Atomic force and near-field scanning microscopy of solid zFP538 films

摘要

We had previously reported about high aggregation number of the yellow fluorescent protein zFP538, as wasshown by gel-filtration and dynamic light scattering. In this study we used atomic force microscopy (AFM) and nearfieldscanning optical microscopy (NSOM) to image zFP538 in solid state. Height and phase measurements of thesample were taken using NT-MDT AFM operated in tapping mode. Height data provides three-dimensionaltopographical information on the sample while the phase data, which measures the phase shift in cantilever oscillation,responds to attractive and repulsive interactions between the cantilever tip and the sample. This signal can be related tothe stiffness of the sample. Unlike EGFP, which crystallizes upon drying of water solution, zFP538 forms ring-like filmsdue to its surfactant properties. According to AFM these films are comprised of ellipsoidal protein granules, with themajor axis of the granules lying in the range from 50 to 300 nm and the minor axis – in the range from 30 to 130 nm.Average volume of these granules is about 19500 times higher than volume of zFP538 monomer, calculated as in thecase of GFP molecule (a cylinder with a height of 4.2 nm and 2.4 nm diameter). NSOM with a 532 nm laser emissionfrom CW Nd:YAG shows that fluorescence of zFP538 is retained in solid state. A ratio of maximum emission toshoulder at 580 nm is 0.65 for solid zFP538 and only 0.25 for water solution.