Frequently Asked Questions about Atomic Force Microscopy

by Peter Eaton

This FAQ was originally created for clients of the AFM, i.e. those whose samples I scan.
However, it's grown a lot, and should also answer many questions of people planning to use the AFM themselves, or researching the technique. Its contents include a description of AFM suitability to various samples, sample preparation, tips for scanning and data processing, and a short bibliography. There is also a guide to recognising artifacts in AFM.

All the material here is discussed in greater detail in the book "Atomic Force Microscopy".

 


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. How do I optimise the feedback parameters?
21. What kind of artifacts can occur in AFM images? How can I avoid getting artifacts in my images?
22. I need to get an accurate height measurement. Should I calibrate?
23. 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. This article gives a brief summary of the operation of AFM. A more detailed description can be found in Atomic Force Microscopy - to be published in early 2010. Also, look at the end of this document where I recommend some external sources.

But, briefly:
AFM is analogous to a surface profiler, where a sharp tip is dragged over the sample, and the movement 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.

The cantilever deflection 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 its 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 the 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, these often 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), which 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 can be analysed by AFM!
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 covalently bound to a surface: e.g. self-assembled monolayers, and others
  • Molecules that are 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 surfaces: e.g. leaves, fruit
  • Paper


In short, if it has a surface, and it's solid, AFM can image it.
There has even been some work reported on gel and liquid surfaces.


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). Using some dynamic modes, true atomic resolution is also possible in AFM in non-vacuumt conditions. This issue is discussed in further detail in the forthcoming book.

**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, that 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 sometimes possible to just put a drop of water on your sample, and scan in that. Usually water, or buffer are used but organic liquids are also possible, as long as they don’t damage the liquid cell, and can be held on the sample.

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, Asylum MFP, Topometrix explorer, most Agilent scopes) 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!).

Sample-scanning microscopes (such as the Veeco multimode) are designed for small samples, will limit the sample size even more. Remember, AFM is a very high resolution technique, and is not really appropriate for very big samples. 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 x 1mm (height). Thickness must 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). See Atomic Force Microscopy for a detailed section on sample preparation.


9. What concentration (of particles) should I use?

I don't know, it depends on so 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 able to calculate an approximate concentration and volume to use. Typically you can fit 3 to 30 microlitres of aqueous solution on a small 8mmx8mm sample.

10. Does my sample have to be clean?

Yes! As clean as possible. If you are diluting in water, use the cleanest water 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 a 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 sources. The way I did it was depositing a drop of water and drying it on freshly cleaved mica. Then the sample was 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)

 2.31

 2.6

 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:

"Atomic Force Microscopy" OUP, 2010, 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, because 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.

The complete updated list of 3rd party software for AFM data processing and analysis can be found here.

13. How do I use this software you recommended?

Read the manual. I don't support the use of these programs.

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. If the image has already been processed, horizontal lines surrounding elevated features are probably an artifact due to horizontal levelling. This is explained further in my book, but to avoid this artifact, exclude the features from the fitting when levelling. You can also use another levelling algorithm such as a plane-fit.


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
Z sensor


TAPPING
Height or Topography
Amplitude
Phase
Z sensor

As you can see, topography images are common to both techniques. This is the type of image most commonly published. Z sensor is avialble where the microscope has a calibration sensor in the Z axis, and also represents topography (see Atomic Force Microscopy for explanation of this). Usually the topography images are presented as 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. This is because the deflection and amplitude images are the error signals in AFM.


The Friction, or Lateral Force images, are a map of LATERAL bending of the cantilever in contact mode. In other words, they show 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 some 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 chemical 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?

This is covered in detail in chapter 4 of "Atomic Force Microscopy". However, all AFMs come with a manual or users guide, that takes you through the basics of scanning. Often, the instrument also comes with a standard sample, such as a silicon grid, and the manual has a tutorial showing how to scan this sample. Such a 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! Optimisation of feedback parameters is described in question 21.


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 electronics, 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 means 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" value 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 may tell you what is the best initial value to use for your system.

 20. How do I optimise the feedback parameters?

This is quite a common question. It's difficult to answer, becasue it something you must learn to do. When you see an experienced user changing the feedback parameters while they scan, it can seem like magic - but there IS a method.

 Most AFMs use a PI controller. This is a kind of simplified PID controller. To control the feedback circuits, you change the P and I values. P stands for proportional and I for Integral. However, commonly in the AFM software they are referred to simply as P and I. To understand what thesereally mean, look at PID controllers on wikipedia, or look at chapter 3. In some instruments, D (derivative) may also be avialable.
For both P and I values, increasing the value increases the amount of the input signal (form the photodetector) which is fed back into the output signal (the z piezo). SO, the higher the values, the faster the AFM will react to changes in topogprahy in the sample. Thus, the higher you cna have them, the better. The problem is that if tey become TOO high, feedback oscillations will result (see figure 4.6 of my book, which shows the effect of having the PID values too high and too low). So, the trick is to set them as high as possible while avoiding oscialltions in the image. Typically, I is increased first, followed by P.  As Sandhya suggests, the setpoint is related to how well the feedback behaves as well. In fact, PID values, setpoint scan size and scanning speed all affect the correct sample tracking by the AFM, and are interelated with each other.

There is a more detailed explanation of this in Chapter 4 of my book.


21. 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.

22. I need to get an accurate height measurement. Should I calibrate?

 If the height measurement you get is important (it nearly always is!), then yes, you should calibrate. Since the calibration is scale-dependent, when you want to make a very specific measurement, the best thing is to calibrate with a height standard that has a step of height close to the value you are interested in. So, in the case of atomic steps, you ideally want a sub- nanometre step. A suitable sample would typically be other monoatomic steps. On the other hand, if you are measuring things in the range of 100's of nanometers, a silicon grid with similarly sized steps is more suitable. Look at Appendix A in my book for descriptions of appropriate samples. A more up to date (but less detailed) list is also available here. Look also in appendix B for recalibration procedures. They might also be described in your instrument manual.

23. Educational Resources and References

 This answer is rather out of date. I recommend you look for the answer in other parts of this site, especially, "Links", and "AFM Manufacturer List".


AFM Tutorials


Manufacturer Sites

See the  Full list of AFM Manufacturer websites


DI Digest

Unfortunately, the spm digest has been discontinued. The replacement for the old "DI Digest" is Bruker's "nanoscale world", which has forums with similar material.


Software

Full ist of AFM software here: SPM Software List


Books

Of course, my book is the best! But, there are some other good ones. Here are a few recomended ones.

Atomic Force Microscopy by Peter Eaton, and Paul West, OUP, 2010.


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.



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This document was written by, and is maintained by Peter Eaton (This email address is being protected from spambots. You need JavaScript enabled to view it.)
Reproduction or distribution not allowed without my permission.

Copyright (2010) Peter Eaton.

Please feel free to email me comments / questions / answers.
Document last updated on 15th April 2010.