Endocytosed nanogold fiducials for improved in-situ cryo–electron tomography tilt-series alignment

• Published in: Journal of structural biology 2021-03, Vol.213 (1), p.107698-107698, Article 107698
• Main Authors: Berger, Casper, Ravelli, Raimond B.G., López-Iglesias, Carmen, Peters, Peter J.
• Format: Article
• Language: English
• Subjects: Bovine serum albumin bsa-gold; Cryo-electron microscopy; Cryo-electron tomography; Cryo-focused-ion beam lamella; Nanogold fiducials; Tilt-series alignment
• ISSN: 1047-8477; 1095-8657
• DOI: 10.1016/j.jsb.2021.107698

[Display omitted] •Endocytosis of bovine serum albumin (BSA)-gold by mammalian cells.•Presence of BSA-gold in endosomal organelles in focused ion beam (FIB)-milled lamellae.•Improved alignment of cryo–electron tomography tilt-series recorded on FIB-lamellae with BSA-gold compared to fiducial-less patch-tracking. Cryo–electron tomography (CET) on cryo–focused ion beam (FIB)–milled lamellae is becoming a powerful technique for determining the structure of macromolecular complexes in their native cellular environment. Prior to tomogram reconstruction, CET tilt-series recorded on FIB lamellae need to be aligned. Traditionally, CET tilt-series alignment is performed with 5–20 nm gold fiducials, but it has thus far proven difficult to apply this to FIB lamellae of eukaryotic cells. In here, we describe a simple method to allow uptake of bovine serum albumin (BSA)-gold fiducials into mammalian cells via endocytosis, which can subsequently be used as fiducials for tilt-series alignment of cryo-FIB lamellae. We compare the alignment of tilt-series with BSA-gold fiducials to fiducial-less patch-tracking, and find better alignment results with BSA-gold. This technique can contribute to understand cells at a structural and ultrastructural level with both cryo- and room-temperature electron tomography. Furthermore, fluorescently labeled BSA-gold has the potential to be used as fiducials for correlative light and electron microscopy studies.