The Application of NanoWizard ULTRA Speed AFM to Study Dynamics of Living KPG7 Fibroblasts

Over the last three decades, the atomic force microscope (AFM) has become an indispensable tool for the high-resolution structural analysis of biological specimens ranging from single molecules to complex systems. Nevertheless, relatively slow acquisition times and other limitations of conventional AFM techniques have historically complicated AFM-based life science research -- particularly on soft or topographically inhomogeneous samples or for the study of structural features of rapidly changing and very active living cells. More recent advances in AFM technology have diminished these limitations; with the correct equipment, researchers can now (1) gain additional information about the interactions between not only single cells, but also organelles and macromolecular protein complexes and (2) pursue potential future opportunities to image dynamic biopolymerization processes that underlie the formation of a number of key macromolecules taking place both in vivo and in vitro.

To demonstrate these capabilities, in this app note, the NanoWizard ULTRA Speed AFM was successfully applied to monitor two examples of dynamic biological processes in living KPG7 fibroblasts in nearly physiological buffer conditions. Both cytoskeletal reorganization and cell membrane protrusion events were studied on the time scale of 0.1-0.2 frames per second. Note that such events are typically impossible to observe with conventional AFM due to its low temporal resolution and slow feedback response.

Readers can expect to learn about:

• How the fast-scanning NanoWizard ULTRA Speed AFM can be applied for studying weak and challenging signals, including dynamic cellular processes in near-physiological liquid conditions;
• The full compatibility of the NanoWizard ULTRA Speed system with high-resolution optical techniques for correlative microscopy; and
• The use of these technical combinations to collect even more molecular information from various molecular or immunostains with high spatiotemporal resolution.

_{KEYWORDS: Dynamic Cellular Processes; Fast-Scanning AFM; Fluorescence Microscopy; High-Speed AFM; Fibroblasts; Live Cell Research}