Labeling conditions were selected to minimize background noise and the microscope configuration was selected to reduce bleed-through between imaging channels to negligible levels. In order to ensure optimal imaging performance, instrument alignment was performed at regular intervals by Zeiss. Multi-coloured TetraSpeck florescent beads were used to monitor point spread functions and correct chromatic shift; maximum tolerated shifts were 50 nm in X-Y and 100 nm in Z. To minimise chromatic aberrations, great care was also taken to Sorafenib Raf inhibitor balance labeling intensities in different imaging channels. Confocal sections were collected through a 1006 lens and 3-D images generated using Z stacks and processed in ImarisH software. For LSM510 image acquisition the following channel settings were used: green 2488 nm laser line at 2% intensity with a BP 500�C530 IR filter; red �C 543 nm laser line at 32% of intensity and LP 545 filter. 4-D time-lapse imaging was performed using either a DeltaVision microscope with a CoolSNAP-HQ2 camera and Olympus objective or Zeiss LSM510META confocal microscope using the settings detailed above. The Deltavision system was used for long-term imaging experiments , with the intensity of light during imaging kept to 32% using an acquisition speed of 100�C200 ms. The conditions used allow imaging for at least 2 days without influencing cell viability or cell cycle parameters. Because of the zoom facilities, the Zeiss system was used when foci-level resolution was required . As above, the light intensity was reduced to the minimum required to resolve individual foci and the imaging conditions used were shown not to prevent subsequent cell division. For detailed co-localization analysis , confocal imaging was performed using a Zeiss LSM710 microscope using instrument setting equivalent to those detailed above to minimize bleedthrough between channels and background levels. Z-stacks were acquired for each sample with voxel dimensions of 0.860.860.34 microns, for X, Y and Z respectively with an XY resolution of 9886988 pixels and a pinhole setting of 1.0 Airy unit. Amplifier and detector gain and offset were optimally chosen by the instrument for each field acquired. For the Alexa-488 channel an EF1 filter set was used with a SPI wavelength range from 493�C543 nm. For the Cy3 channel an EF2 filter set was used with a SPI wavelength range from 566�C681 nm. Such images are artificial and while providing an MG132 Proteasome inhibitor accurate representation of the positions of foci are not intended to provide a realistic representation of the foci themselves. For high-throughput image analysis, in-house scripts were developed using Fiji software with the aid of the suite of 3- D filters . Co-localization analysis was performed with JACoP and co-localized volumes estimated by multiplying the number of co-localized voxels by the volume covered by a single voxel.