AFM In-Situ Characterization of Supported Phospholipid Layers Formed by Vesicle Fusion

Abstract

Atomic force microscopy (AFM) is a powerful tool for direct visualization of supported biological membranes [1]. Moreover, in-situ AFM measurements permit investigations of biological phenomena in real time and in physiological environments. In a previous work, we have studied the morphology and stability of supported phospholipid layers prepared by solution spreading (casting) on mica [2]. The images were acquired in the contact or contact-intermittent modes and the samples analyzed ex-situ just after solvent evaporation and after a hydration step, and in-situ with immersion in a buffer solution. Contact-mode imaging is less suitable for soft or weakly attached materials, since the tip can often scrape or drag the membranes during scanning, a disadvantage that can be overcome by applying intermittent methods. However, studies have also demonstrated that by adjusting the operative force it is possible to use contact-mode to obtain AFM images of soft phospholipids layers [3]. In the present work, we applied successfully in-situ AFM contact-mode to characterize phospholipid layers of 1,2-dimyristoyl-sn-glycero-3-phosphatitidylcholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), as well as a binary mixture of these phospholipids. The supported membranes were prepared on mica substrates by vesicle fusion method.