CHANGING SURFACE STRUCTURES DURING IMAGING
Unlike optical microscopy or electron microscopy, atomic force microscopy uses physical force to image the surface ”by feel” and not by electromagnetic radiation or electrons. This mechanical force can often be destructive to the surface itself. Lateral forces because of the rastering of the tip over the surface can cause surface objects to be displaced during imaging, whereas vertical forces can cause indentation of soft surfaces.
Displacement During Imaging
As the tip is rastered across the surface, there is a lateral force applied to the object being imaged. This lateral force can remove the object from the surface if the object is not adhered strongly to the surface. If the object is removed during imaging, the structure will suddenly disappear from the image. The image may show just part of the surface structure (imaged before it disappears). It is possible to observe this more clearly by first imaging a small area at a high force for some time and then imaging a larger area with a smaller force. The middle of Fig. 8 shows an area that has been imaged for some time. All the particles in this area have been removed because of imaging.[19]
The lateral interaction between the tip and the surface can be minimized by imaging with tapping-mode atomic force microscopy instead of contact-mode atomic force microscopy, or by decreasing the amount of force used while imaging. In tapping-mode atomic force microscopy, the tip is not in constant contact with the sample surface. Instead, the tip is vibrated close to the resonance frequency of the AFM probe. When the vibrating tip strikes the surface, some of the energy is absorbed by the surface. This causes a decrease in the vibrational amplitude of the probe. The piezo adjusts the distance between the surface and the tip to maintain constant vibrational amplitude.
Although tapping-mode atomic force microscopy will reduce lateral forces on the surface objects, it is often necessary to take more steps to ensure that the objects are not displaced. One approach is to ensure that the objects are strongly anchored to the surface. This is particularly important for imaging negatively charged objects on a negatively charged surface, which is the case for most biological species on silica or mica. For example, Shibata-Seki et al.[19] showed that liposomes can be pushed out of the way at a force >4.2 nN. Similarly, SK-N-SH (human neuroblastoma cells) and AV12 (Syrian hamster cells) can be moved during imaging, especially if they are alone.[20]
Fig. 7 Scanning electron micrographs of impurities on AFM tips. (A) Multiple contaminants and imperfections. (B) Cantilever tip located under cantilever coating. (C) Off-centered probe. (D) Unused probe showing particulate contamination with protein-like globules (arrows). (E) Contaminated probe with protein. (F) Used probe showed doubling images.
One solution is to modify the surface so that it can covalently bond with the object. This can be accomplished by using amino-silanization[21,22] on silica. In this method, silane trimethoxylsilyl-propyl-diethylenetriamine func-tionalizes the glass. The object can then bond to the func-tionalized surface via an amide bond linkage.
Another solution is to render the surface cationic using a cationic polymer. These polymers include spermine,[23] polylysine,[24] polyethyleneimine (PEI),[25,26] chitosan,[25] and diethyl-aminoethyl dextran (DEAE—dextran).[25] A complete study of the adhesion of Escherichia coli as well as polystyrene spheres via the above surface modification showed that PEI is the most effective adhesion mecha-nism.[25] When using cationic polymers, care should be taken to remove all the nonadhered polymers, as non-adhered polymers may stick to and contaminate the tip during imaging.
Another option for imaging bacteria is to trap them in holes in a filter to prevent them from moving around.[27] You et al.[20] showed that bacteria adhered to the surface via cell-EPS interactions or cell-cell interactions may be strong enough to prevent displacement. Finally, studies on DNA have shown that imaging in air[23] or propanol[28] prevents water-tip interactions and makes imaging easier.
Indenting During Imaging
Tips can cause not only lateral forces but also vertical forces that can deform the surface. This is particularly seen on soft surfaces such as polymers, and biomaterials such as bacteria. Fig. 9 shows a bacterium in an aqueous solution being irreversibly ”deflated” because of tapping-mode atomic force microscopy imaging. The arrows indicate the direction of scanning.[6] At first, the image of the bacterium shows a height of 1 mm (Fig. 9A and B). As scanning continues, the height of the image suddenly decreases, as shown on the top of Fig. 9C and D. Finally, in Fig. 9E and F, the bacterium has been completely deflated. Another deflated bacterium shown in Fig. 9G and H shows a rectangular indent in the top left corner of the image. Other biological species such as tobacco mosaic virus (TMV)[23] and human neuroblastoma cells (SK-N-SH)[20] have been irreversibly damaged during imaging.
Fig. 8 Demonstration of AFM artifacts because of high forces. In situ AFM images of liposomes on substrates taken at different load forces over the same region. Images were taken with a standard Si3N4 cantilever tip (spring constant of 0.12 N/m). (A) Load force was approximately 0.72 nN. (B) Load force was approximately 4.2 nN. (C) Load force was approximately 2.2 nN.
This indentation will occur if the effective spring constant of the object is similar to the spring constant of the cantilever. If the surface is a Hookien elastic material, the deformation, or indentation, of the surface i and the deflection of the cantilever d can be expressed by Eq. 1:
where kc is the spring constant of the cantilever and ks is the spring constant of the surface.
The first step to avoid this indentation is to use tapping-mode atomic force microscopy. However, as shown in Fig. 10, this does not completely eliminate the indentation. Another solution is to use a cantilever with a smaller spring constant. This will decrease kc and decrease i in Eq. 1 but may also sacrifice some resolution. In both contact-mode atomic force microscopy and tapping-mode atomic force microscopy, it is possible to image with a lower force, which can decrease the indentation.
Fig. 9 Creation of a deflated bacterium because of high vertical forces. (A), (C), (E), and (G) are height images, whereas (B), (D), (F), and (H) are deflection images. The bacterium is deflated in (C) and (D) at the dotted line. A similar deflated bacterium is shown in (G) and (H).
Fig. 10 Linearity and x-y calibration of the AFM piezo. (A) Test pattern. (B) Image produced when the piezoelectric scanner is not linear.
Finally, in some cases, it may be possible to ”stiffen” the surface structure. In the case of biological cells, this can be performed by exposing the cells to glutaraldehyde.[26,29,30] This makes imaging easier because the spring constant of the bacteria increases; however, the procedure may render the cells more hydro-phobic.[31] Another option is to image the soft surface in a media other than water, such as air[28] or propa-nol.[29,32] These techniques will provide better images but may not accurately represent the image topography in an aqueous solution.
SCANNER ARTIFACTS
The scanner head on the AFM can introduce artifacts while imaging. These artifacts result from nonlinearity in the horizontal and vertical movements of the scanner, vertical overshoot because of the control system, and scanner drift.
Fig. 11 Tapping-mode atomic force microscopy images of E. coli bacteria with a scan angle of 0° (A), 180° (B), and 90° (C). In (D), the scan angle is 180° and the tip direction is from left to right. The dark spots of the image are always on the back side of the scanning probe direction. This is because of an overshoot in the feedback control system.
Fig. 12 Artifact caused by scanner drift. (B) A zoomed-in area of (A). The pattern is distorted because of scanner drift.
The piezoelectric scanner moves the tip across the surface in both horizontal (x-y) and vertical (z) planes. It is critical that the scanner is moving linearly with time and that it is calibrated. If the scanner is not moving linearly, features on one side of the image will appear smaller than the other side (Fig. 10).[33] If the scanner is moving linearly but not calibrated, the dimensions in the horizontal dimension will not be accurate. Most AFM packages come with a standard grid that can be used to check the linearity and accuracy of the scanner. Similar compensation software tools can be used to calibrate the scanner in the vertical plane.
Another common artifact because of the scanner is the presence of ”shadows” on the opposite side of the direction of scanning (Fig. 11). These shadows are because of hysteresis in the vertical control of the piezo-scanner. The scanner overshoots the edge and shows a decrease in height, even in the line profile. Decreasing the scan speed can reduce this effect, whereas reversing the scan direction can reveal the true edge of the object.
The scanner is sensitive to temperature changes. These temperature changes can cause drifts in the scanner and distortions of the image. This most commonly appears at the beginning of a scan of a zoomed-in region of an image (Fig. 12).[33] For this reason, an area should be scanned multiple times to make sure there are no changes.
ELECTRONIC ARTIFACTS
While imaging with the AFM, it is important that the equipment is isolated from external vibrations. These can include acoustic vibrations from sound or mechanical vibrations of the floor. These artifacts typically appear as periodic patterns in the image. The equipment can be isolated from the room by using an isolation table, or by suspending the apparatus from a tripod.
Electronic noise can also cause periodic patterns in the image. If the gains of the control system are set too high, the system control system will become unstable. Reducing the gains can eliminate this problem. Similar effects can be observed with faulty electronics.
USING ARTIFACTS TO YOUR ADVANTAGE
Although the artifacts discussed should be avoided to obtain the most accurate image possible, the identification of many of these artifacts has led to breakthroughs in new ways of using the AFM. These breakthroughs include nanomanipulation and nanoindentation.
Although it is often not desired to move the object to be imaged, researchers have recently taken advantage of the interaction between the tip and the objects on the surface to manipulate surface objects. The ability of the AFM tip to move with angstrom-level precision makes it ideal for nanomanipulation. The surface can be imaged using weak attractive forces. The tip can then be brought into stronger contact with individual objects to manipulate them.[34] This technique has been used to build two-dimensional and three-dimensional structures of nanoparticles,[34,35] as well as to manipulate biomolecules such as DNA and proteins.[36-38]
Researchers have also taken advantage of vertical forces applied during imaging using the AFM for nano-indentation. Nanoindentation allows measurements of mechanical properties of various surfaces including polymers surfaces[39-42] and biological surfaces.[8,21,26,43-46] For polymeric surfaces, the indentations made with a high spring constant cantilever (100-300 N/m) made at various forces are imaged via tapping mode. The depth of the indentations is used to determine the spring constant of the surface according to Eq. 1. For more elastic surfaces, such as biological surfaces, the mechanical properties of the surface can be determined by analyzing the AFM force curves.[8,21,26,43-46]
CONCLUSION
The AFM is a powerful tool for imaging surfaces. With proper care, accurate images can be obtained and can provide important quantitative information about surfaces on the nanometer scale. However, it is imperative that researchers using this tool are aware of the many artifacts that can be present in the created images. These artifacts may be because of the shape and size of the AFM tip, the lateral or vertical interaction between the tip and the surface, or the electronics of the AFM system. Once aware of these artifacts, corrections can be made to create more accurate images of the surface structures of interest.
