Anatomical study of the otosclerotic stapedial footplate.
Explore the source record for details and available documents.
Ear Ossicles↗
SEARCH · Search PubMed
Results for “MICROMANIPULATION”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Computer-assisted system combining image analysis and chromosome dissection.
Explore the source record for details and available documents.
Chromosomes↗
Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light.
A quadrant photodiode placed in the back-focal plane of the microscope of a laser trap provides a high-resolution position sensor. We show that in addition to the lateral displacement of a trapped sphere, its axial position can be measured by the ratio of the intensity of scattered laser light to the total amount of the light reaching the detector. The addition of the axial information offers true three-dimensional position detection in solution, creating, together with a position control, a photonic force microscope with nanometer spatial and microsecond temporal resolution. The measured position signals are explained as interference of the unscattered trapping laser beam with the laser light scattered by the trapped bead. Our model explains experimental data for trapped particles in the Rayleigh regime (radius a <0.2lambda) for displacements up to the focal dimensions. The cross-talk between the signals in the three directions is explained and it is shown that this cross-talk can be neglected for lateral displacements smaller than 75 nm and axial displacements below 150 nm. The advantages of three-dimensional single-particle tracking over conventional video-tracking are shown through the example of the diffusion of the GPI-anchored membrane protein Thy1.1 on a neurite.
Animals↗
Distinct cytogenetic alterations in squamous intraepithelial lesions of the cervix revealed by laser-assisted microdissection and comparative genomic hybridization.
BACKGROUND: It has been established that comparative genomic hybridization (CGH) on Papanicolaou-stained cervical smears can be used to identify chromosomal imbalances. METHODS: In this study, the authors identified normal and dysplastic squamous epithelial cells cytologically, eliminated surrounding bacteria or leukocytes by a ultraviolet laser microbeam under microscopic control, and scraped out the cell groups of interest by a microdissection system. In 3 cases of squamous intraepithelial lesions (SIL), a total of 9 samples of dysplastic (n = 6) and nontumorous cells (n = 3) were investigated, each of them consisting of 3-40 cells. The DNA was amplified by degenerate oligonucleotide primed PCR (DOP-PCR) and used for CGH. RESULTS: Analyses of all nontumorous cell groups resulted in fluorescence ratio profiles that showed no deviation from the normal range, confirming that no methodologic artefacts have been produced. The CGH profiles from dysplastic cells, however, showed various chromosomal imbalances affecting six to nine different chromosomes. The most frequent gains in DNA were observed on chromosomes 1p, 2q, 4, and 5, whereas losses were found on chromosomes 6q and 13q. CONCLUSIONS: The results of this study demonstrate the feasibility and reliability of CGH on microdissected cell samples of routinely processed cervical smears. To the authors' knowledge, this is the first study reporting the use of CGH on cervical routine smears. This approach offers the opportunity to investigate sequence copy number changes in small, morphologically well-defined groups of dysplastic cells. It may, therefore, serve as a cytogenetic screening test for identifying chromosomal aberrations in precancerous lesions that are associated with a high risk for progression to invasive cancer.
Chromosome Aberrations↗
Feasibility study of magnetic resonance imaging-guided intranasal flexible microendoscopy.
Interventional magnetic resonance imaging (MRI) offers potential advantages over conventional interventional modalities such as X-ray fluoroscopy, ultrasonography, and computed tomography (CT). In particular, it does not use ionizing radiation, can provide high-quality images, and allows acquisition of oblique sections. We have carried out a feasibility study on the use of interventional MRI to track a flexible microendoscope in the paranasal sinuses. In this cadaver study, high-speed MRI was used to track a passive marker attached to the end of the endoscope. Automatic image registration algorithms were used to transfer the coordinates of the endoscope tip into the preoperative MRI and CT images, enabling us to display the position of the endoscope in reformatted orthogonal views or in a rendered view of the preoperative images. The endoscope video images were digitized and could be displayed alongside an approximately aligned, rendered preoperative image. Intraoperative display was provided in the scanner room by means of an liquid crystal display (LCD) projector. We estimate the accuracy of the endoscope tracking to be approximately 2 mm.
Algorithms↗
Laser-guided direct writing of living cells.
To perform their myriad functions, tissues use specific cell-cell interactions that depend on the spatial ordering of multiple cell types. Recapitulating this spatial order in vitro will facilitate our understanding of function and failure in native and engineered tissue. One approach to achieving such high placement precision is to use optical forces to deposit cells directly. Toward this end, recent work with optical forces has shown that a wide range of particulate materials can be guided and deposited on surfaces to form arbitrary spatial patterns. Here we report that, when we use the light from a near-infrared diode laser focused through a low numerical aperture lens, individual embryonic chick spinal cord cells can be guided through culture medium and deposited on a glass surface to form small clusters of cells. In addition, we found that the laser light could be coupled into hollow optical fibers and that the cells could be guided inside the fibers over millimeter distances. The demonstration of fiber-based guidance extends by 2 orders of magnitude the distance over which optical manipulation can be performed with living cells. Cells guided into the fiber remained viable, as evidenced by normal cell adhesion and neurite outgrowth after exposure to the laser light. The results indicate that this particle deposition process, which we call "laser-guided direct writing," can be used to construct patterned arrays of tens to hundreds of cells using arbitrary numbers of cell types placed at arbitrary positions with micrometer-scale precision.
Animals↗
Single-molecule manipulation of double-stranded DNA using optical tweezers: interaction studies of DNA with RecA and YOYO-1.
By using optical tweezers and a specially designed flow cell with an integrated glass micropipette, we constructed a setup similar to that of Smith et al. (Science 271:795-799, 1996) in which an individual double-stranded DNA (dsDNA) molecule can be captured between two polystyrene beads. The first bead is immobilized by the optical tweezers and the second by the micropipette. Movement of the micropipette allows manipulation and stretching of the DNA molecule, and the force exerted on it can be monitored simultaneously with the optical tweezers. We used this setup to study elongation of dsDNA by RecA protein and YOYO-1 dye molecules. We found that the stability of the different DNA-ligand complexes and their binding kinetics were quite different. The length of the DNA molecule was extended by 45% when RecA protein was added. Interestingly, the speed of elongation was dependent on the external force applied to the DNA molecule. In experiments in which YOYO-1 was added, a 10-20% extension of the DNA molecule length was observed. Moreover, these experiments showed that a change in the applied external force results in a time-dependent structural change of the DNA-YOYO-1 complex, with a time constant of approximately 35 s (1/e2). Because the setup provides an oriented DNA molecule, we determined the orientation of the transition dipole moment of YOYO-1 within DNA by using fluorescence polarization. The angle of the transition dipole moment with respect to the helical axis of the DNA molecule was 69 degrees +/- 3.
Bacteriophage lambda↗
The micro-robotic laboratory: optical trapping and scissing for the biologist.
With the addition of tightly focused laser beams, microscopes have been turned into elaborate preparative tools that permit not only allow detailed observation of a specimen but also the capture, displacement, and microdissection of biological samples in vitro with astonishing ease and accuracy. Laser-Tweezers are used to capture and manipulate cells and organelles. LaserScissors are used to perform microdissections at the submicrometer level. After a short technical description of the instrumentation and its principles of operation, several examples of applications are given relevant to the field of clinical research that could only be achieved using such modern technology. For instance, LaserTweezers and LaserScissors offer a unprecedented means to study the immune response to cancer, to control the growth of nerve cells, or expand the significance of assisted reproductive technologies. It is suggested that newly developing procedures and assays using laser-assisted technologies will prove beneficial for future clinical laboratory testing.
Animals↗
Cost-effective screening methods for various single gene defects in single cells using high magnesium and total ionic strength and restriction enzymes.
A reliable cost-effective protocol for the diagnosis of various defective genes in single blastomeres from preimplantation embryos has been established. Single cells were lysed in alkali buffer followed by neutralization and addition of a solution containing a high concentration of sulfhydryl reducing agents and MgCl(2) in relatively high ionic strength (0.45) (solution M) with or without restriction enzyme(s). The reaction mixture was incubated at 37 degrees C for 15 min followed by heat denaturation at 95 degrees C for 10 min. Respective polymerase chain reaction (PCR) mixture was then added to amplify each designated DNA region. The treatment of neutralized single cell lysate with adequate restriction enzyme(s) which do not cleave the target DNA sequences but shortens the genomic template DNA strands. This may facilitate primer-template annealing. The subsequent heat denaturation of the cell lysate in solution M indeed gave better signals of amplified DNA fragments on polyacrylamide gels. Defects in Tay Sachs exons 11 and 12, CF-DeltaF508 and CF-N1303K, and genomic sequences of ZFX/ZFY were successfully detected on gels after one-step PCR amplification, especially those cell lysates treated with restriction enzymes. In conclusion, a cost-effective one-step PCR method for amplifying various specific genomic regions containing a single gene defect in single cells has been established. This protocol may be applied to genetic screening for many single defective genes of biopsied single blastomeres from preimplantation in vitro fertilization (IVF) embryos.
Blastocyst↗
Sucrose pretreatment for enucleation: an efficient and non-damage method for removing the spindle of the mouse MII oocyte.
Oocytes enucleated at metaphase II stage can support reprogramming of transferred nucleus and further developing to term. However, the first polar body in mice sometimes migrates away from the original place of expulsion, so the chromosomes of the oocyte will displace from the first polar body. Thus, it is not always possible to successfully enucleate according to the position of the first polar body. Here we use sucrose treatment to visualize metaphase spindle fibers and chromosomes with standard light microscopy. In the manipulation medium containing 3% sucrose, oocytes of poor quality become shrunken, deformed or fragmented, while oocytes of good quality in the same medium would show a swelling around the metaphase chromosomes and a transparent spindle area, shaped like "infinity" and "0". So it is easy to remove the well-distinguished spindle and chromosomes in oocytes of good quality. Re-examined by Hoechst 33342 stain under the UV light, the enucleation rate was 100%. There was no significant difference in IVF and cleavage rates between the sucrose treatment and the control group. In conclusion, this study demonstrated that 3% sucrose pretreatment can give a method for evaluating embryo quality and more importantly, it can, under a common microscope, allow the visualization of the spindle and chromosomes in oocytes of good quality and hence efficiently improve enucleation rate without any harm.
Anaphase↗
Nanofabrication: conventional and nonconventional methods.
Nanofabrication is playing an ever increasing role in science and technology on the nanometer scale and will soon allow us to build systems of the same complexity as found in nature. Conventional methods that emerged from microelectronics are now used for the fabrication of structures for integrated circuits, microelectro-mechanical systems, microoptics and microanalytical devices. Nonconventional or alternative approaches have changed the way we pattern very fine structures and have brought about a new appreciation of simple and low-cost techniques. We present an overview of some of these methods, paying particular attention to those which enable large-scale production of lithographic patterns. We preface the review with a brief primer on lithography and pattern transfer concepts. After reviewing the various patterning techniques, we discuss some recent application issues in the fields of microelectronics, optoelectronics, magnetism as well as in biology and biochemistry.
Biomedical Technology↗
Combined laser tweezers and dielectric field cage for the analysis of receptor-ligand interactions on single cells.
A new technique based on the combination of optical and chip-based dielectrophoretical trapping was developed and employed to manipulate cells and beads with micrometer precision. The beads were trapped with optical tweezers (OT) and brought into contact for defined times with cells held in the dielectrophoretic field cage (DFC). The well-defined ligand-receptor system biotin-streptavidin was used to study the multiple interaction between biotinylated live cells and streptavidin-coated beads. The biotin density on the cell surface was varied down to a few single bonds (3 +/- 2 bonds/microm2) to control the valency of the binding. The quantitative relationship between the contact area, ligand density and its diffusion rate in the outer membrane of the cell could be demonstrated. The increase of the strength of the cell-bead adhesion was strictly dependent on the increase of individual bond numbers in the contact area. This is in part due to accumulation of ligands (D approxiamtely (0.5 +/- 0.1) 10(-8) cm2/s) in the contact area as seen by confocal laser scanning microscopy. Individual receptor-ligand rupture forces were evaluated and are compatible with values obtained by biomembrane force probe techniques. To summarize, the combination leads to a new powerful microsystem for cell handling and pN-force measurements on the single-cell level.
Animals↗
High-speed separation system of randomly suspended single living cells by laser trap and dielectrophoresis.
We developed a new system for random separation of a single microorganism, such as a living cell and a microbe, in the microfluidic device under the microscope by integrating the laser-trapping force and dielectrophoretic (DEP) force. An arbitrarily selected single microbe could be isolated in a microchannel, despite the presence of a large number of microbes in solution. Once the target microbe is trapped at the focal point of the laser, we can easily realize exclusion of excess microbes around the target by controlling the electric field, while keeping the target trapped by the laser at the focal point. To realize an efficient separation system, we proposed a new separation cell and produced it by microfabrication. Flow speed in the microchannel is adjusted and balanced to realize high-speed and high-purity extraction of the target. Some preliminary experiments are conducted to show the effectiveness. The target is trapped by the laser, transported, and is taken out from the extraction port. Total separation time is less than 20 s. Our method is extremely useful in the pure cultivation of the cell and will be a promising method for biologists in screening useful microbes.
Cell Separation↗
Mass spectrometry meets chip technology: a new proteomic tool in cancer research?
DNA chip technologies are the most exiting genomic tools, which were developed within the last few years. It is, however, evident that knowledge of the gene sequence or the quantity of gene expression is not sufficient to predict the biological nature and function of a protein. This can be particularly important in cancer research where post-translational modifications of a protein can specifically contribute to the disease. To address this problem, several proteomic tools have been developed. Currently the most widely used proteomic tool is two-dimensional protein gel electrophoresis (2-DE), which can display protein expression patterns to a high degree of resolution. As an alternative to 2-DE, a preliminary study using a new technique was employed to generate protein expression patterns from whole tissue extracts. Surface-enhanced laser desorption/ionization (SELDI) allows the retention of proteins on a solid-phase chromatographic surface (ProteinChip Array) with direct detection of retained proteins by time of flight-mass spectrometry (TOF-MS). Using this system, we analyzed eight cases of renal cell carcinoma (RCC) including normal, peripheral and central tumor tissue as well as four microdissected cases of cervical intraepithelial neoplasia (CIN) and three microdissected cases of cervix uteri carcinoma. Differentially expressed proteins were found by comparing the protein expression patterns generated using SELDI-based TOF-MS of tumor tissue with normal and neoplastic tissue, respectively. By applying this fast and powerful ProteinChip array technology it becomes possible to investigate complex changes at the protein level in cancer associated with tumor development and progression.
Carcinoma, Renal Cell↗
Pattern formation in ciliary organelle systems of ciliated protozoa.
Genetic, morphometric, and microsurgical investigations of the pattern of ciliary organells in ciliate protozoa support the view that ther are two types of developmental process responsible for the positioning of these organelles. The first is exemplified by the propagation of ciliary rows through localized addition of new ciliary units along the axis of the row, a process which is responsible for the maintenance of the pre-existing number of rows in clonal lineages over a large number of fissions. The second is illustrated by two examples: (1) Ciliary units are distributed among ciliary rows of Euplotes minuta according to an invariant geometrical pattern that is independent both of the total number of units and of the number of rows. (2) Microsurgical alteration of the topographical contours of a related ciliate, Paraurostyla weissei, brings about a shift in the sites of formation of certain specific ciliary rudiments to new positions that are determined in relation to the newly constructed form. The two modes of pattern formation in ciliates are discussed from both genetic and developmental viewpoints. The localized positional mechanisms within the ciliary rows allow for a 'configurational heredity' shich is, however, subject to constraints of nuclear genic control both of the stable range of number of rows and of the positioning mechanism itself. The large-scale systems of pattern determination are probably more closely related to the field properties of developing multicellular organisms. In ciliates such systems are almost certainly located in the cell membrane or in the relatively fixed cytoplasmic layer just beneath the membrane.
Animals↗
Marker chromosome 21 identified by microdissection and FISH.
A child without Down syndrome but with developmental delay, short stature, and autistic behavior was found to be mosaic 46,XX/47,XX,+mar(21) de novo. The marker was a small ring or dot-like chromosome. Microdissection of the marker was performed. The dissected fragments were biotinylated with sequence-independent PCR as a probe pool for fluorescence in situ hybridization (FISH). FISH results suggested an acrocentric origin of the marker. Subsequent FISH with alpha-satellite DNA probes for acrocentric chromosomes, and chromosome-specific 21 and 22 painting probes confirmed its origin from chromosome 21.
Child, Preschool↗
Ultrastructure of the embryonic stem cells of the 8-day pig blastocyst before and after in vitro manipulation: development of junctional apparatus and the lethal effects of PBS mediated cell-cell dissociation.
Ultrastructural examination of 8-day hatched pig blastocysts (large and small), their cultured inner cell mass (ICM), and cultured epiblast tissue (embryonic stem cells) was undertaken to assess the development of epiblast cell junctions and cytoskeletal elements. In small blastocysts, epiblast cells had no desmosomes or tight junction (TJ) connections and few organized microfilament bundles, whereas in large blastocysts the epiblast cells were connected by TJ and desmosomes with associated microfilaments. ICM isolation by immunodissection damaged the endoderm cells beneath the trophectoderm cells but did not appear to damage the epiblast cells or their associated endoderm cells. Epiblast cells in cultured ICMs were similar in character to those in the intact large blastocyst except that perinuclear microfilaments were observed. Isolated pig epiblasts, cultured for approximately 36 hr on STO feeder layers, formed a monolayer whose cells were connected by TJ, adherens junctions and desmosomes with prominent microfilament bundles running parallel to the apical cytoplasmic membranes. Perinuclear microfilaments were a consistent feature in the approximately 36 hr cultured epiblast cells. A feature characteristic of differentiation into notochordal cells, i.e., a solitary cilium, was also observed in the cultured epiblast. Exposure of the cultured epiblast cells to Ca(++)-Mg(++)-free phosphate buffered saline (PBS) for 5-10 min resulted in extensive cell blebbing and lysis. The results may indicate that pig epiblast cells could be more easily dissociated from early blastocysts ( approximately 400 microm in diameter) if immunodissection damage to the ICM can be avoided. It may be difficult, however, to establish them as embryonic stem cell lines because the cultured pig epiblast cells were easily lysed by standard cell-cell dissociation methods.
Animals↗
Microdissection and microcloning of human chromosome regions in genome and genetic disease analysis.
Explore the source record for details and available documents.
Animals↗