A Method for Increasing Contrast of Mitochondrial Inner Membrane Spheres in Thin Sections of Epon-araldite Embedded Tissue

The particles of 90 A diameter on the inner membranes of mitochondria, first discovered by negative staining by Fernândez-Morân (2) and later described in various other mitochondria by Parsons (9), Stoeckenius (20), Smith (18), and Sjöstrand (16), have been the center of controversy for nearly a decade. Doubts as to the identity of these inner membrane spheres have been expressed by Sjöstrand et al . (17) and Wainio (22) . The isolation of inner membrane spheres and the morphological and biochemical reconstitution of the membranes led to the identification of these spheres as mitochondrial ATPase, which is also called coupling factor 1 (F 1 )I (7, 13, 14) . The supporting evidence was the concomitant loss of radioactive F 1 , ATPase activity, and number of spheres from membranes treated with several agents which dissociate F 1 molecules into subunits (7) . F 1 has a mol wt of 284,000 (10) and the content of F 1 was estimated to be about 10 percent of the total proteins of the inner membrane . Ogawa and Mayahara (8) supported the above biochemical data by reporting the intramitochondrial localization of ATPase activity by a histochemical method and by demonstrating its relationship to the inner membrane spheres in rodent cardiac mitochondria . Reports of the presence of the spheres on cristae of mitochondria embedded in plastic have been few (1, 4) . Fleischer et al. (3) have described 90-A particles in thin sections of cristae of bovine heart mitochondria depleted of neutral lipids . The embedded submitochondrial particles had few if