Widefield ti2

Images were taken on confocal laser scanning microscopes (Leica SP5, Leica SP8, Nikon A1Rsi+, Nikon Spinning Disk Confocal CSA-W1 SoRa), or on a widefield microscope (Nikon Widefield Ti2). Images were obtained with a 10× air, and 20×-, 40×-, or 63× oil immersion objectives (Leica: HCX PL APO 20×/0.7, HCX PL APO 63×/1.20W motCORR CS; Nikon 20× Plan Apo, Air, 0.8 NA, 1.000 DIC N2 VC, Nikon 40× Plan Flour, Oil, 1.3 NA, 200WD, DIC N2 H; Nikon 40× Plan Flour, Sil, 1.25 NA, 300WD, SR HP DIC N1 λS OFN25; Nikon 10× Plan Fluo Air, 0.3 NA, 15.200 WD, Ph 1 DL; Nikon 10× Plan Fluo Air, 0.45 NA, 4.000 WD, DIC N1 λ OFN25). Fast blue was imaged with a 405 nm, GFP with a 488 nm, and tdTom, mCherry, or RFP with a 561 nm laser (bandwidth, Leica, 425/70, 515/25, 590/70; Nikon, 447/60, 450/50, 525/50, 595/50; Nikon widefield, 435/33, 519/26, 595/31).

For microscopy, cells were seeded on glass bottom #1.5H µ-slides (IBIDI, Germany) or glass bottom #1.5H-N 96-well plates (Cellvis, USA). Imaging was performed on a Nikon Widefield Ti2 equipped with a sCMOS, PCO.edge camera and a live-cell incubator. Image acquisition was done in Flurobrite medium (GIBCO) supplemented with 2% FBS, 1:100 PenStrep, and 1x GlutaMax at 37 °C and 5% CO₂. The following light sources (LEDs) and emission filters were used for the different channels: CFP (cerulean): excitation 438/29, emission 473/24 nm; GFP (dClover2): excitation 475/28 nm, emission 520/26 nm; YFP (mClover3, dClover2): excitation 511/16 nm, emission 540/30 nm; RFP (mScarlet-I) excitation 555/28 nm, emission 642/80 nm. Objectives: 40x ApoFluor, NA 0.95, WD 250 µm; 20x Plan Apo, NA 0.8, WD 1 mm. Illumination time for CFP was usually 500 ms and 2 s for all other channels. To synchronize the circadian rhythms, cells were either washed twice with cold PBS for 2 min (Fig. 1f, Supplementary Figs. 3 and 7a) or treated with 1 µM dexamethasone for 30 min followed by washing with warm PBS (Figs. 2–4), or by exchange to pre-warmed imaging medium (Supplementary Figs. 5 and 7b, c). Imaging started 2 h after synchronization with a regular imaging interval of 1 h.