Our 2014 AFM course has had all the places reserved. I am pleased to see we have 16 students from all over the world, including the USA; Malaysia,  Germay, Spain, the Czech Republic, Poland, and here in Portugal.

Meanwhile,  I will be teaching on another upcoming course, in July 2014, at Kent State University, in Ohio. This course will be 5 days, with full 5 afternoons of instrument time. Places are very limited. More details can be found here: http://afmworkshop.com/atomic-force-microscope-workshop.php. The AFMWorkshop website also hosts a PDF flyer.

Up until now, this website has been funded by Google text adverts that appear on the right of the page. I don’t control what ads appear there, except that I can remove some categories of ads. Beginning soon, I expect to have actual ads from AFM companies appearing there.

The advertisers do not influence any of the other text I write here. Although I do work with some AFM manufacturer’s equipment more than others (and my co-author on the book, Paul West has been owner/CEO of various AFM companies), I do not really favour instruments of one manufacturer over others. I have used many (more than ten) different AFM instruments over the years and this has led me to think that -

 

ALL AFM instruments can produce great results.


What is necessary to get great results are a certain level of skill on the part of the operator, a good probe, careful sample preparation, patience, use of the right modes and settings, and sometimes, a dash of luck! While newer instruments certainly offer amazing new modes, and in some cases lower noise levels, increased ease of use, or faster scanning, in my experience 99% of AFM could actually be done on just about any instrument. In my teaching, I hope I explain things that are useful to users of all instruments. Furthermore, although I am happy to get new listings, and factual corrections for the “Where to buy instruments” and “Probes” and “Calibration artifacts” pages, I do not accept copy written by the companies for inclusion on those pages. Any inaccuracies, or opinions are mine alone.

While many other procedures are important for full determination of the performance of an AFM instrument, the Z noise floor is often used as a simple parameter to quantify instrument performance, since it indicates the lower limit of what the precision that can be reached in the z axis in that instrument, and is also simple to measure.noise-floor

It can be essential to know the noise floor of the AFM instrument to assure that high resolution measurements are meaningful. This can be particularly important for measurements of very small features (i.e. < 5nm), and for high resolution force spectroscopy. Measuring the noise floor can also help in optimizing instrument setup and vibration isolation. It is important to know the noise floor when using only the z piezo in the z feedback loop, as well as the noise floor of the z calibration sensor if there is one in the instrument. In most instruments, the noise floor of the z calibration sensor will be much higher than that of the z piezo.

In order to get reproducible results, all scan parameters should be maintained the same when compaing two results. Some factors, such as the PID values vary greatly from instrument to instrument, so the specific values to use cannot be suggested here. In each case, standard values should be established such that a fair comparison can be made.

Note that the procedure below is adapted from general guidelines given in Appendix B, page 195 of Eaton and West “Atomic Force Microscopy”. For a outline of a procedure that’s generally applicable to any model of AFM, take a look at the procedure below. Click here to find a specific procedure for measuring the z noise floor on a TT-AFM from AFM workshop.

 

Measuring noise floor in the z piezo signal

a) Place a flat, clean sample in the instrument. Use a new probe.

b) Scan a small image on the sample to verify cleanliness and optimize the PID parameters.

c) Set the instrument to make a zero size scan such that the probe does not move in   the x and y axis.

 

d) Measure an image without probe motion in x or y, i.e. an image with a scan size of 0 nm, at a 1 Hz scan rate. A 128 x 128 pixel image is adequate. The data from the z piezo voltage should be used. This may be labelled height, or topography. The z scale should be in nanometers.

 

e) It may be necessary to flatten the data before the measurement, e.g. by a 1st order horizontal line levelling routine.

 

f)  Calculate the RMS roughness (Rq, see chapter 5) of the image, this is the noise floor.


The achievable noise floor varies from one instrument to another, as well as depending on the noise in the environment, the measurement parameters, and the vibration isolation, but typically a sub-Ångström noise floor can be achieved. An example of type of image you should get is shown in the image above.

 This article contains a small extract from Chapter 7 of “Atomic Force Microscopy”. Chapter 7 contains descriptions of applications of AFM in materials science, chemistry and physics, biology and the life sciences, nanotechnology, and in industry. This short section describes some examples of applications of AFM in bacteriology. References lists, and the second figure can be found in the full book.

AFM is a highly suitable tool to examine bacteria, and has been widely applied to their study. Bacteria are commonly studied by optical microscopy, which can give an overall idea about gross cell morphology (via a two-dimensional projection), and is also useful for cell-counting studies. In comparison, AFM is slower, and thus is less useful for quantitative cell-counting, but allows measurement of a variety of other cellular properties, particularly by nanoindentation and force spectroscopy experiments [611]. In addition, the greatly increased resolution of AFM allows for the imaging of finer details of cell morphology and sub-cellular features such as pili and fimbriae [612]. The three dimensional information from AFM can also be useful in differentiating morphologies which would look the same in optical microscopy [6]. Various other micro-organisms have been studied by AFM such as spores [178, 613–615], fungi [616, 617], including yeasts [171, 618], viruses [287, 619], and others [620] but here we concentrate on bacteria for the sake of brevity.

fig 7.20

Fig. 7.20. Studies of bacterial morphology. Top left: Streptococcus, showing typical linear clusters. Top right: large clusters of Staphylococcus aureus. Bottom left: Salmonella biofilm showing pili-like fimbrial structures. Bottom right: E. coli. All these images were measured in air. Reproduced with permission from [624] (top left) and [626] (bottom left). Right hand images the author's own work.

TT-AFM noise floor measurement

1. Place a clean silicon calibration sample on the scanner.
2. Place a new probe in the instrument.
3. For this comparison, use vibrating mode. Setup the optical  alignment and Tune frequency as normal.
4. Select parameters to test are listed below. note that for a fair comparison, you can use parameters relevant to your typical measurements. The parameters described here, will give you an “ideal” value, i.e the best result possible.

 

Suggested parameters 

Parameter

Suggested value

X Gain %

0

Y Gain %

0

XY HV Gain

Initially 1 (note it should be 0 for the actual measurement)

Z HV Gain

1

Image Add

Off

Z Feedback values

Values you typically  use, for example Gain 1.5, Proportional 150, Integral 1500

Lines

128

speed

1 Hz

Left Image

Z_DRIVE

Samples/Pixel

25

 

Note that you can use any different parameters you like for this, and they can and will alter the results that you get. Also, the instrument should be properly calibrated in z to allow comparison with any other values. Be aware that tip approach with Z HV gain at 1 will be very slow, and some post-approach adjustments might be necessary.(i.e Jog Down).

 

5. Go into feedback, and scan an image of a small area of the surface (XY HV Gain of 1). The image should be clean, with very few features visible. Ensure you end the scan with the probe on a flat part of the sample.

6. Withdraw probe.

7. Set scan size to 0, by using XY HV Gain = 0.

8. Approach surface again.

 

NOTE: It is important that you go into feedback on the surface in the same way as you did when you scanned an image. If you go into false feedback (probe almost but not quite, on the surface), you will not make a valid noise floor measurement.

9. Scan another image. No sample features should appear, as scan size is zero. The image may look something like the image below, or have some regular patterns in it.

noise-floor
1
0. Save the file, and open the Left image -  z piezo drive file (height image) in gwyddion.
11. Apply a 1st order polynomial levelling (fit linear).
12. Use “Statistical Quantities” and record the RMS average roughness (Sq). This is the noise floor

You should get a value of <1 Angstrom (gwyddion may report this as <100 pm).

If you do not get a satisfactory value, try removing sources of external vibration (other machinery, acoustic noise, unnecessary cables) from the instrument. Ensure probe and sample are grounded. Ensure vibration isolation system is being used properly.

Typical values found in a TT-AFM in an acoustically shielding box, with bungee-cord type isolation, in vibrating mode would be 0.3 to 0.5 Angstrom.

You should be able to achieve at least the value specified by AFM workshop on the instrument spec. sheet which was delivered with your instrument.


All materials on this website are copyright 2010-2014 Peter Eaton.

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