Assembly of Centromere Chromatin for Characterization by High-Speed Time-Lapse Atomic Force Microscopy

Atomic force microscopy (AFM) is an imaging technique that enables single molecule characterization of biological systems at nanometer resolution. Imaging in ambient conditions can provide details of the conformational states and interactions of a population of molecules which is well complemented by single-molecule imaging of the systems dynamics using time-lapse AFM imaging, in which images are capture at rates of 10-15 frames per second in an aqueous buffer. Here we describe the assembly and preparation of nucleosomes containing centromere protein A (CENP-A) for AFM imaging in both static and time-lapse modes. The AFM imaging and data analysis techniques described enable characterization of the extent of DNA wrapping around the histone core and time-resolved visualization of the systems intrinsic dynamic behaviors.