RESUMO
In this paper, we explore the development of the Cryo-Lift-Out (cryo-LO) technique as preparation tool for cryogenic transmission electron microscopy (cryo-TEM). What started in early work defying what was considered 'practically impossible' has developed into state-of-the-art practical reality. This paper presents the key hardware, basic principles and key considerations for the practical usage of cryogenic Lift-Out for those new to the field. Detailed protocols and in-depth description of considerations and points for further development are presented. The authors have attempted to formalise everything known about the technique gathered together from their expertise gained in the development of this approach. LAY DESCRIPTION: A major challenge in electron microscopy is the production of suitable samples from hydrated biological and soft-matter materials for subnanometre resolution imaging in a cryo-Transmission Electron Microscope (TEM). A well-known solution for room temperature materials is called (in situ) Lift-Out. It uses a fine needle that picks up a tiny section called a lamella. Lamellae are made by a Focused Ion Beam (FIB). In this paper, we seek to set out the beginnings of Lift-Out sample preparation conducted under cryogenic conditions and the development of this approach as applied to frozen, hydrated biological and soft-matter samples. We discuss the required basic hardware and provide a thorough description of developed protocols. We aim at those new to the field of cryo-Lift-Out to fully educate them in the finer points of hardware setup and practical considerations when attempting to perform cryo-Lift-Out and to demonstrate what has been achieved thus far. We also discuss areas of further improvement and talking points for the future direction of this promising sample preparation technique.
