Mind the crack: Crack-arrest holes and soft-suspension support integration in cryo-lamella preparation for improved resistance to crack formation, fracture and deformation

Abstract

Preparing electron-transparent cryo-lamellae is inherently a serial and low-throughput process. Once the lamellae are milled, these thin structures endure both mechanical and thermal stress, and as a result many valuable lamellae crack or even disintegrate entirely. This loss is often regarded as a "lamella tax", i.e. an unavoidable cost of working with such fragile specimens. In this work, we introduce two modifications to the standard lamella-preparation workflow aimed at improving lamella mechanical resistance to crack formation and external stress. The first modification involves milling arrays of perforations directly within the lamella body. These perforations are designed to function as crack-arrest holes, intercepting cracks as they appear and preventing, or at least delaying their further propagation. By slowing crack growth, these features increase the likelihood that the lamella remains intact long enough to complete cryo-TEM imaging. The second modification replaces the conventional rigid attachment of the lamella to the surrounding cellular bulk material with a softer suspension using ring-shaped springs formed by ion beam milling. Mounting the lamella on smooth annular springs provides mechanical compliance both across and along the lamella axis, as well as at intermediate angles and in the out-of-plane direction. This flexibility allows the lamella to accommodate larger stresses and deformations without reaching its mechanical failure threshold. We fabricated a series of test lamellae incorporating different crack-arrest hole geometries, as well as lamellae suspended on soft annular springs. We performed high-resolution cryo-TEM imaging to characterise the perforations themselves and characterised the captured crack geometry within the lamellae at the highest level of detail achieved to date. TEM imaging shows crack interception and guided, non-catastrophic failure paths, while simulations confirm lowered stress in suspended lamellae.