Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5)

Autor: Matthias Geissbuehler, Theo Lasser, Marcel Leutenegger, Iwan Märki, Thiemo Spielmann, Aurélie Formey, Kai Johnsson, Boris Hinz, Dimitri Van De Ville
Předmět:
Optics and Photonics
Rhodamines/chemistry
Myocytes
Smooth Muscle
Kinetics
Spectroscopy
Imaging
and Other Techniques
Intracellular Space
Video Recording
Analytical chemistry
Biophysics
chemistry.chemical_element
Ascorbic Acid
Fluorescence
Optics and Photonics/*methods
ddc:616.0757
Oxygen
Cell Line
Oxygen Consumption
Image Processing
Computer-Assisted
Animals
Vasoconstrictor Agents
Myocyte
Triplet state
Quenching (fluorescence)
Rhodamines
Chemistry
Intracellular Space/metabolism
Ascorbic Acid/chemistry
beta-Galactosidase
Ascorbic acid
Rats
Muscle Contraction/drug effects/*physiology
Arginine Vasopressin
Myocytes
Smooth Muscle/drug effects/*physiology
Oxygen/chemistry/metabolism
Models
Chemical
Vasoconstrictor Agents/pharmacology
Calibration
Beta-Galactosidase/chemistry/metabolism
Linear Models
Limiting oxygen concentration
Algorithms
Arginine Vasopressin/pharmacology
Muscle Contraction
Zdroj: Oxygen Transport to Tissue XXXIII ISBN: 9781461415657
Biophysical Journal
Biophysical Journal, Vol. 98, No 2 (2010) pp. 339-349
ISSN: 0006-3495
Popis: 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 β-galactosidase with SNAP-tag labeled with tetramethylrhodamine. Oxygen consumption in single smooth muscle cells A7r5 during an [Arg8]-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.
Databáze: OpenAIRE
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