An optical multifrequency phase-modulation method using microbeads for measuring intracellular oxygen concentrations in plants

Abstract

A technique has been developed to measure absolute intracellular oxygen concentrations in green plants. Oxygen-sensitive phosphorescent microbeads were injected into the cells and an optical multifrequency phase-modulation technique was used to discriminate the sensor signal from the strong autofluorescence of the plant tissue. The method was established using photosynthesis-competent cells of the giant algae Chara corallina L., and was validated by application to various cell types of other plant species.

FIGURE 1

Microinjection of the microbeads in a Chara cell (CC) using a glass microcapillary (M). The scale bar represents 500 μm.

FIGURE 2

Measurement setup: the built-in sinusoidal frequency generator of the lock-amplifier drives a cyan LED, which illuminates the sensor-containing cell. An optical glass fiber collects the sensor light and guides it to a photo multiplier. Optical filters minimize the excitation light intensity. A preamplifier converts the current signal into a voltage. Comparing the signals of detector and sinusoidal frequency generator, the lock-in amplifier evaluates the phase shift. A PC converts the phase shift into an oxygen concentration. The blue LED is used to stimulate photosynthesis.

FIGURE 3

(A) Luminescence spectra of the oxygen-sensor dye PtPFPP embedded in polystyrene microbeads. (Black line, excitation curve, with emission at 650 nm; dotted line, emission curve, with excitation at 509 nm; and dashed line, emission curve, with excitation at 390 nm.) (B) Luminescence spectra of a Chara corallina cell. (Black line, emission curve, with excitation at 509 nm; and dotted line, excitation curve, with emission at 687 nm.) For the oxygen measurements, the cells were illuminated with green light (540 ± 30 nm bandpass filter is put in front of the excitation LED), which keeps the autofluorescence to a minimum.

FIGURE 4

(A) Correlation of sensor phosphorescence lifetime and oxygen content. Albumin-preincubated PtPFPP microbeads were suspended in desalted water at 23°C and aerated with various oxygen concentrations. The plotted lifetimes are average values of four single measurements. The reproducibility of a single measurement was in the range of ±1 μs. The adapted Stern-Volmer equation Eq. 1 was fitted to the data for x = 0.75 (black line). The inset shows the temperature dependency of the sensor's phosphorescence lifetime determined at 100% air saturation oxygen content. (B) Measurements in Chara corallina cell sap yielded the same lifetimes as in desalted water. The number within the circles show the corresponding oxygen content (% air saturation) at which the phosphorescence lifetime was determined.

FIGURE 5

(A) Shows the phosphorescence lifetimes of the oxygen sensor at different pH values, determined at 100% oxygen air saturation, and 24°C. (B) Effect of carbohydrates, determined at 100% oxygen. Sucrose at 23°C (squares), glucose at 25°C (triangles). (C) Effect of protein concentration, determined at 100% oxygen air saturation and 24°C. (D) Illustration of the irreversibility of protein adsorption onto the microbeads. First, phosphorescence lifetime was measured using microbeads that were not pretreated with a protein incubation (1). When these microbeads were then used to measure τ in a 1.5 mg ml⁻¹ albumin solution with the same oxygen concentration as before, the phosphorescence lifetime increased (2). Once the beads were incubated in a protein solution, the phosphorescence lifetime of the beads became independent of the protein concentration of the solution (3). Measurements were done at 57% oxygen air saturation and 24°C.

FIGURE 6

Intracellular oxygen concentrations of a Chara corallina cell (A) and a cell of an orange fruit (B). In addition to the modulated green light used to excite the sensor, both cell types were temporarily exposed to unmodulated blue light to stimulate photosynthesis. In the photosynthesis competent Chara cell, the internal oxygen concentration rapidly increases when the blue light source was switched on. In the nonphotosynthetic orange cell, blue light had no effect on the internal oxygen concentration. The temperature was 23°C.