Because of the complexity of nanomedicines, analysis of their morphology and size has attracted considerable attention both from researchers and regulatory agencies. The atomic force microscope (AFM) has emerged as a powerful tool because it can provide detailed morphological characteristics of nanoparticles both in the air and in aqueous medium. However, to our knowledge, AFM methods for nanomedicines have yet to be standardized or be listed in any pharmacopeias. To assess the applicability of standardization of AFM, in this study, we aimed to identify robust conditions for assessing the morphology and size of nanoparticles based on a polystyrene nanoparticle certified reference material standard. The spring constant of the cantilever did not affect the size of the nanoparticles but needed to be optimized depending on the measurement conditions. The size analysis method of the obtained images affected the results of the analyzed size values. The results analyzed by cross-sectional line profiling were independent of the measurement conditions and gave similar results to those from dynamic light scattering. It was indicated that approximately 100 particles are required for a representative measurement. Under the optimized conditions, there were no significant inter-instrument differences in the analyzed size values of polystyrene nanoparticles both in air and under aqueous conditions.
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