ANALYSIS OF MITOCHONDRIAL MORPHOLOGY AND FUNCTION WITH NOVEL FIXABLE FLUORESCENT STAINS

Investigation of mitochondrial morphology and function has been hamper ed because photostable, mitochondrion-specific stains that are retaine d in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H-2-CMXRos dyes were more p hotostable than rhodamine 123. In addition, fluorescence and morpholog y of mitochondria stained with the CMXRos and CMXRos-H-2 dyes were pre served even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H-2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of c ytochrome c oxidase, indicating that the dyes specifically stain mitoc hondria. Confocal microscopy of these mitochondria yielded colored ban ding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine str ucture. We also used poisons that decrease mitochondrial membrane pote ntial and an inhibitor of respiration complex II to show by now cytome try that the fluorescence intensity of CMXRos and H-2-CMXRos dye stain ing responds to changes in mitochondrial membrane potential and functi on. Hence, CMXRos has the potential to monitor changes in mitochondria l function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the mi crotubule network to concomitantly evaluate multiple features of cell morphology.