Volume 77, Issue 5 p. 341-347
Research Article

Microscopic oxygen imaging based on fluorescein bleaching efficiency measurements

Martin Beutler

Corresponding Author

Martin Beutler

bionsys GmbH, Fahrenheitstraße 1, 28359 Bremen, Germany

Correspondence to: Martin Beutler, bionsys GmbH, Fahrenheitstr. 1, 28359 Bremen, Germany. E-mail: [email protected]Search for more papers by this author
Ines M. Heisterkamp

Ines M. Heisterkamp

Max Planck Institute for Marine Microbiology Bremen, Celsiusstraße, 28359 Bremen, Germany

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Bastian Piltz

Bastian Piltz

Max Planck Institute for Marine Microbiology Bremen, Celsiusstraße, 28359 Bremen, Germany

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Peter Stief

Peter Stief

Department of Biology, Nordisk Center for Jordens Udvikling (NordCEE), Campusvej 55, 5230 Odense M, Denmark

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Dirk De Beer

Dirk De Beer

Max Planck Institute for Marine Microbiology Bremen, Celsiusstraße, 28359 Bremen, Germany

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First published: 07 March 2014
Citations: 4

REVIEW EDITOR: Prof. George Perry

ABSTRACT

Photobleaching of the fluorophore fluorescein in an aqueous solution is dependent on the oxygen concentration. Therefore, the time-dependent bleaching behavior can be used to measure of dissolved oxygen concentrations. The method can be combined with epi-fluorescence microscopy. The molecular states of the fluorophore can be expressed by a three-state energy model. This leads to a set of differential equations which describe the photobleaching behavior of fluorescein. The numerical solution of these equations shows that in a conventional wide-field fluorescence microscope, the fluorescence of fluorescein will fade out faster at low than at high oxygen concentration. Further simulation showed that a simple ratio function of different time-points during a fluorescence decay recorded during photobleaching could be used to describe oxygen concentrations in an aqueous solution. By careful choice of dye concentration and excitation light intensity the sensitivity in the oxygen concentration range of interest can be optimized. In the simulations, the estimation of oxygen concentration by the ratio function was very little affected by the pH value in the range of pH 6.5–8.5. Filming the fluorescence decay by a charge-coupled-device (ccd) camera mounted on a fluorescence microscope allowed a pixelwise estimation of the ratio function in a microscopic image. Use of a microsensor and oxygen-consuming bacteria in a sample chamber enabled the calibration of the system for quantification of absolute oxygen concentrations. The method was demonstrated on nitrifying biofilms growing on snail and mussel shells, showing clear effects of metabolic activity on oxygen concentrations. Microsc. Res. Tech. 77:341–347, 2014. © 2014 Wiley Periodicals, Inc.