Using Atomic Force Microscopy for imaging biomolecules immediately exposes us to some challenges: imaging in salty liquids, and most importantly dealing with live organisms that need to be kept happy in their environment and still need to be attached firmly to a support to make AFM imaging possible. Several methods for sample preparation are presented and discussed.
Atomic Force Microscopy (AFM) has a unique advantage over other microscopy techniques, like electron microscopy for instance, as we are able to fully mimic the natural conditions and environment of our sometimes very delicate biological entities. At the same time, we are facing some key requirements and difficulties when it comes to biological imaging:
A. Imaging in salty buffer solution is required — which is a corrosive environment — that might lead to scanner damage for instance. A top-down scanner design is preferred here! In addition, in order to change the environment, like temperature or the imaging solution or atmosphere, a low-noise pump design and good sealing of the fluid cell is required!
B. Life means dynamics and movement, which per se is a big challenge for AFM! We are constantly confronted with loose attachment of molecules, weak binding, or moving organisms on the surface. Gently imaging modes, like magnetic AC mode, or off-resonance tapping mode with soft cantilevers is one way to be successful, apart from preparing the substrate and using the right treatments of the substrate to bind biomolecules to the surface. Here are the key points to remember:
1. Cells and biomolecules must be placed on a flat surface prior to AFM imaging, there should be a stable connection between the molecules and the surface. Also remember that there is a roughness limit of around 10µm, not only given by the limited z-range of the piezo used for scanning, but also restricted by the height of the actual AFM tip!
2. Surface properties of the molecules define the selection of the right substrate and/or substrate treatment and modification via charges, functional groups etc.
3. The size of the molecule or cell has less influence than the functional groups they have
4. Choose a preparation method that is just needed, you do not always have to go for the most advanced/sophisticated/complex binding protocol.
5. Always consult literature, or your colleagues before you start, don’t re-invent the wheel, there are a lot of protocols and papers out there already that is perfectly right for you.
6. A path from basic/simple to advanced sample preparation looks like this:
a. Biosample dried from solution on glass or graphite, and imaged in air
b. Physisorption techniques: charge attraction, ions, hydrophobic binding
c. Chemisorption: covalent linking, organic/inorganic complex formation
d. Biological interaction: avidin-biotin, His6-NTA, antibody antigen, DNA binding
Here are some examples of biomolecules and their proposed binding protocol:
· DNA can be firmly attached to negatively charged, and perfectly flat, mica surfaces by treating the surface with high concentrations of bi-valent cations (like magnesium, nickel or zinc) and thereby bridging and the negative charges from the negatively charged phosphate backbone of DNA to this negatively charged mica substrate
· Proteins have a big variation in functional groups, this means we have to look at the amino acid composition, the pI (isoelectric point) and possible anchor groups very carefully. Sometimes physisorption is good (avidin binds directly to mica for instance due to its positive charge at physiological pH), in some cases covalent binding via amino- or thiol groups and crosslinkers is required to get a firm attachment, sometimes complex formation via biotin or histidine-tags is the method of choice, or antibody-antigen or ligand-receptor linking is required.
· Bacteria are imaged in air in many cases, by drying a sample from solution, for imaging in liquids, the challenge is that most bacteria are not well adherent to surfaces, at least before they form a biofilm, so surface coating with poly-lysine or gelatin is required.
· Imaging Living cells is regarded as one of the highest arts in AFM imaging, a few things to consider:
o Fixed cells are easier to image than live cells
o Surface contact is crucial, well adherent cells are preferred, a confluent layer of cells is better than single cells, assistance by optical microscopy is needed to judge the quality of attachment and healthiness of a cell
o Different cell types require different suface coating: plain glass, poly-lysine coating, gelatin matrix, Cell-tak™, or surface cell receptors, look in literature!
o What is a good prep for fluorescence experiments might not be sufficient for AFM imaging in terms of tight binding of the cells to the surface.
o Soft cantilevers, low force imaging, and a controlled environment are important
Our article on imaging DNA and RNA in “Biomaterials” can be found here:
Sample preparation is an important topic in our review article in “Nanotoday”:
A beautiful article in “Ultramicroscopy” on imaging bacteria in liquid is here: