Mechanical measurements with Cellular Force Microscopy (CFM)
Plant cells are under very high internal pressure (2-10 bars) which is borne by their stiff cell wall. The cell wall material has to yield (deform plastically) under the turgor pressure in order for the cell to grow. Little is known of the relationship between turgor pressure, elastic properties of the cell wall and growth, especially in land plants. Our recent work shows a non-straightforward link between elasticity and growth: the more the cells are strain-stiffened with increasing turgor pressure, the less they grow (Kierzkowski 2012). Hence, in order to understand cell wall mechanics, one has to measure the cell wall elasticity under various levels of cell turgor pressure.
Cellular Force Microscopy (CFM) is a new micro-indentation technique (Routier-Kierzkowska 2012) that we developed in collaboration with Brad Nelson’s group (ETH Zurich) and the company Femtotools. As in other micro-indentation techniques (Geitmann 2006), the sample stiffness is measured by indenting a thin probe connected to a force sensor. The recorded force and displacement are then used to determine the stiffness: for a same indentation depth, the stiffer the sample the higher the force. Similarly to Atomic Force Microscopy (AFM) (Milani 2011, Peaucelle 2011), CFM is automated and provides high-resolution stiffness maps as well as height maps (fig. 1). The main advantage of CFM resides in the very large range of forces and displacements that it can measure. The CFM system is also highly flexible and is used in combination with various optical microscopes (fig. 2).
In plant cells, the stiffness measured by micro-indentation reflect both the turgor pressure and cell wall elasticity. Stiffness measured with small indentation mainly reflect the turgor pressure, similar to the ball tonometry technique (Linthillac 2000). The idea of CFM is to indent deep enough into a the cell, so that the cell wall is stretched and cell wall elasticity can be deduced from stiffness measurements. The cell also has to be pressurized (non-plasmolized) during the measurement, so that the sample will remain mechanically stable during the indentation. CFM sensors can measure a large range of forces (fig. 3), enabling deep indentation of turgid cells, up to the point of breaking the cell wall (fig. 4). The sensor smooth spherical tips (diameter 0.1 to 3 um) indent the cell wall without penetrating it or applying a too localized stress.
We use Finite Element Modeling (FEM) based simulations to interpret micro-indentation results, taking into account the sample geometry, pressure, boundary conditions. We also use FEM to determine the best experimental conditions to reach the experiment purpose (e.g. measure pressure and/or wall elasticity).
CFM can be used in a large range of applications:
- rapid, non-invasive measure of cell turgor pressure
- mapping of cell wall elasticity within a tissue
- precise single cell ablation by puncture
- measure of cell wall strength (force needed for rupture)
- study of cell response to mechanical stimulation
- force sensor range: ± 160 µN and ± 2000 µN (for sensor type FT-S540 and FT-S270)
- force sensor resolution: 0.05 µN and 0.4 µN at 100Hz (for sensor type FT-S540 and FT-S270)
- displacement range: 3 to 5 cm
- displacement resolution: around 1 nm
- probe tip diameter: 0.1 um, 1 um, 2 um or 3 um
Weber A, Braybrook S, Huflejt M, Mosca G, Routier-Kierzkowska AL, Smith RS (2015). Measuring the mechanical properties of plant cells by combining micro-indentation with osmotic treatments. Journal of Experimental Botany, erv135. http://dx.doi.org/10.1093/jxb/erv135
Routier-Kierzkowska AL, Smith RS (2013) Plant Cell Morphogenesis – Methods and Protocols. Methods in Molecular Biology 1080, 135-146. http://dx.doi.org/10.1007/978-1-62703-643-6_11
Routier-Kierzkowska AL, Smith RS (2013) Measuring the mechanics of morphogenesis. Current Opinion in Plant Biology 16,25–32. http://dx.doi.org/10.1016/j.pbi.2012.11.002
Routier-Kierzkowska AL, Weber A, Kochova P, Felekis D, Nelson B, Kuhlemeier C, Smith RS (2012) Cellular Force Microscopy for in vivo measurements of plant tissue mechanics. Plant Physiology 158, 1514-1522. http://dx.doi.org/10.1104/pp.111.191460
Geitmann A (2006) Experimental approaches used to quantify physical parameters at cellular and subcellular levels. American Journal of Botany 93: 1380–1390. http://dx.doi.org/10.3732/ajb.93.10.1380
Milani P, Gholamirad M, Traas J, Arneodo A, Boudaoud A, Argoul F, Hamant O (2011) In vivo analysis of local wall stiffness at the shoot apical meristem in Arabidopsis using atomic force microscopy. Plant Journal 67: 1116–1123. http://dx.doi.org/10.1111/j.1365-313X.2011.04649.x
Peaucelle A, Braybrook S, Le Guillou L, Bron E, Kuhlemeier C, Hofte H (2011) Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis. Current Biology 21, 1720-1726. http://dx.doi.org/10.1016/j.cub.2011.08.057
Lintilhac PM, Wei C, Tanguay JJ, Outwater JO (2000) Ball tonometry: a rapid, nondestructive method for measuring cell turgor pressure in thin-walled plant cells. Journal of Plant Growth Regulation 19: 90–97. http://link.springer.com/article/10.1007/s003440000009
sensor manufacturer: http://www.femtotools.com
positioner manufacturer: http://www.smaract.de