Fig. 4. Release of cyt c-GFP following partial irradiation. (A) Confocal images showing cells stably expressing cyt c-GFP loaded with CMXRos. One cell that had adequate mitochondrial cyt c-GFP localisation was subjected to partial irradiation, the region of irradiation indicated by the dashed box. Other adjacent cells that had diffuse cyt c-GFP localisation were not irradiated. Images were collected before irradiation, immediately after irradiation (0 minutes) and at various times after partial irradiation (25 minutes and 30 minutes). Gain levels for CMXRos signal at 25 minute and 30 minute time points were increased manually to enable visualisation of mitochondria. Note that at 30 minutes the irradiated cell had begun to detach from the growth substrate, causing it to reside in a different focal plane from that of fully adherent non-irradiated cells. Therefore, the CMXRos and cyt c-GFP fluorescence in such non-irradiated cells were not visualised when the image was captured at the particular optical Z-plane of focus of the irradiated cell. Release of cyt c-GFP is indicated by a weaker diffuse GFP fluorescence throughout the cell, the mitochondrially localised fluorescence becoming greatly reduced. This release is representative of that in 17 of 31 cells analysed up to 2.5 hours after irradiation (cf. Fig. 5). (B) Images of control cyt c-GFP expressing cells not irradiated were collected similarly to those in A. The retention of GFP fluorescence in mitochondria indicating failure to release cyt c-GFP was observed in all of 21 cells analysed. Bars, 10 µm.