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.
- Bovine serum albumin bsa-gold
- Cryo-electron microscopy
- Cryo-electron tomography
- Cryo-focused-ion beam lamella
- Nanogold fiducials
- Tilt-series alignment
- GOLD NANOPARTICLES