Fig 1.
GeminiSEM 300 with a 3View2XP setup.
(A) 3View2XP stage setup. Insets show the retractable nozzle for the Focal Charge Compensation system (FCC) and the sample block at higher magnification. (B) Picture of the retractable ultramicrotome system in the 3View2XP stage.
Fig 2.
Key steps in the workflow for SBF-SEM of adherent tissue culture cells.
(A) Cell pellet workflow and (B) en face with CLEM workflow. Step numbers on the Fig correspond to step numbers in the step-by-step protocol in the S1 File and on protocols.io (doi.org/10.17504/protocols.io.e6nvwdz5zlmk/v2).
Fig 3.
SBF-SEM imaging concept and key imaging parameters.
(A) Schematic of the SBF-SEM sample and imaging setup. FCC (Focal Charge Compensation). (B) Table of the key imaging parameters and their definitions. (C) Table describing how increasing (up arrow) or decreasing (down arrow) key parameters impacts properties of the resulting image, including signal-to-noise, charging and cutting artifacts during imaging.
Fig 4.
Effect of different imaging parameters on image quality.
(A) Table showing the imaging parameters tested. (B) A representative slice from the block face. Insets highlight the ultrastructure of different host cell organelles ‐ mitochondria, magenta; ER, yellow. (C) Slices 1–6 from Condition 1. Cyan arrowheads highlight charging and cutting artifacts. (D) Slices 1–6 from Condition 2. Insets show the overlay of mitochondria segmented in magenta (preceding slice) and green. No change in mitochondria morphology is observed from slice 3 to slice 4 and slice 5 to slice 6, highlighted by a complete overlap of the magenta and green annotations. This indicates a cutting defect between slices 3 and 4, and between slices 5 and 6. (E-F) Slices 1–6 from Condition 3 (E) and Condition 4 (F). Magenta and green annotations do not overlap from slice 1 to slice 6 in both these conditions. Continuous changes are expected from one slice to the next, showing that cutting defects do not occur. Also see Movies 1–4. Scale bars: 1 μm.
Table 1.
Table of advantages and disadvantages of the cell pellet versus en face embedding of adherent tissue culture cells.
Fig 5.
Representative imaging results for cell pellet and en face embedded samples.
(A, D) Tables showing the imaging parameters used to achieve good signal-to-noise and avoid charging artifacts in the cell pellet sample (A) or en face sample (D). (B, E) Representative slices from the cell pellet (B) or en face (E) samples using the imaging parameters listed in (A) and (D), respectively. Insets highlight the ultrastructure of different host cell organelles ‐ nucleus, blue arrowhead; mitochondria, magenta arrowhead; ER, yellow arrowhead. (C, F) Slices 1–4 of the cell pellet (C) or en face (F) samples. Also see Movies 5–6. Scale bars 1 μm.