Fluorescence lifetime imaging (FLIM) of Myoglobin (Myo)-mCherry, is used for sensing oxygen partial pressure (pO2) in the intracellular environment. Herein, we present the potential sources of lifetime error such as sample oversaturation or dimeric Myo-mCherry configurations resulting in self-quenching fluorophores. We also provide a correction protocol for Myo-mCherry expression, adjusting parameters to account for second harmonic generation (SHG) components and dark counts that result in accurate mean lifetime values and pO2 in the cellular environment.
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