Volocity

HeLa cells stably expressing mitochondria-targeted R-GECO1.2 (mito-R-GECO1.2), which acts as mitochondria Ca²⁺ sensor and emits red fluorescence, or cyto-GCaMP6s, which acts as cytosol Ca²⁺ sensor and emits green fluorescence, were used for the Ca²⁺ imaging experiments. Ca²⁺ imaging was performed with a spinning-disk UltraVIEW VoX imaging system (Perkin Elmer) equipped with a 20 × /0.75 NA objective lens (Nikon).
For histamine-induced Ca²⁺ dynamics, cells were washed twice with Hank’s balanced salt solution, followed by the addition of Ca²⁺ buffer (150 mM NaCl, 5.4 mM KCl, 20 mM HEPES, 10 mM glucose, 1 mM MgSO₄, and 1.8 mM CaCl₂, pH 7.4), immediately before imaging. Cells were excited at either 561 nm (for mitochondria Ca²⁺) or 488 nm (for cytosol Ca²⁺), and images were acquired every 5 s for 5 min. Approximately 30 s after the start of the experiment, histamine was added at a final concentration of 100 μM. Images were post-processed with Volocity (Perkin Elmer).
For mitochondria Ca²⁺ upon DNA damage, cells were treated with 1 μM cpt for 10 h before imaging. Images were acquired at 30 points randomly, and post-processed and analyzed with Volocity (Perkin Elmer).

Live animals and whole-mount in situ hybridization samples were photographed using a microscope (SteREO Discovery.V20; Carl Zeiss) equipped with a Plan Apochromat 1.0× objective and a digital microscope camera (AxioCam HRc; Carl Zeiss) automated by AxioVision Rel.4.8 software (Carl Zeiss). Confocal images were captured on a Leica SP5 confocal microscope with a 20×, 40×, or 63× objective. All the quantifications were using the Measurement program of Volocity (Perkin Elmer) and normalized by the quantified animal area. For H3P quantifications, all the mitotic events were determined by counting nuclei labeled with the anti-H3P antibody. For quantification of notum⁺, wnt1⁺ or fst⁺ cells, all the probe signals surrounding the nucleus in the vicinity of the wounds were calculated. For cells under apoptosis quantifications, we obtained all the TUNEL signal using confocal microscope (about fifty 1 μm stacks), then we used Volocity software (PerkinElmer) to build 3D image for these stacks and quantify TUNEL⁺ nuclei in our region of interest (ROI), and finally the number of TUNEL⁺ nuclei was normalized by the area of the upper surface of ROI.

Mitotic spreads were prepared as described [58] (link) with the exception that 5% Lipsol (Dynalab) was used as a detergent. Wild-type and mutated Smc6-3×HA-expressing cells were arrested in G2/M after a synchronous S-phase at 35° before preparation of spreads. Monoclonal rat-anti-HA (Roche) was used as the primary antibody followed by Cy3-conjugated goat-anti-rat (Invitrogen) to detect Smc6-3×HA on spreads. Each image was acquired under identical exposure conditions using a Leica microscope and 100× objective. Image analysis was carried out in Volocity (Perkin Elmer). Signals from >50 chromosome spreads were quantified using the analysis tools provided by the Volocity software (Perkin Elmer), and background staining in adjacent regions of the same size were subtracted. Box plots were made using standard statistical tools and represent all values measured between the maximum and the minimum. Statistical analysis to measure significance of differences between strains was done using a two-tailed T-test, with Welch's correction, which was used because the two populations compared had unequal variance. P-values greater than or equal to 0.05 were considered insignificant.

Images were acquired as a series of 2D tiff (FIB-SEM) or dm3 (3view) files. In order to compile a 3D tiff file format, the images were registered in Fiji (Schindelin et al., 2012 ) (http://fiji.sc/Fiji; Plugin Registration > StackReg > Translation) or IMOD (Kremer et al., 1996 (link)) (http://bio3d.colorado.edu/imod/; tiltxcorr algorithm). Representation of orthogonal views and/or threshold-based segmentation was done in Imaris (BitPlane, www.bitplane.com) or Fiji’s 3D Viewer. Manual segmentation and visualization of 3D data was done using 3DMOD (http://bio3d.colorado.edu/imod/doc/3dmodguide.html).
3D reconstructions of confocal images were done using Volocity (PerkinElmer, Volocity">www.perkinelmer.co.uk/Volocity).