Dr. Alexandra Brown Profile

Dr. Alexandra Brown

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Proceedings Article | 4 March 2022 Poster + Presentation + Paper

metmyoglobin detection in mitochondria using fluorescence lifetime imaging of Citrine-Myoglobin-mCherry

Katie Link, Rozhin Penjweini, Alexandra Brown, Greg Alspaugh, Jay Chung, Jay Knutson

Proceedings Volume 11965, 119650K (2022) https://doi.org/10.1117/12.2626315

KEYWORDS: Fluorescence resonance energy transfer, Fluorescence lifetime imaging, Oxygen, Luminescence, Hypoxia, Sensors, Iron, Signal processing, Proteins, Microscopes

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The biological relevance of nitric oxide (NO) in cells to processes of signaling, metabolic regulation, and disease treatment has become abundantly clear. NO or reactive oxygen species (ROS) can oxidize myoglobin to the met state (metMb; the Fe³⁺ state of myoglobin), a change accompanied with an altered absorbance profile in the visible region. Recent studies show that metMb has a broad functional role in metabolic pathways, oxidative/nitrative regulation and gene networks of many cells. Thus, real-time monitoring of the different charge states of myoglobin is a promising field of research. We previously introduced a Förster resonance energy transfer (FRET) sensor, EYFP-Myoglobin-mCherry, to measure the deoxygenation, oxygenation and met states of myoglobin, creating a simultaneous oxygen (O₂) and NO sensor. In this sandwich probe, the mCherry binary chimera lifetime responds to oxygenated vs. deoxygenated myoglobin, while the yellow fluorescent protein (YFP) lifetime selectively responds to metMb (while indifferent to O₂ concentration). We now use Citrine, a more robust YFP, in place of EYFP and append a mitochondrial targeting peptide sequence to specifically target mitochondria. We use fluorescence lifetime imaging (FLIM) of this mtCitrine-Myoglobin-mCherry sandwich probe while monitoring both oxygenation level and NO-induced met formation in mitochondria of mouse embryonic fibroblasts. We also test the NO response of Citrine alone to verify that the met sensitivity is specific to the Mb sandwich probe and not Citrine alone.

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