Morphological difference between fibronectin sprayed on mica and on PMMA.

We have imaged with scanning force microscopy in air fibronectin (Fn) molecules sprayed on mica and on polymethylmetacrylate (PMMA), the latter being extensively used as biomaterial for implants. On mica we can observe small aggregates as well as individual molecules whose shape is influenced by the tip interaction during the scanning process, most of the isolated molecules showing a V-shape oriented in the scan direction. This indicates that the arms of the molecules are relatively free to move and the binding to the mica substrate is located near the disulfide bridge between the two subunits of the molecule. On the other side, when Fn molecules are sprayed on PMMA under the same conditions as for mica, we observe a thin network which we interpret as Fn molecules bound to each other. We relate our observation to the fact that mica is known to be strongly hydrophilic, which could reduce the Fn binding properties by interacting relatively strongly with molecules. On the other side, PMMA being hydrophobic, would interact less with molecules, leaving more binding sites for inter-molecular attachment.

Scanning force microscopy and cryo-electron microscopy of tobacco mosaic virus as a test specimen.

In this study, tobacco mosaic virus (TMV) provides a resolution criterion for specimen preparation methods as well as for imaging parameters of the scanning force microscope (SFM). We present scanning force microscopic images of the virus embedded in 0.5% buffered phosphotungstic acid solution adsorbed on a freshly cleaved mica surface, and imaged under atmospheric conditions. Individual TMV particles were clearly identified with a characteristic shape of long rods of about 300 nm long and 60-70 nm in apparent width due to the geometric parameters of the tip. The structure of the virus was compared with cryo-electron microscopic data of vitrified suspensions observed to a resolution of 1.15 nm. Uncoated TMV particles were also deposited on evaporated titanium thin films and imaged by SFM.

Imaging of DNA by scanning force microscopy.

The scanning probe microscopies applied to the sequencing of DNA is a challenging goal attempted by several groups. But one limitant parameter has been the sample preparation of DNA molecules. Here we report how to hold DNA molecules fixed on mica substrate and we show the three-dimensional configuration of double-stranded DNA obtained with our scanning force microscope. We can image DNA under negative supercoiling, a feature of general importance controlling the activities of DNA. We compared the electron micrographs of a carbon replica of the same DNA specimen with scanning force images which demonstrates well the feasibility and accuracy of our scanning probe measurements.

A small scanning tunnelling microscope with large scan range for biological studies.

We have developed a scanning tunnelling microscope specially designed for biological application presenting some new features: the scanner tube is mounted parallel to the surface of the sample which enables a high resolution optical microscope to be brought close to the sample when working in air or liquids. The maximum scan range is 5x20 microm with a vertical range of 20 microm and the total size of the system does not exceed 10x40 mm. The piezo-sensitivity of the scanner tube versus applied voltage was analysed by interferometry measurements and by using scanning tunnelling microscopes. We found a value for the piezoelectric constant d13 of -1.71 A/V at low voltages (under a few volts) going up to -2 A/V for higher voltages. Large-scale images of a carbon grid showed a surprisingly good linearity of the scanner tube.