Mind the Bend: Curved and Corrugated Cryo-Lamella for Improved Mechanical Resilience

Abstract

Preparing electron-transparent cryo-lamellae is inherently a serial, low-throughput process. During sample handling, milling, and transfer, cryo-fixed cells and their supporting films are subjected to mechanical forces as well as thermal stresses caused by temperature fluctuations. After milling, these extremely thin lamellae remain vulnerable to both mechanical and thermal stress, often leading to cracking or complete disintegration. Consequently, the loss of valuable lamellae is frequently an unavoidable aspect of working with such fragile specimens. In this work, we reconsider the conventional lamella geometry, which is typically a flat, thin cross-sectional slab. During milling, lamellae often become unintentionally bent, complicating the final polishing step required to achieve uniform thinning across their width. To address this limitation, we propose deliberately fabricating lamellae in a pre-bent configuration, i.e. specifically, adopting an arch-shaped profile instead of the traditional flat geometry. The arch shape is intrinsically more mechanically stable than a flat structure, thereby reducing lamella loss due to mechanical failure. Moreover, pre-bent milling patterns facilitate uniform thinning of bent lamellae, which is difficult to achieve using conventional flat milling approaches. In addition to the arch geometry, we investigate corrugated lamellae, characterised by a sinusoidal variation around the plane of a conventional flat lamella. Similarly to the arch shape, the corrugated design offers enhanced mechanical stability compared to traditional flat lamellae. We fabricated a series of test lamellae incorporating both arches and corrugations. High-resolution cryo-TEM imaging was performed to evaluate these structures, demonstrating that non-flat geometries do not compromise cryo-electron tomography performance. Furthermore, finite element method (FEM) simulations were conducted to provide insight into stress distributions within bent and corrugated lamellae.