Cryo-plasma FIB/SEM volume imaging of biological specimens

Dumoux, Maud; Glen, Thomas; Ho, Elaine M. L.; Perdigão, Luı́s M. A.; Klumpe, Sven; Yee, Neville B.-y.; Farmer, David A.; Smith, Julie; Lai, Pui Y A; Bowles, William; Kelley, R. J.; Plitzko, Jürgen M.; Wu, Liang; Basham, Mark; Clare, Daniel K.; Siebert, Christopher; Darrow, Michele C.; Naismith, J.H.; Grange, Michael

doi:10.1101/2022.09.21.508877

Cited by 4 publications

(16 citation statements)

References 46 publications

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“…Charging artefacts often appear when insulating materials interact with the electron beam (2,4) . This effect is normally minimized by adjusting beam energy or scanning parameters, and its severity depends on the target substance being imaged.…”

Section: Charge Artifact Mitigationmentioning

confidence: 99%

“…In our recent cryogenic pFIB/SEM study (2) , calibration was performed using images of polystyrene beads of 1 m diameter (Abvigen, USA). The images show the same field-of-view at 1.12, 2.25, and 4.5 nm pixel size.…”

Section: Calibrationmentioning

confidence: 99%

“…Cryogenic serial pFIB/SEM is an emerging microscopy technique that is used to visualise 3D structures of biological features at mesoscale resolutions (2) . This technique requires common post processing of data such as alignment and charge mitigation to enable robust segmentation and analysis.…”

Section: Impact Statementmentioning

confidence: 99%

“…With the development of cryogenically stable stages and reduced rates of ice formation it is possible to image cryogenically prepared samples providing structural information of samples in near native states. Cryogenic serial FIB/SEM has been done using modified room temperature plasma ion beam milling microscopes which were designed for physical science applications and more recently has been further enabled through the development of fit-for-purpose commercial options, some of which offer multiple plasma generation gases (2,(8)(9)(10)(11)(12)(13) .…”

Section: Introductionmentioning

confidence: 99%

“…Example data used throughout this manuscript are available on EMPIAR and their sample preparation and FIB/SEM data collection has been described in Dumoux et al (2) .…”

Section: Introductionmentioning

confidence: 99%

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Okapi-EM – a napari plugin for processing and analysing cryogenic serial FIB/SEM images

Perdigão

Cheng

et al. 2022

Preprint

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An emergent volume electron microscopy (vEM) technique called cryogenic serial plasma focused ion beam milling scanning electron microscopy (pFIB/SEM) can decipher complex biological structures by building a three-dimensional picture of biological samples at mesoscale resolution. This is achieved by collecting consecutive SEM images after successive rounds of FIB milling that expose a new surface after each milling step. Due to instrumental limitations, some image processing is necessary before 3D visualisation and analysis of the data is possible. SEM images are affected by noise, drift, and charging effects, that can make precise 3D reconstruction of biological features difficult. This paper presents Okapi-EM, an open-source Napari plugin(1) developed to process and analyse cryogenic serial FIB/SEM images. Okapi-EM enables automated image registration of slices, evaluation of image quality metrics specific to FIB-SEM imaging, and mitigation of charging artefacts. Implementation of Okapi-EM within the Napari framework ensures that the tools are both user- and developer-friendly, through provision of a graphical user interface and access to Python programming. Napari also hosts a variety of other image processing plugins so Okapi-EM tools can be integrated into and combined with other workflows. Okapi-EM can be downloaded freely at https://github.com/rosalindfranklininstitute/okapi-em, or installed from Python package index (PyPI).

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Section: Charge Artifact Mitigationmentioning

confidence: 99%

Section: Calibrationmentioning

confidence: 99%

Section: Impact Statementmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Example data used throughout this manuscript are available on EMPIAR and their sample preparation and FIB/SEM data collection has been described in Dumoux et al (2) .…”

Section: Introductionmentioning

confidence: 99%

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Okapi-EM – a napari plugin for processing and analysing cryogenic serial FIB/SEM images

Perdigão

Cheng

et al. 2022

Preprint

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Okapi-EM: A napari plugin for processing and analyzing cryogenic serial focused ion beam/scanning electron microscopy images

Perdigão¹,

Ho²,

Cheng³

et al. 2023

Biol. Imaging

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An emergent volume electron microscopy (vEM) technique called cryogenic serial plasma focused ion beam milling scanning electron microscopy (pFIB/SEM) can decipher complex biological structures by building a three-dimensional picture of biological samples at mesoscale resolution. This is achieved by collecting consecutive SEM images after successive rounds of FIB milling that expose a new surface after each milling step. Due to instrumental limitations, some image processing is necessary before 3D visualisation and analysis of the data is possible. SEM images are affected by noise, drift, and charging effects, that can make precise 3D reconstruction of biological features difficult. This paper presents Okapi-EM, an open-source napari plugin (1) developed to process and analyse cryogenic serial pFIB/SEM images. Okapi-EM enables automated image registration of slices, evaluation of image quality metrics specific to pFIB-SEM imaging, and mitigation of charging artifacts. Implementation of Okapi-EM within the napari framework ensures that the tools are both user-and developer-friendly, through provision of a graphical user interface and access to Python programming. Impact statementCryogenic serial pFIB/SEM is an emerging microscopy technique that is used to visualise 3D structures of biological features at mesoscale resolutions (2) . This technique requires common post processing of

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Plasma FIB milling for the determination of structures in situ

et al. 2023

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Structural biology studies inside cells and tissues require methods to thin vitrified specimens to electron transparency. Until now, focused ion beams based on gallium have been used. However, ion implantation, changes to surface chemistry and an inability to access high currents limit gallium application. Here, we show that plasma-coupled ion sources can produce cryogenic lamellae of vitrified human cells in a robust and automated manner, with quality sufficient for pseudo-atomic structure determination. Lamellae were produced in a prototype microscope equipped for long cryogenic run times (> 1 week) and with multi-specimen support fully compatible with modern-day transmission electron microscopes. We demonstrate that plasma ion sources can be used for structural biology within cells, determining a structure in situ to 4.9 Å, and characterise the resolution dependence on particle distance from the lamella edge. We describe a workflow upon which different plasmas can be examined to further streamline lamella fabrication.

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Cryo-plasma FIB/SEM volume imaging of biological specimens

Cited by 4 publications

References 46 publications

Okapi-EM – a napari plugin for processing and analysing cryogenic serial FIB/SEM images

Okapi-EM – a napari plugin for processing and analysing cryogenic serial FIB/SEM images

Okapi-EM: A napari plugin for processing and analyzing cryogenic serial focused ion beam/scanning electron microscopy images

Plasma FIB milling for the determination of structures in situ

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