This single-cell level of analysis results in a better understanding of the role of astrocytes to maintain homeostasis in the CNS, particularly in the sensing and removal of debris in damaged or pathologic nervous tissue. Immediately following laser irradiation irradiated cells shown with yellow ROI , PI enters the irradiated cell and intercalates into the DNA of the dead cell. In this simulation we use the same parameters as in figure 3. The astrocyte located in the top right in the Pre Laser-Irradiation image was targeted with the laser along the ROI indicated by the yellow line. Response based on the closest distance between plasma membranes of the laser-irradiated cell and the responding cell. Nobes CD, Hall A. The femtosecond laser was utilized at an average optical intensity of 3. In another study on living cells, the temperature rise was measured to be less than 1.
Simulation model of trapped chromosomes Chromosomes at metaphase have a cylindrical structure 31 , 37 , 38 of densely packed chromatin. Imaging intracellular viscosity of a single cell during photoinduced cell death. To view a copy of this license, visit http: The simulation used the optical tweezers toolbox with the T-matrix method to calculate the optical forces on microsphere in the trap 29 , As the astrocyte crawls away, no visible debris remains of the dissected growth cone. Berns b, 1, 3. Biophys J 99 , —
In neighboring astrocytes, dramatic changes in cell morphology were observed, including increased amounts of membrane ruffling and the formation of filopodia projecting toward the irradiated cell or its debris. Over time, we observe vesicle formation via phase contrast imaging. Ejection mechanics and trajectory of the ascospores of Gibberella zeae anamorph Fuarium graminearum.
Therefore, most features of the trap are identical between the experiments inside and outside the cell: J Cell Biol 97— Astrocyte response to laser induced cell lysis in a multicellular cluster.
Phagocytic response of astrocytes to damaged neighboring cells
The parameter Q is the dimensionless trapping efficiency mofie indicates the fraction of the incident light hkatibzadeh transferred to the trapped object and varies from 0 to 2 18 The astrocyte reaches the active growth cone 90 minutes following laser irradiation.
Eukaryot Cell 9— A mixed population astrocytes and neurons of primary cells was isolated from the cerebral cortices of embryonic day Our results showing a higher incidence of phagocytosis in a responding astrocyte when the nucleus of the target cell was irradiated, suggests that DNA one of the DAMP molecules or other possible nuclear factors can enhance the phagocytic response. Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation.
The trapping forces on the chromosomes linearly increased with the trapping powers at each individual viscosity as shown by linear regressions represented by the dashed red lines on Fig. Prior to laser exposure, PI 0. Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian khatibzadehh.
Parametric study of the forces on microspheres held by optical tweezers. Light from an arc lamp source was collected by the microscope condenser to illuminate khatibzaxeh sample.
To confirm the identity of responding cells, cells that formed endocytic vesicles were fixed, stained for GFAP, and imaged.
Low viscosity in the aqueous domain of cell cytoplasm measured by picosecond polarization microfluorimetry. In addition, the escape path of khatibzadeu trapped particle affects the escape force 26and this can be affected by the rate at which the ratio of viscous drag to optical force increases.
Alireza Khatibzadeh –
To determine if the substrate affects the phagocytic response of astrocytes in vitro, cells from the immortalized Ast-1 line were plated on both glass and matrigel-coated glass.
Nobes CD, Hall A. The resulting focused laser khattibzadeh at the microscope image plane was near-diffraction-limits, approximately 0. Discussion In this study we demonstrate that a single laser damaged or killed astrocyte or neuron induces a nearby astrocyte to initiate phagocytosis.
If unattached to the irradiated cell, the responding astrocyte first migrates toward, and subsequently phagocytoses the debris of the irradiated cell.
This is an open access article distributed under the terms of the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Proc Natl Acad Sci U. Fluorescence imaging was performed on a Zeiss Axiovert using a Cy3 filter set.
Fluorescent images acquired confirm that some of the endocytic vesicles correspond to an increased fluorescence signal at vesicle positions Fig 8. Therefore, the data of all the astrocyte studies from both laser systems were combined. Two different connections between cells were observed: Often dull chromosomes were floating horizontally in the suspension media prior to trapping, and usually they fulp to a vertical orientation when the trap was activated.
Once a physical connection is established between the plasma membrane of the astrocyte and the previously severed growth cone, the responding astrocyte engulfs the remaining growth cone and subsequently forms endocytic vesicles. Proc SPIE— These forces can be calculated by measuring the in situ trapping powers required to stop the chromosomes, as long as the trapping efficiency Q of the chromosomes is known.
The shaded region of the force curves illustrates the variation in the trap strength with refractive index between 1. Since the experimental measurements and simulations of the escape of chromosomes are in reasonable agreement, with the differences attributable to uncertainties in the size and optical properties of the chromosomes, the measurements of escape force for khatibzadhe are considered reliable.
They are closer to the theoretical values of 0. Similarly two vesicles with corresponding fluorescence are visible at 1 hour and 13 minutes and 2 hours and 15 minutes post laser irradiation.
Phagocytic response of astrocytes to damaged neighboring cells
Astrocyte response to a laser dissected neuron process. S7 Fig Astrocyte response to laser targeted cell body of a neuron. Biophys J 71— The initial equilibrium orientation is khatibxadeh and the cylinder refractive index of 1. By 58 minutes post-laser, large vesicles have formed in the responding astrocyte, and continue to form over a minute period.
Next, chromosomes from Chinese hamster ovary CHO cells were isolated using standard procedures 27 and optically trapped with a range of in situ trapping powers. Vernon PJ, Tang D. Force of single kinesin molecules measured with optical tweezers. This calibration procedure to find the escape force or escape Q was performed using the same optical tweezers system as used for the measurements on chromosomes within the cell.
A similar response was observed between immortalized Ast-1 astrocytes and those isolated from primary cortical tissue Fig Exp Cell Res—