Molecular oxygen is an important reporter of metabolic and physiological status at the cellular and tissue level, and its concentration is used for the evaluation of many diseases (e.g.: cancer, coronary artery disease). The development of accurate and quantitative methods to measure O2 concentration ([O2]) in living cells, tissues and organisms is challenging and is subject of intense research. We developed a protein-based, fluorescent oxygen sensor that can be expressed directly in cells to monitor [O2] in the intracellular environment. We fused Myoglobin (Myo), a physiological oxygen carrier, with mCherry, a fluorescent protein, to build a fluorescence resonance energy transfer (FRET) pair, Myo-mCherry. The changes in the spectral properties of Myoglobin upon oxygen binding result in changes of the FRETdepleted emission intensity of mCherry, and this effect is detected by monitoring the fluorescence lifetime of the probe. We present here the preparation and characterization of a series of Myo-mCherry variants and mutants that show the versatility of our protein-based approach: the dynamic range of the sensor is tunable and adaptable to different [O2] ranges, as they occur in vitro in different cell lines, the probe is also easily targeted to subcellular compartments. The use of fluorescence overcomes the most common issues of data collection speed and spatial resolution encountered by currently available methods for O2-monitoring. By using Fluorescence Lifetime Imaging Microscopy (FLIM), we show that we can map the oxygenation level of cells in vitro, providing a quantitative assessment of [O2].
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.
Oxygen (O2) is one of the most important biometabolites. In abundance, it serves as the limiting terminus of aerobic respiratory chains in the mitochondria of higher organisms; in deficit, it is a potent determinant of development and regulation of other physiological and therapeutic processes. Most knowledge on intracellular and interstitial concentration ([O2]) is derived from mitochondria isolated from cells or tissue biopsies, providing detailed but nonnative insight into respiratory chain function. The possible loss of essential metabolites during isolation and disruption of the normal interactions of the organelle with the cytoskeleton may cause these data to misrepresent intact cells. Several optical methodologies were also developed, but they are often unable to detect heterogeneity of metabolic characteristics among different individual cells in the same culture, and most cannot detect heterogeneous consumption within different areas of a single cell. Here, we propose a noninvasive and highly sensitive fluorescence lifetime microscopy probe, myoglobin-mCherry, appropriate to intracellular targeting. Using our probe, we monitor mitochondrial contributions to O2 consumption in A549 nonsmall cell lung cancer cells and we reveal heterogeneous [O2] within the intracellular environments. The mitochondrial [O2] at a single-cell level is also mapped by adding a peptide to target the probe to the mitochondria.
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