4 April 2005 Atomic force and near-field scanning microscopy of solid zFP538 films
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We had previously reported about high aggregation number of the yellow fluorescent protein zFP538, as was shown by gel-filtration and dynamic light scattering. In this study we used atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) to image zFP538 in solid state. Height and phase measurements of the sample were taken using NT-MDT AFM operated in tapping mode. Height data provides three-dimensional topographical 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 to the stiffness of the sample. Unlike EGFP, which crystallizes upon drying of water solution, zFP538 forms ring-like films due to its surfactant properties. According to AFM these films are comprised of ellipsoidal protein granules, with the major 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 the case of GFP molecule (a cylinder with a height of 4.2 nm and 2.4 nm diameter). NSOM with a 532 nm laser emission from CW Nd:YAG shows that fluorescence of zFP538 is retained in solid state. A ratio of maximum emission to shoulder at 580 nm is 0.65 for solid zFP538 and only 0.25 for water solution.
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Nadya N. Zubova, Nadya N. Zubova, Artem A. Astafyev, Artem A. Astafyev, Andrew N. Petrukhin, Andrew N. Petrukhin, Oleg M. Sarkisov, Oleg M. Sarkisov, Alexander P. Savitsky, Alexander P. Savitsky, } "Atomic force and near-field scanning microscopy of solid zFP538 films", Proc. SPIE 5704, Genetically Engineered and Optical Probes for Biomedical Applications III, (4 April 2005); doi: 10.1117/12.590226; https://doi.org/10.1117/12.590226

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