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Combined atomic force microscopy and side-view optical imaging for mechanical studies of cells

Abstract

The mechanical rigidity of cells and adhesion forces between cells are important in various biological processes, including cell differentiation, proliferation and tissue organization. Atomic force microscopy has emerged as a powerful tool to quantify the mechanical properties of individual cells and adhesion forces between cells. Here we demonstrate an instrument that combines atomic force microscopy with a side-view fluorescent imaging path that enables direct imaging of cellular deformation and cytoskeletal rearrangements along the axis of loading. With this instrument, we directly observed cell shape under mechanical load, correlated changes in shape with force-induced ruptures and imaged formation of membrane tethers during cell-cell adhesion measurements. Additionally, we observed cytoskeletal reorganization and stress-fiber formation while measuring the contractile force of an individual cell. This instrument can be a useful tool for understanding the role of mechanics in biological processes.

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Figure 1: Concept of side-view AFM.
Figure 2: Measurement of adhesion between leukocyte and endothelial cell.
Figure 3: Contraction of U2OS cell against load.
Figure 4: Contractile force of U2OS cell exceeds force of adhesion to bottom surface.

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Acknowledgements

We thank J.W. Shaevitz, M. Van Duijn, M.J. Rosenbluth, A. Crow and all members of the Fletcher laboratory for helpful discussions, and B. Zuchero (University of California San Francisco) for the gift of GFP-transfected U2OS cells used in initial experiments. This work was supported by a US National Science Foundation graduate research fellowship (O.C.); a US National Institutes of Health National Research Service Award, Hammond research fellowship and Hartwell biomedical research fellowship (W.A.L.), and a National Institutes of Health R01 grant (D.A.F.).

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Correspondence to Daniel A Fletcher.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–2 (PDF 3214 kb)

Supplementary Video 1

Side-view video of leukocyte shape during leukocyte cell-endothelial cell adhesion measurement. This video is taken from the experiment described in Figure 2. Video is composed of 41 images taken in intervals of 1 second. Scale bar, 10 μm. (MOV 2232 kb)

Supplementary Video 2

Side-view video of U2OS contraction. This video is taken from the experiment described in Figure 3. Images of GFP-actin are taken approximately every 30 seconds over 17 minutes. Scale bar, 10 μm. (MOV 4624 kb)

Supplementary Video 3

Side-view video of U2OS contraction experiment showing rupturing of adhesion to surface. As in the experiment described in Figure 4, the U2OS cell pulls the cantilever down towards the sample surface as it contracts, resulting in an increasing tensional force applied by the cantilever across the cell. Images of GFP-actin are taken approximately every 15 seconds over 63 minutes. Fibers are observed to form in the video, and de-adhesion occurs due to rupture of the adhesion between the U2OS cell and the fibronectin-coated surface. Scale bar, 10 μm. (MOV 2788 kb)

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Chaudhuri, O., Parekh, S., Lam, W. et al. Combined atomic force microscopy and side-view optical imaging for mechanical studies of cells. Nat Methods 6, 383–387 (2009). https://doi.org/10.1038/nmeth.1320

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