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Two-point calibration protocol for the Förster Resonance Energy Transfer indicator Pyronic in neurons
Felipe Baeza-Lehnert1, Yasna Contreras-Baeza2, Camila Aburto2
1Leipzig University, Carl-Ludwig-Institute of Physiology, Faculty of Medicine, Leipzig, Germany.
Neurophotonics
|October 27, 2025
Summary
Researchers developed a new protocol to measure cytosolic pyruvate concentration in neurons. This method uses the Pyronic fluorescent indicator for quantitative analysis of cellular metabolism.
Area of Science:
- Cellular Metabolism
- Neuroscience
- Biochemistry
Background:
- Pyruvate is a key metabolic intermediate, linking glycolysis to respiration.
- Accurate measurement of pyruvate is crucial for understanding cellular energy production.
- Existing methods lack single-cell resolution or quantitative accuracy.
Purpose of the Study:
- To present a detailed protocol for quantifying cytosolic pyruvate concentration in neurons.
- To achieve single-cell resolution using a Förster Resonance Energy Transfer (FRET)-based indicator.
- To establish a minimally invasive, two-point calibration approach.
Main Methods:
- Utilized the genetically encoded fluorescent indicator Pyronic.
- Employed a noninvasive pharmacological two-point calibration method.
- Determined Pyronic's dynamic range (ΔRMAX) by substrate manipulation and inhibition.
- Incorporated in vitro Kd values for Pyronic.
Main Results:
- Enabled the measurement of absolute cytosolic pyruvate concentrations in neurons.
- Provided a quantitative parameter for comparing pyruvate metabolism across diverse conditions.
- Demonstrated reliable calibration of Pyronic in neurons.
Conclusions:
- The developed protocol offers a robust and quantitative method for studying pyruvate metabolism.
- This approach is suitable for various physiological and pathological scenarios in neurons.
- Facilitates comparative analyses of pyruvate metabolism with high precision.
