Quantitative Cristae Ultrastructure Analysis

Cells were washed in PBS, fixed with 2.5% glutardialdehyde and 2% formaldehyde in a buffered solution and postfixed in 1% osmium tetraoxide that had been reduced with 1% potassiumhexacyanoferrate^{34 (link)}. The cells were dehydrated in an ascending ethanol series, embedded in TAAB embedding resin, and sectioned on a Leica Ultracut 7 ultramicrotome using a Diatome diamond knife. The sections were counter stained using platinum blue (IBIlabs) and lead citrate (Leica) and visualized in an FEI Tecnai 20 transmission electron microscope. They were photographed at ×27,000 magnification with a Gatan ultrascan 1000 camera. To quantitatively analyze the electron microscopically images a line was drawn in imageJ manually into the cristae starting with the cristae junction proceeding into the cristae volume as far as the image quality was sufficient for image quantification, the curvature of the cristae was not crossing the line or a maximum of 130-nm length was reached. Along the drawn line every 2 nm orthogonally line plots with a width of 10 nm were measured with an ImageJ macro starting from the CJ. Each line plot was halved and the position of the minimal intensity was determined for both sides. The distance of both minimal intensities was set as the distance between opposing sides of the cristae.