Atomic Force Microscopy Imaging Artifacts Part 1 (Nanotechnology)

INTRODUCTION

The invention of the atomic force microscope (AFM; also known as scanning probe microscope, SPM) in 1986[1] has revolutionized the way researchers study surfaces.[2] Material surfaces such as metallic or polymeric thin films can be imaged under ambient conditions (air, liquids) with nanometer resolution. Furthermore, it is now possible to image biological species such as DNA, proteins, and bacteria in their natural environment. However, researchers using the AFM for imaging must do so with great care to avoid the many artifacts that can be present during imaging. It is the purpose of this article to identify these artifacts and to present ways to avoid these artifacts while using the AFM. Interestingly, the discovery of many of these artifacts has opened doors to new uses of the AFM. This will be discussed as well.

OVERVIEW

Before discussing the artifacts present in the AFM, a brief discussion of the AFM mechanism is warranted. Fig. 1 shows the mechanism of a typical AFM. The primary components are: 1) helium-neon laser; 2) AFM probe; 3) piezoelectric scanner; and 4) photodiode detector. Laser light is reflected off the top of a probe and is detected by a photodiode detector. The probe consists of a sharp tip attached to a flexible cantilever beam. The piezoelectric scanner controls either the vertical position of the surface (Fig. 1), or the vertical position of the probe. The piezoelectric scanner is able to move in three dimensions with angstrom-level precision. When the surface and the tip are brought closer together, the interaction between them causes the tip to be deflected either toward the surface (because of attractive forces), or away from the surface (because of repulsive forces). This deflection of the tip is recorded as the change in the position of the laser on the detector. The piezo responds to this change by increasing or decreasing the height of the sample to maintain a constant distance between the tip and the surface. Images can be recorded as either the deflection on the detector (deflection image), or the vertical distance of the piezo (height image). The vertical resolution of AFM images is dictated by the interaction between the tip and the surface. The lateral resolution is determined by the size of the tip, as will be discussed below.

Fig. 2 shows a diagram of a typical pyramidal AFM tip on a cantilever. In most systems, the cantilever is tilted approximately 12° toward the surface. At a 0° scan angle, the tip moves across the surface in the direction of the arrow in Fig. 2. The AFM tips have traditionally been pyramidal in shape, but can also be of other shapes, such as a nanowire or a colloidal sphere.

Because the functionality of the AFM depends on the interaction between the tip and the surface, most of the artifacts that exist with the AFM are because of the size, shape, and cleanliness of the tip. Artifacts are also introduced when the tip interacts with the surface in a way that changes the surface itself. Finally, there are artifacts that exist because of the electronics of the control system. Table 1 shows a summary of each artifact along with the solution to the problem. Details of each artifact are discussed below.

ARTIFACTS BECAUSE OF FINITE SIZE AND PYRAMIDAL SHAPE OF TIP

The image produced by the AFM is a convolution of both the shape of the surface being imaged and the tip geometry. The rule of thumb is that the image will accurately reflect the surface structure if the difference in the image of the radius of curvature of the tip is one-tenth the radius of the imaged structure.1-3-1 The exception to this rule occurs if the height of the imaged structure is comparable to the height of the tip.

Broadening of Surface Features

For any object on the surface with a finite height, the side of the tip will interact with the side of the surface feature. For the surface structures shown in Fig. 3, the side of the tip interacts with the surface structure when it is a distance of b away from the edge of the object. This will produce a broadening of the object in the image by a distance of b on both sides of the surface structure. If the lateral dimension a of the object is large and the height of the object is small (Fig. 3A), this effect is small. However, if the lateral dimension of the surface structure is small, or the height of the surface structure is large (Fig. 3B and C, respectively), the interaction of the side of the tip with the surface structure will result in significant broadening of the object in the image. For these reasons, this artifact will be predominant in images of nanoparticles (including proteins or DNA) as well as images of large particles such as bacteria

Fig. 1 Schematic of AFM.

An experimental example of the broadening of a small object because of the tip size (similar to Fig. 3B) has been observed when imaging DNA. Images obtained with a tip, with a radius of 30-50 nm, indicate that the diameter of the DNA strand is 14 nm whereas the true diameter is 2 nm.[4,5] In this case, the height of the image (as determined by the line plot) will be correct and only the width will be distorted.

Fig. 2 Typical shape of AFM tip and cantilever relative to the surface. The tip is square pyramidal and the cantilever is tilted toward the surface at an angle of 12°. The arrow indicates the direction of scan (at 0° scan angle).

An effect similar to Fig. 3C can be observed while imaging large bacteria in aqueous media (Fig. 4). Although the radius of the bacteria (500 nm) is much larger than the radius of curvature of the AFM tip (5-40 nm), the object is also very tall compared to the tip. The diagonal lines represent the interaction of the diagonal edges of the tip with the side of the bacterium or sphere. Note that the lines appear mostly on one side because of the 12° tilt of the cantilever.[6] Similar effects have been observed in tapping-mode images of P. chrysosporium spores[7] and images of a fixed liver endothelial cell (LEC).[8]

There have been many attempts to correct this image artifact. One way is to use a standard tip and then reconstruct the image to determine the true size of the surface structure.[9-15] In most cases, these reconstruction techniques require knowledge of the shape and size of the AFM tip. Information such as the radius of curvature of the AFM tip can be obtained from the manufacturer but is best determined experimentally by the technique of reverse imaging. In this technique, the surface structure is smaller than the tip itself, so that the image reflects mostly the shape of the tip. The surface structure can be a grading of spikes,[15] circular depressions,1-13-1 or close-packed spheres.[10] Digital Instruments® also has a Tip Estimation software program that can be used to determine the shape of the tip. Blind restoration, or blind reconstruction, can also be used to deconvolute the images without information about the tip shape.[4,16] This is performed via mathematical morphology operations (e.g., dilation and erosion) that can estimate the shape of the tip from the image.

Table 1 How to identify and avoid common AFM imaging artifacts

Artifact	Cause	Solution
Broadening of features	Interaction of surface with side	Use deconvolution techniques
	of tip (structure above surface is	Use sharpened tip (higher aspect ratio)
	too small laterally, or too
	high vertically)
	Contamination on tip	Clean tip with UV/ozone or piranha solution Test for cleanliness by taking a force curve before and after imaging Image tip with SEM before and after imaging
	Bluntness of tip	Use new tip image tip Image tip with SEM to ascertain shape Use deconvolution techniques to determine actual surface structure
Smaller features	Interaction of surface with side of	Use deconvolution techniques
	tip (surface structure below surface	Use sharpened tip (higher aspect ratio)
	is too small laterally, or too deep)
Surface structures	Lateral forces between tip and surface	Use tapping mode
that disappear	are too large and objects are pushed	Minimize force during imaging
while imaging	away via AFM tip	Bond surface structures to surface
Destruction of	Vertical forces between tip and	Use tapping mode
soft surfaces	surface are too high	Use smaller force during imaging Use cantilever with smaller spring constant Stiffen surface
Appearance of ”shadows”	Overshoot of control system	Slow down imaging speed
on opposite side of		Change scan direction
tip movement
Distortion after image zoom	Drift in piezoelectric scanner	Rescan area of interest
Periodic ”noise” in image	Mechanical or electrical noise	Remove vibrations from room Decrease gains on control system

Another solution to the broadening effect is to use a tip with a high aspect ratio such as a nanowire (Fig. 5). The high aspect ratio reduces the interaction of the side of the tip with the surface structure, but one must be cautious when using a sharpened tip because it can create large pressures on the surface that are more likely to damage soft samples. For large objects on the surface, adjusting the angle between the cantilever and the surface so that they are parallel can minimize artifacts because of high vertical dimensions.

Features Appear Too Small

If the probe needs to go into a feature that is below the surface, the surface structure in the image may appear too small because of the interaction between the tip and the inside of the structure. This artifact has been observed in images of germanium surfaces bombarded by ions.[9] Atomic force microscope images showed the presence of columnar structures present because of the shape of the tip and did not appear in electron microscopy images. In this case, the tip may not be able to image the corner of the feature (Fig. 6A), or may not be able to reach the bottom of the structure (Fig. 6B). This will be particularly noticeable if the hole is small laterally (Fig. 6A) and/or deep (Fig. 6B).

Again, this artifact may be removed by using a tip with a higher aspect ratio (such as a sharpened tip, or a nanowire). Reconstruction of the image can also be performed, although the deconvolution algorithm cannot reconstruct parts of the tip that did not make contact with the sample.[12]

Artifacts Because of Contamination of Tip or Misshapen Tips

Because the lateral resolution of the image depends on the radius of the tip, any changed to the tip that cause the tip to become broader will sacrifice this resolution. These changes to the tip may be because of contaminants on the tip, or because of tip damage.

Fig. 3 Schematic of image artifacts because of the size and shape of the tip. In all cases, the side of the tip interacts with the surface object with a lateral dimension of L and a height of h at a distance of d away from the object. This causes a distortion in the image, indicated by the dotted line. In (A), the radius of the tip is much smaller than the radius of the object being imaged, and the artifact is minimized. In (B) L is small and the distortion is more noticeable. In (C), the height of the object h causes a greater distortion of the image.

Scanning electron microscopy has shown that many tips have defects and have been contaminated before they are even used (Fig. 7).[17] These contaminants may include silicone oils from packaging,[18] or irregular apexes. More commonly, the tip can pick up contamination during imaging.[17] The presence of these contaminants, or defects, can change the shape of the image and result in an irregularly shaped image, or a broadening of the image. The presence of contaminants is particularly problematic for AFM applications where the tip is chemically modified because these techniques rely on a clean tip surface.

There are multiple suggestions for cleaning tips before using them. The most common cleaning procedure is to use UV/ozone cleaning.[18] This destroys organics on the tip and can be performed easily using an UV/ ozone cleaner. Another more rigorous approach is to soak the tips for 30 min in piranha solution (70:30 H2SO4/ H2O2).[17] These cleaning procedures ensure only that the tip is clean before it is used, but does not prevent the tip from becoming contaminated during imaging.

Fig. 4 Atomic force microscope line artifacts because of the interaction of the side of the tip with a tall surface structure (Fig. 3C). (A) and (B) are tapping-mode amplitude and phase images of 1-mm-high E. coli. The white bar represents 1 mm. The vertical scale for the height images is 1 mm. The scan direction is from right to left, with a 0° scan angle. The lines highlighted in black show the artifact, which appears as lines ±27° to the scan direction. The line artifacts appear on the right side of the image because of the tilt of the cantilever relative to the surface.

One suggestion to test for the cleanliness of the tip during imaging is to measure the force between the tip and a clean surface (such as clean glass or mica) before and after imaging your surface of interest. A change in force indicates a chemical change in the tip.[17] Another suggestion is to image the tip via SEM before and after use. Although this is a time-consuming step, it will identify any contaminant that adheres while imaging.

Fig. 5 Multiwall nanotube attached to a silicon tip as an example of a high aspect ratio AFM tip.

Fig. 6 Schematic of image artifacts because of imaging a surface structure with a lateral dimension of L and a depth of h below the surface. In (A), the tip is not able to image the corner of the structure, whereas in (B), the tip is not able to image the bottom of the structure. The dotted line represents the image produced by the AFM.

A tip may become misshapen either during manufacturing of the tip, or during imaging. This effect is similar to the presence of a contaminant and will result in a strangely shaped image. The new shape of the tip can be identified by using a deconvolution program (discussed above), or by imaging with an SEM.