Single‐Molecule Graphene Liquid Cell Electron Microscopy for Instability of Intermediate Amyloid Fibrils

Single‐molecule techniques are powerful microscopy methods that provide new insights into biological processes. Liquid‐phase transmission electron microscopy (LP‐TEM) is an ideal single‐molecule technique for overcoming the poor spatiotemporal resolution of optical approaches. However, single‐molecule LP‐TEM is limited by several challenges such as electron‐beam‐induced molecular damage, difficulty in identifying biomolecular species, and a lack of analytical approaches for conformational dynamics. Herein, a single‐molecule graphene liquid‐cell TEM (GLC‐TEM) technique that enables the investigation of real‐time structural perturbations of intact amyloid fibrils is presented. It is demonstrated that graphene membranes significantly extend the observation period of native amyloid beta proteins without causing oxidative damage owing to electron beams, which is necessary for imaging. Stochastic and time‐resolved investigations of single fibrils reveal that structural perturbations in the early fibrillar stage are responsible for the formation of various amyloid polymorphs. The advantage of observing structural behavior in real time with unprecedented resolution will potentially make GLC‐TEM a complementary approach to other single‐molecule techniques. Graphene liquid cell transmission electron microscopy (GLC‐TEM) is a novel single‐molecule technique offering valuable insights into molecular processes. GLC‐TEM provides an extension of the observable limit for biomolecules and demonstrates molecular structural variations. Using GLC‐TEM, stochastic and time‐resolved investigations of single amyloid fibril reveal structural perturbations during the initial fibrillar stage.