The variety of samples investigated using Atomic Force Microscopy in biology includes the smallest biomolecules proteins, lipids, DNA, RNA and other nucleic acids, as well as larger objects like living cells, viruses and platelets.
|AFM in Life Science|
|Nucleic acids||Platelets||Bone cement|
In its main microscopy function AFM provides real-space three-dimensional images with high vertical and lateral resolution well beyond the 2D-projections of particles obtained by TEM. Additionally, high-resolution visualization of various biological species by AFM allows for the evaluation of local nanomechanical properties. Furthermore, its applications can be extended to the mapping of local surface charges and variations in electric properties.
AFM studies of biological samples require special conditions due to their general softness, as well as a desire to examine them in an aqueous environment.
Probes for imaging
Regarding the stiffness of biological objects, only bone material and some macromolecular assemblies (such as collagen) have an elastic modulus in the GPa range, whereas the majority of biological samples, such as cells, have an elastic modulus in the kPA range. This very low modulus makes the imaging and mechanical probing of biological samples challenging. Imaging using soft AFM probes is preferable. In contact mode probes with stiffness of 0.1 N/m or lower are most suibtale. Although regular Si3N4 probes usually satisfy this requirement, they are not very sharp (apex diameter is in the 20 - 30 nm range). Therefore, soft silicon probes such as HQ:CSC17 (k=0.1 N/m) are recommended for high-resolution imaging of biological macromolecules in contact mode.
Investigation of biological samples with oscillatory modes is generally less destructive, but the use of soft probes (stiffness about 1 N/m or lower, e.g. HQ:NSC18 and HQ:NSC19) is also preferable, especially when imaging in buffers or water. AFM studies in liquid environments are useful due to the fact that the absence of capillary forces common with imaging in air allow the tip-sample interactions to be examined at smaller force levels. At the same time, measuring in liquids is much more complicated than in air. There are a number of instrumental difficulties for imaging in a closed liquid environment and most commercial liquid accessories do not provide a clean mechanic excitation of the AFM probe at its resonance frequency.
|Contact mode||Oscillatory modes|
|Soft samples||k~0.1N/m HQ:CSC17||k~0.5 N/m f~65 kHz HQ:NSC19|
|Hard samples||k~0.1 N/m HQ:CSC17||k~5 N/m f~150 kHz HQ:NSC14|
Along with direct imaging of biological objects, AFM plays a significant role among numerous biophysical methods for the investigation of specific and non-specific molecular interactions. These are protein-protein, enzyme-substrate, antigen-antibody, receptor-ligand interactions, drug-target associations, a diverse number of biocomplexes and many others. The highest sensitivity reached so far allows force measurements between individual biomolecules and complexes. Single-molecule atomic force spectroscopy is becoming a typical application.
The AFM cantilever can be used as the main sensitive element of a biosensor. Such force measurements are usually performed using the AFM probe functionalized with a biomolecule of interest and its complementary molecule immobilized onto the sample surface. The functionalizing of AFM probes by different chemical and biological species is a non-trivial custom procedure that requires reliable tip characterization. Soft Si3N4 and Si probes are usually chosen for chemical and biological modifications.
The sample preparation for biological samples can also be a challenge. Adsorbing biological species to a substrate to avoid their displacement by AFM probes during scanning is not easily done. There are different approaches for the fixation of these objects on substrates, from specific chemical modifications of substrates to ensure the sample adhesion to the use of porous substrates that help species immobilization.
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HQ:CSC probes for contact mode
Probes with stable reflective coating
In aggressive liquid media
Probes with chemically stable coating
HQ:NSC probes with soft cantilevers
HQ:NSC probes with stable reflective coating
Agressive liquid environment
Probes with chemically stable coating