Table of Contents
Future Applications of Rapid AFM Imaging
There are three key application areas that benefit from a
high-bandwidth Atomic Force Microscope (AFM) with identical data
quality, operating cost, force control and convenience of use as a
typical AFM. These can be classified under screening,
survey and dynamics. For
each of these categories, the Dimension FastScan was applied.
A survey of a material is typically undertaken to understand the
representative morphologies of a
heterogeneous, unknown sample. This is a very typical situation when
using an AFM (or any microscopy technique) on a new sample. Especially
for complicated (e.g., biomaterial) samples, the major part of imaging
time is often spent looking at enough sample surface to understand what
is important, rather than capturing the final images that represent the
sample. Covering a larger area of the sample, with enough detail and
within an acceptable amount of time enables a better, more balanced
view of the parts and their respective roles.
Applying a higher bandwidth AFM
toward this goal can be done in the
- On a rough sample, more sites can be engaged and imaged in a
shorter amount of time.
- The NanoScope software’s MIRO image overlay capability can be
utilised to keep track of all the scans within one context, and in
relation to an overview optical image.
- On a fairly flat sample, another way to survey the sample is to
capture a very large scan area with very high pixel resolution. The
data can then be zoomed into and analyzed (even without using further
tool time) and representative areas can be magnified and published.
- A key advantage of this method is that it is possible to decide
on the best scale and framing after taking all the data. The data shown
in figure 1 comprises one 16-megapixel image of a 20-micron scan range
on a PTFE polymer film, acquired in 8 minutes, with data zooms of
various interesting morphologies, as well as phase data for two of
Figure 1. 20mm,
16MP image of PTFE polymer film (left), acquired in 8 minutes.
Right: Multiple data zooms showing detail and phase data. Surveying a
sample means to explore and understand it’s representative
morphologies, and document them in publication quality images. On
sufficiently flat samples, one survey method is to take a large,
high-resolution scan that can be explored offline for representative
morphologies, which can then be magnified and published.
It is easy to understand the space of possible phenomena in
screening applications, however in order to understand the dependency
of an input parameter or a process parameter and a nanoscale morphology
or property must be understood and quantified. It is important to image
a number of sites on multiple samples and to effectively analyse and
quantify the property or morphology. Imaging speed is essential as also
multi-sample loading and automation, reliable operation without
intervention of the user, data management and batch image analysis are
Figure 2 shows an example of AFM
screening from the pharmaceutical
industry. Here the active pharmaceutical ingredient (API) is combined
(formulated) with an (inactive) excipient to form an amorphous solid,
with the objective of maximizing the API’s solubility after ingestion.
At room temperature, the amorphous formulation is solid (frozen) but
otherwise it would phase separate.In order to observe the possible
phase separation with bulk techniques, relatively macroscopic (~100nm)
separation and re-crystallization of the API must first occur. The
FastScan AFM can obtain the indicators of instability on a
much smaller size scale, much earlier.
Figure 2. Screen of twelve amorphous drug
formulation candidates (fractured film, 3mm
scans, five sites per candidate). Batch analysis shows material
specific roughness with tight error bars; excipients with API load are
smoother than blanks. This type screen is used to verify compound
compatibility, and to rapidly predict stability/shelf life, after brief
stress aging. (Samples courtesy of M.E. Lauer, F. Hoffmann-La Roche,
The “typical” discipline for high-speed AFM
is the time-resolved
study of dynamic processes on the scale of proteins and DNA. This
application is responsible for a large part of the initial
understanding of how to make AFMs
faster, while maintaining
non-destructive tip-sample forces. It was discovered that it was
essential to make cantilevers smaller. It then becomes necessary to
enable the use of smaller cantilevers, to scan faster, and to capture
data faster. In this hunt for speed, it was found that the achievable
frame rate scales roughly with the dimensions of the cantilevers. It
also scales with the data quality, with the number of lines, and with
the acceptable pixel blur caused by loose tracking (parachuting).
Achieving frame rates more than 1fps is typically achieved by
increasing imaging bandwidth, and by trading image quality for speed.
In order that the FastScan to be more than a single-purpose movie
machine, it was important to have full AFM performance at increased
bandwidth, but to be able to further trade off resolution for speed in
the way of other high-speed AFMs, and to maintain superior control of
tip-sample forces at high scan rates.
Figure 3 shows three frames from a time sequence of 2100 frames,
captured at a rate of 1 frame per second, of DNA in buffer solution,
loosely bound to and diffusing on an APS-treated mica substrate.One can
see different motions of the DNA, including a “sliding” motion of the
DNA along its contour, and approximately perpendicular to the scan
direction. This shows that the DNA’s binding to the substrate is loose
enough to allow it to move, and diffusion is not dominated by the
back-and-forth scan motion of the AFM tip. This should lay a good
foundation for the observation of more complex sample systems, such as
DNA-protein complexes, ATP-driven systems, etc.
Figure 3. DNA loosely bound to mica treated by
APS-method. TappingMode in buffer solution. Probe: Broadband-C. 1
frame/s. Shown are 3 of 2100 frames, showing the diffusion of the DNA
over 35 minutes. This study of sample dynamics demonstrates 1 frame/s
imaging, with the typical, project- specific trade-off of frame rate
and image quality. Good tracking must be maintained to minimize tip
impact on the loosely bound, fragile sample. (Sample courtesy of Y.
Lyubchenko, Univ. of Nebraska Medical Ctr., USA.)
Future Applications of Rapid AFM Imaging
The idealistic notion of faster AFM
imaging is almost as old as the
AFM itself. A number of implementations for specific applications have
shown that great increases in AFM imaging speed are possible. Higher
speed AFM has not been approached as a certain set of applications, by
certain fields of research and on certain samples, but with the faith
that one would rather always image faster, however not at the expense
of quality, sample size or delicacy, usability, or operating cost. We
do expect that a faster AFM will open up new areas of investigation
over the full range of applications, from routine industrial to
molecular biophysics. Significantly it will enable researchers to
quickly and efficiently look at and understand a sample at the
nanoscale, using the breadth and content richness of the AFM technique.
Nano provides Atomic Force Microscope/Scanning Probe Microscope
(AFM/SPM) products that stand out from other commercially available
systems for their robust design and ease-of-use, whilst maintaining the
highest resolution. The NANOS measuring head, which is part of all our
instruments, employs a unique fiber-optic interferometer for measuring
the cantilever deflection, which makes the setup so compact that it is
no larger than a standard research microscope objective.
This information has been sourced, reviewed and
adapted from materials provided by Bruker AXS.
For more information on this source, please visit Bruker