The most common imaging mode is the constant force mode. In this case, the AFM tip is brought in contact with the sample surface, and set to scan the sample in a x-y raster pattern. A feedback loop maintains a 'pre-set' constant deflection (force) of the cantilever in respect with the sample surface by moving the z scanner for each x-y coordinate. This change in z axis corresponds with the topographical height at the sample at each given point. By adjusting the pre-set force accordingly, the image contrast can be varied and the damage to the sample can be minimized.
Movie 3. Contact mode imaging of a live cell shown as a cut-off profile. The laser diode beam shines on top of the cantilever and is bounced towards the photodetector to record the cantilever deflection. In the same time the height of the piezoelectric element driving the AFM probe is recorded.
Press the lower left corner button to play.
The 3D image of the sample is reconstructed by combining the information on all three axes in an image called 'height image'. The height image preserves the true height information of the sample. Also, a 'deflection' image can be recorded by monitoring the cantilever deflection from a straight line. This image is not recorded at constant force, the tall features in the image representing regions of higher force i.e. higher degree of cantilever bending. This type of imaging looses the true height information, but presents more fine details of the sample than the height image because the feedback loop response is faster for correcting the position of the small cantilever in comparison with correcting the position of the piezoelectric element.
The contact mode images of a live smooth muscle cell in culture are shown. The tutorial allows choosing the height or deflection images (lower button from upper left corner). Click the mouse anywhere on the image and a cross-section of the cell will be shown at right on x- or y-axis (the axis can be chosen using the top button from upper left corner).
For very soft samples contact mode might not be the best choice due to the friction between AFM tip and the sample surface. In tapping mode, the cantilever vibrates at its resonant frequency (bounces up and down) under an external electrical excitation. While rastering the sample in x-y, the AFM tip briefly touches the sample at the bottom of each swing, producing a decrease in the oscillation amplitude (Figure 3).
Similar with the pre-set constant force in contact mode, the feedback loop keep this decrease in oscillation amplitude at a constant value by moving the piezoelectric element, and a height image can be recorded. Also, a 'phase' image can be recorded in tapping mode. When the tip touches the sample at the bottom of its swing, the phase of oscillation is disturbed, inducing a phase difference between the tip and the electrical oscillator that is driving it. The contrast of a phase image is directly dependent on the elastic properties of the sample.
Figure 3. Tapping mode imaging. The tapping mode images of a fixed fibroblast cell are shown (height image left and amplitude image right). Figure courtesy of Veeco Instruments.
For force measurements the AFM is operated in force mode (Figure 4). The piezoelectric scanner is set to drive the cantilever to touch and retract over a predefined distance in the z-axis at a fixed xy position, in such a way that the z-axis movement of the piezoelectric element and the deflection signal from the cantilever are recorded in a force curve.
Figure 4. Diagram of generic force curves. Approach force curve (blue line) and retraction force curve with adhesion events (red line) are presented. The x-axis represents the piezoelectric element displacement and the y-axis represents the force. When the probe is extended towards the cell surface (1), a contact point is established with the cell (2) and thereafter the cell surface is indented. Due to cell stiffness, further probe extension causes an opposing force of increasing magnitude to be generated along with increasing indentation in the cell membrane (2-3). The upward deflection of the cantilever as it bends in response to this force, results in an increasing deflection signal. When the probe retracts from the sample (3-4), the force between probe and sample gradually decreases and if adhesions occurred between the probe and sample surface, the force causes the cantilever to bend downward, and the deflection signal will be lower than the original value (5). When all adhesions are broken, the cantilever returns to the original position (1), and the deflections (unbinding of adhesion events) are recorded on the retraction force curve. The adhesion force is calculated as the product between the deflection associated with the unbinding event and the spring constant of the cantilever.
Movie 4. The movie shows the measurement of adhesion forces between the functionalized AFM tip and cell surface receptors. The AFM tip movement in respect with the cell surface is correlated with the corresponding force curve. Representation not at scale.
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The tutorial shows the retraction part of the force curve correlated with the AFM tip position in respect with the cell. From the upper left corner, two or three adhesion events can be chosen. Move the mouse from left to right on the force curve to see the AFM tip position and the rupture of adhesion events. Representation not at scale.
On soft samples such as cells, the force application results in an indentation of the cell, that is a measure of the local elastic properties of the cell. If the sample is considered rigid, then for each 1 nm of piezoelectric element movement in the negative z direction, there would be 1 nm of probe deflection in the positive z direction caused by bending of the cantilever. However, due to the elastic properties of the cell membrane, only a portion of the piezoelectric element movement causes deflection of the probe; the remainder results in indentation of the cell membrane. The difference between the experimental trace of deflection vs. displacement and the theoretical line expected for an inelastic solid gives a measure of the indentation. The degree of bending or curvature of the displacement curve following cell surface contact describes the local elastic properties of the cell (e.g. the softer the cell the less the curve bends upward and away from the horizontal pre-contact part). The membrane indentation part of the force curve could be analyzed using different models in order to determine the local values of the Young modulus of elasticity as a measure of the local apparent elasticity of the cell at the point of contact.
The tutorial shows the approach part of the force curve correlated with the AFM tip position in respect with the cell. From the upper left corner, different approach curves can be chosen, dependent upon the local cell stiffness at the point of investigation: stiff, soft, and very soft. Move the mouse from right to left on the force curve to see the AFM tip position and the amount of cell indentation in respect with the local stiffness. Representation not at scale.
The force between the AFM tip and the sample varies as the tip scans the sample. The change in the approach curve from force measurements at every point into an image can provide 3D data regarding the stiffness variations from point to point along the sample surface. In this force volume mode the AFM is driven in contact or tapping imaging mode combined with recording the approach force curves for every point in the sample. In this way, one can record stiffness maps of the cell surface.
Figure 5. Force volume mapping. Stiffness measurements were performed on a live fibroblast cell over a16x16 points array. Approach force curves were acquired and processed. The cell stiffness map shows that the cell body is softer than the surrounding substrate. Left - raw data acquired by the AFM software. Right - pseudocolored left image in Photoshop software. The pseudocolor scale bar shows purple-to-red as stiff to soft sample. Figure courtesy of Veeco Instruments.
The AFM can be further combined with optical methods to cover a much larger range of applications:
Figure 6. The same cell was imaged in DIC, fluorescence (blue, DNA) and tapping mode AFM. The image represents the overlay of the three images. MIRO software was used for correlating AFM and optical images. Figure courtesy of Veeco Instruments.
More to come!!!