The basics of imaging biomolecules
Atomic force microscopy has been used successfully to image a wide range of biomolecules. AFM is a mechanical surface characterization technique, and while the forces the tip exerts on the sample are very small, they are still enough to move things around. It is essential to adhere the molecules of interest firmly to the substrate of choice and to make sure that there are no contaminants in solution that might cover the structures of interest. Imaging dry is usually the first step as it addresses many of these issues and also confirms if the solution is pure enough to deliver the desired information. A next possible step is imaging in liquids and force measurements on or between molecules. The most common substrate for molecular characterization is muscovite mica, an atomically flat, sheet-like mineral with a negative surface charge, to which molecules can be bound to electrostatically.
Imaging DNA
DNA is the most common biomolecule sample in AFM imaging for multiple reasons. It is at the root of many biological questions, relatively easy to obtain in high concentrations, difficult to image by transmission electron microscopy (TEM), and suitable for imaging by AFM both dry and in liquid. There is excellent literature on preparing and imaging DNA, including advanced methods with choices of substrates, probes and modes.
DNA imaging requires a high-quality starting material (highly purified), clean handling and reagents, and high-quality mica. The most straightforward method is the use of divalent cations (magnesium or nickel) in the solution to bind the DNA to the negatively charged mica, followed by washing and drying.
Additional data here: Specialized Application – Imaging DNA with AFM
Other biomolecules
Biomolecules vary dramatically in size and stability and range from large macromolecules to individual proteins. Sample preparation plays a key role in obtaining reliable data and needs to be taken into consideration when interpreting the data. Again, the first test should be done on a dried sample, with the caveat that structures decay to varying degrees during fixation, washing and drying. To demonstrate this we have analyzed some readily available yet surprisingly challenging biomaterials.
Collagen is an example of a large, polymeric and highly relevant biomolecule that is very accessible by AFM. We have found it serendipitously in the eggshell membrane, a great biomaterial for educational outreach programs. With closer inspection we found that the entire surface was still covered by egg white proteins which are readily visualized because of their homogenous appearance (ovalbumin makes up 54% of egg white proteins).
Casein micelles in milk are another readily available though much more fragile and complex macromolecular complex for which AFM imaging is a perfect method. We tested a sample of 2% cow’s milk by filtering the solutes and imaging the filter cake. In this case we expect the drying process to significantly disrupt the structure of the emulsified particles but were able to image what appear to be casein micelles.
Collagen I network found in an eggshell membrane facing the egg white. See Specialized Application – Of Eggshells and Serendipity
Egg white proteins on the eggshell membrane, mostly ovalbumin. See Specialized Application – Of Eggshells and Serendipity
Solutes in 2% cow’s milk enriched by filtration showing casein micelles and fragments. See Specialized Application – Filtration Membranes
