1. Fix a sample on stiff support. Fixation method is individual for a type of sample and experiment.
2. Run you AFM and prepare to force-volume mode.
3. Chose cantilever to your sample. General rule is softer sample>>softer cantilever. Generally it must be find experimentally.
4. Calibrate the cantilever spring constant k (N/m) in air. For calibration, thermal tune is most often used. The resonance frequency ω from the thermal tune put to the equation k/k_n = ω²/ω_n², where k_n and  are nominal spring constant and nominal resonance frequency ω_n. It should be done in several replicates.
5. Calibrate a deflection sensitivity of the cantilever in the same media as for experiment. Calibration may be done on glass and is expressed in nm/V. It should be done in several replicates.
6. Go with cantilever above the sample and approach.
7. Capture the force curves. It may be done in a matrix to have a force map of the sample.
8. Fit the Hertz-Sneddon model  (equation below) to experimental force-indentation to determine the Young’s modulus E. F – force, d - indentation, a - the tip opening angle, n - the Poisson ratio

homogenization

- HAAKE Mars rheometer (Thermo Scientific, Karlsruhe, Germany).

  - parallel plate measuring geometry

- The plates were serrated in order to prevent the sample slippage.

- A sample was placed on the lower plate, and the upper plate was lowered until the gap of 1.0 mm was reached. The excess of sample was trimmed and the edges were sealed with a paraffin oil to prevent the sample from drying during measurements. The sample was left to rest for 10 min before measurements, so that residual stresses could relax. All experiments were performed at 30 °C in triplicates. Frequency sweeps test was carried out from 0.1 to 50 Hz, under a 5-Pa strain level, which was within a linear viscoelastic region of all tested samples.

The images were acquired by default settings for brightness and contrast, saved in tiff format and cropped using ImageJ software (National Institutes Health, Bethesda, MD) to obtain the largest possible field of view representing 40× 50 mm of the slice area.

Cropped colour images were converted into an 8-bit greyscale images, whilst the threshold method used for differentiating gas cells and non-cells was carried out by means of the Otsu algorithm.

Bruker AVANCE III solid state spectrometer (300MHz)

The flour suspension for fundamental rheological tests was prepared with 12.4 g (calculated on 14 % moisture basis) of flour and 70 ml of distilled water in order to obtain the same flour–water ratio as in the standard Amylograph method. Fundamental rheometric measurements were taken using the HAAKE MARS—Modular Advanced Rheometer Systems (Thermo Scientific, Germany). The measuring geometry consisted of measuring cup Z40 (43.4 mm diameter, 8 mm gap) and FL2B paddle-shaped rotor with 2 blades. In order to prevent moisture evaporation from the sample, the solvent trap Z40 DIN was used. Two methods for determination of pasting properties of wheat flour were developed: so-called slow and rapid procedure. Slow procedure comprised the complete simulation of Amylograph test with a heating rate of 1.5 C min-1 from 30 to 95 C and maintaining the temperature of 95 C for a period of 600 s. A shear rate of 10 s-1 was applied, which corresponded to 95 rpm. The rapid procedure comprised the tempering of flour suspension at 50 C for 180 s, heating from 50 to 95 C at a heating rate of 3 C min-1 and subsequent maintenance of temperature of 95 C for 300 s to ensure starch gelatinization. Three parameters were recorded in duplicate: pasting temperature, peak viscosity and peak temperature.