Thinner is not always better: Optimising cryo lamellae for subtomogram averaging

  • Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyse biomolecules in situ by subtomogram averaging (STA). Specimen thickness is a key factor affecting cryo-ET data quality. Cells that are too thick for transmission imaging can be thinned by cryo-focused-ion-beam (cryo-FIB) milling. However, optimal specimen thickness for cryo-ET on lamellae has not been systematically investigated. Furthermore, the ions used to ablate material can cause damage in the lamellae, thereby reducing STA resolution. Here, we systematically benchmark the resolution depending on lamella thickness and the depth of the particles within the sample. Up to ca. 180 nm, lamella thickness does not negatively impact resolution. This shows that there is no need to generate very thin lamellae and thickness can be chosen such that it captures major cellular features. Furthermore, we show that gallium-ion-induced damage extends to depths of up to 30 nm from either lamella surface.

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Author:Maarten W. TuijtelORCiD, Jan Philipp KreysingORCiD, Sonja WelschORCiDGND, Gerhard HummerORCiD, Martin BeckORCiDGND, Beata TuroňováORCiD
Parent Title (English):bioRxiv
Document Type:Preprint
Date of Publication (online):2023/08/02
Date of first Publication:2023/08/02
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2023/08/09
Page Number:27
Biochemie, Chemie und Pharmazie / Biochemie und Chemie
Angeschlossene und kooperierende Institutionen / MPI für Biophysik
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Licence (German):License LogoCreative Commons - CC BY-NC-ND - Namensnennung - Nicht kommerziell - Keine Bearbeitungen 4.0 International