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

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-...

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Autores principales: Parkhurst, James M., Crawshaw, Adam D., Siebert, C. Alistair, Dumoux, Maud, Owen, C. David, Nunes, Pedro, Waterman, David, Glen, Thomas, Stuart, David I., Naismith, James H., Evans, Gwyndaf
Formato: Online Artículo Texto
Lenguaje:English
Publicado: International Union of Crystallography 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10161776/
https://www.ncbi.nlm.nih.gov/pubmed/36952226
http://dx.doi.org/10.1107/S2052252523001902
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author Parkhurst, James M.
Crawshaw, Adam D.
Siebert, C. Alistair
Dumoux, Maud
Owen, C. David
Nunes, Pedro
Waterman, David
Glen, Thomas
Stuart, David I.
Naismith, James H.
Evans, Gwyndaf
author_facet Parkhurst, James M.
Crawshaw, Adam D.
Siebert, C. Alistair
Dumoux, Maud
Owen, C. David
Nunes, Pedro
Waterman, David
Glen, Thomas
Stuart, David I.
Naismith, James H.
Evans, Gwyndaf
author_sort Parkhurst, James M.
collection PubMed
description 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.
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spelling pubmed-101617762023-05-06 Investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae Parkhurst, James M. Crawshaw, Adam D. Siebert, C. Alistair Dumoux, Maud Owen, C. David Nunes, Pedro Waterman, David Glen, Thomas Stuart, David I. Naismith, James H. Evans, Gwyndaf IUCrJ Research Papers 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. International Union of Crystallography 2023-03-24 /pmc/articles/PMC10161776/ /pubmed/36952226 http://dx.doi.org/10.1107/S2052252523001902 Text en © James M. Parkhurst et al. 2023 https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.
spellingShingle Research Papers
Parkhurst, James M.
Crawshaw, Adam D.
Siebert, C. Alistair
Dumoux, Maud
Owen, C. David
Nunes, Pedro
Waterman, David
Glen, Thomas
Stuart, David I.
Naismith, James H.
Evans, Gwyndaf
Investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae
title Investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae
title_full Investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae
title_fullStr Investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae
title_full_unstemmed Investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae
title_short Investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae
title_sort investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae
topic Research Papers
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10161776/
https://www.ncbi.nlm.nih.gov/pubmed/36952226
http://dx.doi.org/10.1107/S2052252523001902
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