Optical oxygen micro- and nanosensors for plant applications

Abstract

Pioneered by Clark's microelectrode more than half a century ago, there has been substantial interest in developing new, miniaturized optical methods to detect molecular oxygen inside cells. While extensively used for animal tissue measurements, applications of intracellular optical oxygen biosensors are still scarce in plant science. A critical aspect is the strong autofluorescence of the green plant tissue that interferes with optical signals of commonly used oxygen probes. A recently developed dual-frequency phase modulation technique can overcome this limitation, offering new perspectives for plant research. This review gives an overview on the latest optical sensing techniques and methods based on phosphorescence quenching in diverse tissues and discusses the potential pitfalls for applications in plants. The most promising oxygen sensitive probes are reviewed plus different oxygen sensing structures ranging from micro-optodes to soluble nanoparticles. Moreover, the applicability of using heterologously expressed oxygen binding proteins and fluorescent proteins to determine changes in the cellular oxygen concentration are discussed as potential non-invasive cellular oxygen reporters.

Keywords: biosensors; dual-frequency phase‐modulation; endogenous sensor proteins; microsensors; nanosensors; oxygen sensor; phosphorescence quenching; plant science.

Figure 1.

Jablonski Diagram describing the possible states of the indicator molecule, i.e., a luminophore in the ground state (S) and after absorption of radiation (hv) to higher energetic electronic states, namely excited singlet (S*) and excited triplet states (T*). To return to the ground state, the excited molecule emits light of short lived (fluorescence) or long lived emission (phosphorescence), the latter involving a change in the electron spin, a radiationless process termed intersystem crossing (ISC). Molecules in the triplet state are prone to interact with other molecules, like oxygen, and during the process of collisional quenching energy is transferred to the oxygen molecule (O₂^T), resulting in singlet oxygen (O₂^S).

Figure 2.

Structural similarities of porphyrin, chlorophyll a and the oxygen probe PtPFPP.

Figure 3.

3D-plot of the oxygen concentration in a cell culture of chondrocytes after four days of growth. Every sphere represents one microprobe located within the sample. The colors of the spheres display the measured oxygen concentrations at their positions. The circulary arranged small black dots mark the position of the upright cylindrical cell carrier.