Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Abstract

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

A sex-specific switch in a single glial cell patterns the apical extracellular matrix

“…Samples were sectioned using an ATS knife (Diatome, Switzerland) mounted on a Leica UC7 ultramicrotome (Leica, Austria). Approximately 100 to 300 sections were obtained per animal and transferred to a 2x4 cm silicon wafer (Burel et al, 2018; Franke and Kolotuev, 2021). Wafers were dried at ambient temperature by evaporation and subsequently incubated in a 60°C oven for further fixation as previously described (Burel et al, 2018; Franke and Kolotuev, 2021; Santella et al, 2022).…”

“…Approximately 100 to 300 sections were obtained per animal and transferred to a 2x4 cm silicon wafer (Burel et al, 2018; Franke and Kolotuev, 2021). Wafers were dried at ambient temperature by evaporation and subsequently incubated in a 60°C oven for further fixation as previously described (Burel et al, 2018; Franke and Kolotuev, 2021; Santella et al, 2022). Wafers were analyzed with a Helios SEM microscope (Thermo Fisher Scientific) at 2keV landing energy and 0.8 nA beam current at 2 mm distance using a Mirror Detector (MD-BSA) (Burel et al, 2018; Franke and Kolotuev, 2021).…”

“…Wafers were dried at ambient temperature by evaporation and subsequently incubated in a 60°C oven for further fixation as previously described (Burel et al, 2018; Franke and Kolotuev, 2021; Santella et al, 2022). Wafers were analyzed with a Helios SEM microscope (Thermo Fisher Scientific) at 2keV landing energy and 0.8 nA beam current at 2 mm distance using a Mirror Detector (MD-BSA) (Burel et al, 2018; Franke and Kolotuev, 2021). Images were collected manually or automatically with 4-6 µs dwell time using Maps 3.11 software (Thermo Fisher Scientific) (Burel et al, 2018; Franke and Kolotuev, 2021; Santella et al, 2022).…”

“…Wafers were analyzed with a Helios SEM microscope (Thermo Fisher Scientific) at 2keV landing energy and 0.8 nA beam current at 2 mm distance using a Mirror Detector (MD-BSA) (Burel et al, 2018; Franke and Kolotuev, 2021). Images were collected manually or automatically with 4-6 µs dwell time using Maps 3.11 software (Thermo Fisher Scientific) (Burel et al, 2018; Franke and Kolotuev, 2021; Santella et al, 2022). Images were collected at 3 mm/s (1024x886) or 5 mm/s (6084x2044) to generate 20 nm and 5 nm resolution images, respectively.…”

“…Images were collected at 3 mm/s (1024x886) or 5 mm/s (6084x2044) to generate 20 nm and 5 nm resolution images, respectively. To cover a larger area of the sample, several images were collected at a given resolution and stitched together using Maps 3.17 software (Thermo Fisher Scientific) (Burel et al, 2018; Franke and Kolotuev, 2021). Finally, serial sections were aligned using the IMOD program (University of Colorado) (Kremer et al, 1996).…”