An Optimized Protocol for High-Quality AFM Imaging of Amyloid Fibrils.

Characterizing the morphology of amyloid proteins is an integral part of studying neurodegenerative diseases. Such morphological characterization can be performed using atomic force microscopy (AFM), which provides high-resolution images of the amyloid protein fibrils. AFM is widely employed for visualizing mechanical and physical properties of amyloid fibrils, not only from a biological and medical perspective but also in relation to their nanotechnological applications. A crucial step in AFM imaging is coating the protein of interest onto a substrate such as mica. However, existing protocols for this process vary considerably. The conventional sample preparation method often introduces artifacts, particularly due to deposition of excess salt. Hence, an optimized protocol is essential to minimize salt aggregation on the mica surface. Here, we present an optimized protocol for coating amyloid proteins onto mica using the dip-washing method to eliminate background noise. This approach improves the adherence of protein to the mica surface while effectively removing residual salts. Key features • The protocol introduces a new method to coat protein samples onto mica sheets for AFM imaging. • It presents a dip-washing technique aimed at removing excess salt deposited on the mica sheet, thereby minimizing imaging artifacts. • This protocol can be used for analyzing amyloid fibrillation mechanisms as well as capturing time-dependent fibrillation dynamics under various physiological conditions. • It also provides clear stepwise washing instructions that balance the salt removal and retention of protein fibrils on the mica.