Tin Nanospheres: A Tip Qualification Tool

Using a titanium sample for tip qualification is a standard procedure for certifying tip quality and for estimating the effective probe radius for nanomechanial measurements. This has been discussed in a previous post and while a reliable and quantitative method, it suffers the risk of damaging the probe in process of tip qualification as the titanium sample is quite hard. Another non-quantitative method is to use tin nanospheres on HOPG. Tin is quite soft and by controlling the substrate temperature nucleation can be controlled quite precisely. A sample with approximately 20 nm spheres is ideal for identifying a host of tip problems.  In truth they aren't "spheres" as they are only 8-10 nm tall and 20-ish nm in diameter

In this study several ScanAsyst Air SiN probes that "failed" were used to image this sample sample. PeakForce tapping was used with ScanAsyst, but any imaging mode would work. Because of the symmetric point-like nature of the objects, and because the radius of the Sn spheres (~ 20 nm) is close to the probe radius (~ 2 nm, but < 20 nm).

This allows one to effectively directly image the functional part of the probe. In the first two images one finds objects that look at least half-normal.

In the first image the spheres are all surrounded by something of a halo. The spheres are always in the left margin of this halo. In general, any image with self-similar features can be assumed to reflect imaging artifacts.

The second also reflects self-similar features. The objects all have a similar shape with a tail extending to the top left. What is interesting is that the density of objects is much higher as well. The objects all have multiple companions showing that the functional image surface is split into multiple parts.

While the first two images could be glossed as normal, the third and fourth images show radically damaged probes.

The last image shows a probe so broadened in radius that no objects are visible.

What Happens When A Probe Fails

There are many parameters that determine the performance of an AFM probe. These include the tapping frequency, spring constant, and probe radius. The spring constant is particularly important to control probe normal forces to mitigate shear forces or to provide the required normal force to deform surfaces in a specific range of modulus. Probe stiffness is also important in image quality when imaging soft materials.

The probe radius is the primary design parameter related to resolution. The process of tip qualification to measure the probe radius using a sample with sharp features has been discussed in a previous blog entry. An effective probe radius can be also be measured using controlled deformations while measuring the modulus of a material with known modulus. These methods of measuring probe radius are required for mechanical measurements as well as certifying image quality.

The degradation of the probe radius will impact image resolution as the surface features are convoluted with a larger than anticipated probe radius. A "sharp" probe might have a radius of 3-5 nm, so a blunting to 30-50 nm will result in the dramatic loss of image quality. Probes coated with conducting material, such as platinum or aluminum, will have additional artifacts in such modes as TUNA or conductive AFM in the current channels due to the loss of conducting contact area with the sample.

In this post visible apex AFM probes were imaged in a field emission SEM. This probe, the Bruker OTESPA, is called a visible apex probe because the functional part of the probe is at the very end of the cantilever, allowing one of locate objects of interest in the optical microscope of the AFM very easily. The probes were imaged with a 2 kV beam using the through lens detector and field immersion. The larger top image shows a good probe. Even at 2 kV parts of the probe appear to be "transparent" as SE1 secondary electrons are generated near the edges of the probe, while SE2 secondary electrons are generated from the thicker body of the probe.

It is common for probes to fail not only by being blunted or worn-- from their radius being drastically increased-- but from picking up objects. The smaller images show three examples of failed OTESPA probes. The probes showed lower resolution and in all cases either self-similar structures or features with companions. These SEM images show objects attached to the probe. In the second image a blunt object is stuck near the end of the probe, and the shape of this object where it contacts the surface would be convoluted with the sample features. In the third image a sharp object is attached just behind the probe. This can act as an companion probe that contacts features and probes companions or doubles to sample features. The last image shows deposition of material across much of the probe, and the attachment of a significantly large foreign object. An object of this size will not only degrade image quality by make the probe very unstable as it interacts with the surface.

The point of this post is to show that things really do go wrong with a probe when image quality degrades.

Power Spectral Density: Collagen D-bands (Part 2)

In a previous post, collagen D-bands were measured by sectioning an image. One sample of the D-band period was found to be 54 nm-- much less than the 67 nm found in TEM of stained histological specimens. While some variation is expected depending upon hydration, this is a significant discrepancy.

A fast Fourier transform (FFT) of the image is shown. A circular ring reflects the periodicity of the D-band spacing. This ring in the FFT is also broken reflecting the fiber orientation. Most of the intensity is between 11 and 1 o'clock as this reflects the orientation of the fibers with this D-band period. Another portion is around 2 o'clock representing the fibers at the top of the image that are diagonal in the image field. This is one useful application of the FFT: quantitating the orientation of structures in the image field.

The second image is 2D isotropic power spectral density. This reflects the power of image signal strength as a function of spatial frequency. The first maximum is at 64 nm and reflects the D-band spacing. As this includes spatial frequency information from the entire image, this is a much more robust method of estimating the D-band spacing than manually sectioning a couple D-band in an image.

Image Sectioning: Collagen D-bands (Part 1)

Images can be sectioned using the sectioning tool. In this case an image of human collagen was taken with PeakForce tapping. The image was flattened to remove large scale spatial relationships not related to the structure of individual collagen fibrils.

The D-bands arising from the staggering of tropocollagen structural units is clearly visualized on individual collagen fibrils. By drawing the sectioning tool across individual fibers, an effective digital cross section of the fiber along that line is produced. In this case the spacing between two specific bands is found to be 54 nm.  While the D-band period is a function of such environmental parameters as hydration, this is far less than the expected 67 nm seen in TEM of stained fibrils.

Application of the sectioning tool can be problematic as measurements are then biased according to the operator's selection of targets and placement of the dimensioning cursors. It is human nature of select features that are the least ambiguous and simplest to dimension by interacting with the image. As an example in this case I chose to section a long fiber in a cluster of long fibers. Removing operator bias would require a sampling methodology which covered more of the image field, including less "attractive" looking fibers, and statistically combining these measurements.

Human Collagen: PeakForce Tapping

The primary advantage of tapping AFM is that it is a non-contact AFM technique. While it "taps" on the sample surface, the tapping is intermittent and as such there is minimum shear force. This is ideal in imaging soft materials, including biological systems, as significant shear forces modify the sample surface while it is being imaged.

That tapping is generally done at the resonant frequency of the probe. A stiff silicon tapping probe has a resonant frequency on the order of 300 kHz, and so a tapping frequency just below resonance is selected for tapping AFM. Constraining the tapping causes the probe frequency to increase, and this will cause the probe frequency to move towards resonance not off resonance.

While this mode of imaging produces minimal shear force, one disadvantage is that it produces an indeterminate maximum normal force. While one can image in soft tapping mode by reducing the tapping amplitude, it is a non-trivial task to estimate the maximum peak normal force in tapping AFM. It is sometimes also desirable to know the maximum force exerted on a sample to quantitate sample deformation.

Bruker's PeakForce tapping combines the best of tapping and contact AFM imaging modes. What it does is perform complete force curves at 1-2 kHz at every image point. The force curve is triggered at the maximum applied normal force according to these force curves, and it is this "PeakForce" that is the set-point or parameter maintained constant during imaging. The second image, borrowed from Bruker, shows force curves as a function of time and position, and the PeakForce set-point is point C on both curves. A further innovation is ScanAsyst technology which dynamically and intelligently monitors and optimizes the scan rate, PeakForce set-point, gains, and Z-limit to produce the best image quality.

In this image human collagen from cadaver skin was imaged using a ScanAsyst Air probe in ScanAsyst mode. This image of a fairly soft biological specimen was imaged with little operator interaction beyond aligning the scanner and focusing on the sample. An additional advantage of the PeakForce tapping imaging mode is that force curves are available at every image point, and these can be used to perform nanomechanical measurements-- what is called PeakForce QNM.