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

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Abstract

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 tiltseries 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 tiltseries alignment of cryo-FIB lamellae. We compare the alignment of tilt-series with BSA-gold fiducials to fiducialless 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.

Original languageEnglish
Article number107698
Number of pages7
JournalJournal of Structural Biology
Volume213
Issue number1
DOIs
Publication statusPublished - Mar 2021

Keywords

  • Bovine serum albumin bsa-gold
  • Cryo-electron microscopy
  • Cryo-electron tomography
  • Cryo-focused-ion beam lamella
  • Nanogold fiducials
  • Tilt-series alignment
  • GOLD NANOPARTICLES
  • ENDOSOMES
  • ELECTRON
  • ARCHITECTURE
  • ALBUMIN

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