Worm Breeder's Gazette 11(4): 115

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Scanning Tunneling Microscopy (STM) of C. elegans Microtubules

Shahid Siddiqui, Stuart Hameroff, Bob McKuskey, Dror Sarid and Sam Ward

Figure 1

We have been studying C.  elegans microtubules from cellular and 
genetic aspects.  Since scanning tunneling microscopy (STM) can 
provide atomic resolution of metals and semiconductive surfaces; our 
objective is to obtain images of C.  elegans microtubules resolved at 
the atomic level.  However, application of STM to biomolecules is 
faced with a variety of problems, including poor electrical 
conductivity, flexible stability under STM imaging conditions.  In 
spite of limitations, recently, impressive images of DNA biomolecules 
have been obtained at atomic resolution.
Microtubules (MT) were isolated from 30g of freshly grown N2 worms 
according to the procedure of Aamdot and Culotti 1986, and Siddiqui et 
al 1989, using cycles of temperature dependent assembly and 
disassembly (Shelanski, et al.  1973).  Assembled MT were taken in MT 
isolation buffer (containing 0.1mM GTP, 20 M taxol, and 2.0 mM DTE), 
diluted tenfold and fixed in glutaraldehyde (0.1%).  The samples were 
kept frozen at -20 C, for 4-5 days.  The STM imaging samples were 
thawed, and a drop was smeared onto a chip of freshly cleaved HOPG (
highly oriented pyrolitic graphite, Union Carbide), and allowed to air 
dry at room temperature.  The STM images were obtained using 
'Nanascope II Digital Instruments, California (Sam Howell and C.  Lee, 
kindly helped with the STM settings).  All STM imaging was conducted 
at atmospheric pressure and at room temperature (22 C).  Two different 
STM images of C.  elegans MT are shown here.
[See Figure 1]
Most of our STM scans of MT correspond to one or two microtubules, 
but they appear flattened and bent along the seams of the 
protofilaments.  The dimensions of alpha and  -tubulin dimer subunits 
are about 8 nm, and each protofilament has a width of 4.6 nm.  The 
flattening and buckeling of protofilaments along the seams could arise 
due to the fixation conditions, or caused by the STM current flow 
through the specimen.  At present, we are unable to attribute this 
observation to any single factor, and the images of MT do not provide 
any higher resolution than conventional electron microscopy using 
negative staining.  But it is increasingly possible that one may be 
able to resolve individual amino acids or even atoms using STM.  We 
plan to continue looking for improved conditions for better STM or AFM 
images of C.  elegans MT.  
Brief description of 
Scanning tunneling microscopy (STM) traces three dimensional images 
of surfaces - even at the level of individual atoms.  Gerd Binnig & 
Heinrich Rohrer of IBM Zurich received the Nobel Prize in 1986 for STM 
(Refer, e.g.: Scientific American, August,1985).  Briefly in STM, the 
'aperture' is a small tungston probe, its tip extremely fine (about 0.
2 nm in width, and may consist of a single atom).  Piezoelectric 
controls move the tip within a nanometer or two of the surface of the 
conducting sample, allowing an overlap between the electron clouds of 
the atom at the probe tip and the closest atom of the sample placed on 
the surface.  As a small voltage is applied to the tip, electrons 
'tunnel' across this gap, creating a tiny tunneling current.  The 
strength of the tunneling current is exponentially sensitive to the 
width of the gap (about an order of magnitude per angstrom).  
Depending on the substrate, typical currents and voltages are in the 
range of nanoamperes and millivolts.  A servo system uses a feedback 
control that keeps the tip to substrate gap constant, by modulating 
the voltage across a piezoelectric positioning system (Piezoceramic 
materials expand or shrink extremely small distances (i.e.  angstroms )
in response to the applied voltage.  As the tip scans the surface, 
variations in this voltage, when plotted correspond to surface 
I wish to thank members of Ward lab (Alicia, Andrea, Bill, Bonnie, 
Bruce, Jacob, John and Paul); Hameroff Lab (Dolly, Larry, Mohammad, 
and Richard); and Sarid Lab (Sam and Lee).

Figure 1