Stiffness

Tuning the detection of the point of contact

The first step to compute the stiffness of a sample is to accurately detect the point of contact between the AFM tip and the indented sample.

The detection of the point of contact can be tuned with tools present in the “Parameters” tab.

../_images/Parameters_poc_detection.png

The detection of the point of contact is done by a linear fit of a portion of the off-contact part of the curve.

The size of the portion used for the fit can be controlled by changing the “Fit segment size” value. 1 means the totality of the curve, 0.5, the half, 0.3 is one third, ...

An algorithm moves the segment selection along the curve, finding the optimal one. In some case, it is necessary to block this feature. This is done by the “Limit sliding” check box.

By clicking on “Adjust sensitivity” on the “Point of contact” section, a vertical scrollbar appears on the curve window. Moving this scrollbar adjusts the noise dependency of the point of contact detection.

Computing Young’s modulus

In order to compute the Young’s modulus, go to the “Parameters” tab to set the computation parameters.

../_images/Parameters_stiffness.png

The sphere and cone models are selectable from the “Model” combo box. The tip caracteristic is specified in the “Tip size” spin button. The sphere models needs the tip radius (in nm) and the cone models the tip semi-opening angle (in rad). For dnp cantilevers, nominal values are 20nm for the radius and 0.35 rad for the semi-opening angle. The Poisson ratio is also accessible. Standard value is 0.3 for biological samples.

Once the parameters are set, the computation can be launched by the “Compute stiffness” button in the “Compute” tab.

../_images/compute_stiffness.png

The number and size of indentation curve segments can be modified here (see stiffness tomography [1] publication for more informations). If “re-compute point of contact” is checked, OpenFovea will compute the point of contact, erasing previously computed data. This box can be safely unchecked if the point of contact is correctly detected.

[1]Roduit, C. et al. Stiffness Tomography by Atomic Force Microscopy. Biophysical Journal 97, 674-677 (2009).

Displaying the results

After computing the stiffness, you can plot the results. In “Display” tab are located the button to display the stiffness map, stiffness tomography, mean stiffness tomography and OpenGL 3D view.

../_images/display_stiffness.png

The “Stiffness” button displays a map of the computed stiffness.

../_images/Stiffness_map.png

A slice of this data set can be displayed by moving the right slider. As the slider moves down, the displayed slice deepens. The display of the color scale can be turned on/off by clicking on the “Color scale” button on the lower right corner of the window. The color lookup table can be modified by moving the horizontal sliders.

The “Stiffness tomography” button displays slices of the reconstructed volume.

../_images/Stiffness_tomography.png

The slices in the x, y and z direction are shown in the bottom left, top left and bottom right panels respectively. The slices can be changed by modifying the values in the spin button on the top of the window, or by clicking on the maps. The color lookup table can be modified by moving the horizontal sliders.

The “OpenGL” button displays 3D view of the reconstructed volume.

../_images/Stiffness_3D_view.png

The volume can be moved (zoom, slide, rotate) with the mouse (scroll, right-click, left-click respectively). By pressing the x, y or z keyboard and scrolling with the mouse, the user can slice the volume at his/her wish. Pressing simultaneously the MAJ and x, y or z will slice the volume the opposite direction. The OpenGl display use the color lookup table defined with the stiffness tomography window.

Adjusting tip properties

In some case, the spring constant, or the deflection sensitivity are not well detected. OpenFovea permits to modify these parameters.

Go to the menu “Edition” and choose “Preferences”.

../_images/Edition_preferences.png

The opened dialog contains several tabs. Choose the “Configuration” tab.

../_images/Dialog_experiment_properties.png

OpenFovea reads and display the spring constant that was measured during the experiment capture in the first line. This section permits to modify this value in the case where the spring constant was measured after the experiment.

The “Glass stiffness” represents a correction factor of the “deflection sensitivity”. If the measurement of the deflection sensitivity was inaccurate, OpenFovea permits to adjust it using the “Glass stiffness” value.

In the ideal case, the on-contact part of a indentation curve recorded on stiff area (glass or petri dish) will appears vertical. Adjusting the “Glass stiffness” value updates the displayed indentation curve, allowing to fine-tune this parameter.

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