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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 |
| Biomolecules |
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Functional units |
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Biomaterials |
| Proteins |
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Cells |
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Dental implants |
| Lipids |
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Viruses |
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Contact lenses |
| Nucleic acids |
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Platelets |
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Bone cement |
| etc... |
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etc... |
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etc... |
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 best.
Although regular Si3N4 probes usually satisfy
this requirement, they are not very sharp (apex diameter is the
20-30 nm range). Therefore soft silicon or diamond probes such as SCD17 (k=0.1 N/m)
are recommended for high-resolution imaging of biological macromolecules
in contact mode.
Examination 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. SCD19) 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 the 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 resonant frequency.
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Contact mode |
Oscillatory modes |
| Soft samples |
k~0.1N/m SCD17 |
k~1 N/m f~50 kHz CSC37
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| Hard samples |
k~1 N/m SCD17 |
k~5 N/m f~150 kHz SCD14
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Biosensors
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 practice.
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.
Sample preparation
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 insure the sample
adhesion to the use of porous substrates that help species immobilization.
Further reading
Cells
Viruses
Biomolecules
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