We establish photoconversion of green fluorescent protein (GFP) as an optical `highlighter' to investigate the continuity of the mitochondrial matrix in living budding yeast (Saccharomyces cerevisiae). Photoconversion of GFP resulting in a marked shift of the absorption and emission spectra to longer wavelengths is elicited, under low oxygen conditions, by irradiation with blue light. Photoconversion induced a several 100-fold increase in red fluorescence of matrix targeted GFP without affecting cell viability. The color changing facilitates simple and effective regional optical marking in a conventional ftuorescence microscope. We found the mitochondrial compartment of S. cerevisiae to generally consist of one luminally continuous large part and occasionally some additional smaller fragments. Separated fragments fuse within a few minutes to the large part, resulting in a rapid intermixing of the entire mitochondrial matrix compartment. In Deltafis1 and Deltadnm1 mutants restricted in outer membrane fission, the mitochondria are still luminally continuous, suggesting a tight coupling of inner and outer membrane fissions. Matrix constrictions frequently occurring in wild type cells as well as in Deltafis1 and Deltadnm1 mutants do not interfere with luminal continuity. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.