Sensor principles and calibration

In contrast to CO₂, Clark-type O₂ microsensors have long been available to plant scientists, enabling continuous measurements of tissue O₂ with high temporal and spatial resolution (Clark, 1956; Armstrong, 1979; Revsbech, 1989). CO₂ microsensors of the Severinghaus type (Severinghaus and Bradley, 1958) are available, but these are based upon indirect measurements of CO₂ via changes in pH using a pH-sensitive transducer embedded in a small reservoir with bicarbonate and carbonic anhydrase (CA) to speed up hydration of CO₂ (Caflisch et al., 1979; De Beer et al., 1997; Zhao and Cai, 1997). Due to the pH-sensitive transducer, these sensors are sluggish and respond to external CO₂ in a logarithmic fashion with poor resolution at high CO₂ concentrations.

In the present study, leaf tissue CO₂ and O₂ were measured using custom-built microsensors. The novel CO₂ microsensor with a tip diameter of 35 μm consisted of an outer casing sealed at the very tip with a gas-permeable silicone membrane (Fig. 1A). Behind the membrane, there was a reservoir holding a chemical O₂ scavenger (1 mol CrCl₂ L^–1 in 0.1 mol HCl L^–1) to prevent O₂ interference with CO₂. The tip of the CO₂ transducer was positioned within the outer casing about 80 μm behind the outer membrane. The microsensor was polarized at –1.2 V and had a linear response to CO₂ in the external medium up to at least 1400 μmol L^–1 (4 kPa), and was insensitive to O₂ (Fig. 1B). When the microsensor was not used, the tip was stored in an alkaline ascorbate solution (zero O₂ and CO_2; see calibration of O₂ microsensor) to keep the zero current low and to extend the life time of the chemical O₂ scavenger.

Calibrations of the CO₂ microsensor were carried out at 20 °C at three different CO₂ concentrations in artificial lake water (always at 10 % concentration; for composition see ‘Plant material’) of low and high pO₂ to check possible interference from O₂. A calibration solution of zero CO₂ and zero O₂ was obtained by bubbling the artificial lake water without any inorganic carbon added and at pH >11 with high purity N₂ for 1 h; at high pH, all inorganic carbon is converted into CO₃^2– (although inorganic carbon was not added to the solution, it might still have dissolved from atmospheric air) and the N₂ removes (purges out) dissolved O₂ and also CO₂. A solution with zero CO₂ but with approx. 40 kPa pO₂ was obtained by mixing solutions purged with high purity N₂ or O₂ and adjusted to pH >11. Calibration solutions with either zero O₂ or approx. 40 kPa pO₂ but with a CO₂ concentration of about 700 or 1400 μmol L^–1 (corresponding to approx. 2085 and 4071 Pa pCO₂) were prepared by injecting known amounts of KHCO₃ into acidified (pH <2 using HCl) artificial lake water prepared as above. The CO₂ microsensor used in the present study did not show any interference with O₂ within the range tested (0–40 kPa pO₂, Fig. 1B). The CO₂ microsensor was calibrated prior to each experiment, and calibration and O₂ sensitivity were again checked after each experiment, at 24–30 h after the first calibration depending on the experimental design. If calibrations before and after measurements differed, corrections were performed by assuming linear drift in signal over time.

The O₂ microsensor (tip diameter = 25 μm) used for tissue O₂ measurements was constructed according to Revsbech (1989) and polarized at –0.8 V. It was calibrated at zero O₂ (approx. 2 g of ascorbate in 100 mL of deionized H₂O in 0.1 N NaOH) and at air equilibrium (284 mmol O₂ m^–3 at 20 °C, corresponding to 20.6 kPa pO₂). The sensor has previously been shown to respond linearly to O₂ up to 101 kPa pO₂ (Revsbech, 1989).

The CO₂ and the O₂ microsensors were connected to a picoampere meter (Field Multimeter, Unisense A/S, Denmark) and the sensor signals were collected with a frequency of one sample per minute using data acquisition software (SensorTrace Suite 2.8, Unisense).