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.
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.
The extraction of fluorophore lifetimes in a biological sample provides useful information about the probe environment that is not readily available from fluorescence intensity alone. Cell membrane potential, pH, concentration of oxygen ([O2]), calcium ([Ca2+]), NADH and other ions and metabolites are all regularly measured by lifetime-based techniques. These measurements provide invaluable knowledge about cell homeostasis, metabolism and communication with the cell environment. Fluorescence lifetime imaging microscopy (FLIM) produces spatial maps with time-correlated singlephoton counting (TCSPC) histograms collected and analyzed at each pixel, but traditional TCSPC analysis is often hampered by the low number of photons that can reasonably be collected while maintaining high spatial resolution. More important, traditional analysis fails to employ the spatial linkages within the image. Here, we present a different approach, where we work under the assumption that mixtures of a global set of lifetimes (often only 2 or 3) can describe the entire image. We determine these lifetime components by globally fitting precise decays aggregated over large spatial regions of interest, and then we perform a pixel-by-pixel calculation of decay amplitudes (via simple linear algebra applied to coarser time-windows). This yields accurate amplitude images (Decay Associate Images, DAI) that contain stoichiometric information about the underlying mixtures while retaining single pixel resolution. We collected FLIM data of dye mixtures and bacteria expressing fluorescent proteins with a two-photon microscope system equipped with a commercial single-photon counting card, and we used these data to benchmark the gDAI program.
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