Cellular force microscopy for in vivo measurements of plant tissue mechanics

Abstract

Although growth and morphogenesis are controlled by genetics, physical shape change in plant tissue results from a balance between cell wall loosening and intracellular pressure. Despite recent work demonstrating a role for mechanical signals in morphogenesis, precise measurement of mechanical properties at the individual cell level remains a technical challenge. To address this challenge, we have developed cellular force microscopy (CFM), which combines the versatility of classical microindentation techniques with the high automation and resolution approaching that of atomic force microscopy. CFM's large range of forces provides the possibility to map the apparent stiffness of both plasmolyzed and turgid tissue as well as to perform micropuncture of cells using very high stresses. CFM experiments reveal that, within a tissue, local stiffness measurements can vary with the level of turgor pressure in an unexpected way. Altogether, our results highlight the importance of detailed physically based simulations for the interpretation of microindentation results. CFM's ability to be used both to assess and manipulate tissue mechanics makes it a method of choice to unravel the feedbacks between mechanics, genetics, and morphogenesis.

Figure 1.

Comparison of tip sizes and forces used in nano-indentation and CFM studies on plants. AFM (in blue) was used by Milani et al. (2011) with a pyramidal indenter of 40 nm diameter and maximal forces of approximately 100 nN. Peaucelle et al. (2011) extended the working range of AFM in plant tissue (dotted blue line) by using stiffer cantilevers (up to 110 N m⁻¹) on which glass beads of 5 μm in diameter were glued manually. We used CFM (in green) for stiffness mapping with forces around 4 to 10 μN and hemispherical tip diameter of 2 to 3 μm. The use of rounded tips allows avoiding stress concentration inherent to sharp indenters. Puncture of the cell wall was obtained with a tip of 1 μm in diameter and forces up to 1 mN. Bar = 3 μm.

Figure 2.

Effect of slope on the apparent stiffness. A, Microscopic view of a turgid onion epidermis during the measurements. The shadow of the indenter is visible in the top part of the photograph. The black rectangle indicates the scanned area of the tissue. Bar = 40 μm. B, Color map of the angle (°) formed between the cell surface and the indenter probe. C, Color map of the apparent stiffness (N m⁻¹) measured during the scan. D, Color map of the corrected stiffness (N m⁻¹) computed using the measured stiffness, the surface slope, and the bending stiffness of the indenter probe.

Figure 3.

In a fully turgid epidermis, the apparent stiffness is lower over cross walls than on the top of the cells. A, Black rectangles show maps of measured stiffness over two areas of 64 × 52 μm. Note that the surface over the cross walls, in blue, is softer. The red square shows a finer scan of surface of 30 × 30 μm, covering three cross walls. B, The measured stiffness presented in A, after correction for the effect of the surface slope on stiffness. The color scales show stiffness in N m⁻¹. C, Microscopic view of the scanned tissue, with the shadow of the indenter tip. Black rectangles show the area scanned with a lateral step size of 4 μm. The red square shows the area scanned with a 2-μm step size. Bars = 20 μm.

Figure 4.

Changes in topography and measured stiffness in an onion peel recovering from plasmolysis. The left column shows microscopic views of the scanned area, covering three cells. Hours after plasmolysis are indicated. White arrows indicate cytoplasm filling the cell walls. The right column shows color maps of apparent stiffness (N m⁻¹) for the recovering peel. The color scale is the same (2–7 N m⁻¹) for the first four maps but different for the last stiffness map (6–22 N m⁻¹). Bar = 40 μm.

Figure 5.

Sequential steps within a simulated indentation. Epidermal tissue is modeled by FEM using different elements for the anticlinal walls, top wall, and cuticle layer. A, Cut through the model length is shown in its initial state. B, The model is pressurized prior to indentation. C, Simulated indentation experiment. The color scale indicates maximum principal stress in MPa.

Figure 6.

Puncture of the cell wall and effect of the release of turgor pressure on apparent stiffness. A, Light microscopy image and heat maps of the measured stiffness for the scanned region before puncture. B, Light microscopy image and stiffness map of the same region after puncturing the middle cell. The site of puncture is out of the image frame. Bars = 40 μm. C, Force-displacement signal during cell wall perforation. The first noticeable rupture occurs at around 9 μm indentation depth. The color scale indicates apparent stiffness in N m⁻¹.