The principle of operation of the AFM is very similar with that of a stylus profilometer - a sharp cantilever tip interacts with the sample surface sensing the local forces between the molecules of the tip and sample surface (Figure 1). This instrument is not a 'conventional microscope' that collects and focuses light. The word microscope has been associated with this instrument because it is able to measure microscopic features of the sample. The most characteristic property of the AFM is that the images are acquired by 'feeling' the sample surface without using light. In this way, not only the sample topography can be recorded with good resolution, but also the material characteristics and the strength of interaction between the sample surface and the cantilever tip. Due to the fact that no light is involved in acquiring the sample properties, the AFM reaches a resolution far below the diffraction limit offered by the optical microscopy. Its resolution is limited only by the tip radius and the spring constant of the cantilever. The main components of an AFM are (Figure 1):
A short overview of the system components is presented below.
Figure 1. Schematic illustration of a scanned tip AFM system. A flexible cantilever with a tip at the end is rigidly connected with a xyz piezoelectric element. The optical lever consists of a laser diode beam that is focused on the back of the cantilever and bounces off reaching the photodetector.
The most sensitive part of an AFM is the tip that interacts directly with the sample surface. The most common probes are constructed using micro-fabrication techniques from silicon or silicon nitride. Each probe can have multiple integrated cantilevers (Figure 2).The properties and dimensions of the cantilever and tip play a major role in determining the sensitivity and resolution of the AFM. Cantilevers are V-shaped or single beam, gold coated on the upper side, with spring constants ~ 10 pN/nm. The typical AFM tip for live cell imaging has a pyramidal shape with a radius of curvature at the end of pyramid of 50 nm. For special applications the pyramidal tip can be replaced with glass beads with a diameter of 1 - 5 micron. The AFM tip has to be chosen carefully depending on the specific application. In general, the cantilever should be soft enough to be deflected at very small forces and the tip radius should be comparable with the features of the sample. Advances in cantilever manufacturing are offering investigators a variety of probes.
The AFM probe consists of a flat substrate about 1 x 2 mm that has at one end the cantilever with the tip. This AFM probe is mounted into a glass holder that contains the electrical contacts for the piezoelectric scanner. Once the glass holder is rigidly mounted on the scanner, a protective silicon skirt insulates this entire ensemble from the surrounding wet media during the experiment.
Figure 2. Scanning electron microscope images of AFM cantilevers. (a) Typical AFM tip for force measurements. At the very end of the cantilever is a pyramidal shaped tip (4 micron base, 35° half angle and 50 nm radius); (b) The MLCT AFM probe with five different cantilevers; (c) The DNP-10 AFM probe with two different V-shaped cantilevers. Images courtesy of Veeco Instruments.
The optical lever (beam deflection technique) is the most used technique of detecting the cantilever deflection. A laser beam coming from a laser diode is reflected off the back of the cantilever onto a photodetector. The photodetector consists of a split photodiode that senses the changes in light intensity from the reflected beam due to the deflection of the cantilever following tip-sample interaction. This system amplifies a very small cantilever deflection, such that the longer the cantilever-detector beam path, the more amplification is provided at the detector level. The sensitivity of such a system is 0.1 nm.
The main purpose of the feedback loop is to maintain a predefined setpoint that is determined by the instrument gains. The operator has to determine experimentally the optimal proportional, integral and derivative (PID) parameters of the feedback control loop for each application.
The AFM scanners are made from piezoelectric material, which expands/contracts proportional with an applied voltage. The scanner itself is constructed by stacking three independent piezoelectric crystals, each of them being responsible for movements on one axis x, y or z. The piezoelectric scanner can move very precisely with good reproducibility for small displacements, but for displacements > 70% of the full-scale displacement the piezoelectric response is non-linear (hysteresis). The commercially available AFM featuring 'closed loop' feedback systems independently monitor the scanner movement and correct its motion for hysteresis. The scanner configuration consists of a hollow tube with x and y axis controlled by two segments of piezoelectric crystal, and one segment for z axis. There are two types of scanner configurations: scanned tip AFM where the piezoelectric scanner is rigidly attached to the probe and is moved over the sample surface which stands still, and scanned sample AFM where the scanner is attached to the sample and it is moved under the tip. Each of these two designs has advantages and disadvantages. In the scanned tip design, is easier to equip the system with temperature-controlled and liquid flow stages. The scanned tip design could have a potentially higher noise level that the scanned sample and the top view of the sample is blocked due to the scanner that sits on top of it. For the scanned sample AFM, the weight of the sample has to be controlled because it sits on top of the scanner. Also, AFM operation under liquid is not straightforward requiring ingenuous designs of the flow cell and the temperature controller. Both designs could be integrated with optical microscopes or as stand-alone AFM systems, and are commercially available.
Movie 1. Dimension Hybrid Head xyz piezo-scanner movement is shown in detail. Z-sensing and then xy-sensing are shown, together with their internal photodetection systems. Movie courtesy of Veeco Instruments.
Press the lower left corner button to play.
The main electronics configuration of the commercially available AFM consists of a digital signal processor (DSP) that performs all of the signal processing and calculations necessary to drive the AFM in real-time. The analog-to-digital (ADC) and digital-to-analog converters (DAC) are translating both ways the signals between the scanner head and the DSP card. The piezoelectric scanner is driven by a high voltage amplifier. Also, a dual monitor computer interface allows for data display (images or force curves) and instrument parameters control in real-time.
Movie 2. The movie shows the Catalyst Bioscope AFM (Veeco Instruments) combined with a Leica inverted microscope. Fluorescence and AFM measurements such as force, stiffness and protein unfolding are presented. Microscope Image and Registration Overlay (MIRO) software enables sequential acquisition of correlated AFM and optical images. When a region of interest (ROI) is defined in the field of view of the optical image, the AFM further scans that ROI and overlays it in real-time onto the optical image. The example here shows HeLa cells that have been labeled with DAPI (blue, nucleus), phalloidin (red, actin cytoskeleton) and FITC (green, microtubules), and scanned by confocal microscopy and AFM. MIRO also allows AFM force measurements to be targeted optically, eliminating the need for a preliminary AFM imaging due to precise image registration between the two imaging modes. If a point is marked into the optical field, the AFM probe will be offset to that location to perform force measurements. Movie courtesy of Veeco Instruments. Press the lower left corner button to play.