Frequently Asked Questions about Atomic Force Microscopy (AFM FAQ)
by Peter Eaton


IMPORTANT: This document is now outdated.
The latest version of this FAQ is available at my new site, afmhelp.com
-You can find the newest version of the AFM FAQ by following this link.



This FAQ was originally created for clients of the AFM, i.e. those whose samples I scan.
If this applies to you, then I recommend you read it BEFORE preparing your samples.

But it should also be of interest for people just starting to use the AFM themselves,
or for a quick overview of the topic.
Its contents include a description of AFM including suitability to various samples,
tips for scanning and data processing, and a short bibliography.
There is also a guide to recongnising artifacts in AFM.

Contents:
1. How does AFM work?
2. What's the difference between AFM and SPM? What are STM, SFM, etc?
3. What kind of samples can be analysed by AFM? What are the applications of AFM?
4. Can I see individual atoms with the AFM?
5. Can we scan in liquid?
6. Will it take long? (are we there yet?)
7. How big can my sample be?
8. How do I prepare my particulate sample (i.e. a powder)?
9. What concentration should I use to deposit dissolved or suspended particles?
10. Does my sample have to be clean?
11. What do I do with these strange files?
12. What if I want to do the analysis myself?
13. How do I use this software you recommended?
14. My image has weird horizontal lines all over it.
15. My image has weird vertical/diagonal bands lines all over it, or oscillations in the force curve.
16. What are Phase images? What are Amplitude images?
17. How do I use the AFM?
18. How can I see individual atoms with the AFM?
19. What is setpoint? Should I change it?
20. What kind of artifacts can occur in AFM images? How can I avoid getting artifacts in my images?
21. Resources and References


General AFM Questions


1.How does AFM work?

An in-depth explanation of AFM theory is outside the scope of this FAQ,
so check the end of this document where I recommend you to some external sources,
though there are many more internet sites, books, papers, magazine articles about this, for example:

There is a very nice overview of SPM at:
http://www.sci.port.ac.uk/spm/overview1.htm
Introduction to AFM, from Mark J. Waner, Michigan State university:
http://chemistry.jcu.edu/mwaner/research/AFM/
My article on AFM at knol:
Google knol on AFM


But, briefly:
AFM is analogous to a surface profiler, where a sharp tip is dragged over the sample,
and the deflection of the tip is monitored as a measure of sample topography.
However, in AFM, the tip is mounted on a reflective cantilever (the cantilever and tip
together are known as the probe). The deflection of the tip is measured by laser,
reflected off the cantilever onto a split photodiode. This allows vertical AND
horizontal measurement of the cantilever bending. The vertical deflection data tells
us about the interaction between the tip and the sample. This information is fed back
into the scanner, the part that moves the probe over the sample. if the tip is bending
up because the tip has reached a feature, the scanner moves the whole probe upwards,
enough to return the deflection of the cantilever to it's original value. Likewise,
when a "valley" is encountered, the scanner moves the probe downwards. In this way,
the deflection of the cantilever, and hence the tip-sample interaction force is kept
constant.
The amount he scanner had to move to maintain the deflection is equivalent to sample
topography, and is recorded by the computer. This is contact-mode AFM.


2. What's the difference between AFM and SPM? What are STM, SFM, etc?

AFM is Atomic Force Microscopy, or the Atomic Force Microscope. AFM was developed after initial work on STM
- Scanning Tunneling Microscopy. Later, AFM spawned its own variations, such as Magnetic Force Microscopy
(MFM), Lateral Force Microscopy (LFM), Scanning Nearfield Optical Microscopy(SNOM), etc, etc.
There are LOTS of such techniques that usually use the same method as the AFM of scanning a probe
close to the sample surface, but can differ in the properties they measure.
Together, these related techniques together are referred to as Scanning Probe Microscopy(SPM).
Another term sometimes used is Scanning Force Microscopy(SFM), this is a synonym for AFM.

3. What kind of samples can be analysed by AFM? What are the applications of AFM?

Well, almost anything that is solid!
Here is a short list of SOME samples, in no particular order:

Polymers
Metals
Fibres: Hair, synthetic fibres, nanotubes
Particles: micro-, nanoparticles, quantum dots
Molecules bound to a surface: e.g. self-assembled monolayers
Molecules NOT bound to a surface, e.g. nucleic acids, proteins, many others
Minerals
Langmuir-Blodgett films, Lipid bilayers
Cells: Mammalian, Bacteria, Plant, etc.
Viruses
Ceramics
Plant surface: e.g. leaves, fruit
Paper

In short, if it has a surface, and it's solid, AFM can image it.
There has been some work reported on gel , and liquid surfaces.
This is quite difficult, though!

4. Can I see individual atoms with the AFM?

Sort of! It is sometimes reported that contact mode AFM has "atomic resolution", as
it is possible, with some samples, to get images which show the atomic lattice. However,
it has been shown that these images show not the individual atoms, but just the lattice,
i.e. they show "atoms" where, on average, the atoms are but cannot show, for example,
vacancies (missing atoms) in that lattice. Nevertheless, such images can still be
useful and interesting. Typically, to get "true" atomic resolution, we must use STM,
or AFM under special conditions(.e.g. in UHV, cryo-AFM).

**See *:How can I see individual atoms with the AFM?

5. Can we scan in liquid?

Yes. This is one of the principle advantages of AFM, it can be used in vacuum, in air,
or in liquid. A liquid cell is USUALLY required. Top-down AFMs, for example, don’t
really need one, it's possible to just put a drop of water on you sample, and san in
that. Usually water, or buffer are used but organic liquids are also possible, as long
as they don’t dissolve the liquid cell.

6. Will it take long? (are we there yet?)

Yes, it will take a long time. AFM is slow. New, fast AFMs exist. We don’t have one. Go get a coffee.
Seriously, though, if you sit and watch someone else do AFM, you will get bored.
Images take about 5-10 minutes each. On a good day. With sample mounting, and instrument
set-up this equates to about 1 sample an hour, 6 samples a day. This assumes you
only want 3 images per sample, and we are just getting pretty pictures, no fancy
stuff! Rougher samples take longer, as do dirty samples.


Sample Preparation

7. How big can my sample be?

The long answer:
It depends a lot on the particular system to be used. Some systems (e.g. Veeco
dimension, Topometrix explorer, Molecular Imaging PicoPlus(now known as the
Agilent something) are designed for relatively "large" samples (but we're not
talking about bricks, here - perhaps you could scan a sample with height
< 2cm, diameter laterally of 5cm ). These are typically "top down" AFM models,
i.e. the entire microscope sits on top of the sample. This allows for larger samples.
In these cases, it depends on the specifics of the "sample holder". Usually,
such microscopes can even scan WITHOUT the sample holder, though, so you are
almost unlimited in sample size. DO remember though, that the sample must be
flat.(Hence, no bricks!)

Other microscopes (including the Veeco Multimode), are designed only for small
samples. Remember, AFM is a very high resolution technique. Also, it typically
only scans very small areas (less than 100 micrometers x 100 micrometers usually),
so there's little point in having a very big sample.
So, ask the operator, but for a typical sample size see below.

The short answer: A "typical" size for the multimode is 8mm x 8mm. Thickness should
be less than 4 mm. The maximum possible size for the multimode is a circle of 15 mm
diameter, but only the central part will be available

8. How do I prepare my particulate sample (i.e. a powder)?

Dry powders are a problem for AFM. If your particles are loose on flat surface,
they will most likely move when the AFM tip bumps into them. Usually, resuspending
in water, followed by dilute dispersion onto freshly cleaved mica, then thorough
drying will result in particles fixed to the surface.
Other possible fixing techniques are fixing on a membrane, dispersion onto a
glue that you later cure, even flaming (for bacterial cells).

9. What concentration should I use?

I don't know, it depends on many factors. But the image size is usually 1
micrometer x 1 micrometer to 10 micrometers x 10 micrometers. If you want
to see, say, 100 particles per images, you should be bale to calculate an
approximate concentration, and volume to use.


10. Does my sample have to be clean?

Yes! As clean as possible. If you are diluting in water, use the cleanest
you can find.(I can supply some, if you're preparing it for me).I buy Sigma
"Water for Molecular Biology". This is better than most "milliQ" water.
Blowing off unattached crud with filtered dry nitrogen or argon is highly recommended too.
Remember, AFM is a SURFACE microscopy technique and it shows EVERYTHING at the surface.
If your 3mm thick sample has only a 3nm contamination layer,
you might see NONE of the sample!

Here are a few results on water quality. It’s not based on la statistically significant number of samples, and is based on MY water supply, so your mileage may vary, but it should give you an idea of the RELATIVE qualities of various water treatments.

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.

Sample

Mean Ra/nm

Mean Rq/nm

Tap water

0.25

0.54

DI water (Filtered in our lab)

2.31

2.60

MilliQ water

0.15

0.35

SIGMA Water for Molecular Biology

0.07

0.1













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.


Data Analysis

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 don’t 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.

CONTACT
Height or Topography
Deflection
Friction

TAPPING
Height or Topography
Amplitude
Phase
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?

RTFM.
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
published images.
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

AFM Tutorials

Written by James Smith from the University of Portsmouth:
An Overview to Scanning Probe Microscopy

http://www.sci.port.ac.uk/spm/overview1.htm
(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:
http://www.pacificnanotech.com/afm-tutorial_single.html

Introduction to AFM, from Mark J. Waner, Michigan State university
http://chemistry.jcu.edu/mwaner/research/AFM/

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.
http://www.mobot.org/jwcross/spm/

How AFM works, on the US navy website:
http://stm2.nrl.navy.mil/how-afm/how-afm.html

Manufacturer Sites

http://www.veeco.com
http://www.molec.com/
http://www.asylumresearch.com/

DI Digest

For in-depth discussion of AFM and SPM, see this mailing list:
http://spmlist.di.com/pipermail/spm/

Software

http://www.nanotec.es
http://www.imagemet.com
http://www.gwyddion.net

Books

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:
http://www.asmicro.com/Applications/phase.htm

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 (peter.eaton@fc.up.pt)
Reproduction or distribution not allowed without my permission.
Please feel free to email me comments / questions / answers.
Document updated on 5th August 2008.