The laser dye rhodamine 123 is shown to be a specific probe, for the localization of mitochondria in living cells.By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment.Since the classical investigations of the. mitochondrion some 90 years ago, much work has been carried out on the structure and function of this complex organelle. Extensive biochemical studies of mitochondria have proved that they play a cardinal role in the generation of energy essential for the survival and proliferation of eukaryotic cells (1-4). As intracellular organelles, mitochondria show remarkable plasticity, mobility, and morphological heterogeneity (5-13). Mitochondrial morphology is influenced by the metabolic state of the cell, cell cycle, cellular development and differentiation, and by pathological states (14)(15)(16)(17)(18)(19)(20). In addition, both morphological and functional changes in mitochondria have been shown to occur in conjunction with neoplastic transformation (21).Although isolated mitochondria and mitochondria in fixed cells have been extensively investigated, much less attention has been paid to mitochondria in living cells. Previous investigations of mitochondria in living cells have been hampered by the lack of techniques allowing high-resolution visualization of these organelles. Use of Janus Green B, a dye relatively specific for mitochondria, aids somewhat in their recognition but also causes mitochondrial distortion and cytotoxic effects (5,22 sponsible for mitochondrion-specific staining in such a preparation has been characterized as rhodamine 3B (unpublished data). This finding prompted us to screen various rhodamine compounds for mitochondrion-specific staining. The results reported here describe the use of the laser dye rhodamine 123 as a specific fluorescent probe for mitochondria in living cells. Rhodamine 123 stains mitochondria directly (without passage through endocytic vesicles and lysosomes) and provides lowbackground high-resolution fluorescent images of mitochondria without apparent cytotoxic effects. MATERIALS AND METHODSThe purified laser dye rhodamine 123 (Eastman) was dissolved in double-distilled water at a concentration of 1 mg/ml and subsequently diluted to 10 ,g/ml in Dulbecco's modified Eagle's medium (GIBCO). Cultured cells grown on 12-mm round glass coverslips (Rochester Scientific, Rochester, NY) were incubated with rhodamine 123 (10 ,ug/ml) for 30 min in a 10% CO2 incubator at 37°C. Coverslips were then rinsed through three 5-ml changes of medium (5 min per rinse) and mounted in medium supplemented with 10% fetal calf serum (GIBCO) on a live-cell observation chamber prep...

Permeant cationic fluorescent probes are shown to be selectively accumulated by the mitochondria of living cells. Mitochondria-specific interaction of such molecules is apparently dependent on the high trans-membrane potential (inside negative) maintained by functional mitochondria. Dissipation of the mitochondrial trans-membrane and potential by ionophores or inhibitors of electron transport eliminates the selective mitochondrial association of these compounds. The application of such potential-dependent probes in conjunction with fluorescence microscopy allows the monitoring of mitochondrial membrane potential in individual living cells. Marked elevations in mitochondria-associated probe fluorescence have been observed in cells engaged in active movement. This approach to the analysis of mitochondrial membrane potential should be of value in future investigations of the control of energy metabolism and energy requirements of specific biological functions at the cellular level.

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O n c of the distinctive features of cancer cells in vivo is their antisocial behavior in a sea of well-organized normal cells. The symptonis o f such ahnormality include cell division at the wrong time and in the wrong place. penetration to neighboring tissues, travel in the circulation, which should normally be confined to only a few cell types, and adhesion to forbidden tissues. Numerous factors contribute to the antisocial behavior of cancer cells. O n e of them agreed upon by many investigators, is the alteration in the cell surface. Among the alterations on the cell surface that can potentially contribute to the antisocial behavior of cancer cells are changes in composition of the lipid bilayer, in glycolipids, in glycocalyceal components, in topographic structure, and in the motile properties of the cell surface. Each of these aspects is being intensively investigated. The current status of this field has recently been reviewed. '-4 Among numerous alterations in cancer cells, a reduction in one of the cell surface components seems to warrant further investigation, because it may be relevant to the antisocial behavior of cancerous cells. This component is thc large, external. transformation-sensitive (LETS) protein. LETS protein was first detected in fibroblasts by a variety of techniques and has since been termed as band I ,5 galactoprotein a,6 fibronectin,' zeta,' and cell surface protein, CSP.9 It is a glycoprotein with a molecular weight of 220-250,000 daltons. I t is significantly reduced in many virally transformed fibroblasts (for review. see Rcferences 3 and 4). LETS protein was first partially purified by Yamada and Weston' from chick embryo fibroblasts by mild urea extraction. The addition of such purified LETS protein restores the normal morphology of transformed cells.lO.ll 11 has also been suggested by Ruoslahti and Vaherilz that LETS protein is closely related t o a plasma protein. cold-insoluble g l~b u l i n . '~"~ It is likely that cold-insoluble globulin in the blood circulation is the modified shedding product of cell surface LETS protein.Around the spring of 1974 in Dr. J . M. Buchanan's laboratory a t Massachusetts Institute of Technology (MIT), we found that thrombin, a highly specific protease responsible for blood clotting, is a potent mitogen for cultured