High Content Ratio Imaging for Apoptosis Detection Figure 2. Schematic overview of the high-content imaging methodology. The method utilizes 96 well plate ratio imaging as the primary readout of caspase activity in cells stably expressing caspase cleavable FRET probe as described earlier. The pathway Bio imager was configured with ECFP and EYFP FRET filter as described in the materials and methods. The cells were seeded on 96 well glass bottom plates at the desired density and image acquisition was done in an automated on-the-fly ratio mode with desired montage option at regular time intervals. Post acquisition analysis was done using Attovision software after automated segmentation into nuclear and cytoplasmic channels, followed by conversion of qualitative data to quantitative data. The quantitative ROI information is converted in to list mode that can be analysed in any flow cytometer based software. The cumulative information helps in the identification of positive hits. doi:10.1371/journal.pone.0020114.g002 accompanied with MMP loss, except in the case of Mevinolin where, FRET ratio loss happened without much reduction in the MMP loss. Thus, this screening platform integrates signals of multiple parameters associated with apoptosis signaling from individual cells so as to generate a final picture of the response of the population as a whole. Drug resistant cells execute caspase 9 independent cell death One of the greatest features of the multi-well plate imaging tool used here is the option of revisit imaging of the same well for longer periods of time, thereby enabling precise fate analysis of the surviving fraction. We identified cell colonies that survived drug PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e20114