Recent Advances in Gas Injection System-Free Cryo-FIB Lift-Out Transfer for Cryo-Electron Tomography of Multicellular Organisms and Tissues

Abstract

Abstract:Cryo-electron tomography (cryo-ET) enables visualization of protein complexes within their native cellular environment at molecular resolution. Most cells and all tissues, however, are too thick to be imaged directly by transmission electron microscopy (TEM). Overcoming this limitation requires the production of thin biological sections called lamellae. The procedure to obtain lamellae of cells, either seeded or grown directly on electron microscopy grids, requires cryo-focused ion beam (cryo-FIB) milling to thin the samples. This method faces an additional challenge when dealing with tissues and multicellular organisms, as these samples must be high-pressure frozen, which embeds the sample in a thick layer of ice. Nonetheless, lamellae can still be prepared from such samples by extracting a small volume and transferring it to a receiver grid for lamella preparation, a process called lift-out. Here, we describe the available workflows to produce lamellae by lift-out at cryogenic conditions and recent developments in gas injection system (GIS)-free approaches to the lift-out transfer. These advances expand the applications of cryo-ET, enabling the investigation of tissues and whole organisms in situ at molecular resolution.

Translation dynamics in human cells visualized at high resolution reveal cancer drug action

“…The combined improvements in energy filtering (36), image processing (Warp-RELION-M pipeline) (22) and tight control over specimen thickness (37,38) enable high resolution, as long as the number of particles that can be obtained is sufficient. Further improvements in the automatization of specimen preparation techniques may thus be instrumental in pushing the resolution for other macromolecular assemblies analyzed inside cells (38)(39)(40)(41)(42)(43).…”

Translation dynamics in human cells visualized at high-resolution reveal cancer drug action

“…(35), image processing (21), and tight control over specimen thickness (36,37) enable close-to-atomic resolution, as long as the number of particles that can be obtained is sufficient. Further improvements in the automatization of specimen preparation techniques may thus be instrumental in pushing the resolution for other macromolecular assemblies analyzed inside cells (37)(38)(39)(40)(41)(42).…”

Serial Lift-Out – Sampling the Molecular Anatomy of Whole Organisms

“…The study of multicellular organisms and tissues by cryo-electron tomography, however, holds an enormous potential for biological discovery and technical advances are thus needed. We anticipate the combination of recent hardware and workflow improvements (Klumpe et al 2022, Parmenter & Nizamudeen 2021, Schreiber et al 2018 with the increased throughput of Serial Lift-Out to make cryo-ET data acquisition from high-pressure frozen material more attainable. In addition, plasma focused ion beam technology, while available for some years, was recently introduced to the cryo-FIB community (Berger et al 2023, Martynowycz et al 2023, Sergey et al 2018 and may further improve cryo-lift-out throughput by increased ablation rates.…”

“…Cryo-lift-out refers to the extraction of the material for lamella preparation from bulk HPF sample and subsequent transfer and attachment to a lift-out receiver grid, conventionally a half-moon shaped grid (Giannuzzi et al 2005). Two main types of micromanipulator devices are currently available for cryo-lift-out: sharp needles and a cryo-gripper (Klumpe et al 2022). Initially, trenches are milled around the area of interest leaving it connected to the bulk material on a single side.…”