As the tip moves up and down in the z-axis as it traverses the specimen, the movements are recorded on the diode as movements of the reflected laser beam. A piezoelectric device under the specimen is activated in a sensitive feedback loop with the diode to move the specimen up and down so that the laser beam is centered on the diode. As the tip dips down into a depression, the piezoelectric device moves the specimen up to compensate, and when the tip moves up over an elevation, the device compensates by lowering the specimen. The current to the piezoelectric device is interpreted as the z-axis, which along with the x-and y-axes renders the topography of the specimen at a molecular, and sometimes an atomic, resolution (Fig. 1.14).
A major advantage of the AFM for examining biologic specimens is that, unlike high-resolution optical instruments (i.e., TEM or SEM), the specimen does not have to be in a vacuum; it can even be in water. Thus, it is feasible to image living cells and their surrounding environments.
FIGURE 1.13 • Diagram of the atomic force microscope (AFM). An extremely sharp tip on a cantilever is moved over the surface of a biologic specimen. The feedback mechanism provided by the piezoelectric scanners enables the tip to be maintained at a constant force above the sample surface. The tip extends down from the end of a laser-reflective cantilever. A laser beam is focused onto the cantilever. As the tip scans the surface of the sample, moving up and down with the contour of the surface, the laser beam is deflected off the cantilever into a photodiode. The photodiode measures the changes in laser beam intensities and then converts this information into electrical current. Feedback from the photodiode is processed by a computer as a surface image and also regulates the piezoelectric scanner. In contact mode (left inset), the electrostatic or surface tension forces drag the scanning tip over the surface of the sample. In the tapping mode (right inset), the tip of the cantilever oscillates. The latter mode allows visualization of soft and fragile samples while achieving a high resolution.
FIGURE 1.14 • Atomic force microscopic image of a single DNA molecule. This image was obtained in the contact mode in which the sharp scanning tip “bumps” up and down as it is moved back and forth over the surface of the sample. The sample lies on an ultrasmooth mica surface. An individual molecule of DNA easily produces enough of a bump to be detected. Thickenings along the DNA molecule are produced by proteins bound to the molecule, and these thickenings produce an even larger movement of the scanning tip. The scan field measures 540 nm by 540 nm. The length of the DNA molecule ranges from 0 to 40 nm. 185,000. (Courtesy of Dr. Gabriela Bagordo, JPK Instruments AG, Berlin, Germany.)