Pectin Nanostructure Visualization by Atomic Force Microscopy

Materials and Reagents

1. Tri-distilled butanol (VWR International, catalog number: 20810.323 )
   
2. Pectin fractions from cell wall extracts
   Notes:
   
   1. Cell wall extraction protocol is described in Posé et al. (2013).
      
   2. Pectin fractions from cell wall material are obtained by sequential extractions with CDTA buffer followed by sodium carbonate buffer, to solubilize a cell wall fraction enriched in ionically and covalently bound pectins respectively, as described in Posé et al. (2013) (see Recipes).
      
   3. Both pectin fractions (i.e., one extracted with CDTA and the other with sodium carbonate) were extensively dialyzed and stored until required at -20 ºC as aqueous solutions. Important: in order to prevent possible aggregation, any freeze-drying step must be avoided. It is recommended to aliquot the samples to avoid freeze-thawed successive cycles.
3. Ammonium bicarbonate (FLUKA, catalog number: 09830 )
   Note: Currently, it is “Sigma-Aldrich, catalog number: 09830”.
   
4. Trans-1,2-diaminocyclohexane-N,N,N'N'-tetra-acetic acid monohydrate (Sigma-Aldrich, catalog number: D 319945 )
   
5. Sodium carbonate (Sigma-Aldrich, catalog number: S2127 )
   
6. NaBH₄ (Sigma-Aldrich, catalog number: D 452882 )
   
7. 10 mM ammonium bicarbonate buffer (pH 8) (see Recipes)
   
8. CDTA buffer (see Recipes)
   
9. Sodium carbonate buffer (see Recipes)
   

Equipment

1. Acoustic-isolated and temperature-controlled room (Figure 1A-1)
   
2. Low-power light microscope, equipped with a television camera, was used to roughly position the AFM probe onto the top of the sample (Figure 1A-2)
   
3. Atomic Force Microscope (East Coast Scientific Limited, Cambridge, UK) (Figure 1A-3; Figure 2A). Any AFM of suitable resolution can be used, although the details of the software and liquid cell will vary
   
4. Anti-vibration table under the AFM microscope (Figure 1A-4)
   
5. Photodiode amplifier (Figure 1A-5)
   
6. Oscilloscope (Figure 1A-6; Figure 3)
   
7. Digital control system composed by computer, DAC (digital-to-analog converter) box, laser driver and high voltage amplifier. (for more details see Morris et al., 2010) (Figure 1A-7)
   
8. PYREX^® Culture Tubes with Rubber Liner Screw Caps (Thomas Scientific, catalog number: 9212C21 ) and Teflon-lined caps
   Note: The screw-capped glass tubes were acid washed using a 1% hydrochloric acid solution overnight then rinsed with water.
   
9. Sheets of mica (Elektron Technology, Agar Scientific, model: G250 ) cleaved with adhesive tape (3M, model: Magic Tape ) (Video 1)
   
10. Short tip variety AFM probe model contact cantilevers (Budget Sensors SiNi, Bulgaria)
    Note: The tip is mounted on the edge of a V-shaped cantilever, the typical geometry used for topographical imaging (Figure 1B).
    
11. Tip holder and open bucket liquid cell (Figure 1C; Vdeo 2)
    
12. Basic equipment: pipettes, vortex, sonicator bath, heating block
    
    
    Figure 1. Atomic force microscopy (AFM) equipment. A. Photograph including an overview of AFM room set-up. The numeric labels are in accordance with the equipment list description. B. Scheme of a sharp tip located at the free end of a cantilever. C. Detail of tip holder (left) and liquid cell (right).
    

Software

1. AFM software supplied with the instrument (SPM 6.01, ECS, Cambridge, UK)
   
2. For the length measurements, images were converted to TIFF files using Paint Shop Pro v5.00 software (http://web.archive.org/web/19980514080113/http://jasc.com/)
   
3. Image contrast and 3D effects were optimized using Gwyddion software v2.32
   
4. AFM images were analyzed off-line using Image J v1.43u software (http://imagej.nih.gov/ij/index.html)
   
5. Gwyddion is free and open source software, covered by GNU (General Public License) (http://gwyddion.net/)
   

Procedure