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The laser microbeam trap as an optical tool for living cells.

Pulsed ultraviolet lasers coupled into a microscope can be used for micromanipulation of cells and subcellular structures. In addition, continuous infrared lasers can be used as ultrafine optical tweezers (or synonymously: optical trap). The pulsed UV lasers (for example excimer lasers or nitrogen lasers) can be used as optical scalpels for the preparation of protoplasts from plant root hairs. The precise microdissection of chromosomes with the laser microbeam provides access to chromosome segments where a specific gene is supported to be localized. From such segments, specific DNA libraries can be prepared for the search after such genes or markers in their environment. With the optical trap contact between effector cells of the immune system and their target cells can be established in a very simple and gentle way. The kinetics of the attack of a natural killer on an erythroleukemia cell can be studied from the first seconds after contact. Isolated plant cells as well as cells in a plant embryo tissue can be perforated and DNA or fluorescent molecules can be injected. From the temperature dependence of laser induced membrane lesions one can obtain predictions on laser induced cell fusion, which can be performed at slightly modified irradiation conditions under total microscopic control. Since focusing into the depth of a cell with an accuracy of better than a micrometer is possible, one can work on subcellular structures in the interior of a cell without opening it. For example, in rapeseed protoplasts subcellular structures such as mitochondria or chloroplasts can be perforated or moved out of their original position. Interestingly, such structures find their way back into the original position after the laser is switched off. From their speed one can obtain estimates on intracellular viscoelasticity.

Animals↗

[Stretched polytene chromosomes--model for studying the functional organization of eukaryotic chromosomes].

To localize functional loci on cytological maps of polytene chromosomes we propose to use 10-100 times stretched chromosomes. Three different ways of stretchening are briefly considered: the squash tissue preparation, when chromosomes are stretched by hydrodynamical forces; the treatment of isolated polytene chromosomes in 10-minus 4M EDTA OR 0.8M NaCL with subsequent change of these solution for saline when abrupt structural changes occur in chromosomes and they become morphologically homogeneous threads (Gruzdev and Belaya, 1973); and, finally, the use of microneedles of the micromanipulator. After an intense (ca. 100 times) stretchening, the autoradiography is sufficient to localize the loci within one micron length of double helical DNA molecule.

Chromosome Mapping↗

Laser tweezers are sources of two-photon excitation.

The most important application of continuous wave (cw) near infrared (NIR) microbeams in cellular and molecular biology are single-beam gradient force optical traps, also called "laser tweezers". Laser tweezers have been used for optical picoNewton force determination as well as for 3D cellular and intracellular micromanipulation, such as optical spermatozoa transportation in laser-assisted in vitro fertilization. Light intensities in the MW/cm2 range are necessary to confine motile spermatozoa in the optical trap. The enormous photon concentration in space and time results in non-resonant two-photon excitation of endogenous and exogenous absorbers with electronic transitions in the ultraviolet and visible spectral range. Trap-induced two-photon excitation of intracellular flurorophores can be used to study metabolism and vitality of motile cells without additional fluorescence excitation sources. Therefore, laser tweezers as sources of two-photon excitation may act as novel non-linear tools in cell diagnostics. The far red/NIR trapping radiation, in particular <800 nm, may also excite endogenous absorbers such as NAD(P)H, flavins, porphyrins and cytochromes. Excitation of these cellular absorbers may result in oxidative stress via type I and type II photooxidation processes. Severe non-linear-induced cell damage in a variety of cells confined in <800 nm traps was found. Two-photon induced destructive effects are enhanced in multimode traps due to longitudinal mode-beating phenomena. Pulsed laser sources are not suitable for safe optical trapping of living cells. The use of single frequency long-wavelength NIR traps (800 nm-1200 nm) for vital cell handling is recommended.

Animals↗

Systemic hyperoxia decreases vascular endothelial growth factor gene expression in ischemic primate retina.

OBJECTIVES: To determine whether systemic hyperoxia can reverse retinal hypoxia and decrease vascular endothelial growth factor (VEGF) gene expression in ischemic nonhuman primate retina. METHODS: Six eyes of 3 cynomolgus monkeys were studied. Retinal ischemia was induced via laser vein occlusion and confirmed with fluorescein angiography. Animals were randomly assigned to treatment with either 21% or 100% inhaled oxygen. Arterial PO2 was monitored while systemic acid-base status was maintained. An oxygen microelectrode on a micromanipulator was used to measure preretinal oxygen concentrations in ischemic and nonischemic retina in situ. RNA was isolated from fresh whole retinas, and VEGF messenger RNA levels were quantified with Northern blotting. RESULTS: The preretinal PO2 in ischemic retina was less than the PO2 in nonischemic retina in animals breathing 21% oxygen (intervascular zone PO2, 14.3+/-0.53 vs 21.8+/-0.55 mm Hg; P=.002). After 8 hours of systemic hyperoxia (arterial PO2, 512+/-18 mm Hg), the preretinal PO2 in ischemic retina increased to 166.2+/-15.6 mm Hg (21.8% oxygen) and retinal VEGF messenger RNA levels were reduced by an average of 55%. CONCLUSIONS: These data demonstrate that systemic hyperoxia can lower retinal VEGF gene expression and reoxygenate ischemic adult primate retina.

Animals↗

Carbon dioxide laser serial microtrapdoor flap excision of subglottic stenosis.

The recent development of microspot micromanipulators for the carbon dioxide laser with 250-microns spot sizes has greatly facilitated the extension of endolaryngeal procedures to children and neonates. It is possible to accurately develop microtrapdoor flaps within the limited exposures in the pediatric subglottis. We have used serial microtrapdoor flaps in the excision of subglottic scar tissue in eight children. In five children, the stenosis was improved with relief of stridor. In three children, the flaps were used as an adjunct to laryngotracheoplasty. The surgical techniques of this procedure are detailed in this report.

Child↗

Oxygen tension measurements of head and neck cancers.

OBJECTIVE: There is continued investigation of agents capable of overcoming hypoxic cell radioresistance. The evaluation of tumor oxygenation, therefore, assumes great importance. Previous efforts to measure tumor oxygen tension (PO2) using polarographic methods were limited by the size of the electrodes and efficiency of measurements. A new system employing fine-needle electrodes and a computerized micromanipulator was evaluated. DESIGN: Prospective study. PATIENTS: Sixteen patients with cancers of the head and neck, seen at Stanford (Calif) University Medical Center and Santa Clara Valley Medical Center, San Jose, Calif, between March 1, 1991, and July 31, 1991. Of the 16 tumors, 14 were squamous cell carcinomas; 11 were previously untreated. In two patients, measurements were repeated during radiation therapy. In three patients, measurements were taken under conditions of varied inspired oxygen during anesthesia. In seven patients, subcutaneous tissue (SQ) measurements were made for comparison. RESULTS: The mean (+/- SD) PO2 of all tumors was 25.6 +/- 20.2 mm Hg, with a range of 2.3 to 76.4 mm Hg. When only squamous cell carcinomas were considered, the mean tumor PO2 was 22.7 +/- 16.0 mm Hg. The mean SQ measurement was 57.2 +/- 12.8 mm Hg. In all cases (seven of seven) in which SQ data were available, the tumor PO2 was lower than the SQ PO2 by an average of 41.8 mm Hg. The mean tumor PO2 increased in two of three cases of hyperoxia and decreased in the the third. The mean tumor PO2 increased in one patient after 25 Gy of radiotherapy and remained the same in the other. CONCLUSIONS: These measurements suggest that there is significant interindividual variability in the PO2 of head and neck cancers. Squamous cell cancers are generally less well oxygenated than normal SQ tissue. The PO2 histograph identified patients with a low mean tumor PO2.

Aged↗

High-sensitivity analysis and sequencing of peptides and proteins by quadrupole ion trap mass spectrometry.

This paper describes experience with the commercially available LCQ quadrupole ion trap mass spectrometer applied to the off-line analysis of peptides and proteins. The standard front end of the electrospray probe was replaced with a micromanipulator which, with the aid of a magnifying device, allowed the use of a variety of miniaturized spraying interfaces. The low sample consumption and extended analysis times of these devices were ideally suitable to obtain improved results in terms of sensitivity and mass accuracy. This needed a careful optimization of the number of ions stored inside the trap (ion target parameter) and required spectrum averaging of many scans. A method is presented for the mathematical fitting of ZoomScan spectra to theoretical isotopic distributions, which allowed the mass determination of large peptides with more accuracy than that achieved by conventional deconvolution algorithms. A very simple on-line desalting configuration is also described which needed no external micro-high-performance liquid chromatographic pumps, and can be easily mounted using the built-in syringe delivery system of the LCQ. This set-up allowed extended analysis times of 'in-gel' protein digests in subpicomole amounts. Finally, the multiple fragmentation capabilities of the ion trap were found to be extremely useful for the analysis of peptide modifications such as phosphorylation and for sequencing individual peptides from highly complex MHC-bound peptide pools.

Animals↗

Practical histological microdissection for PCR analysis.

Recovery of cells by histological microdissection is increasingly used for analysis by polymerase chain reaction (PCR) or microchemical techniques. This paper describes techniques of histological microdissection. Sections of archival formalin-fixed, paraffin-embedded tissue up to 15 years old were mounted on plain glass slides. Sections 6-7 microns in thickness stained with toluidine blue were dissected under proteinase K buffer solution, using an electrolytically sharpened tungsten needle in a bacteriological loop-holder and a Leitz mechanical micromanipulator (model M). Detached cell groups were recovered in a silicone-coated pipette tip for PCR analysis after digestion in 25-50 microliters of proteinase K (500/ml) in TRIS-HCl buffer (pH 8.3). Consistent amplification and analysis of microsatellite loci were obtained from 2 microliters of crude lysate using 28-30 cycles of PCR incorporating a 32P 5'-end-labelled primer, electrophoresis under denaturing conditions on 6 per cent polyacrylamide gels, and autoradiographic detection.

Alleles↗

Applications of the FIB lift-out technique for TEM specimen preparation.

A site-specific technique for cross-section transmission electron microscopy specimen preparation of difficult materials is presented. A focused ion beam was used to slice an electron transparent membrane from a specific area of interest within a bulk sample. Micromanipulation lift-out procedures were then used to transport the electron-transparent specimen to a carbon-coated copper grid for subsequent TEM analysis. The FIB (focused ion beam) lift-out technique is a fast method for the preparation of site-specific TEM specimens. The versatility of this technique is demonstrated by presenting cross-sectioned TEM specimens from several types of materials systems, including a multi-layered integrated circuit on a Si substrate, a galvanized steel, a polycrystalline SiC ceramic fiber, and a ZnSe optical ceramic. These specimens have both complex surface geometry and interfaces with complex chemistry. FIB milling was performed sequentially through different layers of cross-sectioned materials so that preferential sputtering was not a factor in preparing TEM specimens. The FIB lift-out method for TEM analysis is a useful technique for the study of complex materials systems for TEM analysis.

Ceramics↗

Hypo-osmotic swelling can accurately assess the viability of nonmotile sperm.

Viable, healthy sperm are preferred for oocyte fertilization with intracytoplasmic sperm injection. Currently, motility is the most widely applied measure of sperm viability. However, with this criterion, viable but immotile sperm are overlooked as candidates for micromanipulation. Supravital stains identify viable sperm but may be toxic to the gamete or embryo. We tested the hypothesis that hypo-osmotic swelling, developed to assess sperm membrane integrity, can accurately determine sperm viability. Thawed sperm from 12 fertile donors were exposed to a hypo-osmotic solution and to two supravital stains. A total of 2,010 sperm were assessed for tail coiling after a 30-min exposure to hypoosmotic solution. By supravital stains, 31% of thawed sperm were viable; 23% showed tail coiling. Among coiled-tail sperm, 100% were viable by supravital stain. As a measure of viability, tail coiling exhibited a sensitivity of 30%, specificity of 100%, and positive predictive value of 100% compared to supravital stains. With a 60-min hypo-osmotic incubation, the specificity (89%) and positive predictive value (78%) decreased significantly (P < 0.05). Therefore, hypo-osmotic swelling accurately detects viable sperm among a nonmotile population. Assay accuracy, however, is very sensitive to the incubation time in hypo-osmotic solution.

Benzimidazoles↗

Nuclear transfer of porcine embryos using cryopreserved delipated blastomeres as donor nuclei.

Nuclear transfer protocol for the pig using cryopreserved delipated four- to eight-cell and morula stage embryos as nucleus donors was developed. Donor embryos, which had been delipated by micromanipulation following centrifugation for polarizing cytoplasmic lipid droplets, were cryopreserved with 1.5 M 1,2-propanediol and 0.1 M sucrose. Recipient cytoplasts were prepared from ovulated oocytes. Activation of oocytes could be induced more efficiently when electric stimulation was given 53 hr after the hCG injection or later (66-83%), compared with 52 hr or earlier (11-16%, P < 0.05), suggesting that aging after ovulation may be required for in vivo matured porcine oocytes to be activated by electric stimuli. Membrane fusion rates between donor blastomeres and enucleated oocytes were 88% (127/144) and 97% (56/58, P > 0.05) for the four- to eight-cell and morula stage embryos, respectively. In vitro developmental rates to the two-cell (53/100 vs. 35/65), four-cell (34/100 vs. 26/65), and morula stage (17/100 vs. 18/65) were the same between the nuclear transfer embryos with four- to eight-cell and morula nuclei. However, more embryos reconstituted with morula nuclei developed to blastocysts (15% vs. 6%, P < 0.05). These data demonstrated that blastomeres of cryopreserved, delipated porcine embryos can be used as donor nuclei for nuclear transfer. Frozen-thawed, delipated blastomeres can be efficiently isolated and fused, and therefore provide a useful source of donor nuclei.

Analysis of Variance↗

Effect of developmental stage on bovine oocyte plasma membrane water and cryoprotectant permeability characteristics.

Knowledge of bovine oocyte plasma membrane permeability characteristics at different developmental stages in the presence of cryoprotective agents (CPAs) is limited. The objective of this study was to determine the oolema hydraulic conductivity (Lp), cryoprotectant permeability (P[CPA]), and reflection coefficient (sigma) for immature (germinal vesicle stage, GV) and in vitro-matured (metaphase II, MII) bovine oocytes. Two commonly used cryoprotective agents, dimethyl sulfoxide (DMSO) and ethylene glycol (EG), were studied. Osmometric studies were performed using a micromanipulator connected to an inverted microscope at 22 +/- 2 degrees C. Each oocyte was immobilized via a holding pipette, and osmotically induced volume changes over time (dv/dt) were recorded. The Lp values for GV and MII oocytes in DMSO (L(p)DMSO) were 0.70 +/- 0.06 and 1.14 +/- 0.07 microm/min/atm (mean +/- SEM) and in EG (L(p)EG) were 0.50 +/- 0.06 and 0.83 +/- 0.07 microm/min/atm, respectively. Estimates of P(DMSO) for GV and MII oocytes were 0.36 +/- 0.03 and 0.48 +/- 0.03 microm/sec, and PEG values for GV and MII oocytes were 0.22 +/- 0.03, 0.37 +/- 0.03 microm/sec, respectively. The values for GV and MII oocytes in DMSO (sigma[DMSO]) were 0.86 +/- 0.03 and 0.90 +/- 0.04 and in EG (sigma[EG]) were 0.94 +/- 0.03 and 0.76 +/- 0.04, respectively. These data demonstrate that bovine oolema permeability coefficients to water and cryoprotectants change after in vitro maturation. Furthermore, the bovine oocyte P(DMSO) is higher than the P(EG). These results may provide a biophysical basis for developing criteria for choosing optimal CPAs and for minimizing damage during addition and removal of the CPAs. Additionally, these data support the hypothesis that different procedures may be required for optimal cryopreservation of different oocyte developmental stages.

Animals↗

In vitro and early in vivo development of sheep gynogenones and putative androgenones.

Genomic imprinting, where only one of the two parental genes is expressed, occurs in many phyla. In mammals, however, this phenomenon has been primarily studied in mice, and to a lesser extent, in humans. To understand how genomic imprinting may affect development in other species, particularly those with a different mode of placental development from mice and humans, 339 sheep zygotes were micromanipulated to contain either 2 large (presumptive male) or 2 small (presumptive female) pronuclei. One hundred and twenty-seven of these embryos and 86 manipulated and nonmanipulated control embryos were transferred to recipient ewes over 3 breeding seasons. Twenty-one control and 7 experimental conceptuses were recovered on day 21. Four of these conceptuses derived from zygotes with 2 small pronuclei were identified by karyotyping to be gynogenones (maternal-derived genome). While the gross morphology of the embryos appeared no different to those of normal controls, the extra-embryonic tissue from the conceptuses showed some hypertrophy and hypervascularization. Preliminary Northern blots of mRNA from allantoic and trophoblast tissue showed an overexpression of H19 and an underexpression of IGF2. Although the sheep gynogenetic phenotype contrasts with that seen in mice, these two genes appear to be similarly differentially expressed.

Animals↗

Temperature-dependent osmotic behavior of germinal vesicle and metaphase II stage bovine oocytes in the presence of Me2SO in relationship to cryobiology.

Plasma membrane permeability coefficients and their activation energies (Ea) for water (Lp) and dimethyl sulfoxide (PMe2SO) as well as the reflection coefficient (sigma) were determined for germinal vesicle (GV) and metaphase II (MII) bovine oocytes. A micropipette perfusion technique was used with a temperature controlled circulation chamber, which was adapted to a micromanipulator. Experiments were performed at five different temperatures (30, 20, 10, 4 and -3 degrees C). The Kedem and Katchalsky model was assumed and L(p), P(Me2SO) and sigma were estimated. Estimated permeability values from the experimental temperatures were then applied to Arrhenius plots In(Lp) or In(PMe2SO) vs 1/Temperature (K) to estimate the activation energies (Ea) for L(p)Me2SO and P(Me2SO). The estimated E(a) for L(p)Me2SO for GV and MII oocytes were 23.84 Kcal/mol and 8.46 Kcal/mol, respectively. The E(a) for P(Me2SO) were 21.0 Kcal/mol and 23.20 Kcal/mol, respectively. The correlation (r2) for these linear regression plots for GV oocytes were 0.83 and 0.95 for L(p)Me2SO and P(Me2SO), respectively. For MII oocytes, r2 values were 0.95 and 0.99 for L(p)Me2SO and P(Me2SO), respectively. There was a possible discontinuity detected in the Arrhenius plot for L(p)Me2SO for GV oocytes. A significant decrease of the reflection coefficient was observed at 10 degrees C compared to other experimental temperatures. These data provide a fundamental basis that should be taken into account for low temperature preservation of bovine oocytes in the presence of Me2SO.

Animals↗

Smooth muscle electromyography of the urinary bladder.

To elucidate smooth muscle activity of the urinary bladder, we utilized an optimized animal model and a specially developed, computer-aided data acquisition and analysis system for bioelectrical signals. Twenty-five Wistar rats were pharmacologically paralyzed and artificially respirated. The urinary bladder was exposed by a suprapubic midabdominal incision, and both ureters were ligated to prevent physiological filling of the bladder. The bladder was initially emptied by slight manual pressure and was then filled via a transurethral catheter in 0.1-ml steps to a maximum of 0.45 ml with physiological saline. A custom-made, gold-plated needle electrode was tangentially guided by a micromanipulator to the smooth muscle of the bladder dome, and the recordings commenced. Furthermore, smooth muscle EMG recordings of the bladder were performed after pharmaco-stimulation of the detrusor with carbachol. Initial results demonstrate that, with the animal model presented here, it is possible to record reproducible and almost artifact-free smooth muscle activity from the urinary bladder. All experiments displayed a stochastic distribution of similar electrical events, increasing in appearance and amplitude with increased bladder volume and after pharmacostimulation with carbachol. Two-dimensional power spectrum analysis revealed a main signal frequency below 1 Hz.

Animals↗

In vitro fertilization by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is the latest, and by far the most efficient, variant of micromanipulation-assisted fertilization, whereby a single spermatozoon is selected, aspirated into a microinjection needle and injected to the oocyte cytoplasm. The development of this technique is mainly linked to application in human assisted reproduction for which it enables fertilization with defective spermatozoa that would not otherwise be able to penetrate an oocyte by their proper means. Because ICSI by-passes many steps of the natural fertilization process, it offers an extremely interesting model for the study of basic mechanisms underlying fertilization. This is particularly true for oocyte activation, whose mechanism needs to be revisited in light of the current ICSI research. The massive application of ICSI in human infertility treatment also represents a huge laboratory in which the impact of different genetic and epigenetic anomalies of the male gamete on fertilization and embryonic development can be studied.

Female↗

Single sperm typing demonstrates that reduced recombination is associated with the production of aneuploid 24,XY human sperm.

To account for the increased proportion of paternal nondisjunction in 47,XXY males as compared to other trisomies, it has been suggested that the XY bivalent, with its reduced region of homology, is particularly susceptible to nondisjunction. Molecular studies of liveborn Klinefelter syndrome (47,XXY) individuals have reported an association between the absence of recombination in the pseudoautosomal region and nondisjunction of the XY bivalent. In this study we examined single sperm from a normal 46,XY male to determine if there is any alteration in the recombination frequency of aneuploid disomic 24,XY sperm compared to unisomic sperm (23,X or Y). Two DNA markers STS/STS pseudogene and DXYS15 were typed in sperm from a heterozygous man to determine if recombination had occurred in the pseudoautosomal region. Individual unisomic sperm (23,X or Y) were isolated using a FACStar(Plus) flow cytometer into PCR tubes. To identify disomic 24,XY sperm, 3-colour FISH analysis was performed with probes for chromosomes X,Y and 1. The 24,XY cells were identified using fluorescence microscopy, each disomic sperm was scraped off the slide using a glass needle attached to a micromanipulator and then put into a PCR tube. Hemi-nested PCR analysis of the two markers was performed to determine the frequency of recombination. A total of 329 unisomic sperm and 150 disomic sperm have been typed. The frequency of recombination between the two DNA markers was 38.3% for the unisomic sperm, similar to frequencies previously reported. The 24,XY disomic sperm had an estimated recombination frequency of 25.3%, however, a highly significant decrease compared to the unisomic 23,X or 23,Y sperm (chi(2) = 10.7, P = 0.001). This direct analysis of human sperm indicates that lack of recombination in the pseudoautosomal region is a significant cause of XY nondisjunction and thus Klinefelter syndrome.

Aneuploidy↗

Microvascular anastomoses performed in rats using a microsurgical telemanipulator.

OBJECTIVE: To determine the feasibility of performing microsurgical procedures with a remote telemanipulator using a rat femoral artery anastomosis model. MATERIALS AND METHODS: A remote telemanipulator system was developed that enabled precision movements to be performed at up to 30x magnification. Ten 1-mm femoral artery anastomoses were performed in rats using the telemanipulator, and results were compared to those from a control group in which the procedure was performed with conventional microsurgical techniques. Study endpoints included anastomosis completion time, short-term patency, and procedural complications. Statistical analysis was performed using Student's t-test. RESULTS: All anastomoses performed by remote telemanipulation and by conventional microsurgery were completed successfully. Anastomosis completion times were 100.0 +/- 18.6 minutes in the telemanipulator group and 38.8 +/- 5.0 minutes using conventional techniques (p < 0.001). Patency in both groups at 5 minutes and at one hour was 100%. No intraoperative complications were encountered. Postmortem ex vivo examination of the excised arterial segment revealed no technical defects in either group. CONCLUSIONS: Complex procedures requiring a high degree of precision and dexterity can be performed using an electromechanical interface specifically designed for micromanipulation. Performance limitations are similar to those previously reported for remote surgical teleoperation, and most likely reflect incompletely characterized restrictions on multi-sensory information.

Anastomosis, Surgical↗