Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies

Abstract

Spatially-controlled cell adhesion on electron microscopy (EM) supports remains a bottleneck in specimen preparation for cellular cryo-electron tomography. Here, we describe contactless and maskfree photo-micropatterning of EM grids for site-specific deposition of extracellular matrix-related proteins. We attained refined cell positioning for micromachining by cryo-focused ion beam milling.Complex patterns generated predictable intracellular organization, allowing direct correlation between cell architecture and in-cell 3D-structural characterization of the underlying molecular machinery. MainIn parallel to the ongoing resolution revolution in cryo-electron microscopy for macromolecular structure determination 1 , cryo-electron tomography (ET) has matured to reveal the molecular sociology in situ sensu stricto 2-4 . Yet, cryo-ET of adherent mammalian cells can only be directly performed on their thin peripheries (< 300 nm). To reveal function-related structural variation, conformational states and function-related assemblies of macromolecules at the cell interior, thinning by advanced cryo-focused ion beam (FIB) has proved an optimal, artifact-free preparation method 2,5,6 .Specimen preparation for cellular cryo-ET, whether performed directly on thin cellular peripheries or following cryo-FIB micromachining, involves seeding of adherent cells directly on EM grids made of a biocompatible metal, e.g. gold. Standard EM grids are 3 mm diameter metal meshes overlaid with a delicate perforated thin film. Cells are typically allowed to spread, subjected to genetic or molecular perturbation to represent different physiological settings to be examined in molecular detail, that are then arrested by vitrification 7 . For cells to be thinned by cryo-FIB, they must be positioned roughly at the center of an individual grid square (Fig. 1a) 6 , within a few squares away from the grid center. Whilst the first requirement is necessary to allow access to cellular material for ablation by the FIB, the latter is posed by the subsequent requirement of stage tilt in the transmission electron microscope (TEM) for

The promise and the challenges of cryo‐electron tomography

“…For cellular structural studies, cells are grown on grids which must be non‐cytotoxic. To have some control over where cells grow, avoiding, for example, areas obstructed by grid bars, micropatterning methods can be used [19] (Fig. 2).…”

3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy

“…Since EM grid micropatterning shows great promise for further simplified and more efficient cell seeding (Engel et al, 2019;Toro-nahuelpan et al, 2019) we designed grid holders that are also compatible with micropatterning approaches using a standard, noncommercial setup. Specifically, high-throughput patterning on EM grids requires grid holders to provide a low, stable and even support of grids for controlled ablation of the Polyethylene glycol(PEG)-brush layer, which is applied to EM grids to render them nonadherent.…”

“…Hence, a significant level of user experience and a steady hand are required to achieve sufficient throughput, an appropriate cell density and to ensure minimal damage to the grids during specimen preparation. A notable improvement for controllable cell seeding conditions on grids was recently introduced by EM grid compatible substrate micropatterning approaches (Engel et al, 2019;Toro-nahuelpan et al, 2019). Here, only defined areas on grids are made adherent or are specifically functionalized with extracellular matrix (ECM) proteins, allowing one to precisely control the positioning, shape, and size of adherent cells.…”

Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies

“…The proof of principle and broader applications of this protocol in the context of biological studies by means of state-of-the-art cryo-electron microscopy pipeline (i.e., sample vitrification, cryo-correlative light and electron microscopy, cryo-FIB milling and cryo-electron tomography) are discussed in detail in the associated manuscript 10 .…”