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PubMed2003

Open-dish incubator for live cell imaging with an inverted microscope.

Here we describe the design and fabrication of an inexpensive cell culture incubator for the stage of an inverted light microscope for use in live cell imaging. This device maintains the temperature of the cell culture at 37 degrees C with great stability and, after reaching equilibrium, provides focal stability of an image for 20-25 min with oil-immersion lenses. We describe two versions of the incubator: one for use with standard 60-mm plastic culture dishes, and the other version for imaging of cells on glass coverslips. Either can be made for less than $400. Most components are widely available commercially, and it requires only simple wiring and 3 h to assemble. Although the device is generally useful for live cell imaging on an inverted microscope, it is particularly suitable for work in which instruments are introduced into the culture, such as electrophysiology or micromanipulation. The design is based on the principle that control performance is limited by the lag time between detection and response. The key element of the design is a heated, temperature-controlled aluminum ring serving as a mini-incubator surrounding the culture vessel. For this reason, we call our design a "ringcubator."

Animals↗

PubMed2001

Prenatal screening of single-gene disorders from maternal blood.

Fetal cells and cell-free fetal DNA can be found circulating in maternal blood. Fetal cells recovered from maternal blood provide the only source of pure fetal DNA for noninvasive prenatal DNA diagnosis. Fetal nucleated erythrocytes (NRBCs) are considered the most suitable maternally-circulating fetal cells for this purpose, because they are not commonly found in the peripheral blood of healthy adults and are most abundant in the fetus during early gestation. Because fetal cells in maternal blood are extremely rare, a definitive separation method has not yet been established. Fetal NRBCs can be enriched from maternal blood via fluorescence- or magnetic-activated cell sorting, density gradients, immuno-magnetic beads or micromanipulation. Fetal cells are identified by Giemsa staining, hybridization with Y-chromosome specific probes, PCR-detection of a specific paternal allele, or immunostaining for fetal cell antigens. Amplification of fetal DNA sequences by primer extension preamplification and PCR has allowed prenatal screening for Duchenne muscular dystrophy and the fetal RhD blood type. Sequence-specific hybridization has been used to detect sickle cell anemia and beta-thalassemia prenatally in heterozygous carriers of these disorders. The use of cell-free fetal DNA in maternal plasma for the diagnosis of single-gene disorders is limited to disorders caused by a paternally inherited gene or a mutation that can be distinguished from the maternally inherited counterpart. At present, fetal gender can be determined from maternal plasma. When a pregnant woman is a heterzygous carrier of an X-linked disorder, the determination of fetal gender is clinically very informative for first-step screening to avoid invasive amniocentesis. The non-invasive prenatal diagnosis of genetic disorders should be applied to pregnant women with a definite risk for a specific single-gene disorder.

Cell Separation↗

PubMed1992

Effects of mechanical stretch on membrane currents of single ventricular myocytes of guinea-pig heart.

A technique to record whole cell membrane current during stretching single cardiac myocytes was developed. Ventricular myocytes were dissociated by treating guinea-pig hearts with collagenase. One end of the cell was fixed either to a microglasstool tip or to a glass plate, while the other end was attached either to a microglasstool tip or to a suction pipette, which was mounted on a micromanipulator. A time-independent current showing a reversal potential of -15 +/- 4 mV (n = 7) was activated when the myocytes were stretched more than 20% of the length between two fixed point. The current gradually relaxed during the maintained stretch, and disappeared on releasing the stretch. We failed to detect any consistent change in either the L-type Ca2+ current or the inward rectifier K+ current. For comparison, current changes induced by inflating the cell using a hypo-osmotic solution were recorded. The inflation was not accompanied by any change in the time-independent current. Instead, the delayed rectifier K+ current was increased to 170 +/- 48% control by the 70% hypo-osmotic solution. Thus, the effect of mechanical stretch on the time-independent current is different from those of hypo-osmotic cell inflation. The stretch-induced time-independent current is compared with reported current changes induced by the intracellular microinjection of Ca2+.

Animals↗

PubMed1990

Development and clinical usefulness of a new neuroendoscope system for CT-guided stereotactic brain surgery.

Since computed tomography (CT)-guided stereotactic surgery is essentially blind surgery, it always involves the risk of injuring viable brain tissue and vessels, and it is difficult to obtain a sufficient amount of biopsy specimen from cystic or necrotic lesions. Moreover, the direct observation of the lesion would provide extremely valuable information. The authors have therefore developed a new subminiature rigid endoscope and an ultrasonic aspirator for use in stereotactic surgery, as well as a new micromanipulative system to attach the equipment to the stereotactic frame so that they can be accurately inserted into the optimum position from any direction. The new neuroendoscope includes a graded refractive index glass rod only 1.0 mm in diameter (Selfoc, Nippon Sheet Glass Co., Osaka), which provides a focal depth from 1 mm to infinity, as well as an extremely bright and wide visual field with an angle of 70 degrees. Furthermore, because the outer probe and endoscopic sheath share the outer rigid metal tube 4.5 mm in outer diameter, a large internal channel of 2.1 mm in diameter allows the introduction of various microinstruments such as an ultrasonic aspirator probe, laser probe, and biopsy forceps. When a miniaturized video camera is attached, the surgical procedures can be controlled using the video monitor and photographed. The new probe for the ultrasonic aspirator has been developed in various types suitable for fragmentation and aspiration of hematomas and brain tumors, so under endoscopic control even the harder coagulants and tumors can be removed.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1987

Production of sheep-goat chimeras by inner cell mass transplantation.

Embryos were surgically flushed from goats and sheep on d 6 and 7, respectively, following the first day of estrus (d 0). After enzymatic removal of the zonae pellucidae, inner cell masses were isolated from caprine blastocysts by immunosurgery. The intact inner cell masses were injected into ovine blastocysts with the aid of a micromanipulator. Twenty-two manipulated blastocysts were surgically transferred into 12 ovine recipients. Nine ewes gave birth to a total of 13 young (59% embryo survival). Ten were classified by serum electrophoretic assays or karyotypes as lambs, one as a kid, and two as interspecific chimeras.

Animals↗

PubMed1993

Outpatient CO2 laser excisional conization for cervical intraepithelial neoplasia under local anesthesia.

AIM AND OBJECTIVE: To evaluate the feasibility of CO2 laser excisional conization in patients with cervical intraepithelial neoplasia (CIN) in an outpatient setting under local anesthesia. DESIGN: A prospective study of women with cervical intraepithelial neoplasia over one year recording early and late complications, cure rate, and patients' compliance. The procedure was performed with a Coherent CO2 laser connected to the colposcope with a micromanipulator. The equipment attaining 19,700 W/cm2, spot diameter 0.43 mm, focus distance 300 mm, and continuous beam. RESULTS: The material included 81 women. Seventy-eight women were treated as outpatients, while three women were admitted for 24 hours observation due to surgical and technical complications during the treatment. Sixty-six percent of the women had bleeding less than 10 ml during the treatment. One woman was admitted 24 hours after surgery due to bleeding requiring suturing. Median operation-time was 11 minutes (range 3-60). All specimens contained CIN. In 76% of the cases the pre- and postoperative diagnosis were identical. Twelve specimens had CIN in one or both margins. Abnormal cytology after conization was found in three cases (4.2%). High patient compliance was found as 92% of the treated women would choose the same kind of treatment if it had to be repeated. CONCLUSION: High success rate, few complications and high patient compliance make the procedure adequate as a routine procedure in treatment and diagnosis of CIN.

Adult↗

PubMedDate not supplied

Studies on the sensory hairs of receptor cells in the inner ear.

The crista ampullaris of the semicircular canal in the frog can be isolated and mounted in a chamber so that the sensory hairs can be observed under high magnification in interference-contrast. The cupula is removed and the sensory hairs can be manipulated and their mechanical properties investigated by a microprobe held in a micromanipulator. The hairs appear quite stiff and pivot around their base. When subjected to force they break as if they are brittle. All the cilia within a bundle move together as if joined to one another. Labelling for electron-microscopy with polycationic ferritin reveals that the membrane surrounding the cilia has a surface coat of negatively charged molecules. When the organ is incubated with polycationic ferritin before fixation the sensory hairs agglutinate. Fusion of the membrane surrounding individual sensory hairs also occurs.

Animals↗

PubMed1988

Treatment of severe subglottic stenosis in children with the CO2 laser. A preliminary report on a few successful cases.

Subglottic stenosis in children is difficult to resect even with the CO2 laser. The smallest laser bronchoscope is usually too large to pass through the glottis. In cases where it is possible, the precision may not suffice. A micromanipulator used together with a laryngoscope will permit a far better control of the laser beam. The commercially available instruments are not sufficiently small for use in a small stenotic larynx. As only small parts of the stenosis should be removed at one time, there has been a need for delicate instruments to protect parts of the laryngeal interior and at the same time permit access to the nether area of the subglottic region. Instruments designed by the author is described and the preliminary results of hitherto five consecutive cases are reported. Ages at the beginning of the treatment varies from 2 months to 4 years and 4 months. The most severe case has been treated six times. Three children are living without breathing problems. One child is about to be decannulated, and the treatment has just begun in the most recent case. The observation time after treatment is 2 years at most.

Child, Preschool↗

PubMed1993

Preimplantation diagnosis of genetic disease.

If a couple is known to be at risk of transmitting a severe genetic disease to their offspring, the fetus may be tested by chorionic villus sampling or amniocentesis during pregnancy. If the fetus proves to be affected, the couple has the option of terminating the pregnancy. Recently, an alternative approach has been developed, i.e. to screen embryos produced by in vitro fertilization before implantation. Diagnostic analysis is carried out on a single cell removed by micromanipulation from the 8-cell embryo or on the polar body from the unfertilized egg. Preimplantation diagnosis is now a feasible alternative to screening during pregnancy and may be the preferred route for some couples.

Blastocyst↗

PubMed1997

Assisted reproductive technology: the state of the ART.

At least one in ten couples of reproductive age is affected by infertility. Tubal disease, ovulatory defects, endometrosis and abnormal sperm physiology are the most common causes of failure to conceive. Many of these disorders can be treated successfully with surgery, ovulation induction or intrauterine insemination, but in selected cases, or where there is long-standing intractable infertility, assisted reproductive technology (ART) becomes the treatment of choice. We provide an overview of the techniques for assisted reproduction, including in vitro fertilization, gamete intrafallopian transfer and other related procedures. Indications for treatment, patient evaluation and advances in reproductive technology including embryo cryopreservation, micromanipulation and donor gametes are also reviewed.

Cryopreservation↗

PubMed2000

The rationale for requesting a second consecutive sperm ejaculate for assisted reproductive technology.

We assessed the value of requesting a second semen sample provided within 1 hour of the first in cases of unacceptable ejaculate quality (compared to previous occasions) or very poor semen characteristics. The study population consisted of 109 males with severe oligo-terato-astenospermic (OTA) syndrome in an assisted reproduction program. Semen volume, sperm count and sperm motility were recorded in the first and second samples with a Makler Counting Chamber before sperm processing, and total motile sperm in the ejaculate was calculated. Differences in sperm parameters between the consecutive samples were determined by paired-sample t test. In 36 cases (33%) the second sample was found to be superior in quality to the first and was therefore used for the fertilization process. Of the 12/109 cases in which the first ejaculate demonstrated no motile sperm, five (41%) had detectable motile sperm in the second (total 0.1 x 10(6), 0.2 x 10(6), 0.3 x 10(6), 8.4 x 10(6) and 20.8 x 10(6)). We conclude that a request for second ejaculate immediately after the first in males with poor semen quality or no detectable motile sperm can yield a better sample in a significant percentage of cases. Using this method, clinicians can avoid the utilization of elaborate sperm processing techniques and the need for unnecessary micromanipulative fertilization.

Ejaculation↗

PubMed1994

Cell surface antigen expression in human erythroid progenitors: erythroid and megakaryocytic markers.

This review summarizes the changes in cell surface antigen expression during proliferation and differentiation of human erythroid progenitors. The content is based on our experimental data obtained from complement-mediated cytotoxicity assays against hematopoietic progenitors and a combined technique of sequential micromanipulations of paired daughter cells derived from erythroid burst-forming units (BFU-E) and immunostaining with a panel of monoclonal antibodies, as well as from current information. BFU-E has CD34, CD41a (platelet glycoprotein[GP]IIb/IIIa) and CD41b(GPIIb) antigens. Paired daughter cells derived from BFU-E have CD41a, CD41b, CD71 (transferrin receptor) and HLA-DR antigens, but not CD34 or CD33 antigen. The CD36 antigen (thrombospondin receptor or GPIV) is first expressed on the cells after 5 days of culture, in agreement with the report that the anti-CD36 positive fraction contained a greater part of the erythroid colony-forming units (CFU-E). The blood group A antigen is first expressed on cells from aggregates derived from BFU-E after 5 days of culture. Glycophorin A is expressed on cell surface after 7 days of culture when proerythroblasts first appear. Hemoglobin alpha is expressed after 8 days of culture and coincides with the first appearance of basophilic erythroblasts. This review provides useful information on the identification of leukemic cells from poorly differentiated acute leukemias such as early erythroblastic leukemia and acute megakaryoblastic leukemia, and is useful in the understanding of the commitment and differentiation of erythroid and megakaryocytic progenitors in normal hematopoiesis.

Antigens, Surface↗

PubMed1994

Detection of Epstein-Barr virus DNA in Hodgkin- and Reed-Sternberg-cells by single cell PCR.

The Epstein-Barr virus (EBV) can be detected in the majority of lymph nodes involved by Hodgkin's lymphoma using the highly sensitive polymerase chain reaction (PCR). However, the rate of EBV-DNA detection by in-situ hybridisation, which allows allocation of EBV to a defined cell population, i.e. the neoplastic H&RS-cells, is lower. In an attempt to combine the advantages of the high sensitivity of the PCR and the possibility of cellular allocation by in-situ hybridisation, we established a single-cell PCR of Hodgkin- and Reed-Sternberg (H&RS)-cells isolated by micromanipulation from biopsy tissues. We amplified EBV sequences from the BamW-region by single-cell PCR. Using this method we were able to detect EBV-DNA in the H&RS-cells from 4 of 6 patients. In EBV positive cases all H&RS-cells of a given patient were positive, proving the high sensitivity and reproducibility of the method. Other cells in the biopsy tissue involved by EBV-positive H&RS-cells were shown to be negative. This indicates that EBV may have a role in the pathogenesis of many but not all cases of Hodgkin's disease.

Adolescent↗

PubMed1998

Platelet-like particle production from cultured human megakaryocytic cell line.

The human megakaryocytic cell line CMK was positive for the expression of platelet glycoprotein(GP) IIb/IIIa but few cells were positive for GPIb. To establish a GPIb-positive CMK subclone, GPIb-positive CMK cells were purified, micromanipulated and cloned, but resulting sublines were all negative for GPIb expression. The combined presence of interleukin-3 (IL-3) and the anti-virus agent ribavirin strongly induced expression of GPIb on the cells. However, the megakaryocyte growth and development factor (MGDF)/thrombopoietin (TPO) failed to enhance GPIb expression. CMK themselves and particles are negative for GPIb expression. To utilize these particles as platelet substitutes, further appropriate culture systems are necessary.

Blood Platelets↗

PubMedDate not supplied

Nicotinic acetylcholine receptors on hippocampal neurons: distribution on the neuronal surface and modulation of receptor activity.

The recent development of a technique that uses infrared microscopy for the visualization of well-defined areas on the surface of neurons, and a computerized system of micromanipulators led to the discovery that functional nicotinic acetylcholine receptors (nAChRs) are expressed at higher density on the dendrites than on the soma of rat hippocampal neurons. The finding that the expression of alpha-bungarotoxin-sensitive, alpha 7-bearing, nAChRs and dihydro-beta-erythroidine-sensitive, alpha 4 beta 2 nAChRs tends to increase along the dendritic length suggests that these receptors may be highly involved in the integration of synaptic functions in hippocampal neurons. The present report also discusses the finding that ligands such as the anticholinesterase galanthamine can modulate the nAChR activity by binding to a novel receptor site, and that 5-hydroxytryptamine (5-HT) may serve as an endogenous ligand for this site. The ability of 5-HT to modulate the nAChR function in vivo supports the concept that the overall CNS function is determined not only by the neuronal network established by the neuronal wiring, but also by a chemical network established by the ability of a single substance to act as the primary neurotransmitter in one system and as a co-transmitter in another system.

Acetylcholine↗

PubMed2006

Acute microelectrode array implantation into human neocortex: preliminary technique and histological considerations.

OBJECT: Researchers at The Center for Neural Interfaces at the University of Utah have designed and produced a silicon-based high-density microelectrode array that has been used successfully in mammalian models. The authors investigate the ability to transfer array insertion techniques to humans and examine the acute response of human cortical tissue to array implantation. METHODS: Six patients who were scheduled to undergo temporal lobectomy surgery were enrolled in an Institutional Review Board-approved protocol. Before the patients underwent lateral temporal cortical resection, one or two high-density microelectrode arrays were implanted in each individual by using a pneumatic insertion device. Cortical tissue was then excised and preserved in formalin. The specimens were sectioned and stained for histological examination. Pneumatic insertion of a microelectrode array into human cortex in the operating room was feasible. There were no clinical complications associated with implantation and no evidence of significant insertion-related hemorrhage. Tissue responses ranged from mild cortical deformity to small focal hemorrhages several millimeters below the electrode tines. Based on initial results, the insertion device was modified. A footplate that mechanically isolates a small area of cortex and a calibrated micromanipulator were added to improve the reproducibility of insertion. CONCLUSIONS: A high-density microelectrode array designed to function as a direct cortical interface device can be implanted into human cortical tissue without acute clinical complications. Further modifications to the insertion device and array design are ongoing and future work will assess the functional significance of the tissue reactions observed.

Electrodes, Implanted↗

PubMed2003

Clinical application of robotic telemanipulation system in neurosurgery. Case report.

The NeuRobot is a telecontrolled microscopic micromanipulator system designed for neurosurgical procedures. The unit houses a three-dimensional endoscope and three robot arms that the surgeon operates without direct contact with the patient. The authors have successfully performed robotics-assisted neurosurgical procedures by using the NeuRobot in a 54-year-old man who had a recurrent atypical meningioma. Following the usual preparation of craniotomy and opening of the dura mater, a portion of the tumor was removed using the NeuRobot with the aid of microscopic observation. No complication related to the use of the NeuRobot was encountered and the patient's postoperative course was uneventful. Although various kinds of robots have been developed for use in neurosurgery in recent years, a robotic telemanipulation system capable of performing several surgical tasks has not previously been introduced to clinical neurosurgery. This is the first case report in which neurosurgical manipulation by a robotics system is described.

Humans↗

PubMed2001

Direct imaging of single-molecules: from dynamics of a single DNA chain to the study of complex DNA-protein interactions.

Recent years have seen significant advances in the characterization and manipulation of individual molecules. The combination of single-molecule fluorescence and micromanipulation enables one to study physical and biological systems at new length scales, to unravel qualitative mechanisms, and to measure kinetic parameters that cannot be addressed by traditional biochemistry. DNA is one of the most studied biomolecules. Imaging single DNA molecules eliminates important limitations of classical techniques and provides a new method for testing polymer dynamics and DNA-protein interactions. Here we review some applications of this new approach to physical and biological problems, focusing on videomicroscopy observations of individual DNA chains extended in a shear flow. We will first describe data obtained on the stretching, relaxation and dynamics of a single tethered polymer in a shear flow, to demonstrate that the deformation of sheared tethered chains is partially governed by the thermally driven fluctuations of the chain transverse to the flow direction. Next, we will show how single-molecule videomicroscopy can be used to study in real time DNA folding into chromatin, a complex association of DNA and proteins responsible for the packaging of DNA in the nucleus of an eukaryotic cell.

DNA↗