Eclipse ti inverted

Five millilitres of cultures were grown overnight and visualised on an Eclipse Ti inverted microscope (Nikon) at 90× magnification. Images were captured using the NIS-Elements Microscope Imaging Software (Nikon). The area of a minimum of 24 cells was determined for each strain and condition tested using ImageJ (http://imagej.net).

Fluorescent proteins were visualized with a CSU-X1 spinning disk confocal on a Nikon Eclipse Ti inverted microscope with an Andora Clara digital camera using CFI APO 100× oil TIRF objective. Images were collected using differential interference contrast (DIC), 488-nm excitation (GFP), and 561-nm excitation (RFP) and analyzed with NIS-Elements software 4.10 (Nikon) and Fiji [87 (link)].

Cells were grown as described above and fixed with 3.7% formaldehyde for 15 min either before or at indicated times after NaCl addition. Cells were washed two times with 0.1 M potassium phosphate buffer pH 7.5, stained 5 min with 1 μg/mL DAPI (Thermo Scientific Pierce, 62247), and additionally washed two times with 0.1 M potassium phosphate buffer pH 7.5. Cells were imaged on an epifluorescent Nikon Eclipse-TI inverted microscope using a 100x Nikon Plan Apo oil immersion objective. GFP emission was visualized at 535 nm upon excitation at 470 nm (Chroma 49002_Nikon ETGFP filter cube, Chroma Technologies, Bellows Falls, VT, USA). mCherry emission was visualized at 620 nm upon excitation at 545 nm (Chroma 96364_Nikon Et-DSRed filter cube). DAPI emission was visualized at 460 nm upon excitation at 350 nm (Chroma 49000_Nikon ETDAPI filter cube). Nuclear to cytoplasmic intensity values were calculated with customized CellProfiler scripts [121 (link)]. The fraction of cells with nuclear factor before stress was calculated by identifying nuclear masks in Cell Profiler (i.e., identifiable nuclear objects) in that channel that overlapped with DAPI masks and manually correcting miscalls.

Photographs were taken using a Nikon DS-U2-Fi1c camera attached to Nikon’s Eclipse Ti inverted microscope.

Near-TIRF microscopy was used to visualize Fat2-GFP, Sra1-GFP, Abi-mCherry, and F-Tractin-tdTomato (Spracklen et al., 2014 (link)) dynamics at the basal surface. Near-TIRF imaging was performed with a Nikon ECLIPSE-Ti inverted microscope with Ti-ND6-PFS Perfect Focus Unit, solid-state 50 mW 481 and 561 nm Sapphire lasers (Coherent technology), motorized TIRF illuminator, laser merge module (Spectral Applied Research), Nikon CFI 100 x Apo 1.45 NA oil immersion TIRF objective with 1.5 x intermediate magnification, and Andor iXon3 897 electron-multiplying charged-coupled device (EM-CCD) camera. Image acquisition was controlled using MetaMorph software. For two color imaging, frames were collected for each color consecutively with the TIRF illumination angle adjusted in between. Imaging was performed at room temperature. For display, movies were corrected for bleaching using the histogram matching method in Fiji (ImageJ) (Schindelin et al., 2012 (link); Schindelin et al., 2015 (link)). TIRF microscopy was used to acquire the data in Figure 3—figure supplement 2; Figure 3—video 2; Figure 6B and D–F; Figure 6—video 2; Figure 6—video 3; Figure 8B; Figure 8—figure supplement 1; Figure 8—video 1; Figure 8—video 2.

The measurements of fluorescently labeled DNA were conducted on Nikon® Eclipse Ti inverted epifluorescence Microscope (lens ApoTIRF × 100, NA 1.49) equipped with Andor® iXon 897 EMCCD camera and filters providing excitation for SYBR® Green at 465–495 nm and emission at 515–555 nm. DNA was stained by SYBR® Green I Sigma-Aldrich®.