The way I did it was depositing a drop of water and drying it on freshly cleaved mica. Then the sample's scanned with AFM. The Ra value for mica was about 0.05 nm.
water (Filtered in our lab)
Water for Molecular Biology
NOTE: More of this sort of stuff, i.e. advanced tips for sample preparation will appear in my upcoming book:
"Introduction to Atomic Force Microscopy" OUP, 2009, with Paul West.
11. What do I do with these strange files?
AFMs produce the results in proprietary format data files. These are usually
manipulated with the software that came with your instrument. Get the AFM
operator (e.g. me) to process and analyse the data properly for you and produce
images (e.g. .bmp files) that you can insert in your reports.
12. What if I want to do the analysis myself?
It's possible. Be very careful, however, if you dont know what you are doing,
because AFM manipulation packages allow you to change your data radically.
Always keep a backup, DO NOT modify your data, and then save over the original file,
you wont be able to get it back!
If you really want to modify the data yourself, the best thing to do is to get a copy of your AFM manufacturers
software and use that to analyse your data. Only that program is guaranteed to
read the data format of your files correctly. Alternatively, there are third party programs
you can use to view. I list them here, but I reccomend you use them
with caution. It's much safer (and easier!) to get an experienced AFM
user to manipulate your data for you.
1. SPIP, the Scanning Probe Image Processor. This is a program you must buy to use,
but a demo version is available at www.imagemet.com. It looks quite good,
but I haven't used it myself.
2. WSxM. Available at www.nanotec.es. Very powerful software from an AFM manufacturer,
but able to load lots of different formats. Free.
3. Gwyddion. Available at www.gwyddion.net.
Nice, robust, open-source software, opens many formats. Free.
There is an updated list of all 3rd-party SPM software that I maintain at Google knol.
13. How do I use this software you recommended?
RTFM.(I suggest you read the manual.)
14. My image has weird horizontal lines all over it.
Are they broad bands? if so, it just needs levelling horizontally.
If there are single-pixel "scratches", probably the tip skipped as it was scanning
the sample (maybe the feedback was not perfect, or it encountered a movable
obstacle on the surface).
You can't really fix this after finishing scanning, it's best to stick with what
you've got, or re-scan the image. If you are still scanning, try changing
feedback and or setpoint parameters, or clean the sample.
15. My image has weird vertical/diagonal bands lines all over it, or oscillations in the force curve.
This problem is a common artifact in AFM. It is due to the laser light spilling
over the edge of the cantilever, being reflected off the sample, and travelling
back up towards the photodetector. The reflected light interferes with light reflected
from the cantilever, causing typical wave-like oscillations in the image
(oscillations in the fast scan axis, or if you look at the images, bands running
near-vertically in the slow scan axis) or in the zero force line of force curves.
Typically, the wavelength of these oscillations is two wavelengths of the laser
i.e. it is about 1.3 micrometers for a red laser. It is more common with reflective
samples, with high coherence lasers, with narrow cantilevers, and when
the laser alignment is not perfect. The typical way to fix it is to re-align the
laser, trying to make sure the spot is right in the middle of the cantilever.
Newer instruments often have low coherence lasers to reduce this problem. However,
with some instrument/cantilever/sample combinations it is very hard to avoid.
See the Artifacts page for more details about image artifacts in AFM.
16. What are Phase / Amplitude / Friction images?
I assume you know how AFM works (If not, read question 1).
There are a great variety of image types that can be displayed. Below I list the
most common ones for contact and tapping modes.
Height or Topography
Height or Topography
As you can see, topography images are common to both techniques. This is the type
of image most commonly published.
Usually they are a map of differently coloured pixels, with a colour bar relating
the colour to a height.
This is very useful, as on such an image, it's possible to estimate both lateral
(xy) and height(z)measurements. However, one reason other types of image are commonly
shown is that such "height maps" do not really "look like" the object in question,
in other words, the appearance of a certain shape is very different to that it
would have in optical (or electron microscopy).
What this means, is that to the casual observer such images do not display
easily the shape of the features. Ways around this include shading the image, and
more commonly, creating a pseudo-3D image from the height data.
However, an alternative is to show the deflection (or amplitude) image.
Because they are equivalent to a map of the slope of the sample, they often display
the shape of the sample more easily. But bear in mind that the z-scale in deflection
or amplitude is completley meaningless in terms of the sample structure.
All it shows you is how the tip deflected as it encountered sample topography.
It is important to remember too, that the BEST images are obtained when the deflection
(or amplitude) signals are minimised.
The Friction, or Lateral Force images, are a map of LATERAL bending of the cantilever
in contact mode. In other words, how the cantilever twists as it scans across the
sample. This signal can be related to friction between the sample and the tip,
but it also contains topogrpahic contrubutions on a non-flat sample. See references
at the end for more on this.
The Phase images, available in tapping mode, are a map of how the phase of cantilever
oscillation is affected by its interaction with the surface.
The physical meaning of this signal is complicated, (see references) but in addition
to topographic information, the phase can be affected by relative softness / hardness
of the sample, or the cehmical nature of the sample. In general, in mixed (heterogeneous)
samples, it is easy to get a contrast in the phase, but interpretation is more
complicated. Again, there will be some references at the end of this document
to explain this in more detail. Using the AFM
17. How do I use the AFM?
All AFMs that I have used come with a really useful manual, or users guide, that takes you
through the basics of scanning. Often, they also provide a standard sample, such as a
silicon grid, and the manual has a tutorial showing how to scan this sample.
This sample is also very useful for seeing how different parameters affect the results you get.
There is no "magic formula" for optimising AFM conditions. If you watch experts,
you'll see that they all do it slightly differently, even on the same machine, and different
AFMs have wildly different requirements, so it is not possible to describe the
use of the AFM in detail here. So, read your manual, scan the standard sample, and practice!
Finally, for help with specific problems using the AFM you could ask in the DI Digest mailing list
- see the end of this document
18. How can I see individual atoms with the AFM?
Please bear 3 things in mind:
1. Imaging atoms is not as easy as "everyday samples".
2. Imaging atoms is very dependent on the quality of the sample, and of the the tip,
and the noise level in the lab.
3. Imaging atoms is often not very useful!
Having said all that, it's quite fun, and not really difficult! There is a great
tutorial on this subject, explaining everything you need to do available on the net,
currently to be found at: http://web.mit.edu/cortiz/www/AtomicScaleImaging.doc - it's a word document.
This is based on use of a multimode, but the general principles should be applicable to all AFMs.
19. What is setpoint? Should I change it?
The setpoint is basically a measure of the force applied by the tip to the sample.
In contact mode, it is a certain deflection of the cantilever. This deflection
is maintained by the feedback, so that the force between the tip and and sample
is kept constant. In tapping mode, it is a certain amplitude (amplitude of oscillation
of the cantilever), which controls the force with which the tip taps on the sample.
Again, the set amplitude is maintained by the feedback electronics.
Setpoint is expressed differently for different instruments.
So, it is very IMPORTANT that you check your instrument manual to find out how it works in your case.
For some instruments, a small set point, means a low force applied to the sample,
whereas for some, a small set point means a large force.
This apparent contradiction can even change from one mode to the other, on the same system.
A large force applied to the sample, often measn better imaging, but also means more
wear on the tip, and the sample , i.e. lower tip life,
and less chance of getting a complete sample without the tip getting contaminated / broken.
So, generally you should start with a "safe" vlaue of the setpoint
(e.g. just touching the sample) and adjust it slowly until imaging does not improve anymore, then stop.
The "best" setpoint can vary from tip to tip, and sample to sample, please remember
that there is NO "Golden Number" for the setpoint.
If someone tells you a certain value is ideal before you start imaging,
you should take this with a pinch of salt, and instead optimise the value based
on what you see. Having said all that, read your user manual, and it will
tell you what is the best initial approach for your system.
20. What kind of artifacts can occur in AFM images, and how can I avoid artifacts in my images?
A lot of different artifacts can be present in AFM, and are often present even in
See the Guide to Recognising AFM Artifacts page for descriptions and examples of AFM artifacts.
The page also describes how to recognise AFM artifacts, and how to avoid them.
21. Resources and References
Written by James Smith from the University of Portsmouth:
An Overview to Scanning Probe Microscopy
(this curently seems to be missing?) My article on AFM at knol:
Google knol on AFM
A talk on AFM and SPM, originally presented at the SPM Workshop of "Materiais 2007" in Porto, Portugal April 2007,
there's a version of the presentation in .pdf format here:
SPM Fundamentals, Techniques and Instrumentation by Peter Eaton
P Eaton SPM Fundamentals.pdf
Some of my publications on AFM can be found for download on my cv page:
Peter Eaton CV and Publications
From an AFM manufacturer, a guide to AFM theory:
Introduction to AFM, from Mark J. Waner, Michigan State university
Scanning Probe Microscopy (SPM) - Imaging Surfaces on a Fine Scale
By John W. Cross
- another nice explanation of AFM and SPM, with many example images.
How AFM works, on the US navy website:
For in-depth discussion of AFM and SPM, see this mailing list:
Scanning Probe Microscopy and Spectroscopy: Methods and Applications
by Roland Wiesendanger
AFM for Biologists by V. J. Morris
A comprehensive theoretical work is: Scanning Probe Microscopy: The Lab on a Tip
by Ernst Meyer, Hans J. Hug, Roland Bennewitz
References to Imaging Modes:
A quick explanation of phase imaging, with some nice examples of what you can see in phase images:
Theory of phase imaging:
Cleveland, J. P., et al, Appl. Phys. Lett. 1998, 72, 2613-2615.
Friction Force Microscopy (FFM) or Lateral Force Microsopy(LFM):
Ascoli et al, J. of Vac. Sci. & Technol. B: 1994, 12, Issue 3, pp. 1642-1645.
This document was written by, and is maintained by Peter Eaton (firstname.lastname@example.org)
Reproduction or distribution not allowed without my permission.
Please feel free to email me comments / questions / answers.
Document updated on 5th August 2008.