Ix83 fluorescence microscope

All confocal images were acquired on an Olympus IX83 fluorescence microscope equipped with spinning-disk confocal scanner (Yokogawa CSU-W1), a 60× NA 1.49 oil Apochromat objective, an sCMOS camera (Prime 95B), 405/488/561/640 nm lasers (OBIS), and a PIEZO stage (ASI) with stage incubator (Tokai Hit). For live-cell imaging, cells were maintained at 37°C and 5% CO₂ in a humidified chamber. Cells for confocal imaging were plated into 8-well chambered coverglass. All supplementary movies were taken on the spinning-disk confocal microscope. Z stack images were processed by the projection of maximum intensity to generate the movies. Real-time imaging was recorded for 8 h with 4-min intervals to characterize transcriptional bursting of rDNA. Other imaging conditions were described in the legend of corresponding figures.

For static imaging, worms were immobilized using 1 mM levamisole solution and placed on 4% agarose pads, and then imaged using an Olympus IX83 fluorescence microscope equipped with a spinning-disk confocal scanner (Yokogawa CSU-W1), an sCMOS camera (Prime 95B), and a 60x oil Apochromat objective (NA: 1.49). Z stack images were processed by the projection of maximum intensity except for Figs 2A and S5A.
Time-lapse imaging was performed as previously described with some modifications [60 (link)]. Briefly, 2 μl of 1 mM levamisole solution was added into the center of the glass bottom of a microwell dish, then about 20 worms were transferred into the drop of levamisole solution. Next, a 4% agarose pad was gently added onto the animals. All time-lapse movies were taken using the spinning-disk confocal microscope, and Z stack images were processed by projection of maximum intensity except for S10 and S11 Figs, in which single-layer images were shown.
For Stimulated Emission Depletion Microscopy (STED) imaging, worms were immobilized using 1 mM levamisole solution and placed on 4% agarose pads. A Leica TCS SP8 STED fluorescence microscope equipped with 592/660/775 nm lasers, and a HC PL APO CS2 100×/1.40 oil objective was used for imaging. Z stack images were processed by the projection of maximum intensity.