Lsm 700 scanning module

Cell images were acquired on an Axio Observer.Z1 (Zeiss) equipped with an LSM700 scanning module and an Objective Plan-Apochromat 63×/1.46 oil lens (Zeiss) using ZEN 2009 software (Zeiss). For image quantification, z-stack images were acquired with a spacing of 0.22 μm. Maximum-intensity projection of obtained slices was produced with the ZEN software, and ImageJ (NIH) software was used for image quantification.

Lentivirus-transfected RWPE-1 cells were seeded on glass-bottom dishes coated with 0.1μg/μl Retronectin (Takara Bio). Images were acquired every 6–7 minute with a xyzt acquisition mode using an Axio Observer under a Z1 microscope with the LSM 700 scanning module (Zeiss). Cell cultures were maintained at 37°C and 5% CO2 incubator for live imaging.

Confocal microscopic observations were carried out using an Axio observer Z1 microscope with a LSM 700 scanning module and ZEN 2010 software (Zeiss). Excitation of roGFP2 was performed at 488 and 405 nm and a bandpass (BP) 490–555 nm emission filter was used to collect the roGFP2 signal. For background subtraction, the signal was recovered using a BP 420–480 nm emission filter during excitation at 405 nm. Image analyses and quantifications were performed as previously described (Gutscher et al., 2008 (link)), using the public domain image analysis program ImageJ 1.52i (https://imagej.nih.gov/ij/).

For quantification analysis of CENP-A (Fig. 2), z-stack images were acquired with a spacing of 0.22 µm to cover an entire nuclear signal on an Axio Observer.Z1 microscope (Zeiss) equipped with a CSU-X1 confocal scanner unit (Yokogawa), iXon3 DU897E-CS0 camera (Andor) and Plan-Apochromat 100×/1.46 oil lens (Zeiss) using Andor iQ2 software (Andor). Quantitative analysis of acquired images was performed as previously described (Shono et al., 2015 (link)). Other cell images were acquired on an Axio Observer.Z1 (Zeiss) equipped with a LSM700 scanning module and an Objective Plan-Apochromat 63×/1.46 oil lens (Zeiss) using ZEN 2009 software (Zeiss). For quantification analysis in Fig. 4, z-stack images were acquired with a spacing of 0.36 µm, and with total depth of 5.4 µm. Signal intensity was acquired for each pixel in a 22×22 pixel region around the spot from a z-stack maximum intensity projection cell image using Fiji software (Schindelin et al., 2012 (link)). The pixel intensities located in a symmetric position with respect to the x-axis and y-axis were summed up. The integrated pixel intensities at the same distance from the spot were averaged.