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There was no considerable cell damage, suggesting that direct laser ablation is negligible in the cell dissociation. When a single pulse of 230 nJ was focused through a 10× objective lens, some, but not all, of the two-cell aggregates were dissociated. The O f of the fsLP was set at the edge of the interface of two-cell aggregates ( Fig. 2 A). The resultant homotypic cell aggregates were embedded in a viscous bactoagar matrix. These cells were aggregated using a standard procedure. We previously used this assay to examine the adhesive properties of cell adhesion molecule-1 (CADM1, alternatively named Necl-2), a member of the immunoglobulin superfamily, using NIH3T3 fibroblasts that exogenously express CADM1 isoform c ( Fig. S3) ( 13, 14). In the current study, the fsLP-IF was used to dissociate the intercellular adhesions in cell aggregates that formed in the aggregation assay. The reproducibility of the fitting was identical to that of the fitting shown in Fig. 1 B ( Fig. S2 A). Fig. 1 C– E shows the Z-position dependence of F, ω, and α, respectively, when the distance between O f and the top of the cantilever was set to ( X 0, Y 0) = (10,0) μm. 1 was performed, where F, ω, and α were considered variable parameters and k was a constant.
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The transient oscillation was measured as a function of the O f in the optical axis (Z direction). As an approximation, the influence of these phenomena in the calculation was minimized by extrapolating the former vibration prior to the “B” shift by the latter oscillation. The motion before the “B” shift can be interpreted as follows: ( i) the impulsive force cannot be approximated as the delta function-i.e., the impulsive force loading time is not extremely shorter than the vibration cycle (6 μs) and ( ii) the bending modes of the cantilever, except for the fundamental mode with frequency ω, were simultaneously excited and interfered. 1, the oscillation after the second shift “B” was well-reproduced. When the data in Fig. 1 B were fit by Eq.
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When the total impulse at O f is well-defined, fsLP-IF can be used to estimate the force required to break intercellular adhesions in a noncontact manner under biologically relevant conditions. The impulses needed to break leukocyte–endothelial and interepithelial interactions, which were calculated based on the geometrical relationship between O f and the adhesive interface, were on the order of 10 -13 and 10 -12 N-s, respectively. The fsLP-IF was used to break intercellular contacts in two biologically relevant cultures: a coculture of leukocytes seeded over on an endothelial cell monolayer, and a polarized monolayer culture of epithelial cells. These results suggest that fsLP-IF can be used to break intermolecular and intercellular interactions and estimate the adhesion strength.
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The force also broke the interactions between streptavidin-coated microspheres and a biotin-coated substrate with a measurement error of approximately 7%. This impulsive force broke adhesion molecule-mediated intercellular interactions in a manner that depended on the adhesion strength that was estimated by the cell aggregation assay. Based on the magnitude of the vibration and the geometrical relationship between O f and the cantilever, the fsLP-IF generated at O f was calculated as a unit of impulse. In this study, an fsLP-IF was reflected in the vibratory movement of an atomic force microscopy (AFM) cantilever. This force can detach individual adherent cells without causing considerable cell damage. When a femtosecond laser pulse (fsLP) is focused through an objective lens into a culture medium, an impulsive force (fsLP-IF) is generated that propagates from the laser focal point ( O f) in a micron-sized space.
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