+] measurements with a fluorescence measurement system in tissue slices, where upright microscopes are required. It offers the advantage of saving a micromanipulator for the perfusion pipette and facilitates a fast, reproducible and precise positioning of the perfusion system.

Animals↗
PubMed1988

Effect in vitro of bovine viral diarrhea virus on bovine embryos with the zona pellucida intact, damaged and removed.

The in vitro effect of bovine viral diarrhea virus (BVDV) on the survival of day 7 to day 7.5 bovine embryos collected from superovulated donors was studied. Fifty-four experimental embryos with the zona pellucida (ZP) intact, damaged or removed were exposed to 1 X 10(4) TCD50/ml of the NADL cytopathic strain of BVDC at 37 degrees C for 24 hrs and compared to 36 control embryos that were cultured for 24 hr. Seven embryos with the ZP-removed were similarly exposed for 48 hrs and compared to five control embryos. The overall survival rate was 68% for embryos exposed to BVDV for 24 hrs and 77% for embryos not exposed (P greater than 0.05). Extended exposure of the embryos with the ZP removed to virus for 48 hrs did not affect their survival rate compared to controls. Damage to the ZP by cracking or total removal of the ZP by micromanipulation or acidic Tyrode's solution had no effect on subsequent embryonic survival in the presence of BVDV. It was concluded that exposure to BVDV in vitro is not cytopathic for morula and blastocyst stage bovine embryos over a 48 hr period, even when they are not protected by the ZP.

Animals↗
PubMed1985

Parental age and the life-span of zygotes of Saccharomyces cerevisiae.

Isolated cells of Saccharomyces cerevisiae were mated by micromanipulation and the reproductive capacity of the resulting zygotes was determined. The mating frequency was dependent on the age of the parents: conjugations between young cells and cells which had completed more than two thirds of their life-span were very rare events. The life-span of a zygote was very similar to the life-span of its shorter-lived parent. If one of the parent cells had budded several times prior to fusion, the life-span of the zygote was reduced correspondingly, i.e. there was no 'rescue by hybridization.' In four crosses the distribution of buds on both of the parent cells was recorded. In three of these four crosses the buds were evenly distributed, and in one the alpha-parent had three times as many buds as the a-parent.

Female↗
PubMed1985

Exposure and direct stimulation of the semicircular canal cupula.

The ampullary wall of the posterior semicircular canal was isolated from the frog, cut and the cupula exposed in frog Ringer's solution. The cupula was stimulated by depression with a fine glass micropipette. Three points on the cupula were selected for depression: point A, the lowest point on the midline of the cupula surface on the utricular side; point B, the uppermost on the midline of the same surface; and point C, in the middle of the lateral surface of the cupula. Depression at points A and B was toward the canal, whereas depression at point C was toward the centre of the cupula. The amount of depression was controlled by a micromanipulator. At point A, the minimum depression facilitating the posterior canal action potential was 1 micron; at point B, a greater amount of depression was needed to produce the action potential. Even a large amount of depression at point C resulted in only a small action potential, possibly because the direction of the sensory cell polarity is along the long axis of the canal.

Animals↗
PubMed1987

An experimental study demonstrating the physiological polarity of the frog's utricle.

The frog Rana nigromaculata was used as an experimental model. The utricle and its nerve were isolated with the anterior and the lateral semicircular canal ampullae in frog Ringer's solution. The utricular otoconia and the otoconial membrane were then carefully removed. The halved anterior canal cupula was next placed in the tip of a glass microelectrode, which was mounted on a micromanipulator. The cupula was placed in the center of either the medial or the lateral part of the macula and was moved toward or away from the striola along the axis vertical to the striola. When the medial part of the macula was stimulated, 5 micron of striolapetal cupular movement elicited an excitatory nerve discharge. When the lateral part of the macula was stimulated, the striolapetal stimulus likewise induced an excitatory response. These results indicate the existence of physiological polarity on the utricular macula.

Action Potentials↗
PubMed1981

The expression of hex A and hex B isozymes of hexosaminidase in parental and experimental human fibroblast cells and their components.

The expression of the two major isozyme forms of hexosaminidase (EC 3.2.1.30), hexosaminidase A and hexosaminidase B, has been examined. The parental cells and/or cellular components of parental cells are individually fused using inactivated Sendai virus with the aid of a micromanipulator. The progeny cells produced from such hybrids are subjected to a microenzymatic assay which allows measurements at the single cell level. The lysosomal-deficient cells used in this study are Tay-Sachs and Sandhoff fibroblasts, and the normal cells used are WI-38 (fetal lung fibroblasts), amniotic fluid cells (GM 473), and JASD3 (normal human foreskin). The results show that the ratio of cell components which are fused to form the experimental cell affects the percentage of hexosaminidase A expressed in the progeny cells. Furthermore, our results imply the presence of a "factor" in the Sandhoff cell's cytoplasm which, together with the Tay-Sachs nucleus, is necessary for hexosaminidase A expression in the experimental cell's progeny.

Amniotic Fluid↗
PubMed1978

Fluorescence polarization of stretched polytene chromosomes stained with acridine orange.

The molecules of the fluorescent dye acridine orange (AO) bind to DNA in such a way that the absorption and emission dipoles lie on a plane perpendicular to the DNA axis. For this reason, definite fluorescence polarization should correspond to each mode of spatial DNA packing. A chromosome, considered as an axially symmetrical ensemble of DNA, was characterized by two experimental parameters, P parallel and P perpendicular, i.e., by polarizations of fluorescence excited by light polarized parallel and perpendicular to the symmetry axis. In view of the sequential order in the packing levels of DNA fiber in a chromosome, it was suggested that, under mechanical stretching, the highest level is disrupted first, then the others, in the order of their sequence. Isolated chromosomes of Chironomus thummi were stained with AO and stretched with needles of a micromanipulator. From the changes of P parallel and P perpendicular measured during stretching it was concluded the polytene chromosome bands have three, at least, DNA packing levels, tentatively described as 100 A fiber, 250 A coil and chromomere.

Acridines↗
PubMed1987

A polisher for patch pipets.

The design and construction of a novel apparatus to fire polish patch-clamp recording pipets is described. The device positions the polishing filament in the field of view of the high-power polishing objective and uses the mechanical stage of the microscope to hold the electrode, eliminating the need for a micromanipulator to hold either the filament or electrode.

Electrodes↗
PubMed1977

Fabrication of well defined micropipette tips by hydrofluoric acid etching.

Glass tubes were drawn on a two stage pipette puller such that tips with 0.5-2.0 micron inner diameters were obtained as confirmed by scanning electron imaging. The pipettes were attached to an air reservoir with a pressure set at 0.5 bar to prevent fluid from entering the tip. They were lowered into 2% hydrofluoric acid by means of a micromanipulator. When air bubbles began to escape from the tips, they were withdrawn and immersed into 0.5 molar phosphate buffer, pH 7.4, and then rinsed with tap water and ethanol. Etching during 100 s (mean) yielded inner tip diameters of 5 micron (+/- 5% S.D.). The tips were beveled at the same angle at which they had been dipped into the etching fluid. No continuation of the etching process was noticed even after several days.

Glass↗
PubMed1987

Intracellular lucifer yellow staining and electron microscopy of neurones in slices of fixed epitumourous human cortical tissue.

To examine the complete morphology and ultrastructure of lipofuscin-containing human pyramidal cells, epitumourous biopsy tissue was lightly fixed in paraformaldehyde. Cortical slice preparations were immersed in an injection chamber which was transferred to a fixed stage microscope. Electrodes were filled with an aqueous solution of the fluorescent dye Lucifer Yellow and attached to a micromanipulator. Epifluorescence illumination was used to visualize and guide the tip of the Lucifer pipette towards lipofuscin-containing, autofluorescent pyramidal cell somata. After impaling, the neuron was intracellularly stained by iontophoretic injection of Lucifer Yellow. Subsequent graphical reconstructions of injected pyramidal cells revealed complete filling of their dendritic arborizations. Comparison with published Golgi-material prepared for light microscopy revealed no patho-morphological changes in the tissue. Eventually, dye-filled cells were photooxidized in the presence of diaminobenzidine, which resulted in the formation of a homogeneously brown reaction product. Bleached cells were then osmicated and embedded in plastic. Electron microscopy revealed fine electron-opaque label distributed throughout the karyo- and cytoplasm and there were no apparent gross ultrastructural changes. Cytological details, such as organelles and membranes were not obscured by the reaction product. Due to its autofluorescence, the pigment part of the lipofuscin also underwent photoconversion, resulting in a highly enhanced electron-dense matrix. Due to its high selectivity and relative methodological simplicity, the approach presented is considered to be a promising alternative to the gold-toning modification of the Golgi-electron microscope technique.

Brain Neoplasms↗
PubMed1994

Prediction of mechanical damage to animal cells in turbulence.

In previous work a model was proposed for estimation of disruption of animal cells in turbulent capillary flows using information about the hydrodynamics, and cell mechanical properties determined by micromanipulation. The model assumed that the capillary flow consists of a laminar sublayer and a homogeneous turbulent region, and within the latter eddies of sizes similar to or smaller than the cells interact with those cells, causing local surface deformations. The proposed mechanism of cell damage was that such deformations result in an increase in membrane tension and surface energy, and that a cell disrupts when its bursting membrane tension and bursting surface energy are exceeded. The surface energy of the cells was estimated from the kinetic energy of appropriate sized eddies. To test the model, cells were disrupted in turbulent flows in capillaries at mean energy dissipation rates ranging from 800 to 2 x 10(4) Wkg-1. The model assumed that the specific lysis rate is almost independent of the number of passes, which was verified by the experimental data. The implication was that despite the damage the cell mechanical properties did not change markedly during multiple recirculations through the capillaries. On average the model underestimated the cell disruption by about 15%. Although the model gave reasonably good predictions, it lacks proper explanation of the independence of the specific lysis rate on the number of passes. In this paper it is shown that this problem can be resolved in principle by consideration of the localisation of the energy dissipation in turbulent capillary flows. The necessity of further modelling of cell-turbulence interactions is demonstrated.

Animals↗
PubMed1993

In vivo micropuncture of retinal vessels.

Micropuncture has proven to be a valuable tool for the local study of vascular parameters in many organ systems; however, it has not been applied to the study of the circulation of the retina. We report here our extension of micropuncture techniques [4] to use in the intact retina of the anesthetized cat. We use extremely sharp micropipettes with tip sizes much smaller than the diameter of erythrocytes to avoid hemorrhage. The micropipette is held by a microdrive which in turn is mounted on a precision goniometric micromanipulator. We micropuncture retinal arteries and veins with diameters ranging from 20 to 130 microns with no apparent damage to the vessel wall and no observed hemorrhage. During micropuncture we routinely inject nanoliter quantities of dyed saline, which we observe flowing in a plume from the micropipette tip within the lumen of the vessel. Micropuncture techniques may be used in the laboratory to study retinal autoregulatory mechanisms by microinfusion of vasoactive substances and by measuring blood pressure in retinal microvessels. In the clinic micropuncture may be useful for treating disorders such as retinal vascular occlusion.

Animals↗
PubMed1989

The hemizona assay (HZA): a predictor of human sperm fertilizing potential in in vitro fertilization (IVF) treatment.

The hemizona assay (HZA) was developed to assess human sperm fertilizing potential. This blinded study investigated the relationship between sperm binding to the hemizona and in vitro fertilization (IVF) success (36 patients). Nonliving human oocytes were recovered from excised ovaries and stored. Each zona pellucida was cut into equal hemispheres by micromanipulation. For the HZA, one droplet exposed a hemizona to abnormal spermatozoa, while the control droplet contained the matching hemizona and spermatozoa from normal semen. After 4 hr, the number of tightly bound spermatozoa was counted. Binding to the hemizona was significantly higher for those having IVF success (mean of 36.1 +/- 7, versus 10.4 +/- 4 from the failure group; P less than 0.05). Fewer sperm from the failure group had a strictly normal morphology (3.2 versus 12.7%; P less than 0.05, Kruger method). Tight zona binding was significantly correlated with the percentage motile sperm, percentage normal morphology, and seminal sperm concentration. These results enhanced our confidence that the HZA is diagnostic for identification of patients at high risk of failing to achieve fertilization in vitro.

Female↗
PubMed1979

Two transcripts of an individual Balbiani ring from the salivary gland cells of Acricotopus lucidus (Diptera, Chironomidae).

The polytene chromosomes in main lobe cells of the Acricotopus salivary gland carry two large Balbiani rings (BR1, BR2), which BR1 exhibits the higher incorporation rate of tritiated nucleosides. The size of BR1 varies in conjunction with a subterminal inversion which includes the BR1 site. BR1 in both the homozygous and heterozygous inverted arrangement (BR1A and BR1B, respectively) is consistently smaller than in the homozygous standard arrangement (BR1C). The RNA content of BR1A and BR1C (3.1 and 6.7 pg, respectively) corresponds well with their relative size.--Using the method of micromanipulation of squashed salivary gland chromosomes the RNA transcripts of BR1A and BR1C have been extracted from microdissected Balbiani rings and fractionated by electrophoresis on 1% agarose gels. In BR1A one RNA fraction with a molecular weight of about 40 x 10(6) D is transcribed. The gel electrophoretic pattern of BR1C is characterized by an additional RNA fraction of 5 x 10(6) D. The main part of BR1 RNA is bound to oligo(dT)-cellulose suggesting the adenylation at the chromosome of Balbiani ring RNA.--In the profile of poly(A)-containing cytoplasmic RNA obtained after in vivo labelling two peaks can be detected which correspond in size to the BR1 RNA. These putative BR1 transcripts accumulate to a high concentration in the cytoplasm and show a relatively low stability during in vivo chase experiments.

Animals↗
PubMed1993

Validation of the hemizona assay (HZA) in a monkey model. II. Kinetics of binding and influence of cryopreserved-thawed spermatozoa.

We compared fresh and frozen-thawed cynomolgus monkey spermatozoa tight binding to the zona pellucida under hemizona assay (HZA) conditions. Monkey oocytes were recovered after superovulation and stored in salt solution. Matching hemizonae were obtained by micromanipulation. Semen, obtained by electroejaculation, was used fresh or was cyropreserved, thawed, and washed by swim-up separation. At the standard initial dilution of 500,000 motile sperm/ml (or 5 x 10(4) motile sperm/hemizona), binding was significantly higher for fresh sperm (P = 0.00004). For frozen-thawed samples, there was a linear increase in the number of tightly bound sperm with increasing sperm concentration (r = 0.95). At 1.5 x 10(6) motile sperm/hemizona, binding of frozen-thawed spermatozoa was similar to that of fresh at a standard concentration. Kinetic studies showed peak binding at 1 hr of gametes coincubation. We conclude that, in this monkey model, the HZA is a valuable bioassay for evaluation of sperm binding to the zona pellucida, the initial requisite for fertilization and embryo development.

Animals↗
PubMed1993

Sex determination in single mouse blastomeres by polymerase chain reaction.

In sex determination of mammalian preimplantation embryos, viability of biopsied embryos and accuracy of sexing are together important. In consideration of this point, single blastomeres were mechanically isolated from mouse embryos using a micromanipulator and then sexed by polymerase chain reaction (PCR) using mouse Y chromosome-specific primers. All of 260 embryos biopsied at the four-cell and morula stage survived. Developmental rate of the embryos to normal blastocysts was 93 and 94%, respectively. Sex determination of single blastomeres was performed by amplification of a mouse Y chromosome-specific DNA sequence using PCR technique. The ratio of male to female embryos was 53 and 47%, respectively. The sex-determined embryos were transferred to the uteri of pseudopregnant recipients to test the consistency of the assay system. The sex of 27 of 29 mice developed from male and female embryos agreed with the predicted sex. The method developed for embryo biopsy and sexing could be used for diagnosis of defective genes at the stage of the preimplantation embryos of human and other domestic animals.

Animals↗
PubMed1997

Advances in the evaluation and treatment of the infertile man.

Numerous advances in technology have been made in the last several years in the diagnosis and treatment of the infertile man. Using case presentations, this article highlights these important new developments and reviews the basics of a comprehensive male infertility evaluation. The use of ultrasound as a critical, indispensable, yet noninvasive tool in the evaluation of possible male reproductive tract obstruction is discussed. Since assisted reproductive techniques (ARTs) have become increasingly important in the management of the otherwise untreatable infertile man, specialized testing of sperm function, e.g., the sperm penetration assay (SPA), and strict morphology assessment are often useful prior to the initiation of some of these assisted reproductive procedures such as intrauterine insemination (IUI) or in vitro fertilization (IVF). Testicular touch preparation cytology and image analysis are also important additions to routine testicular biopsy for quantifying the extent of spermatogenesis. Finally, the revolutionary micromanipulation procedure of intracytoplasmic sperm injection (ICSI) has forever changed the practice of andrology by enabling men previously thought to be irreversibly infertile the chance to initiate their own biologic pregnancy.

Humans↗