My name is Peter Eaton, and I have been working with Atomic Force Microscopy for more than 15 years. I am currently a Researcher at the Molecular Medicine Institute (IMM) in the University of Lisboa, Portugal. I am the co-author (with Paul West of AFMWorkshop) of the book Atomic Force Microscopy, published in 2010 by Oxford University Press. I have collaborated with researchers in the UK, France, Spain, Brazil, Argentina, Portugal, the USA and Germany in AFM. I have taught AFM around the globe, and am the coordinator of the Porto AFM Training Workshop (running from 2011 onwards).

In addition to development of research and education in Atomic Force Microscopy, my interests include:

Antimicrobial peptides derived from amphibians

  • Nano particle preparation and characterisation
  • Natural Products
  • Biosensors
  • Antiviral and anticancer peptides
  • Scanning electron microscopy

A complete list of my research papers can be found along with my academic CV here. I have co-authored more than 60 scientific works.

I am happy to collaborate scientifically in interesting projects or carry out scientific consultancy in the field of atomic force microscopy.

I was recently asked what equipment is necessary to set up a new AFM lab, so here goes:

Materials necessary to set up a new AFM lab

Note: Many manufacturers include a lot of these items when you buy an instrument; check before ordering. I have only included estimated prices for items with high cost.



Mandatory Items

1. Calibration/certification product. Usually a silicon grid with repeating features. See extensive list of suppliers here:
SPM References and Standards


Note: This is the most expensive item, but absolutely necessary. The importance of calibration is discussed in my book (especially Appendices A and B). Take care of your calibration artifact once you have it.


Estimated cost: 300-1000 dollars, depending on model and supplier. Traceable standards will be more expensive.


2. Cantilevers / Probes. Of course these are necessary for all work. You typically get a few with a system, and can also beg samples from manufacturers, but you will need more, and soon! See extensive list of suppliers here:
Where to buy : SPM Probes

Estimated cost: 300 dollars for a box of 20


3. Anti-vibration solution. There are many different types of vibration-damping equipment, see chapter 2 of my book. They can usually be bought with instruments, but at additional cost. Suitable vibration / acoustic isolation stages for AFM instruments can cost from 2,000 to 10,000 dollars.

4. Tweezers. These are important for handling samples, and handling probes. I recommend two types, specific AFM probe-handling levers with specially shaped ends, and type 2A (flat, rounded ends, preferably non-magnetic), for handling samples and sample mounting discs.

I often buy from Ted Pella, see here for probe tweezers, and here for type 2A, but most microscopy suppliers carry these, or some equivalent. Note: on a budget, type 2A also can be used for probes, but I highly recommend that you keep a clean pair of tweezers, especially for handling probes.


5. Sample mounting discs. These are simply magnetic steel discs of about 10-15 mm diameter, and 1 mm thick. You can even make them yourself. Most microscopy suppliers sell them in tubes of 50-100. See here for the ones at Ted Pella.


6. Sample mounting adhesive tabs. To stick your sample onto the metal discs (above). See here for example at SPI. Alternatively, can be easily replaced with double sided sticky tape with few problems.

 

Optional Items

7. A source of very clean water. Some labs use milliQ water, and this is fine for most work, but you can also find ultra-pure bottled water, which I mostly use. See chapter 4 of my book.

8. A source of clean gas. Used for removing dust from samples. At a pinch you can use a “blower”, like this: Rocket Air Blaster.

9. Adhesive for when double-sided sticky tape is not enough: 2 part epoxy is the gold standard, superglue (cyanoacrylate) can also be used, but not for work in liquids.

10. Solvents for cleaning, etc.

11. Optical microscope and / or magnifier. Usually AFMs have an integrated optical scope, that can be used to obtain optical images of the sample. Depending on your application, and how good the attached scope is, you may want a separate optical microscope to view samples before putting them in the microscope and possibly to record good optical images. This can help greatly with sample preparation. It is also VERY useful to have a magnifier or small microscope to help with probe seating, i.e. to make sure your probe chip still has a cantilever and that you have placed it in the probe holder of the AFM correctly.

12. Other susbstrates. Depending n the kind of work you need to do, you will want substrates to deposit your samples on. These might be optical glass slides, coverslips, mica, or HOPG. flat silicon samples can also be useful. Most AFMs work with 1cm diameter samples or smaller.

 

Instrument Building / Repair / Diagnostics

For this, you’ll also want many small tools, including Allen (hex) keys, screwdrivers, etc. Glue, including superglue. Multimeter. Good soldering iron and solder, preferably silver solder with a flux core.

 

Room Requirements

For the room you are going to put the AFM in, there are not many real “must-haves”. But it’s recommended you use a small lab, with little foot traffic, or at least one where the people can be expected to keep quiet. Basement labs are preferred to upper stories. These are all due to vibration and acoustic noise issues. It’s useful to have air-conditioning, especially if you expect to have large temperature variations during the day. But you must be able to turn this off in case it interferes with high-resolution work.



As always, suggestions and additions to this are welcome - go HERE for the contact form.

Copyright Peter Eaton 2012-2018

Does an AFM need to be have a technician responsible for it?  Or a senior scientist who can train others to use it? In many research labs, the answer seems to be “no”. Or at least, there is no-one fully responsible for the AFM, who can advise on experiment design, sample preparation, and train users, or even just run users’ samples. In this blog post, I’ll talk about why this situation exists, and what should be done about it.  

 


Image of dsDNA captured by a student on one of our courses. Such images can be challenging to acquire without adequate training.

In research labs in academia, (and, to a lesser extent, also in industry), there are often two kinds of facilities, those that are operated by individual researchers, in their own projects, and those that are run either ONLY by technicians /senior scientists, or only by fully qualified researchers under the close supervision of an expert. For example, in chemistry, a common instrument that is usually left to researchers (such as students) to run by themselves, is a UV/visible spectrometer. This is a pretty simple instrument, that is kind of a “black box”, which requires minimal training to use correctly.This is not to say that some things cannot go wrong, but an occasional prod in the right direction is all that’s required. Typically, if any-one is responsible for the instrument, they often just change the bulb now and again. More complex instruments are often run the same way, up to optical microscopes. But this is not the same for nanometre-resolution microscope, right?

 

Electron microscopes are *rarely* solely user-operated. They nearly always have a technician responsible, who might be the only one to use the microscope at all. When I began to use electron microscopes, it was clear that I was not going to get my hands on the instrument, until after a very thorough training lasting several weeks, and costing a lot of money. After this, I used the instrument for a long period of supervised operation, before finally being considered independent. This makes sense, for several reasons:

  • Electron microscopes are complex, with many controls you need to learn

  • They are easy to misalign, or even damage

  • They usually need careful sample preparation

  • The data from them can need careful interpretation

  • Electron microscopes are (usually) big and expensive instruments

 

So, what about the AFM? Doesn’t all this hold true for an AFM?

 

Surprisingly, there are many many cases where AFM instruments do not have a responsible user, let alone a technician. In the AFM training course I teach, something like 50% of the people who need training say there’s an AFM in the lab and no-one knows how to use it.

 

There are many AFM instruments sitting in lab corners, barely if ever used, or used only by un-trained students.

 

So, why is this? All those bullets points up there kinda hold true for AFM, don’t they? I think they mostly do, but I think the difference is in the last part.

 

An AFM can be a low-cost instrument! And an AFM might be very small indeed...which will often lead to the erroneous idea that it’s a simple instrument to use. Is AFM more difficult than electron microscopy? I don’t think so, but most electron microscopists seem to think so. Then again, perhaps this is because they’ve never been trained properly in use of an AFM.

Let’s look at cost; here’s a rough idea of what it costs to get an instrument with 1 nm resolution:  

 

TEM

SEM

AFM

around 1,000,000 USD

around 400,000 USD

40,000 to 100,000 USD

NOTE: These are “typical purchase costs”, and any of them might costs more or less than this, but not by an order of magnitude….

 

Now, these 3 instruments would not be the same, and I am not saying a 40,000 dollar AFM can do everything a million dollar electron microscope can do, but with these three instruments, you can achieve around 1 nm resolution. To explain what you can really do with them could make another looong blog post...

 

But, wow, it’s a big difference, isn’t it? Add to this the fact that a TEM takes up an entire room to itself, and may even need the ceilings raised to fit in (and it usually won’t fit through your door, either!), and you can start to see why sometimes people take the TEM or SEM more “seriously”...If you have to raise a million dollars in funding to buy a microscope, you are going to make damn well sure that a: It’s not broken by incompetent users, and b), that you get some money back to keep it going by selling services.

 

So there we have our poor little AFM, it’s the new kid on the block, electron microscopists don’t understand it, and no-one ever gets properly trained. Some AFMs are actually installed by technicians who don’t know how to used it.

 

So what can you do?

  • TRAIN your USERS!

  • MAINTAIN the KNOWLEDGE!

  • keep GOOD staff!

 

All of these are difficult, but check out our training courses here: http://afmhelp.com/course

 

I would be interested to hear what other people think about this, it’s something I’ve been telling people a long time, and no-one has contradicted me (audibly) yet! Do people out there who can use  AFMs and EMs think one is more difficult than the other? Modern electron microscopes are also highly automated, partially due to more mature technology, and partly, I guess, to justify their high costs!
What do you think?

 

Incidentally, if you need an AFM expert in your lab after reading this, I am looking for a job! Check out my CV here! ;-)

 

Pete.

 

 

My group recently published a paper in the journal Ultramicroscopy reporting on direct comparison we made between different techniques that can be used to characterise the size of nanoparticles.

 

There are a wide variety of technique available to make these kind of measurements nowadays, however, microscopy is often used, because it is a direct technique (some other techniques measure properties related to size), and because it’s also possible to measure shape at the same time. The size of nanoparticles is extremely important for their properties, and ideally a technique to measure nanoparticle size will have sub-nanometer resolution.

 

Apart from microscopy, light-scattering techniques are probably the most common techniques used. The methods of Dynamic light scattering , or DLS; is extremely popular and used in thousands of labs worldwide. A newer method based on laser light scattering, called nanoparticle tracking analysis, or NTA, is currently growing in popularity.

 

In our project, we prepared nine samples; these we made up of nanoparticles composed of three different, and commonly used materials, a metal, an oxide (silica), and a polymer. We examined each materials in two different sizes, and also tested the ability of each method to distinguish between different populations in mixed samples.

 

We found that unfortunately we were unable to use NTA for our samples  THis technique is very sensitive to sample colloidal stability, and was not able to obtain reasonable results with all our samples.

 

Transmission electron microscopy (TEM), scanning electron microscopy (SEM), and DLS. With all methods, we were able to obtain size values for all samples.

However, we found for SEM, that some samples had to be coated to give decent results. Even with coating, SEM gave the lowest precision of the three microscopy methods, when characterising smaller nanoparticles.

 

Both AFM and TEM had very high precision, and AFM gave the most satisfactory results in separating mixtures of materials. AFM also has the advantage of characterising any type of material, giving high contrast even on soft, organic materials. On the other hand, AFM can be slower than the other two microscopy methods, when measuring large numbers of nanoparticles.

 

As for DLS, we found that it was very poor at separating mixtures, and had much lower precision levels than any of the microscopy methods. On the other hand, since it measures solutions, rather than dried samples, it can give an idea of the dynamic behaviour of the particles in solution.

 

For more details, download the paper HERE, or HERE.

Every year in our AFM training course, we hold a little competition among the students to produce and process an AFM image. The prize is always a liquid product from the city of Porto!

This year, the overall prize was won by this nice treatment of human red blood cells sent in by Akmaral.

 

 
Human erythrocytes, processed and submitted by Akmaral Suleimenova
 
     

 

Thanks to Christie for donating the blood!

 

In addition, this year, we have a special prize for “outstanding image processing”, which goes to Tobias for this unusual image presentation: I can only show you a photo of the image, as he 3D-printed the AFM image of 90nm nanoparticles. Great one, Tobias!

 

          
3D printed image of Nanoparticles from Tobias Burger
 
     

Congratulations to Akmaral and Tobias, your prizes and certificates will be with you soon!

 

Thanks to everyone who entered the competition this year. Hope to see you again!

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