SIM was performed using a custom-built system (61) as modified for live organisms (62). In brief, SGO_0707-YFP cells were cultured for 4 hours at 37°C with the lipid dye FM 4-64FX (F34653, Thermo Fisher Scientific) at a final concentration of 15 ng/μl and then fixed in 1% paraformaldehyde supplemented with Hoechst 33342 (20 μg/ml) for 10 min at 4°C. Cells were then plated on a 25-mm glass coverslip #1.5 with TetraSpeck Microspheres 0.1 μm (T7279, Thermo Fisher Scientific), which were used as fiducials for postprocessing registration. The coverslip was then sealed with another coverslip using ProLong Diamond (P36965, Thermo Fisher Scientific). YFP was excited using the 488-nm laser line and detected using a green fluorescent protein (GFP) filter (BP 525/45). FM 4-64FX was excited using the 561-nm laser line and filtered with a far-red filter (LP 635). Hoechst 33342 was excited with the 405-nm laser line and detected with a 4′,6-diamidino-2-phenylindole filter (BP 480/40). Data acquisition and postprocessing were performed as described (62).

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