Scanning Probe Microscopy: applications to Biophysics

Scanning probe microscopies (SPM) are a relatively new family of imaging techniques whose capabilities are of great interest in several research fields. Based on the interactions arising at very short distance between a very sensitive probe and the surface of a sample, SPM supply the researchers with topographic images of almost any type of samples, organic and inorganic, at a resolution down to molecular, and in some case atomic, scale. When biological samples are the matter of interest, aqueous solutions and physiological conditions become the imaging environment of choice to preserve native conformations and biological activities. While in air only the capillary force has to be considered in addition to the Van der Waals attraction and the orbital-orbital repulsion, in aqueous solutions the Electrostatic Double Layer (EDL) repulsion plays a fundamental role in the processes that lead to the probe-sample, probe-support and sample-support overall interactions. Theoretical fundamentals (DLVO) and the dependence of the EDL force from the electrolyte concentration and pH are discussed. As the resolution of images is strongly influenced by the sample properties, especially when biological samples are investigated, sample preparations suitable for SPM structural investigations are reported. Ionic content of solutions and buffers frequently used to enforce the immobilization of the samples and to reduce the electrostatic repulsion between the probe and the specimens are discussed as well. Topographic images obtained in solution are presented as examples of single protein and thin layers of proteins physically and chemically attached to the support. In the last few years SPMs are evolving in the direction of force sensing techniques, at submolecular scale. Principles of the new techniques such as force mapping and phase mode imaging are described. As a concluding remark, new potential applications of SPM in Biophysics field are outlined.