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Investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae

DOI: 10.1107/S2052252523001902 DOI Help

Authors: James M. Parkhurst (Rosalind Franklin Insititute; Diamond Light Source) , Adam D. Crawshaw (Diamond Light Source) , C. Alistair Siebert (Diamond Light Source) , Maud Dumoux (Rosalind Franklin Insititute) , C. David Owen (Diamond Light Source; Research Complex at Harwell) , Pedro Nunes (Diamond Light Source) , David Waterman (Research Complex at Harwell; CCP4) , Thomas Glen (Rosalind Franklin Institute) , David I. Stuart (Diamond Light Source; University of Oxford) , James H. Naismith (Rosalind Franklin Insititute; University of Oxford) , Gwyndaf Evans (Rosalind Franklin Insititute; Diamond Light Source)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Iucrj , VOL 10

State: Published (Approved)
Published: May 2023

Open Access Open Access

Abstract: Three-dimensional electron diffraction (3DED) from nanocrystals of biological macromolecules requires the use of very small crystals. These are typically less than 300 nm-thick in the direction of the electron beam due to the strong interaction between electrons and matter. In recent years, focused-ion-beam (FIB) milling has been used in the preparation of thin samples for 3DED. These instruments typically use a gallium liquid metal ion source. Inductively coupled plasma (ICP) sources in principle offer faster milling rates. Little work has been done to quantify the damage these sources cause to delicate biological samples at cryogenic temperatures. Here, an analysis of the effect that milling with plasma FIB (pFIB) instrumentation has on lysozyme crystals is presented. This work evaluates both argon and xenon plasmas and compares them with crystals milled with a gallium source. A milling protocol was employed that utilizes an overtilt to produce wedge-shaped lamellae with a shallow thickness gradient which yielded very thin crystalline samples. 3DED data were then acquired and standard data-processing statistics were employed to assess the quality of the diffraction data. An upper bound to the depth of the pFIB-milling damage layer of between 42.5 and 50 nm is reported, corresponding to half the thickness of the thinnest lamellae that resulted in usable diffraction data. A lower bound of between 32.5 and 40 nm is also reported, based on a literature survey of the minimum amount of diffracting material required for 3DED.

Journal Keywords: pFIB milling; crystal lamellae; 3DED; beam damage; 3D electron diffraction

Subject Areas: Technique Development, Biology and Bio-materials

Diamond Offline Facilities: Electron Bio-Imaging Centre (eBIC)
Instruments: Krios I-Titan Krios I at Diamond

Added On: 28/03/2023 22:09


Discipline Tags:

Technique Development - Life Sciences & Biotech Life Sciences & Biotech

Technical Tags:

Imaging Tomography Cryo Electron Tomography (Cryo ET)