Revisiting focused ion beam scanning electron microscopy

Abstract

The need for resolving complex dynamic spatial relationships between and within cells has driven the development of super-resolution microscopic techniques [1.Glancy B. Visualizing mitochondrial form and function within the cell.Trends Mol. Med. 2020; 26: 58-70Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 2.Biazik J. et al.The versatile electron microscope: an ultrastructural overview of autophagy.Methods. 2015; 75: 44-53Crossref PubMed Scopus (28) Google Scholar, 3.De Castro O. et al.Magnetic sector secondary ion mass spectrometry on FIB-SEM instruments for nanoscale chemical imaging.Anal. Chem. 2022; 94: 10754-10763Crossref PubMed Scopus (3) Google Scholar]. The explosion of computational and data storage capabilities has propelled the evolution of fully automated, high-resolution microscopes capable of rendering fine cellular ultrastructure in 3D with high fidelity. Focused ion beam scanning electron microscopy (FIB-SEM) is a technique with resolution <10 nm in all planes, making it ideal for exploring organelle–organelle interactions such as endoplasmic reticulum–mitochondrial contacts sites or resolving fine synaptic features [2.Biazik J. et al.The versatile electron microscope: an ultrastructural overview of autophagy.Methods. 2015; 75: 44-53Crossref PubMed Scopus (28) Google Scholar,4.Xu C.S. et al.Enhanced FIB-SEM systems for large-volume 3D imaging.eLife. 2017; 6e25916Crossref Scopus (174) Google Scholar]. FIB-SEM works by pairing a focused beam of ions to finely ablate the surface of heavily contrasted, resin-embedded samples with a scanning, low-voltage electron beam and backscatter electron detector for surface imaging [2.Biazik J. et al.The versatile electron microscope: an ultrastructural overview of autophagy.Methods. 2015; 75: 44-53Crossref PubMed Scopus (28) Google Scholar]. An array of software are available for 3D reconstruction of features of interest (Table 1) [5.Garza-Lopez E. et al.Protocols for generating surfaces and measuring 3D organelle morphology using amira.Cells. 2022; 11: 65Crossref Scopus (8) Google Scholar,6.Lam J. et al.A universal approach to analyzing transmission electron microscopy with ImageJ.Cells. 2021; 10: 2177Crossref PubMed Scopus (32) Google Scholar]. For more versatility, FIB-SEM can be paired with cryo-capabilities and secondary detectors (Figure 1) [7.Vidavsky N. et al.Cryo-FIB-SEM serial milling and block face imaging: large volume structural analysis of biological tissues preserved close to their native state.J. Struct. Biol. 2016; 196: 487-495Crossref PubMed Scopus (54) Google Scholar,8.Hayles M.F. De Winter D.A.M. An introduction to cryo-FIB-SEM cross-sectioning of frozen, hydrated Life Science samples.J. Microsc. 2021; 281: 138-156Crossref PubMed Scopus (20) Google Scholar].Table 1Examples of 3D reconstruction analysis softwareOpen-source or publicly availableCommercially availableMicroscopy Image BrowserThermo Scientific AmiraReconstructImarisIMODDragonfly Proilastik3D SlicerImageJ/FIJInapari Open table in a new tab Precise, fine sample removal along the z axis. Fine balance of ultra-resolution and volumetric sampling for 3D imaging. Amenable to pairing with a wide array of techniques that can yield localization, topography, and elemental composition with high spatial resolution. Easy access to image processing software. Fully automated. Adjustable, allowing for beam strength to be adjustable and multiple specimens in a small area to be surveyed to ensure efficiency. Cryo-capabilities may aid in fluorescence confocal imaging, the imaging of proteins and molecules in their native state, and avoiding potential damage and morphological changes which may occur with fixation and embedding of traditional FIB-SEM techniques. Unlike transmission electron microscopy (TEM), cannot resolve fine, nanostructural details, such as visualization of viral spike proteins or between intermembrane organellar connections [2.Biazik J. et al.The versatile electron microscope: an ultrastructural overview of autophagy.Methods. 2015; 75: 44-53Crossref PubMed Scopus (28) Google Scholar,9.Baena V. et al.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.Viruses. 2021; 13: 611Crossref PubMed Scopus (16) Google Scholar]. Longer acquisition time and smaller volume sampling capabilities than other volumetric EM techniques. Sample is consumed during image milling and acquisition. Needs large RAM and storage (~TB range) for efficient data transfer and processing for volumetric applications. Contamination may occur with gallium beam, affecting physical and electrical attributes. Learning curve for the analysis software, which varies across different software, may have separate user interfaces, require coding knowledge, or require manual segmentation. High-cost commitment (instrumentation, facility, maintenance, operating, and training costs) and expertise are required, inhibiting ancillary uses of FIB-SEM in many cases; furthermore, cryo-FIB-SEM requires further extensive specialized techniques and materials. We thank Trace A. Christensen, MBA, Mayo Microscopy and Cell Analysis Core, and Jian Shao, PhD, Central Microscopy Research Facility University of Iowa, for their valuable feedback on the manuscript. Funding by the UNCF/Bristol-Myers Squibb E.E. Just Faculty Fund, BWF Career Awards at the Scientific Interface Award, BWF Ad-hoc Award, NIH Small Research Pilot Subaward to 5R25HL106365-12 from the National Institutes of Health PRIDE Program, and DK020593, Vanderbilt Diabetes and Research Training Center for DRTC Alzheimer’s Disease Pilot & Feasibility Program. CZI Science Diversity Leadership grant number 2022- 253529 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation (to A.H.). NSF EES2112556, NSF EES1817282, NSF MCB1955975, and CZI Science Diversity Leadership grant number 2022-253614 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation (to S.A.D.). Image created using BioRender. All representative images are original. The authors have no conflicts of interest to declare.

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