Coupled Broad Ion Beam–Scanning Electron Microscopy (BIB–SEM) for polishing and three dimensional (3D) serial section tomography (SST)

Gholinia, Ali; Curd, Matthew E.; Bousser, Étienne; Taylor, Kevin G.; Hosman, T.; Coyle, S. T.; Shearer, Michael H.; Hunt, John; Withers, Philip J.

doi:10.1016/j.ultramic.2020.112989

Cited by 23 publications

(24 citation statements)

References 29 publications

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“…Very large volumes can be interrogated by TEM-based techniques: the traditional approach of serialsection TEM requires manual collection of consecutive thin (~50 nm) sections of stained, resin-embedded specimens which are then individually imaged [30,31], while GridTape TEM uses a tape-reel to help automate section collection and imaging [32]. Newer plasma focused ion beam (pFIB) or broad ion beam (BIB) [33,34] milling and X-ray microscopy (XRM) [35] approaches are being developed, but to our knowledge have not been shown to resolve viral particles in resin-embedded cell samples. Combinations of these approaches may smartly couple sparse sampling of a large volume with high-resolution imaging of a smaller region-of-interest (ROI) [35,36], but for brevity we do not discuss this here.…”

Section: Volume Electron Microscopymentioning

confidence: 99%

FIB-SEM as a Volume Electron Microscopy Approach to Study Cellular Architectures in SARS-CoV-2 and Other Viral Infections: A Practical Primer for a Virologist

Baena

Conrad

Friday

et al. 2021

Viruses

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The visualization of cellular ultrastructure over a wide range of volumes is becoming possible by increasingly powerful techniques grouped under the rubric “volume electron microscopy” or volume EM (vEM). Focused ion beam scanning electron microscopy (FIB-SEM) occupies a “Goldilocks zone” in vEM: iterative and automated cycles of milling and imaging allow the interrogation of microns-thick specimens in 3-D at resolutions of tens of nanometers or less. This bestows on FIB-SEM the unique ability to aid the accurate and precise study of architectures of virus-cell interactions. Here we give the virologist or cell biologist a primer on FIB-SEM imaging in the context of vEM and discuss practical aspects of a room temperature FIB-SEM experiment. In an in vitro study of SARS-CoV-2 infection, we show that accurate quantitation of viral densities and surface curvatures enabled by FIB-SEM imaging reveals SARS-CoV-2 viruses preferentially located at areas of plasma membrane that have positive mean curvatures.

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Section: Volume Electron Microscopymentioning

confidence: 99%

FIB-SEM as a Volume Electron Microscopy Approach to Study Cellular Architectures in SARS-CoV-2 and Other Viral Infections: A Practical Primer for a Virologist

Baena

Conrad

Friday

et al. 2021

Viruses

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“…In this respect, 3D FIB-SEM is a very good compromise between resolution and volume size. Especially with the recent application of Xe plasma ion beam [18], broad Ar + beam [19], and Femtosecond laser [20], the analysing volumes can be increased largely.…”

Section: Fib-sem Tomographymentioning

confidence: 99%

Accurate post-mortem alignment for Focused Ion Beam and Scanning Electron Microscopy (FIB-SEM) tomography

2021

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Drifts in the three directions (X, Y, Z) during the FIB-SEM slice-and-view tomography is an important issue in 3D-FIB experiments which may induce significant inaccuracies in the subsequent volume reconstruction and further quantification of morphological volume parameters of the sample microstructure. Cross-correlation is frequently applied directly to the cross-section image series for aligning FIB sliced images. This solution is hazardous and can be flawed as it has been easily demonstrated by a dedicated test experiment. As a result, a novel aligning procedure based on the quantification of the topography of the sample surface has been developed. This new approach will be compared to the common cross-correlation methods, as well as another approach consisting in using an artificial reference marker fabricated during the FIB procedure. All these methods will then be discussed in terms of accuracy and liability.

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“…The BIB milling process can be performed on a few millimeters of material and adjusted to the required depth 26 ; therefore, it is suitable for preprocessing cross-sections before SEM. In addition, BIB milling produces cross-sections with very low damage compared to FIB milling 27 , 28 . The setting of specimens on the BIB milling system is very easy, and the cross-section to be processed is positioned protruding from the shield plate several tens of micrometers 28 .…”

Section: Introductionmentioning

confidence: 99%

“…In addition, BIB milling produces cross-sections with very low damage compared to FIB milling 27 , 28 . The setting of specimens on the BIB milling system is very easy, and the cross-section to be processed is positioned protruding from the shield plate several tens of micrometers 28 . The protruded portion is then milled by BIB irradiation.…”

Section: Introductionmentioning

confidence: 99%

A combination of scanning electron microscopy and broad argon ion beam milling provides intact structure of secondary tissues in woody plants

Hatano

Nakaba

Horikawa

et al. 2022

Sci Rep

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The secondary tissues of woody plants consist of fragile cells and rigid cell walls. However, the structures are easily damaged during mechanical cross-sectioning for electron microscopy analysis. Broad argon ion beam (BIB) milling is commonly employed for scanning electron microscopy (SEM) of hard materials to generate a large and distortion-free cross-section. However, BIB milling has rarely been used in plant science. In the present study, SEM combined with BIB milling was validated as an accurate tool for structural observation of secondary woody tissues of two samples, living pine (Pinus densiflora) and high-density oak wood (Quercus phillyraeoides), and compared with classical microtome cross-sectioning. The BIB milling method does not require epoxy resin embedding because of prior chemical fixation and critical point drying of the sample, thus producing a three-dimensional image. The results showed that xylem structures were well-preserved in their natural state in the BIB-milled cross-section compared with the microtome cross-section. The observations using SEM combined with BIB milling were useful for wide-area imaging of both hard and soft plant tissues, which are difficult to observe with transmitted electron microscopy because it is difficult to obtain sections of such tissues, particularly those of fragile reaction woods.

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Coupled Broad Ion Beam–Scanning Electron Microscopy (BIB–SEM) for polishing and three dimensional (3D) serial section tomography (SST)

Cited by 23 publications

References 29 publications

FIB-SEM as a Volume Electron Microscopy Approach to Study Cellular Architectures in SARS-CoV-2 and Other Viral Infections: A Practical Primer for a Virologist

FIB-SEM as a Volume Electron Microscopy Approach to Study Cellular Architectures in SARS-CoV-2 and Other Viral Infections: A Practical Primer for a Virologist

Accurate post-mortem alignment for Focused Ion Beam and Scanning Electron Microscopy (FIB-SEM) tomography

A combination of scanning electron microscopy and broad argon ion beam milling provides intact structure of secondary tissues in woody plants

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