SPM Applications in Biology

     Viruses have been good targets for imaging with AFM especially in early phase of it application to biology. Bacteriophage T4 was first imaged by Kolbe et al. [1537] with a distinction of the head and tail. The result was soon improved by Ikai and his collaborators with a successful imaging of tail fibers of 2 nm in diameter. Zenhausern et al. and Imai et al. imaged tobacco mosaic virus as well as bacteriophages [1538-1541].

     According to Ikai et al. [1538] The dimension of T4 phage in the air dried state was: the head (W=140 nm, H=50-60 nm); the tail (W=40 nm, H=17-20 nm), the tail-fiber (W=7-8 nm, H=1-2 nm). Occasionally phage particles with a very low head height were observed corresponding to "empty" head particles that were devoid of DNA normally packed in the head. One advantage of AFM over other microscopic methods like electron microscopy is its ability to directly give information on the height of the specimens [256].

     Tobacco Mosaic Virus (TMV or Satellite TMV) is the most popular object among other numerous mosaic viruses investigated so far [953, 1045]. Colloidal solutions of TMV behave similar to those of commonly known protein ones so can be studied the same way. TMV microcrystals failed to grow at low supersaturations. It was found that two-dimensional nuclei were the source of growth steps both at high and at rather low supersaturations. However, at higher supersaturations (typically used for TMV crystallization), three-dimensional nuclei provided the major source of growth steps [118, 545, 546]. Another mosaic viruses studied most recently are icosahedral turnip yellow mosaic virus (TYMV) and cucumber mosaic virus (CMV) [783]. Growth of these crystals proceeded by two-dimensional (2D) nucleation. Authors presented highly resolved AFM images of the hexameric and pentameric capsomers of the T=3 capsids on the surface of the individual TYMV virions which are as small as 28 nm in diameter. According to the data obtained by X-ray crystallography the capsid of TYMV is composed of 180 identical protein subunits, each of about 20 kDa, organized into 12 pentameric and 20 hexameric capsomers which project about 40 Å above the surface of the virion. Authors particularly emphasized that the difference between the highest and lowest points on the capsid surface mentioned above is accurately reflected by AFM. CTM virus capsomer structure is much finer and cannot be resolved to appropriate quality as yet. Clear in situ AFM images of structural peculiarities of virus crystal surfaces obtained under various experimental conditions help to interpret X-ray crystallography data. A series of images with scan sizes of 300 x 300 nm² reveals evolution of the surface layer of the (1 0 1) face of the TYMV crystals, when exposed to equilibrium conditions. Structural defects in crystal lattice such as vacancies, individual particles, dislocations and aggregates are excellently discernible.

Fig. 1 AFM image of potato viurs X subjected to phosphorylation. Scan size 2400 x 2400 nm, height 22 nm. (Image cortesy of Prof. I.V. Yaminsky, MSU&ATC)

     Once a living biological specimen is under the AFM tip and in a favorable environment the system can be maintained and studied for hours. Thus, biological processes can be studied in real time. Haberle et al. [1531] studied the viral infection process of mammalian cells. Living monkey kidney cells were reproducibly imaged with a resolution on the 10 nm scale and then a solution of pox viruses was added to the medium. In the first instance the cell membrane was observed to soften for a short period as the viral infection of the cell took place. Exocytosis events were then observed as viral proteins were expelled from the cell. Finally, the emergence of the progeny viruses was witnessed when large temporary protrusions of 200-300 nm cross section appeared in the membrane leaving scars.

     A notable paper that elegantly demonstrates how AFM's high resolution enables the relationship between structure and function to be drawn is that of Müller et al. [1530]. These researchers achieved subnanometer resolution of the f29 bacteriophage headtail connector to provide structural evidence that the connector and its movement play an important role in the packing of the viral DNA.

     A promising AFM technique involving cantilever tips functionalized with specific molecules can be easily extended to the sphere of virology. The prospects of this technique were outlined by R.d.S. Pereira [346]. It was supposed that the use of the enzyme reverse transcriptase from the AIDS virus to modify AFM cantilever make it a powerful tool to test new medications capable of inhibiting this enzyme and inactivating the action of the virus [E001-E004]. Also, Immobilization of one virus particle on the tip of the cantilever opens up the possibility of measuring the force necessary for this virus to infect a single cell [1535]. In this way, a test medication that could weaken these Van der Waals forces could be a potent anti-virus drug and could avoid virus infection.

     Ohnesorge et al. [1536] reported observing exocytosis of a virus from an infected cell in real time. In spite of the low spatial resolution (scan sizes of 3,6 x 2,0 µm) time resolution was relatively high, reaching one image frame per second, so rearrangement of parts of the cytosceleton was quite discernible. The virus itself has also been characterized, with a much higher resolution of ~2-3 nm, enabling the identification of substructural elements on the surface.

     The binding of influenza viruses to supported lipid membrane as well as the effect of the substrate-exposed lipid monolayer on defect structures in self-assembled, fully hydrated bilayers were studied in order to develop a novel biosensor capable of recognition of specific viruses and other macromolecules [1039]. Spherical particles were of about 200 nm in diameter and may be slightly elongated in the scanning direction due to partial deformation by the AFM tip. Lately extended study of influenza hemagglutinin was reported [696].