Rhodamine 800 as a probe of energization of cells and tissues in the near-infrared region: a study with isolated rat liver mitochondria and hepatocytes

To examine the feasibility of optical monitoring of cellular energy states with tissue-transparent near-infrared (NIR) light, the absorption and fluorescence characteristics of Rhodamine 800 in isolated rat liver mitochondria and hepatocytes were investigated. When the dye was incubated with isolated mitochondria, a large red shift of the absorption spectra and quenching of the fluorescence intensity were observed. The absorbance difference at 730 minus 685 nm, or at 730 minus 800 nm, and the fluorescence intensity measured at 692 nm varied linearly with the mitochondrial membrane potential. The spectral changes observed could be explained in terms of the potential-dependent uptake of the dye from the buffer solution into the mitochondrial matrix. The respiration control ratio and oxygen consumption rate were not affected by the addition of Rhodamine 800 at concentrations lower than 5 microM, which was the concentration range mostly employed throughout the present study. In a suspension of hepatocytes, the red shift and fluorescence quenching of Rhodamine 800 characteristic of energized mitochondria were also observed, and these changed to those of the buffer solution with the addition of an uncoupler under normoxia. At the early stage of anoxia, within about 5 min, when cytochrome oxidase was completely reduced, hepatocytes were concluded to be in the fully energized state, since the optical characteristics of Rhodamine 800 were the same as those of energized mitochondria. On the basis of these in vitro data, Rhodamine 800 is concluded to be a possible NIR-active contrast agent, that can be used to monitor the energy states of living tissues, in addition to the tissue oxygenation states, by the use of near-infrared spectrophotometry (NIRS) without harmful effects.