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1.
Figure 2

Figure 2. From: Magnetic nanoparticle-mediated massively-parallel mechanical modulation of single-cell behavior.

Effects of magnetic field gradient and nanoparticle loading on cell response. The image shows an array of tiled cropped images of cells subject to increasing nanoparticle dose and magnetic field gradient. Cells are stained for actin (green), nanoparticles (blue), and DNA (cyan). The cells in the upper right corner display “pull-in” instability. The gradient varies from 2500 to 70000 T m−1. The nanoparticle dose varies from 5 pg to 300 pg cell−1. Scale bar is 10 µm. Note that the maximum intensity threshold for the actin channel was uniformly reduced so that filopodia are more visually apparent. Actin protrusions are not saturated and therefore retain a linear intensity mapping.

Peter Tseng, et al. Nat Methods. ;9(11):1113-1119.
2.
Figure 5

Figure 5. From: Magnetic nanoparticle-mediated massively-parallel mechanical modulation of single-cell behavior.

Nanoparticle-mediated forces bias mitotic spindle orientation. (a) The images show cells dividing after adhesion on the indicated fibronectin shapes and application of force. Red signal indicates fibronectin, green signal shows actin, blue indicates nanoparticles, and cyan indicates DNA stain. (b) The plots show the orientation of the metaphase plate and the subsequent cell division axis for cells subject to force in comparison to control (cells with initially localized nanoparticles but no sustained force). The number of cells per sample is indicated. Shown in magenta are spindle angle histograms for control samples initially localized with nanoparticles but subsequently released to a low holding force, in conjunction with bar plots comparing the distribution of coalesced nanoparticles for both low and high applied force conditions. (c) The plots show orientation from samples with nanoparticle-dosage and magnetic field stimulation as in (b) with inhibition of Src family kinases. Scale bar is 10 µm.

Peter Tseng, et al. Nat Methods. ;9(11):1113-1119.
3.
Figure 4

Figure 4. From: Magnetic nanoparticle-mediated massively-parallel mechanical modulation of single-cell behavior.

Nanoparticle-mediated mechanical tension generates PAK-dependent filopodia. (a) The average intensity of filopodia around regions of induced tension is plotted for three experiments, for the indicated adhesion patterns. Low tension is 0– 0.15 nN µm−1 for square patterns and 0–0.3 nN µm−1 for I and X patterns. High tension is 0.15 or 0.3–2.0 nN µm−1, respectively. Near yield tension is above 2 nN µm−1 for all patterns. The images show typical cellular responses at moderately deforming tensions. The colours are as in Figure 2. (b) The plots are as in (a), testing seven inhibitors of mechanotransductive proteins. Representative images for the indicated inhibitors are shown to the right. (c) Z-slices through two cells with different degrees of filopodial asymmetry displaying the activation of membrane localized phospho-PAK (red). Arrows indicate a band of phospho-PAK that enfolds regions of high deformation. (d) and (e) The images show cells stained for filopodial markers fascin (d) (red, shown by arrows), myosin-x (e) (red, localized to filopodia tips), and actin (e) (green). Staining for DNA (cyan) and nanoparticles (blue) is the same in both images. Scale bars are 10 µm.

Peter Tseng, et al. Nat Methods. ;9(11):1113-1119.
4.
Figure 3

Figure 3. From: Magnetic nanoparticle-mediated massively-parallel mechanical modulation of single-cell behavior.

Nanoparticle tension-dependent asymmetry in actin polymerization. (a) The images show single cells patterned using the indicated fibronectin shapes with the same colour legend as in Figure 2 (b) Scatter plots with overlaid averages (standard deviation represented by error bars) plotting the actin protrusion asymmetry for cells patterned by the fibronectin shapes in (a). The number of cells per sample is indicated. Zero corresponds to symmetric actin across the cell. The gray baseline in the samples is the average asymmetry as determined from control samples (excess nanoparticles under reduced magnetic field). (c) The graph shows the percentage of cells at a given force level with actin asymmetry over 0.7. Coloured arrows denote “protrusion thresholds,” or the tension at which this percentage nears its maximum observed for the separate fibronectin shapes (green corresponds to square, teal to I, and orange to X). (d) Comparison of cell yield tension (the lowest average tension at which nanoparticle clusters are observed to break through the cell membrane) on different adhesive patterns. Yield stress is estimated from yield tension and approximate nanoparticle thicknesses as obtained from confocal microscopy (1.5 to 2.3 µm, with an average of 1.8 µm). The average is used as our approximate thickness. Protrusion threshold is defined from (c). Scale bar is 10 µm.

Peter Tseng, et al. Nat Methods. ;9(11):1113-1119.
5.
Figure 1

Figure 1. From: Magnetic nanoparticle-mediated massively-parallel mechanical modulation of single-cell behavior.

Parallel dynamic localization of magnetic nanoparticle clusters within arrays of cells. (a) Artist’s schematics of the force-generating platform. A permanent magnet remotely magnetizes soft ferromagnetic elements in proximity to fibronectin-patterned cells, coalescing magnetic nanoparticles into force-generating clusters within each cell. (b) Stitched images of patterned and stained cells; the right panel shows an expanded view—actin (green), nanoparticles (red), DNA (blue). (c) Modeled forces on the cell cortex generated by coalesced nanoparticles are plotted as a function of system parameters. Height indicates thickness of the passivation layer above a 9.5 µm thick micro-magnet, distance indicates nanoparticle x-distance from the micro-magnet. The maximum internalized nanoparticle volume was around 100 µm3, or 2 to 3 % of the total HeLa cell volume. The plot assumes an ideal rectangular cluster structure and an external magnetic field (Bt = 0.32, Bn = 0.075 T). (d) The fraction of coalescing nanoparticles (sample thickness: 0.5 µm) is plotted over time. Error bars represent standard deviation (n = 19 cells). (e) The image shows the average localization of nanoparticle ensembles (n = 57 cells). (f) The micrograph shows a single cell in which a small quantity of nanoparticles is localized with high precision by an ultra-fine magnetic tip. Scale bars, 10 µm.

Peter Tseng, et al. Nat Methods. ;9(11):1113-1119.

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