ascorbic-acid and tetramethylrhodamine

The measurement of tissue and cell oxygenation is important for understanding cell metabolism. We have addressed this problem with a novel optical technique, called triplet imaging, that exploits oxygen-induced triplet lifetime changes and is compatible with a variety of fluorophores. A modulated excitation of varying pulse widths allows the extraction of the lifetime of the essentially dark triplet state using a high-fluorescence signal intensity. This enables the monitoring of fast kinetics of oxygen concentration in living cells combined with high temporal and spatial resolution. First, the oxygen-dependent triplet-state quenching of tetramethylrhodamine is validated and then calibrated in an L-ascorbic acid titration experiment demonstrating the linear relation between triplet lifetime and oxygen concentration according to the Stern-Volmer equation. Second, the method is applied to a biological cell system, employing as reporter a cytosolic fusion protein of beta-galactosidase with SNAP-tag labeled with tetramethylrhodamine. Oxygen consumption in single smooth muscle cells A7r5 during an [Arg(8)]-vasopressin-induced contraction is measured. The results indicate a consumption leading to an intracellular oxygen concentration that decays monoexponentially with time. The proposed method has the potential to become a new tool for investigating oxygen metabolism at the single cell and the subcellular level.

Topics: Algorithms; Animals; Arginine Vasopressin; Ascorbic Acid; beta-Galactosidase; Calibration; Cell Line; Fluorescence; Image Processing, Computer-Assisted; Intracellular Space; Kinetics; Linear Models; Models, Chemical; Muscle Contraction; Myocytes, Smooth Muscle; Optics and Photonics; Oxygen; Oxygen Consumption; Rats; Rhodamines; Vasoconstrictor Agents; Video Recording