High resolution spatial investigation of intracellular oxygen in muscle cells.

Molecular oxygen (O 2 ) is one of the most functionally relevant metabolites. O 2 is essential for mito-chondrial aerobic respiration. Changes in O 2 affect muscle metabolism and play a critical role in the maintenance of skeletal muscle mass, with lack of sufficient O 2 resulting in detrimental loss of muscle mass and function. How exactly O 2 is used by muscle cells is less known, mainly due to the lack of tools to address O 2 dynamics at the cellular level. Here we discuss a new imaging method for the real time quantification of intracellular O 2 in muscle cells based on a genetically encoded O 2 -responsive sensor, Myoglobin-mCherry. We show that we can spatially resolve and quantify intracellular O 2 concentration in single muscle cells and that the spatiotemporal O 2 gradient measured by the sensor is linked to, and reflects, functional metabolic changes occurring during the process of muscle differentiation. Highlights: Real time quantitation of intracellular oxygen with spatial resolutionIdentification of metabolically active sites in single cellsOxygen metabolism is linked to muscle differentiation.

Intracellular imaging of metmyoglobin and oxygen using new dual purpose probe EYFP-Myoglobin-mCherry.

The biological relevance of nitric oxide (NO) and reactive oxygen species (ROS) in signaling, metabolic regulation, and disease treatment has become abundantly clear. The dramatic change in NO/ROS processing that accompanies a changing oxygen landscape calls for new imaging tools that can provide cellular details about both [O2 ] and the production of reactive species. Myoglobin oxidation to the met state by NO/ROS is a known sensor with absorbance changes in the visible range. We previously employed Förster resonance energy transfer to read out the deoxygenation/oxygenation of myoglobin, creating the subcellular [O2 ] sensor Myoglobin-mCherry. We now add the fluorescent protein EYFP to this sensor to create a novel probe that senses both met formation, a proxy for ROS/NO exposure, and [O2 ]. Since both proteins are present in the construct, it can also relieve users from the need to measure fluorescence lifetime, making [O2 ] sensing available to a wider group of laboratories.

Developing Analysis Protocols for Monitoring Intracellular Oxygenation Using Fluorescence Lifetime Imaging of Myoglobin-mCherry.

Oxygen (O2) is a critical metabolite for cellular function as it fuels aerobic cellular metabolism; further, it is a known regulator of gene expression. Monitoring oxygenation within cells and organelles can provide valuable insights into how O2, or lack thereof, both influences and responds to cell processes. In recent years, fluorescence lifetime imaging microscopy (FLIM) has been used to track several probe concentration independent intracellular phenomena, such as pH, viscosity, and, in conjunction with Förster resonance energy transfer (FRET), protein-protein interactions. Here, we describe methods for synthesizing and expressing the novel FLIM-FRET intracellular O2 probe Myoglobin-mCherry (Myo-mCherry) in cultured cell lines, as well as acquiring FLIM images using a laser scanning confocal microscope configured for two-photon excitation and a time-correlated single photon counting (TCSPC) module. Finally, we provide step-by-step protocols for FLIM analysis of Myo-mCherry using the commercial software SPCImage and conversion of fluorescence lifetime values in each pixel to apparent intracellular oxygen partial pressures (pO2).

Single cell-based fluorescence lifetime imaging of intracellular oxygenation and metabolism.

Oxidation-reduction chemistry is fundamental to the metabolism of all living organisms, and hence quantifying the principal redox players is important for a comprehensive understanding of cell metabolism in normal and pathological states. In mammalian cells, this is accomplished by measuring oxygen partial pressure (pO2) in parallel with free and enzyme-bound reduced nicotinamide adenine dinucleotide (phosphate) [H] (NAD(P)H) and flavin adenine dinucleotide (FAD, a proxy for NAD+). Previous optical methods for these measurements had accompanying problems of cytotoxicity, slow speed, population averaging, and inability to measure all redox parameters simultaneously. Herein we present a Förster resonance energy transfer (FRET)-based oxygen sensor, Myoglobin-mCherry, compatible with fluorescence lifetime imaging (FLIM)-based measurement of nicotinamide coenzyme state. This offers a contemporaneous reading of metabolic activity through real-time, non-invasive, cell-by-cell intracellular pO2 and coenzyme status monitoring in living cells. Additionally, this method reveals intracellular spatial heterogeneity and cell-to-cell variation in oxygenation and coenzyme states.

Genetically encoded FRET probes for direct mapping and quantification of intracellular oxygenation level via fluorescence lifetime imaging.

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 FRET-depleted 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].