S plan fluor elwd 40

14-bit Images were obtained with an inverted fluorescence microscope (Nikon Eclipse Ti2-E) using a Nikon Plan Fluor 10X or a Nikon S Plan Fluor ELWD 40× objective, a pixel resolution of 0.7373 × 0.7373 µm² or 0.1834 × 0.1834 µm², accordingly, and a Nikon Ds-Qi2 Camera. Excitation of Hoescht was carried out with a Lumencor SOLA II light source and a DAPI filterset.

The plasmid encoding ^ΔNSic1-SULI, ^ΔdbClb2-SULI or Clb2(Δde)^N-SULI-Clb2(Δde)^C was transformed into BY4742 using a standard Li-Ac method. Above clones or clones containing genome-integrated ADE2-SULI or ADE2-mCherry expression cassette were picked and cultured until OD₆₀₀ reached approximately 0.6 under dark conditions. The cells were serially diluted (1:10; first spot was ~2.5 × 10⁴ cells) and cultured in solid medium under light or dark conditions for 3 days. The plates were imaged using a Tanon-5200.
For analysis of the detailed distribution of cell stages, the overnight-cultured cells under light or dark conditions were collected and fixed by incubation with prechilled 70% EtOH at 4 °C overnight. The cells were washed with 50 mM Na citrate buffer twice and resuspended with 50 mM Na citrate (containing 0.1 mg/ml RNase A). After 2 hours at 37 °C, an equal volume of 50 mM Na citrate (containing 8 µg/ml Propidium Iodide) were added and incubated for another 4 hours at 37 °C. Images of cell cycle stages were acquired using an Eclipse Ti inverted microscope system (Nikon) equipped with an S Plan Fluor ELWD ×40, 0.95 numerical aperture (NA) objective, and a digital sight camera.

The WARP setup was integrated into an inverted microscope (Nikon Eclipse Ti). A schematic of the setup and light path is shown in Supplementary Note 10. Collimated light from two identical red LEDs (625 nm centre wavelength, Thorlabs M625L3) was combined onto the same illumination path using a 50–50 beamsplitter. Narrowband filters centred at 632.8 nm with a FHWM of 1 nm (Thorlabs FL632.8-1) were used to select the desired measurement wavelengths from the LED spectra. A tilt of 15° was introduced to the filter at LED 2 to select 628 nm. The LED wavelengths were measured either with a fibre spectrometer, or by matching interference fringes to those produced under illumination with a monochromator coupled to a halogen light source. An achromatic doublet lens (f = 150 mm) focused the light from the two LEDs to the back focal plane of the microscope objective lens (Nikon S Plan Fluor ELWD 40×), generating a collimated beam incident perpendicular onto the microcavity chip. The resulting reflected interference image from the cavity was captured by an sCMOS camera (Andor Zyla 4.2 or Hamamatsu Orca Flash 4.0). A trigger signal from the camera and an IC 4017-decade counter circuit toggled between which LED was used for illumination.