Mitochondria undergo spontaneous transient elevations in matrix pH associated with drops in mitochondrial membrane potential. These mitopHlashes require a functional respiratory chain and the profusion protein optic atrophy 1, but their mechanistic basis is unclear. To gain insight on the origin of these dynamic events, we resolved the ultrastructure of flashing mitochondria by correlative light and electron microscopy. HeLa cells expressing the matrix-targeted pH probe mitoSypHer were screened for mitopHlashes and fixed immediately after the occurrence of a flashing event. (…) Correlation of live/fixed fluorescence and electron microscopy images allowed the unambiguous identification of flashing and nonflashing mitochondria. Three-dimensional reconstruction and surface mapping revealed that each tomogram contained two flashing mitochondria of unequal sizes, one being much larger than the average mitochondrial volume. (…)
How Amira-Avizo Software is used
Amira software was used to align slices and generate three dimensional tomograms, which could then be resliced orthogonally to produce images of the x–y orientation that matched the fluorescence image (…) For this, cristae were automatically segmented from the previously labeled mitochondria by eroding the outer and inner membranes and then applying a contrast-based thresholding on Amira (…) To measure the mitochondrial surface and volume, a semiautomatic segmentation was applied by filling each individual mitochondrion with a paint—and brush—tool in Amira software and by interpolating the mitochondrial contour every five slices.