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Oxygen concentration measurement in 3D cell culture using multifocal optical projection microscopy
Birhanu Belay1,2, Mart Kroon3,2, Kaisla Walls1,2
1Computational Biophysics and Imaging Group, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland. birhanu.belay@tuni.fi.
Analytical Methods : Advancing Methods and Applications
|March 30, 2026
Summary
This study introduces a new method for measuring oxygen in large 3D cell cultures. The technique uses microscopy and fluorescent beads to accurately map oxygen levels deep within cell cultures, aiding research into cell physiology and disease.
Area of Science:
- Biomedical Engineering
- Cell Biology
- Microscopy
Background:
- Precise control of molecular oxygen is crucial for cell culture, impacting physiological functions and disease modeling.
- Existing oxygen measurement methods are often invasive and struggle to assess oxygen distribution in deep, large-volume 3D cell cultures.
Purpose of the Study:
- To develop an adaptable, minimally invasive method for quantifying oxygen levels and gradients in large-volume 3D cell cultures.
- To overcome the depth limitations of current oxygen sensing techniques in complex cell culture models.
Main Methods:
- Utilized multifocal optical projection microscopy combined with fluorescent microsensor beads.
- Acquired fluorescent projection images and simultaneously measured oxygen concentration using an optical fiber sensor.
- Generated a Stern-Volmer calibration curve to convert fluorescence intensity into oxygen concentration.
Main Results:
- Successfully quantified oxygen concentrations at depths exceeding typical 3D cell culture dimensions, up to 21 mm.
- Demonstrated a significant decrease in oxygen concentration with increasing cell density and specimen depth in fibroblast-laden agarose hydrogels.
- Highlighted the necessity of oxygen measurements in 3D cell cultures due to observed oxygen gradients.
Conclusions:
- The developed method is well-suited for minimally invasive oxygen quantification in large-volume hydrogel-based 3D cell cultures.
- This technique enables detailed assessment of oxygen levels and gradients, crucial for understanding cellular behavior in complex environments.
