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.

Figure 1

Concept of the Side-view AFM. (a) Cartoon schematic of the typical layout of an AFM. A cantilever (black) is used to probe a sample (green). The undeflected cantilever position is represented by the dotted line. The cantilever base is held rigidly by a fluid cell. A laser is focused onto the tip of the cantilever, and the reflected beam is captured by a detector. The position of the reflected beam on the detector is used to monitor cantilever position. A standard bottom-view imaging path (the image plane is represented by the dotted blue line) is shown. (b) A typical bottom-view image taken in brightfield. The triangular cantilever is seen, as are several trypsinized U2OS cells (circles). (c) Cartoon schematic of side-view imaging path and desired image of sample (image plane is represented by blue dotted line). (d) Side-view image of a U2OS cell and cantilever under oblique white light illumination. Surface is represented by grey dotted line. (e) A merged image consisting of images taken in epi-fluorescence of the cell membrane (red), GFP-actin (green), and the nucleus (blue) of a U2OS cell. The reflection of the sample from the glass sample surface (grey dotted line) can be seen and identifies the surface boundary. Scale bars are all 20 μm.

Figure 2

Measurement of adhesion between leukocyte and endothelial cell. (a) Cartoon schematic of experiment design. The cantilever is pressed into a leukocyte (red) attached to an endothelial cell (green), then the sample surface is pulled away at a constant rate. (b) Representative force-distance trace for a single cell [AU: correct?] adhesion experiment where distance refers to the distance the sample surface has moved relative to its initial position, and a positive force indicates a compressive force. The surface was moved away at 500 nm/s. (c) Time series of images of the leukocyte labeled with a membrane dye taken at the selected distances. White arrows point to tethers formed between the leukocyte and endothelial cell. (d) A merged epi-fluorescence side-view image of a leukocyte (red) and an endothelial cell (green) immediately after the adhesion measurement. (e) The aspect ratio of the cell (shown as a cartoon in the inset) is measured for all the images taken with the side-view imaging path (images were taken at 10 Hz) and shown in grey. This trace is smoothed with a Savitzky-Golay 2^nd order filter for clarity (black). [AU: Is this a single representative trace? Out of what total n?] Scale bars are 10 μm (c, d).

Figure 3

Contraction of U2OS cell against load. (a) Cartoon of experiment design. Cell width, indicated by blue dotted arrows, is measured from side-view images. (b) Cell length and force of contraction are shown for a single representative experiment [AU: correct?]. Cell length was calculated as the difference between the cantilever deflection and the position of the surface, and was calibrated initially by measuring the height of the cell at its midpoint optically with side-view images (a, black dotted arrow). (c) Cell width (a, blue dotted arrows) was measured for every image taken [AU: is this for a single cell?] during the experiment and shown as the blue trace (top) over time. (d) Side-view images taken at the indicated time points of GFP actin in the cell. The location of the surface and cantilever are represented by the yellow dotted line and blue cartoon respectively. Scale bar is 10 μm.

Figure 4

Contractile force of U2OS cell exceeds force of adhesion to bottom surface. (a) Cell length (red) and contractile force (blue) for a single contracting U2OS cell over time. (b) Series of side-view images taken of GFP actin at the indicated times corresponding to the dotted lines in a. Increase in cell length and decrease in contractile force is coincident with the release of the adhesion (red arrow) to the bottom surface. The location of cantilever surface is represented by the superimposed blue cartoon. Scale bar is 10 μm.