225 research outputs found
Microtubule dynamics in cell division : exploring living cells with polarized light microscopy
Author Posting. © The Author(s), 2008. This is the author's version of the work. It is posted here by permission of Annual Reviews for personal use, not for redistribution. The definitive version was published in Annual Review of Cell and Developmental Biology 24 (2008): 1-28, doi:10.1146/annurev.cellbio.24.110707.175323.This Perspective is an account of my early experience while I studied the dynamic organization and behavior of the mitotic spindle and its submicroscopic filaments using polarized light microscopy. The birefringence of spindle filaments in normally dividing plant and animal cells, and those treated by various agents, revealed: A) the reality of spindle fibers and fibrils in healthy living cells; B) the labile, dynamic nature of the molecular filaments making up the spindle fibers; C) the mode of fibrogenesis and action of orienting centers; and D) force-generating properties based on the disassembly and assembly of the fibrils. These studies, which were carried out directly on living cells using improved polarizing microscopes, in fact, predicted the reversible assembly properties of isolated microtubules
Fabrication of micro-structures for optically driven micromachines using two-photon photopolymerization of UV curing resins
Two-photon photopolymerization of UV curing resins is an attractive method
for the fabrication of microscopic transparent objects with size in the tens of
micrometers range. We have been using this method to produce three-dimensional
structures for optical micromanipulation, in an optical system based on a
femtosecond laser. By carefully adjusting the laser power and the exposure time
we were able to create micro-objects with well-defined 3D features and with
resolution below the diffraction limit of light. We discuss the performance and
capabilities of a microfabrication system, with some examples of its products.Comment: 12 pages, 10 figure
A High-Resolution Combined Scanning Laser- and Widefield Polarizing Microscope for Imaging at Temperatures from 4 K to 300 K
Polarized light microscopy, as a contrast-enhancing technique for optically
anisotropic materials, is a method well suited for the investigation of a wide
variety of effects in solid-state physics, as for example birefringence in
crystals or the magneto-optical Kerr effect (MOKE). We present a microscopy
setup that combines a widefield microscope and a confocal scanning laser
microscope with polarization-sensitive detectors. By using a high numerical
aperture objective, a spatial resolution of about 240 nm at a wavelength of 405
nm is achieved. The sample is mounted on a He continuous flow cryostat
providing a temperature range between 4 K and 300 K, and electromagnets are
used to apply magnetic fields of up to 800 mT with variable in-plane
orientation and 20 mT with out-of-plane orientation. Typical applications of
the polarizing microscope are the imaging of the in-plane and out-of-plane
magnetization via the longitudinal and polar MOKE, imaging of magnetic flux
structures in superconductors covered with a magneto-optical indicator film via
Faraday effect or imaging of structural features, such as twin-walls in
tetragonal SrTiO. The scanning laser microscope furthermore offers the
possibility to gain local information on electric transport properties of a
sample by detecting the beam-induced voltage change across a current-biased
sample. This combination of magnetic, structural and electric imaging
capabilities makes the microscope a viable tool for research in the fields of
oxide electronics, spintronics, magnetism and superconductivity.Comment: 14 pages, 11 figures. The following article has been accepted by
Review of Scientific Instruments. After it is published, it will be found at
http://aip.scitation.org/journal/rs
Evaluation of Dynamic Cell Processes and Behavior Using Video Bioinformatics Tools
Just as body language can reveal a person’s state of well-being, dynamic changes in cell behavior and
morphology can be used to monitor processes in cultured cells. This chapter discusses how CL-Quant
software, a commercially available video bioinformatics tool, can be used to extract quantitative data on:
(1) growth/proliferation, (2) cell and colony migration, (3) reactive oxygen species (ROS) production, and
(4) neural differentiation. Protocols created using CL-Quant were used to analyze both single cells and
colonies. Time-lapse experiments in which different cell types were subjected to various chemical
exposures were done using Nikon BioStations. Proliferation rate was measured in human embryonic stem
cell colonies by quantifying colony area (pixels) and in single cells by measuring confluency (pixels).
Colony and single cell migration were studied by measuring total displacement (distance between the
starting and ending points) and total distance traveled by the colonies/cells. To quantify ROS production,
cells were pre-loaded with MitoSOX Redâ„¢, a mitochondrial ROS (superoxide) indicator, treated with
various chemicals, then total intensity of the red fluorescence was measured in each frame. Lastly, neural
stem cells were incubated in differentiation medium for 12 days, and time lapse images were collected
daily. Differentiation of neural stem cells was quantified using a protocol that detects young neurons. CLQuant
software can be used to evaluate biological processes in living cells, and the protocols developed in
this project can be applied to basic research and toxicological studies, or to monitor quality control in
culture facilities
Vaughan-Jackson-like syndrome as an unusual presentation of Kienböck's disease: a case report
<p>Abstract</p> <p>Introduction</p> <p>Kienböck's disease is a condition of osteonecrosis of the lunate bone in the hand, and most patients present with a painful and sometimes swollen wrist with a limited range of motion in the affected wrist. Vaughan-Jackson syndrome is characterized by the disruption of the digital extensor tendons, beginning on the ulnar side with the extensor digiti minimi and extensor digitorum communis tendon of the small finger. It is most commonly associated with rheumatoid arthritis. We describe a case of a patient with an unusual presentation of Kienböck's disease with symptoms similar to those of Vaughan-Jackson syndrome.</p> <p>Case presentation</p> <p>A 40-year-old man of Indian ethnic origin with no known history of trauma presented to our clinic with a ten-day history of an inability to extend his right little and ring fingers with associated pain in his right wrist. He was being treated with long-term steroids but had no other significant medical history. His examination revealed an inability to extend the metacarpal and phalangeal joints of the right ring and little fingers with localized tenderness over the lunate bone. Spontaneous disruption of the extensor tendons was diagnosed clinically and, after radiological investigation, was confirmed to be secondary to dorsal extrusion of the fragmented lunate bone. The patient underwent surgical repair of the tendons and had a full recovery afterward.</p> <p>Conclusion</p> <p>Kienböck's disease, though rare, is an important cause of spontaneous extensor tendon rupture. The original description of Vaughan-Jackson syndrome was of rupture of the extensor tendons of the little and ring fingers caused by attrition at an arthritic inferior radioulnar joint. We describe a case of a patient with Kienböck's disease that first appeared to be a Vaughan-Jackson-like syndrome.</p
Prime movers : mechanochemistry of mitotic kinesins
Mitotic spindles are self-organizing protein machines that harness teams of multiple force generators to drive chromosome segregation. Kinesins are key members of these force-generating teams. Different kinesins walk directionally along dynamic microtubules, anchor, crosslink, align and sort microtubules into polarized bundles, and influence microtubule dynamics by interacting with microtubule tips. The mechanochemical mechanisms of these kinesins are specialized to enable each type to make a specific contribution to spindle self-organization and chromosome segregation
Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals
© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution 3.0 License. The definitive version was published in Journal of Cell Biology 193 (2011): 1065-1081, doi:10.1083/jcb.201012143.The septins are conserved, GTP-binding proteins important for cytokinesis, membrane compartmentalization, and exocytosis. However, it is unknown how septins are arranged within higher-order structures in cells. To determine the organization of septins in live cells, we developed a polarized fluorescence microscopy system to monitor the orientation of GFP dipole moments with high spatial and temporal resolution. When GFP was fused to septins, the arrangement of GFP dipoles reflected the underlying septin organization. We demonstrated in a filamentous fungus, a budding yeast, and a mammalian epithelial cell line that septin proteins were organized in an identical highly ordered fashion. Fluorescence anisotropy measurements indicated that septin filaments organized into pairs within live cells, just as has been observed in vitro. Additional support for the formation of pairs came from the observation of paired filaments at the cortex of cells using electron microscopy. Furthermore, we found that highly ordered septin structures exchanged subunits and rapidly rearranged. We conclude that septins assemble into dynamic, paired filaments in vivo and that this organization is conserved from yeast to mammals.This work was supported by the National Science Foundation under
grant No. MCB-0719126 to A.S. Gladfelter, the National Institute of Biomedical
Imaging and Bioengineering under grant No. EB002583 to R. Oldenbourg,
a Drexel CURE grant from the State of Pennsylvania Tobacco Settlement Fund,
and National Institute of Neurological Disorders and Stroke grant NS48090-
06A to E.T. Spiliotis
Visualization and Analysis of 3D Microscopic Images
In a wide range of biological studies, it is highly desirable to visualize and analyze three-dimensional (3D) microscopic images. In this primer, we first introduce several major methods for visualizing typical 3D images and related multi-scale, multi-time-point, multi-color data sets. Then, we discuss three key categories of image analysis tasks, namely segmentation, registration, and annotation. We demonstrate how to pipeline these visualization and analysis modules using examples of profiling the single-cell gene-expression of C. elegans and constructing a map of stereotyped neurite tracts in a fruit fly brain
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