Triplet Imaging of Oxygen Consumption During the Contraction of a Single Smooth Muscle Cell (A7r5)

Abstract

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 b-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.

Transient state microscopy probes patterns of altered oxygen consumption in cancer cells

“…Using a high triplet yield dye such as Eosin Y allowed the applied laser irradiances in the TRAST measurements to be reduced by more than a factor of 100 compared with earlier work . This strongly reduces the requirements on the excitation laser, which simplifies and reduces the cost of the instrumentation.…”

“…In this paper, the fluorescence based transient state (TRAST) imaging technique is applied to study the metabolism of breast cancer cells and primary and transformed fibroblasts. In TRAST, the excitation intensity onto a fluorescent sample is modulated in a systematic manner.…”

“…By TRAST, several categories of photo‐induced, long‐lived, dark, transient states can be monitored, including triplet , photo‐ionized and photo‐isomerized states . In particular triplet states are readily quenched by oxygen, and their lifetimes depend strongly on the local oxygen concentration.…”

The Photolytic Activity of Poly‐Arginine Cell Penetrating Peptides Conjugated to Carboxy‐tetramethylrhodamine is Modulated by Arginine Residue Content and Fluorophore Conjugation Site

“…A, 1 O 2 formation by the TMR‐CPPs upon light irradiation was detectable, although very inefficient in comparison to Rose Bengal. These data are consistent with the low values of triplet state quantum yield of TMR (0.001–0.003) reported in the literature . The singlet oxygen quantum yield of TMR thus, should be lower than the reported TMR triplet state quantum yield.…”