Size distribution measurement of vesicles by atomic force microscopy
Section snippets
Materials and methods
Hepatitis B virus surface antigen (HBsAg) L particles were used as vesicles in our study. HBsAg L particles were overexpressed in Saccharomyces cerevisiae AH22R− harboring an expression plasmid pGLDLIIP39-RcT [2]. HBsAg L particles were purified with three sequential ultracentrifugation steps without any chromatographic step [9]. An HBsAg L particle consists of transmembrane HBsAg L proteins (52 kDa) and phospholipids and forms a vacant liposome. The buffer solution used was Dulbecco’s
Results and discussion
Typical AFM images for the low concentration (10 mg/L) of HBsAg L particles are shown in Figs. 1A and B. The images show flattened structures with heights of 1.8–2.5 nm and diameters of 550–1500 nm scattered on the mica surfaces. DLS analysis indicated that these HBsAg L particles have diameters of approximately 80 and 300 nm in solution [9]. Thus, it was considered that HBsAg L particles are adsorbed onto the substrate as a flattened structure. However, the size of HBsAg L particles can be
Conclusions
Atomic force microscopy was demonstrated as a suitable method for examining the vesicle size and the size distribution directly, because AFM facilitates the imaging of molecules one by one. In this study, by using the HBsAg L particles as model vesicles, high and low concentrations of vesicles have been adsorbed onto the substrate and then subjected to AFM observation. At the low concentration, the AFM images reveal that vesicles are generally flattened on the substrate after adsorption. At the
Acknowledgements
This work has been performed under the Center Of Excellence (COE) program supported by the Ministry of education, Science, Sports and Culture, Japan.
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