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Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

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Microscopic oxygen imaging based on fluorescein bleaching efficiency measurements.

Martin Beutler¹, Ines M Heisterkamp, Bastian Piltz

¹bionsys GmbH, Fahrenheitstraße 1, 28359, Bremen, Germany.

Microscopy Research and Technique

|March 11, 2014

Summary

Fluorescein photobleaching, dependent on oxygen, can measure dissolved oxygen using fluorescence microscopy. This method accurately quantines oxygen in aqueous solutions and biofilms.

Keywords:

fluorescein bleaching fluorescence oxygen imaging

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Area of Science:

Biophysics
Analytical Chemistry
Microscopy

Background:

Photobleaching of fluorescein is sensitive to dissolved oxygen levels.
Fluorescence microscopy is a powerful imaging technique.

Purpose of the Study:

To develop a method for measuring dissolved oxygen concentrations using fluorescein photobleaching.
To combine this method with epi-fluorescence microscopy for spatial oxygen mapping.

Main Methods:

Utilized a three-state energy model to describe fluorescein photobleaching kinetics.
Developed a ratio function based on fluorescence decay over time.
Employed a charge-coupled-device (CCD) camera for pixelwise analysis.
Calibrated the system using a microsensor and oxygen-consuming bacteria.

Main Results:

Fluorescence decay rate correlates with oxygen concentration; faster fading at lower oxygen levels.
The ratio function accurately estimates oxygen concentrations across a pH range of 6.5-8.5.
Demonstrated pixelwise oxygen estimation in microscopic images of biofilms.
Observed metabolic activity effects on oxygen concentrations in nitrifying biofilms.

Conclusions:

Fluorescein photobleaching provides a sensitive and quantitative method for dissolved oxygen measurement.
The technique is suitable for microscopic imaging and analysis of biological samples like biofilms.
This approach offers a valuable tool for studying oxygen dynamics in various aqueous environments.