Cargando…
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-...
Autores principales: | , , , , , , , , , , |
---|---|
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 |
_version_ | 1785037562560118784 |
---|---|
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. |
format | Online Article Text |
id | pubmed-10161776 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | International Union of Crystallography |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT parkhurstjamesm investigationofthemillingcharacteristicsofdifferentfocusedionbeamsourcesassessedbythreedimensionalelectrondiffractionfromcrystallamellae AT crawshawadamd investigationofthemillingcharacteristicsofdifferentfocusedionbeamsourcesassessedbythreedimensionalelectrondiffractionfromcrystallamellae AT siebertcalistair investigationofthemillingcharacteristicsofdifferentfocusedionbeamsourcesassessedbythreedimensionalelectrondiffractionfromcrystallamellae AT dumouxmaud investigationofthemillingcharacteristicsofdifferentfocusedionbeamsourcesassessedbythreedimensionalelectrondiffractionfromcrystallamellae AT owencdavid investigationofthemillingcharacteristicsofdifferentfocusedionbeamsourcesassessedbythreedimensionalelectrondiffractionfromcrystallamellae AT nunespedro investigationofthemillingcharacteristicsofdifferentfocusedionbeamsourcesassessedbythreedimensionalelectrondiffractionfromcrystallamellae AT watermandavid investigationofthemillingcharacteristicsofdifferentfocusedionbeamsourcesassessedbythreedimensionalelectrondiffractionfromcrystallamellae AT glenthomas investigationofthemillingcharacteristicsofdifferentfocusedionbeamsourcesassessedbythreedimensionalelectrondiffractionfromcrystallamellae AT stuartdavidi investigationofthemillingcharacteristicsofdifferentfocusedionbeamsourcesassessedbythreedimensionalelectrondiffractionfromcrystallamellae AT naismithjamesh investigationofthemillingcharacteristicsofdifferentfocusedionbeamsourcesassessedbythreedimensionalelectrondiffractionfromcrystallamellae AT evansgwyndaf investigationofthemillingcharacteristicsofdifferentfocusedionbeamsourcesassessedbythreedimensionalelectrondiffractionfromcrystallamellae |