Quantitative Fluorescence Microscopy of Apoptosis Proteins

Experiments were carried out as detailed previously.^{48 (link)} Imaging was performed with a × 40 ELWD Plan Fluor objective (NA: 0.6, Nikon, Melville, NY, USA) on a Nikon Ti-E perfect focus inverted microscope equipped with a spinning disk confocal CSU-X1 (Andor, Oxford Instruments, Belfast, UK), motorized X,Y stage (Nikon), environmental chamber (OkoLab, Pozzuoli, Italy), and iXon3 897 EMCCD camera (Andor, Oxford Instruments), controlled by the NIS-Elements software (Nikon). All analysis was performed using MATLAB (version R2012b, Mathworks, Natick, MA, USA) on 16-bit grayscale images of eGFP-BCL-2 family proteins or mCherry-BH3-only proteins. The mitochondrial compartment was identified as eGFP-localized, small, isolated structures (>~4 μm² and <80 μm²) above local background intensity. In contrast to Wong et al.,^{48 (link)} nuclear-specific areas were not observed. Therefore, the entire cell was identified with a single intensity threshold applied to the eGFP fluorescence, excluding areas identified as mitochondria. Average intensities were reported on an image-wide basis, normalized to the total area of each compartment identified. Ratio of mCherry mitochondrial intensity/cytoplasmic intensity was calculated, and data were normalized and plotted as described.^{48 (link)} Data were analyzed from 10 fields of view per condition in two separate experiments.