Deltavision rt system

Asynchronous cells were grown in filter-sterilized YPD with additional 0.1 mg/l adenine (YPAD) to an OD₆₀₀ of 0.3 at 23°C for 3 hr and then shifted to 37°C for 1 hr. Cells were directly sampled for image acquisition. For the image acquisition, z-stack images with 21 0.3 μm steps (2 × 2 binning) were acquired at 37°C with a DeltaVision RT system (Applied Precision, UK) equipped with FITC (fluorescein isothiocyanate), TRITC (tetramethyl rhodamine isothiocyanate), and Cy5 filters (Chroma Technology, Bellows Falls, VT), a 100x NA 1.4 plan Apo oil immersion objective (IX70; Olympus, Japan), and a CCD camera (CoolSNAP HQ; Roper Scientific, Tucson, AZ). Images were processed and analyzed in ImageJ (NIH).
The quantification of the mean background intensity and mean fluorescence intensity of Spc97-GFP and Spc110-GFP signals at SPBs was performed on planes having SPBs in focus. Spc97-GFP or Spc110-GFP intensity within the 3 × 3 pixel-area covering a single SPB or two unsplit SPBs was measured. For GFP-Tub1 at SPBs, GFP-intensity within a 3 × 3 pixel-area surrounding the SPB was measured. Unsplit SPBs were measured together, whereas split SPBs were measured separately. The standard error (SEM) for each data set (n = 50) and level of significance were determined by one-way ANOVA with Turkey's multiple comparisons' test.

Images were acquired using a DeltaVision RT system (Applied Precision) with a Photometrics CoolSnapHQ camera using a 100× 1.40 NA UPlanSApo objective (Olympus). Images were processed for contrast enhancement and background noise reduction using ImageJ (National Institutes of Health). All image analysis was performed using custom ImageJ macros (see Source code 1). All figures and statistical analyses were generated in R. Reported p-values are the results of a two-tailed t-test.

Cell were fixed on coverslips with methanol at −20 °C for 5 min and the coverslips blocked in 10% FBS, 0.1% Triton-X100 for 30 min. After incubation for 1 h with primary antibody (diluted in 3% bovine serum albumin, BSA (w/v)), cells were incubated with secondary antibody (1:500 dilution in 3% BSA) and 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) and mounted on glass slides with Moviol or ProLong^TM Gold antifade mountant for super resolution microscopy. Cells were extracted with CSK-extraction buffer prior staining with indicated antibodies. The EdU stain detection reaction was accomplished after coverslip blocking and before primary antibody incubation, following the reaction instruction of Click-iT™ Plus EdU Alexa Fluor™ Imaging Kit (555 and 647 Kits, ThermoFisher).
Images were acquired on a DeltaVision RT system (Applied Precision) with an Olympus IX71 microscope equipped with 60X and 100X objective lenses. SIM images were acquired on N-SIM microscope equipped with ×100 objective lenses (Nikon).

HeLa cells were cultured in glass-bottomed culture dishes (MatTek). Cells were co-transfected with shMps1, different shMps1-resistent GFP-Mps1 rescue plasmids, and mCherry-H2B at a ratio of 6:2:1. After 36 h, cells were cultured at 37°C in CO₂-independent medium (Invitrogen) containing 10% FBS and 2 mM glutamine and observed with the DeltaVision RT system (Applied Precision) as previously described (Akram et al., 2018 (link)). Images were prepared for publication using Adobe Photoshop software.

Yeast cells in synthetic media cultured overnight at 30°C were diluted to OD600 = 0.1 and allowed to grow until mid-logarithmic phase (OD600 = 0.5) before fixation with 4% formaldehyde and LD staining with 2 μg/ml BODIPY 493/503. For the automatic quantification of LDs, random microscopy images were acquired using a Delta Vision RT system (Applied Precision). Maximum intensity and integrated intensity projections were created from the deconvolved image stacks using ImageJ. A custom written CellProfiler pipeline (Carpenter et al., 2006 (link)) automatically identified individual yeast cells and measured their number and size of circle shaped LDs. Supersized LDs were defined as the LDs with a diameter above 0.5 μm.