TITLE: FIB SEM protocol for anaerobic ciliates to visualize their prokaryotic endosymbionts and association with MROs
AUTHORS: Johana Rotterova, Ivan Čepička

[STEPS]
?. [Fixation of the cells]
Individually pick several hundreds of cells with a micropipette from 2 ml of a well-grown culture of the targeted ciliate strain. Subsequently, follow fixation protocol by Rotterová et al. (2018) - see steps briefly described below or see full protocol "Transmission electron microscopy protocol for anaerobic ciliates" listed at protocols.io (dx.doi.org/10.17504/protocols.io.85uhy6w).
?. [Fixation of the cells]
Fix picked cells with 2.5% (v/v) glutaraldehyde (Polysciences) and subsequently centrifuge at 800×g for 5 min at 4 °C.
?. [Fixation of the cells]
Carefully replace supernatant with 1 ml of 2.5% (v/v) glutaraldehyde in 0.2M SCB (Sodium Cacodylate buffer, pH 7.2) and incubate 1 hour on ice.
?. [Fixation of the cells]
Then, rinse the pellet carefully with SCB three times per 15 minutes.
?. [FIB SEM sample preparation]
Separate the resin layer with fixed cells and wash thoroughly.
?. [FIB SEM sample preparation]
Select a region containing enough vertically oriented cells within the slice of resin using dissecting microscope with transmitted illumination.
?. [FIB SEM sample preparation]
Subsequently, process the sample following a protocol adapted from Carpenter et al. (2013), Loyola Machado et al. (2017), and Karnkowska et al. (2019) and optimized for larger cells with archaeal endosymbionts (7, 8, 9).
?. [FIB SEM sample preparation]
Cut out square around the region of target cells, glue to the top of blank resin block. Trim and cut out the stub, leaving the height of the block smaller than 5 mm.
?. [3D FIB SEM imaging and acquisition]
Processed the sample using scanning electron microscope with focused ion beam milling (e.g., FEI Helios NanoLab 660 G3 UC). Orient the block using low magnification and secondary electron imaging (30 kV, 0.8 nA).
?. [Fixation of the cells]
Postfix the cells with 1% OsO4 in distilled water for 1 hour on ice.
?. [Fixation of the cells]
Rinse the cells with distilled water, dehydrate them in a graded ethanol series from 30% to absolute ethanol, and infiltrate them with ethanol-acetone mixture, 100% acetone, and acetone-resin mixture.
?. [Fixation of the cells]
Embed cells in absolute EPON resin (Poly/Bed 812, Polysciences) and let the cells get polymerized at 70 ◦C for 48 hours.
?. [Fixation of the cells]
Cut serial ultrathin sections on an Ultracut E ultramicrotome (Reichert) using a diamond knife and stain with lead citrate and uranyl acetate (2–3%).
?. [3D FIB SEM imaging and acquisition]
Apply a protective layer of carbon onto the surface, above the region of interest.
?. [3D FIB SEM imaging and acquisition]
Remove a narrow band of resin from the front of the target region using an ion beam current of 13 - 27 nA at 30 kV.
?. [3D FIB SEM imaging and acquisition]
Mill the target region of the block using a current of 700 pA until no artefacts are present.
?. [3D FIB SEM imaging and acquisition]
Select optimal parameters and take serial images of the targeted cell by automatic slicing (30 kV, 0.79 nA) and imaging (2 kV, 0.2 nA) of backscattered electrons.
?. [3D FIB SEM imaging and acquisition]
Select a region (10 x 10 µm) with sufficient amount of mitochondrion related organelles (MROs) and symbionts for detailed serial imaging with the highest resolution.
?. [FIB SEM sample preparation]
To ensure imaging the correct side, glue the block to an aluminium stub and coat it with a thin layer of platinum with high vacuum sputter coater (e.g., Leica EM ACE600).