Oxygen is one of the most fundamental elements for animals, plants, fungi, and bacterial systems. Real-time measurement of oxygen concentration, transport, and respiration in living cells is crucial to answering physiological questions of development, metabolism, and stress response. There have been significant efforts to measure oxygen transport and metabolism in living cells and tissues1,2,3,4. Early efforts were based on polarograghic electrochemical approaches where a current is measured as a function of oxygen reduction. These electrode-based methods all face limitations because of susceptibility to electromagnetic interference, convective artifacts, and calibration drift, which create high background noise5 and a requirement for constant recalibration. It is possible to partially alleviate convective artifacts from the electrode-based methods by reducing the diameter of an electrode and covering the electrode by a gas-permeable membrane5 but this in turn increases the susceptibility to electromagnetic noise.
The self-referencing technique can provide a reliable solution to measuring the analytes flux associated with living cells. To monitor oxygen flux and respiratory activity in single cells or/and tissues, microelectrode-based electrochemical polarographic methods were adapted in the last decade3,6,7,8. However, the electrochemical microelectrode approach still suffered from experimental artifacts when high sensitivity and accuracy of measurement are required because the electrode sensitivity to electromagnetic noise, fouling, and calibration drift. Therefore, an optical sensor, the so-called optrode (optical electrode), was developed to minimize these disadvantages9,10,11,12.
An optrode is an optical fiber with a specific fluorescence dye (platinum tetrakis pentafluorophenyl porphyrin or PtTFPP in this study), immobilized on the tip of a tapered fiber optic10. The PtTFPP is excited by blue light (505 nm), and the red emission fluorescence signal (640 nm) is conducted through the fiber and recorded by optical equipment. The concentration of analytes, oxygen in this case, changes the lifetime and intensity of the fluorescence signals9,10. These measurable characters of fluorescence signals reflect the concentration of the analyte in a linear relationship (Box 1). The application of an optical fiber prevents corrosion of metallic probes in buffers or physiologic solutions, and the recording of light signals minimizes electromagnetic noise. Furthermore, the measurement of fluorescence duration has significant advantages over measurement of fluorescence intensity, in terms of stability and photobleaching of fluorescence dyes 9,10. Based on applying the principles of frequency domain lifetime approaches, shifts in the phase angle of fluorescence signals are measured for the NMT-OO system.
The NMT can provide non-invasive measurement of the flux of analytes in living cells and tissues. Based on Fick’s law [J = –D(ΔC/ΔX)], we can calculate the flux rate (J) by measuring concentration differences (ΔC) using a microsensor, which oscillates between two positions (ΔX), if the diffusion coefficient (D) is known10. In the current study, we used a newly developed oxygen-specific optrode with high sensitivity and a high signal-to-noise ratio (SNR). Furthermore, when using an optrode with NMS, there is no need to use a reference electrode, and thus the system is simple to construct. This construction helps in decreasing experimental artifacts and errors (Box 2).
The oxygen transport and respiratory activity of plant cell tissues reflect spatial and temporal information about the physiological responses of cell metabolism and stress responses11. Here we compared oxygen metabolism and flux rate on the root surface of wild-type (WT) Arabidopsis and mutant lines of the atrbohD/F double mutant, which lacks the expression of membrane-localized NADPH-oxidase, resulting in a reduced rate of root elongation by inhibition of cell expansion and growth13,14. We also compared rhizosphere oxygen flux rate in light- and dark-grown Arabidopsis seedlings, which show different elongation rates in the elongation zone 15. These results documented differences in respiratory oxygen flux that correlate with root growth, and confirm the NMT-OO approach as a low-cost, easy-to-use instrument for detecting oxygen transport and metabolism with high sensitivity and a high SNR.
The following is a detailed protocol for the complete construction of this optrode-based NMT and measurement of rhizosphere oxygen flux in Arabidopsis seedlings.