Laboratory exercise for studying the morphology of heat‐denatured and amyloid aggregates of lysozyme by atomic force microscopy

Abstract

To facilitate learning advanced instrumental techniques, essential tools for visualizing biomaterials, a simple and versatile laboratory exercise demonstrating the use of Atomic Force Microscopy (AFM) in biomedical applications was developed. In this experiment, the morphology of heat-denatured and amyloid-type aggregates formed from a low-cost and well-characterized model protein, hen egg white lysozyme (HEWL), are compared. Structural differences between the amorphous and ordered particles are quantified using ImageJ for the analysis of AFM images as a postlaboratory assignment. The laboratory exercise allows the direct observation of changes in the protein structure and helps students to understand the operation of AFM, as well as protein folding and misfolding related to many physiological and pathological processes. The described protocol stands alone, but also fits well into a larger module on protein structure and function or microscopic techniques as it can be linked easily to existing laboratory exercises on these topics. It can be easily adapted to the upper level undergraduate laboratory courses with limited lab hours as well as graduate level courses to improve students' research skills. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(2):162-171, 2018.

Eutectic Gallium–Indium Nanoparticles for Photodynamic Therapy of Pancreatic Cancer

“…However, the calculated diameters of the nanoparticles from SEM images were higher (more than doubled) than those determined from AFM size analysis. The AFM methodology is considerably different than SEM as it provides a 3D topographical image, which is used to determine the height as the diameter in the z -direction. − One of the main factors that may explain the size difference is that AFM analysis of EGaPs is done by selecting particles individually, while the SEM software measures the diameter of all particles within an image without individual selection. Another factor is that EGaPs might be flattened (wetting the surface of the substrate) due to the presence of the oxide layer, which would reduce the height and increase the width .…”