Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy

Abstract

The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy.

Actin Organization in Cells Responding to a Perforated Surface, Revealed by Live Imaging and Cryo-Electron Tomography

“…The coordinates of actin rings were determined using MAPS software (Arnold et al, 2016). The autogrids were then loaded into a shuttle and inserted into a cooled stage in the chamber of an FEI DualBeam (FIB/SEM) system (Quanta 3D FEG, FEI Company).…”

The viral replication organelles within cells studied by electron microscopy

“…With the absence of compression and especially crevasses, the sample is better suited for TEM tomography than a CEMOVIS section. However, care needs to be taken to thin the area of interest since a big part of the cell is removed by the FIB (Arnold et al, 2016). A current drawback of this approach is the cost of the equipment.…”

Site-Specific Preparation of Intact Solid–Liquid Interfaces by Label-FreeIn SituLocalization and Cryo-Focused Ion Beam Lift-Out

“…In addition to the inherent limited resolution of the light microscope used, which is worsened by thick or cryogenic specimens (Sartori et al, 2007;Plitzko et al, 2009;Mahamid et al, 2015), this correlation process itself introduces additional uncertainty to the expected positions of the structures (Sartori et al, 2007;Schellenberger et al, 2014). The result is an uncertainty in the final colocalized position of a structure typically on the order of a micron (Sartori et al, 2007;Agronskaia et al, 2008;van Driel et al, 2009), though with added fiducial markers and a more involved correlation process this can now be reduced by an order of magnitude (Kukulski et al, 2011;Schellenberger et al, 2014;Schorb & Briggs, 2014;Arnold et al, 2016). In addition, the added handling and transfer steps introduced by such a two-instrument method increase the likelihood of compromising the sample integrity through contamination or structural damage, especially at cryogenic temperatures (Agronskaia et al, 2008;Faas et al, 2013).…”