Imaging and Measurement of Nanomechanical Properties within Primary Xylem Cell Walls of Broadleaves

Figure 1. Basic principles illustrating how the different nanomechanical properties are extracted from the force curve. The image shows a typical force curve for a primary xylem cell wall sample in the Dutch elm disease-sensitive hybrid 'Groeneveld'. PeakForce QNM mode performs a very fast force curve at every pixel in the image. Analysis of force curve data is done on the fly, providing a map of multiple nanomechanical properties that has the same resolution as the height image. The adhesion is the vertical distance between the base line and the lowest portion of the retraction curve. The deformation is the horizontal distance between the contact point and the turn-away point (representing maximum indentation). The Young’s modulus of elasticity can be extracted by extrapolating the linear portion of the retraction curve after the contact point using a Derjaguin, Muller, Toporov (DMT) model fit. The slope of the linear portion is determined according to the least square method procedure. The energy dissipation can be calculated by integrating the area between the two curves.

Materials and Reagents

1. Leaf midribs from broadleaves (Ulmus spp., Sorbus spp.)
   
2. Glutaraldehyde solution, 25% in H₂O (Grade II) (Sigma-Aldrich, catalog number: G6257 )
   
3. Ethanol solutions (Analytical Reagents)
   Note: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, each of them prepared by diluting of 96% ethanol in a 0.1 M cacodylate buffer, and 100% ethanol.
   
4. 25% glutaraldehyde (Grade I) (Sigma-Aldrich, catalog number: G5882 )
   
5. Xylene (Analytical Reagents) or alternative cleaners with a lower toxicity (for example D-limonene)
   Note: Work with xylene or D-limonene only in a properly operating and certified fume cupboard.
   
6. Paraplast plus (Sigma-Aldrich, catalog number: P3683 )
   
7. (3-Aminopropyl) triethoxy-silane (Sigma-Aldrich, catalog number: A3648 )
   
8. 0.1 M cacodylate buffer (see Recipes)
   
9. Circle glass slides coated with (3-aminopropyl)triethoxy-silane (see Recipes)
   
10. 1x phosphate buffered saline (PBS) (see Recipes)
    
11. Glutaraldehyde fixative solution (see Recipes)
    

Equipment

1. Razor blades (fine tweezers and heated forceps)
   
2. pH meter
   
3. Exicator with a vacuum pump
   
4. Petri dishes
   
5. Laboratory drying chamber
   
6. Retracting base sledge microtome (Series 8000) (Bright Instrument Co.)
   
7. Circle glass slides (11 mm in diameter, 1 mm thick)
   
8. Slide warmer
   
9. Steel AFM sample mounting disks
   
10. Sample adhesive pads
    
11. Silicon probes MPP-12120 (model: TAP150A ) having a 180° rotated tip and the spring constant at least 5 N/m (Bruker AFM Probes)
    
12. Fused silica reference sample (nominal MOE 72.9 GPa) or any other reference sample having the MOE value higher than 70 GPa
    
13. TGT1 commercial tip characterization grating (NT-MDT Co.)
    
14. MultiMode 8 atomic force microscope with a Nanoscope V controller and a PeakForce Quantitative Nanomechanical Mapping mode (Bruker Corporation)
    

Software

1. NanoScope analysis (version 1.40r2) (Bruker Corporation)
   
2. MATLAB (version 7) (MathWorks)
   

Procedure