UV Laser Microbeam Research Articles

GENETIC CHANGES INDUCED IN HIGHER PLANTS BY A UV LASER MICROBEAM

Plant cell walls can be perforated by a UV laser microbeam. Cells and immature pollen grains (microspores) of Brassica napus L. continued to grow in culture after being punctured by a laser pulse. During laser treatment, uptake of buffer into the cytoplasm of cells could be achieved by plasmolyzing cells in a hypertonic solution. Using this approach, plasmid DNA carrying a gene for bacterial glucuronidase was introduced into B. napus cells. The marker gene was expressed by 70% of the treated cells. Likewise, the gene for resistance to the antibiotic hygromycin B was introduced into B. napus cells and was stably integrated into growing cells. Furthermore, DNA was also incorporated into chloroplasts. First, DNA was injected into the cytoplasm and then the organelles were punctured by laser pulses. When a marker gene coding for triazine resistance was introduced into the organelles, transient expression at the level of callus cultures was observed.

Force-feedback joystick as a low-cost haptic interface for an atomic-force-microscopy nanomanipulator

In order to manipulate materials at the nanometer scale, new methods and devices have to be developed. A nanomanipulator interface was designed and implemented in a commercial atomic-force-microscope (AFM) system. With the aid of a positioning joystick, direct positioning of the AFM probe with nanometer precision was possible. A commercial force-feedback joystick served as a haptic interface and provided the user with real-time feeling of the tip–sample interactions. Due to the open design, the manipulator interface could be used with other microscopes of the SPM family. In addition, the nanomanipulator and an UV-laser microbeam for photoablation were combined on an inverted optical microscope. To test the nanomanipulator, human metaphase chromosomes were dissected using both photoablation and mechanical AFM manipulation. The experimental results show that by combining both methods, biological material can be manipulated on different size scales in one integrated instrument. The effects of manipulation on the chromosome were studied in detail by AFM. Sub-400 nm cuts were achieved by photonic ablation. Chromosomal fragments of a size less than of 500 nm could be isolated. By means of mechanical microdissection, different cut sizes ranging from 80 nm to 500 nm could be easily obtained by applying different load forces.

Spindle positioning during the asymmetric first cell division of Caenorhabditis elegans embryos.

Cell division during development in many cases generates daughter cells that differ not only in fate, but also in size. We investigate the mechanisms that ensure proper spindle positioning during such asymmetric divisions using the one-cell stage Caenorhabditis elegans embryo as a model system. We utilized a UV laser microbeam as an in vivo microtubule-severing device to probe the forces driving spindle positioning. Our results indicate that extra-spindle pulling forces acting on the spindle poles dictate spindle position along the anterior-posterior embryonic axis. Importantly, forces acting on the posterior spindle pole appear more extensive than those acting on the anterior one, thus explaining the overall posterior spindle displacement that leads to the asymmetric division of the wild-type one-cell stage embryo. In separate work, we analysed a locus called zyg-8, which plays a key role in ensuring proper spindle positioning. Our data show that zyg-8 is required to promote microtubule growth and/or stability during anaphase. We identified the molecular nature of the zyg-8 locus in the course of a large-scale RNAi-based functional genomics screen. ZYG-8 harbours two notable protein domains: a Ca2+/calmodulin-dependent kinase domain, and a domain related to doublecortin, a human microtubule-associated protein involved in neuronal migration.

Effects of Sodium and Chloride on Neuronal Survival After Neurite Transection

An in vitro investigation was undertaken to study the roles of Na+ and Cl- in mammalian spinal cord (SC) neuron deterioration and death after injury involving physical disruption of the plasma membrane. Individual SC neurons in monolayer cultures were subjected to UV laser microbeam transection of a primary dendrite. Neurons lesioned in modified ionic environments (MIEs) where 50%-75% of the NaCl was replaced with sucrose had higher survival (65%-75%) than neurons lesioned in medium with normal (125 mM) NaCl (28%; p < 0.001). Subsequent experiments found a comparable increase in lesioned neuron survival in MIEs in which only Na+ was replaced with specific ionic substitutes; however, replacement of Cl- was not protective. Electron microscope examinations of neurons fixed <16 min after lesioning showed a dramatic decrease in vesiculation of the smooth endoplasmic reticulum and Golgi apparatus in the low NaCl or low Na+ MIEs. It is hypothesized that Na+ entry after membrane disruption may stimulate elevation of [Ca+2]i leading to ultrastructural disruption and death of injured neurons. The results of these studies suggest that a low NaCl MIE may be useful as an irrigant to limit damage spread and cell death within CNS tissues during surgery or after trauma.

Technical advance: single pollen typing combined with laser-mediated manipulation.

We combined single pollen typing with laser-mediated manipulation. After drilling a hole in the wall of a pollen grain from a dioecious plant (Silene latifolia) with a UV-laser microbeam, the single pollen grain was recovered directly in the cap of a PCR tube, using a non-contact method called laser pressure catapulting. The entire genome of the single pollen grain was then amplified with improved primer-extension-preamplification PCR (I-PEP PCR). Nested PCR with sequence tagged site (STS)-specific primers was used to analyze several loci in the haploid genome. The single copy gene MROS1 was detected in most of the single pollen grains analyzed. Bgl10, which is localized on the Y chromosome, was detected in approximately half of the pollen grains. MROS3 is reported to be localized on the X chromosome. Using inverse PCR, we isolated two genomic clones of MROS3: MROS3A and MROS3B. The single pollen analysis using nested PCR showed that MROS3A and MROS3B are derived from different loci that are not located on the X chromosome. Single pollen typing not only reveals sex chromosome-linkage within the haploid genome, but can also discriminate between alleles and different loci. This method should also be useful for measuring recombination frequencies without genetic crossover analysis.

Laser microdissection and laser pressure catapulting for the generation of chromosome-specific paint probes.

Chromosome-specific paint probes provide a powerful tool with wide applications in cytogenetic analysis. Here, we present a new approach using UV-laser microbeam microdissection in combination with laser-pressure catapulting, which allows the fast isolation of single chromosomes for the generation of chromosome-specific paint probes. To demonstrate the feasibility of this approach, single chromosomes were collected and amplified with degenerate oligonucleotide-primed PCR, hapten-labeled and hybridized onto normal metaphase spreads. Fluorescence in situ hybridization signals revealed specific painting of the respective chromosomes.

Laser microdissection of metaphase chromosomes and characterization by atomic force microscopy

A new experimental setup has been constructed in which a UV laser microbeam and atomic force microscopy (AFM) have been combined on an inverted microscope in order to manipulate and visualize chromosomes with high resolution. The laser beam has been used to dissect Muntjak metaphase chromosomes and was aimed to optimize the physical size of the cuts. The capability of the AFM to visualize biological material with relative ease has been used to characterize the microdissected chromosomes. This work demonstrates that chromosome fiber material can be removed completely at the cut site using appropriate laser power. The minimum cut size achieved with a 337-nm nitrogen UV laser was between 600 and 800 nm. The smallest distance between the cuts was around 500 nm, corresponding to the finest probe for further biochemical use after physical translocation such as the polymerase chain reaction. Limitations on minimizing the cut size due to diffraction-limited focusing and the effects of laser ablation of biomaterial are discussed. © 1997 Society of Photo-Optical Instrumentation Engineers.

Fertilization and early embryology: Zona opening of human embryos using a non-contact UV laser for assisted hatching in patients with poor prognosis of pregnancy

The aim of this study was to examine the safety and the efficiency of a 'non-contact' UV laser to assist hatching through zona opening of human embryos. Between January and November 1995 we performed zona drilling for assisted hatching using a new laser system (PALM UV Laser microbeam), operating in a 'non-contact' mode to create a hole in the zona pellucida of human embryos. In a randomized study, laser zona opening was applied on embryos from two groups of patients with repeated in-vitro fertilization (IVF) failures (two to four attempts): group A was composed of 107 patients who received mixed embryos (216 laser-treated and 223 not treated) and group B of 72 patients who received 218 laser-treated embryos only. Both groups were compared with a control group of 98 patients whose embryos were not laser treated (n = 407) (group C). The mean ages of all groups (38.1, 38.2 and 37.8 years respectively) and the number of IVF attempts (two to four attempts) were similar. The resulting clinical pregnancies were 39 (36.4%) in group A, 32 (44.4%) in group B and 19 (19.3%) in group C. The implantation rates/embryo were 9.3% in A, 16% in B and 5.1% in the control group. In total, 17 normal babies have been delivered (10 in group A and seven in group B). These results show that laser zona drilling increased the pregnancy and implantation rates in all the treated patients. The increase was slight but significant in patients of group A (P < 0.01 and P < 0.02), it was even higher in the patients of group B (P < 0.05).

Fertilization of bovine oocytes induced solely with combined laser microbeam and optical tweezers.

Our purpose was to show that fertilization of oocytes can be obtained solely by laser light-mediated manipulation of gametes. A small channel was drilled into the zona pellucida of bovine oocytes using an ultraviolet (UV)-laser microbeam. Highly diluted cattle sperm were not able to fertilize the laser drilled oocytes. Fertilization was achieved only when three to five cattle sperm were trapped with optical tweezers and inserted directly through the laser drilled hole into the perivitelline space. After 20 hr, 3 of 79 (3.8%) oocytes revealed two pronuclei and a sperm tail within their cytoplasm. Cattle sperm are difficult to catch. Therefore, the gametes had to remain for about 20 min in room atmosphere, which might be the reason for the low fertilization results. The results indicate that a combined UV-laser microbeam and optical tweezers trap can be used successfully for "noncontact" microinsemination procedures.

Responses to localized UV-laser irradiation are different between Bombyx and Drosophila eggs.

It has been found previously that in the silkworm, Bombyx mori, localized ultra-violet (UV) irradiation of eggs at as early as the fertilization stage could induce cuticle defects in resultant larvae. To compare the responses to the UV irradiation between Bombyx and Drosophila, embryos at various stages were irradiated with a scanning UV-laser microbeam (355 nm) over an area of 1.4-1.6% of the total egg surface. The results revealed that: (i) in Bombyx, embryonic mortality after irradiation was always low and independent of the stage of embryos at irradiation, while in Drosophila, the mortality was high at the cleavage stage and decreased as the development proceeded; (ii) in Bombyx, frequency of defective larvae after the irradiation was always high and independent of the stage of embryos at irradiation, whereas in Drosophila, high defect frequencies could be obtained only when irradiations were performed at or after the syncytial blastoderm stage; (iii) in Bombyx, the hypoderm surrounding the site of defect was always fused together to produce a continuous cuticle, while in Drosophila, the surrounding hypoderm was rarely fused together thus producing a hole in the cuticle; (iv) in Bombyx, the extent of the defects was constant from the fertilization through to the cellularization stage, whereas in Drosophila, the extent of the defects was largest at the cleavage stage and decreased as the development proceeded. These results suggest that there may be differences between the two insects in the mechanisms and/or the timing of embryonic pattern specification.

Zona drilling and sperm insertion with combined laser microbeam and optical tweezers

A combined UV-laser microbeam and optical-tweezers trap was used to perform laser zona drilling and subzonal insemination in cattle. Using a precisely focused UV-laser microbeam, a small channel of about 10 microns in diameter was drilled into the zona pellucida. With a three-dimensional optical-tweezers trap, a single sperm was caught and transported through the laser-drilled hole directly into the perivitelline space. Furthermore, the sperm was brought into close contact with the oolemma to facilitate sperm-oocyte fusion. Using the laser-microscope system, noncontact, entirely sterile, and highly selective micromanipulation of gametes can be achieved with no need for mechanical microtools. Laser micromanipulation seems to be less detrimental to the gametes and is comparatively is easy to perform. Thus, the combined UV-laser microbeam and optical tweezers trap may be a helpful tool for IVF procedures.

Astral and spindle forces in PtK2 cells during anaphase B: a laser microbeam study

Rat kangaroo kidney epithelium (PtK2) cells develop prominent asters and spindles during anaphase B of mitosis. It has been shown that severing the spindle at early anaphase B in living PtK1 cells results in a dramatic increase in the rate of pole-pole separation. This result suggested that the asters pull on the spindle poles, putting tension on the spindle, while the spindle acts as a governor, limiting the rate of pole separation. To further test these inferences, we used a UV-laser microbeam to damage one of the two asters in living PtK2 cells at early anaphase B and monitored the effects on individual spindle pole movements, pole-pole separation rates and astral microtubules (MTs). Irradiation at the estimated position of a centrosome greatly reduced its array of astral MTs and nearly stopped the movement of the irradiated pole, whereas the sister pole retained its normal array of astral MTs and actually accelerated. Control irradiations, either close to the estimated position of the centrosome or beside the spindle at the equator, had little or no effect on either spindle pole movements or astral MTs. These results support the inferences that during anaphase B in living PtK cells, the central spindle is under tension generated by pulling forces in the asters (presumably MT-mediated) and that the spindle generates counterforces that limit the rate of pole separation. The results also suggest that the central spindle in living PtK cells may be able to generate a pushing force.

Laser induced cell fusion in combination with optical tweezers: The laser cell fusion trap

A single-beam gradient force optical trap was combined with a pulsed UV laser microbeam in order to perform laser induced cell fusion. This combination offers the possibility to selectively fuse two single cells without critical chemical or electrical treatment. The optical trap was created by directing a Nd:YAG laser, at a wavelength of 1.06 microns, into a microscope and focusing the laser beam with a high numerical aperture objective. The UV laser microbeam, produced by a nitrogen-pumped dye laser (366 nm), was collinear with the trapping beam. Once inside the trap, two cells could be fused with several pulses of the UV laser microbeam, attenuated to an energy of approximately 1 microJ/pulse in the object plane. This method of laser induced cell fusion should provide increased selectivity and efficiency in generating viable hybrid cells.

Application of laser optical tweezers in immunology and molecular genetics

Optical tweezers, based on a compact diode pumped Nd:YAG laser providing 350 mW at 1,064 nm coupled into a Zeiss IM 35 microscope, were used to sort CD4+ T cells into a capillary for further mechanical handling and to establish contact between single human natural killer (NK) cells and human erythroleukemia cells (K562) as targets. After contact and a lag phase of a few tens of seconds, the target cell starts to change its morphology and membrane blebbing occurs. The kinetics of the attack of the NK cell on K562 cells is not straightforward but governed by temporal oscillations in the shape of the target cell (zeosis). In a second application, the optical tweezers are combined with a UV laser microbeam based on a pulsed UV laser and with flow cytometry and sorting. With the pulsed laser, segments of sorted chromosome 1 of the chinese hamster karyotype (CHV 79) can be easily micro-dissected and subsequently collected using the optical tweezers. This allows preparation of a few hundred chromosome segments per day without mechanical contact and in an absolutely sterile way and thus may provide an interesting basic technique in any type of genome sequencing project.

Laser Microdissection in the Search for the Molecular Basis of Diseases A Molecular Library from a Human Chromosome Segment

AbstractIt is shown that a UV Laser microbeam can replace mechanical microdissection for the preparation of human chromosome segment specific DNA libraries. 450 DNA fragments are prepared from segment q22‐32 of human chromosome 7. 220 of them are of low copy number. 20 of the clones analyzed by gel electrophoresis have an average molecular weight of 3.8 kb. Three out of a sample of four clones can be localized to chromosome bands 7q22‐q32.

Telomeric sequences derived from laser-microdissected polytene chromosomes

Telomeric fragments from salivary gland squashes of Drosophila melanogaster Oregon R. were produced by a new microdissection technique, UV laser microbeam dissection. Microdissection, an essential step in microcloning procedures, is usually performed using micromanipulators and microneedles. Recently it has been shown that microdissection can be improved to very high precision if a laser coupled into a microscope is used. A laser microbeam, generated by an excimer pumped dye laser, allows chromosomes to be cut into slices of less than 0.5 micron. Here it is shown, that single copy DNA probes prepared from Drosophila chromosomes by laser microdissection and microcloning relocalize to the chromosomal regions from which they are derived. The combination of laser technique and microcloning provides an advantageous approach for rapid genetic analysis with potential for the study of genetic diseases and genome mapping.

Laser spectroscopy for studying chemical processes

In recent years, various methods have been developed to observe and to influence the course of chemical reactions using laser radiation. By selectively increasing the translational, rotational, and vibrational energies and by controlling the relative orientation of the reaction partners with tunable infrared and UV lasers, direct insight can be gained into the molecular course of the breaking and re-forming of chemical bonds. As exmaples for the application of lasers in chemical synthesis the production of monomers and catalysts is discussed. The application of linear and nonlinear laser spectroscopic methods, such as laser-induced fluorescence (LIF), Coherent anti-Stokes Raman Scattering (CARS), infrared-absorption measurements with tunable diode and molecular lasers is described for non-intrusive observation of the interaction of transport processes with chemical reactions used in industrial processes with high temporal, spectral and spatial resolution. Finally the application of a UV laser microbeam apparatus in genetic engineering for laser-induced cell fusion, genetic transformation of plant cells as well as diagnosis of human diseases by laser-microdissection of chromosomes is described.

Microperforation of plant tissue with a UV laser microbeam and injection of DNA into cells

Microperforation of plant tissue with a UV laser microbeam and injection of DNA into cells

Cytoplasmic damage gradients in dendrites after transection lesions.

Ultrastructural changes in the cytoplasm of transected dendrites have been studied with serial section electron microscopy 10 to 12 min after the lesion. Dendrites from large multipolar neurons in mouse spinal monolayer cultures were selected 4 weeks after seeding and cut with a pulsed UV laser microbeam. The data reveal cytoplasmic damage gradients which decrease in severity with distance from the lesion. The damage is expressed primarily in cytoskeletal disruption, the swelling and vacuolation of mitochondria (MC), and the swelling and vesiculation of the endoplasmic reticulum (ER). For a 3-4 microns diameter dendrite lesioned 150 microns from the soma and fixed 10 min after transection, the following results can be reported. The number of microtubules (MT) decreases almost linearly with a slope of 40 MT/10 microns until MT completely disappear 20 microns from the lesion. Neurofilaments (NF) decrease at approximately 50 NF/10 microns, with 20% of these structures still intact at or near the lesion. MC show both gradual and explosive swelling in the damaged process. The onset of these two phases of MC swelling are correlated with the initial and total loss of MT respectively. MT and NF densities in cut dendrites are drastically different from those in control processes and suggest a possible retrograde redistribution of these organelles. Damage from external shockwaves that cause process distortion but not transection results in a less severe but nevertheless similar loss of MT and NF.

Gene transfection by laser cell surgery.

The exposure to a uv laser microbeam can modify a selected portion of cell membrane to be temporarily permiative, thereby allowing a foreign substance to enter living cell. As a first application of laser microbeam to the field of bionicis, the method was employed in incorporating the exogenous gene into cells. The method was found to offer a marked advantage over the existing ones in the success rate of gene incorporation, effi-ciency to large throughput, and in versatility to broad cell-type.