Biological Applications of AFM


One of the advantages of AFM is that it can image the non-conducting surfaces. So it was immediately extended to the biological systems, such as analyzing the crystals of amino acids and organic monolayers. Applications of AFM in the biosciences include: DNA and RNA analysis; Protein-nucleic acid complexes; Chromosomes; Cellular membranes; Proteins and peptides; Molecular crystals; Polymers and biomaterials; Ligand-receptor binding. Bio-samples have been investigated on lysine-coated glass and mica substrate, and in buffer solution. By using phase imaging technique one can distinguish the different components of the cell membranes.

Little sample preparation is required for bioimaging with the AFM. In most cases it is as simple as spotting a few microliters of solution on mica or glass. Of course contaminations that cover surface features have to be avoided or removed. First the substrate-adsorbate should be rinsed with a large excess of buffer. The following procedures are dialysis, centrifugation and homogenization. In order to get good contrast and to reduce mechanical damage of the soft biological materials, the samples can be stabilized by adding covalent cross-linking agents or certain cations that are able to link the constituents of the sample to each other or to the substrate. Cooling can also stiffen the sample. Nevertheless, all these methods have significant influence on the properties of biomolecules. The sharper tip now is available commercially and really does help a lot.

One area of significant progress is the imaging of nucleic acids. The ability to generate nanometer-resolved images of unmodified nucleic acids has broad biological applications. Chromosome mapping, transcription, translation and small molecule-DNA interactions such as intercalating mutagens, provide exciting topics for high-resolution studies. The first highly reproducible AFM images of DNA were obtained only in 1991. Four major advances that have enabled clear resolution of nucleic acids are: Control of the local imaging environment including sample modification; TappingMode scanning techniques; Improved AFM probes( such as standard silicon nitride probes modified by electron beam deposition and Oxide Sharpened NanoProbes ) and Compatible substrates( such as salinized mica and carbon coated mica ).

Cell biologists have applied the AFM's unique capabilities to study the dynamic behavior of living and fixed cells such as red and white blood cells, bacteria, platelets, cardiac myocytes, living renal epithelial cells, and glial cells. For example, plasma membrane in migrating epithelial cells has been imaged in real time. The dynamic membrane invagination process was observed in the presence of calcium and when calcium levels were reduced the process was prevented. 30nm lipidic pore formation could also be resolved during calcium reduction. AFM imaging of cells usually achieves a resolution of only 20-50 nm, not sufficient for resolving membrane proteins but still suitable for imaging other surface features, such as rearrangements of plasma membrane or movement of submembrane filament bundles. The requirement for the imaging buffer is not restrictive, as long as the buffer does not severely affect the integrity of the cells. EDTA should be avoided because cells will detach from the substrate in the absence of divalent cations. Cultured cells normally adhere well to the substrate and are not displaced by modest probe forces.

There has been recent success imaging individual proteins and other small molecules with the AFM such as collogen. Smaller molecules that do not have a high affinity for common AFM substrates have been successfully imaged by employing selective affinity binding procedures. Thiol incorporation at both the 5' and 3' ends of short PCR products has been shown to confer a high affinity for ultraflat gold substrates. A similar approach was used to immobilize antibodies (IgG1) on treated mica. In this case, the low affinity that IgG molecules have for mica was overcome by cloning a metal-chelating peptide into the carboxy terminus sequence of the IgG's heavy chain. The recombinant sequence was transformed into cells that expressed the complementary light chain. The purified IgG containing the metal-chelating peptide was shown to bind in a regiospecific manner to nickel-treated mica. Covalent binding of biological structures to derivatized glass substrates has also enabled high resolution imaging of some samples that are not stable on untreated glass substrates. New approaches in AFM have provided a solid foundation from which research is expanding into more complex analyses. Higher resolution imaging of a variety of small molecules is improving at a rapid pace.

The recent innovation, such as Digital Instruments BioScope system, which combines the high resolution of AFM with the ease of use and familiarity of inverted optical microscopes, has further added to the attractiveness of AFM for biological imaging. Bright-field, flourescence and other optical techniques can be used to identify structures of interest while the AFM simultaneously generates nanometer-resolved images of the sample surface.

Countless biological processes - DNA replication, protein synthesis, drug interaction, and many others - are largely governed by intermolecular forces. AFM has the ability to measure forces in the nanonewton range. This makes it possible to quantify the molecular interaction in biological systems such as a variety of important ligand-receptor interactions. Another application of AFM force measurements is to image or quantify electrical surface charge. The dynamics of many biological systems depends on the electrical properties of the sample surface. In addition to measuring binding forces and electrostatic forces, the AFM can also probe the micromechanical properties of biological samples. Specifically, the AFM can observe the elasticity and, in fact, the viscosity of samples ranging from live cells and membranes to bone and cartilage.

AFM gives scientists a key tool to investigate the real world.


Author: Hong-Qiang Li: email: hli@uoguelph.ca
Curator: Dan Thomas email: <thomas@chembio.uoguelph.ca>
Last Updated: Thursday, April 24 1997