Material and methods

Two-dimensional (2D) oxygen measurements Planar oxygen optodes

The transparent and semi-transparent optodes consisted of 2 layers: a transparent polyester support foil (125 µm thick, Goodfellow) and a sensing layer spread on the support foil by knife-coating. The sensing layer was made of 10 mg of the luminescent oxygen indicator platinum(II) meso - tetra (pentafluorophenyl) porphyrin (Pt-PFP) (Porphyrin Products), 490 mg Polystyrene (Sigma-Aldrich) and 3 ml Chloroform (Merck). For semi-transparent optodes, 330 mg of titanium dioxide (TiO2) particles (< 5 µm, Aldrich) were added to the matrix solution. By this process, the optode loses its transparency and the sensor appears milky, however, the sensor matrix becomes more robust (Klimant et al. 1995). In the preparation of optically isolated optodes, an additional layer of black silicone was knife-coated on top of the dry sensing layer (drying in air for ~ 5 - 10 min). The thicknesses of the dry sensing layer and the black silicone layer were approx. 15 - 30 µm and 20 - 40 µm, respectively. The planar optodes were glued with transparent silicone (Elastosil® E4, Wacker) onto flat transparent walls of diverse setups

(e.g., wave tanks, aquaria, square sediment corers) to facilitate oxygen measurements in the studied samples.

Oxygen imaging system

2D oxygen measurements were conducted with the modular luminescence lifetime imaging (MOLLI) system (Holst et al. 1998; Holst and Grunwald 2001). Blue light emitting diodes (LEDs: HLMP-CB 15, λmax = 475 nm, Agilent, or Luxeon V Star Blue, λmax = 470 nm, Lumileds) were used to create an approximately homogeneous excitation light field to illuminate the planar optodes. The red luminescence (λmax = 647 nm) emitted by the optode was filtered by a red optical filter (Deep Golden Amber, LEE-Filters) to remove most of the reflected/scattered excitation light. A fast gateable CCD camera (SensiCam, PCO) with a resolution of 640 × 480 pixels recorded the luminescence images (Fig. 1).

Figure 1: Scheme of the general oxygen imaging system.

Oxygen scanning system

Apart from illuminating the planar optode with a light field from LEDs, blue light from a laser-diode module (PVLS-3000, λ= 404 nm, Toptica Photonics) was also used. The laser light was shaped by line optics (OZ Optics) to form a thin laser-line (~ 200 µm wide and ~ 4 cm long in the distance of ~ 10 cm). With the help of a motorised micro-positioner

excitation light field transparent setup wall sensing layer

support foil

array of blue LEDs emitted luminescence Computer

SensiCam

of the Stern-Volmer equation (Klimant et al. 1995; Holst et al. 1998). All experiments were conducted in the dark to minimise the effects of background light on the oxygen measurements. Further image processing was carried out with either a custom-made program developed in IDL® (Holst and Grunwald 2001) or Matlab® (Polerecky et al. 2005).

Calibration

Images of 0 and 100 % air saturation (AS) were taken before and after the experiments and served as calibration images. In contrast to a pixel-by-pixel calibration procedure (Glud et al. 1996; Wenzhöfer and Glud 2004), lifetime values averaged over several pixels (n > 100) served as calibration values. This was done because it was impossible to fix the used setups in such a way that the optode (e.g., fixed at a wall of an aquarium) and the camera remained precisely aligned during both the calibration procedure and the experiments.

One-dimensional (1D) oxygen measurements

For comparison, 1D high spatial resolution oxygen concentrations and oxygen microprofiles were measured with Clark-type oxygen microelectrodes (Revsbech 1989). The sensors had tip diameters of 10 – 20 µm, stirring sensitivities of < 1.5 % and response times of < 0.5 seconds. The microelectrodes were connected to a high-precision picoammeter whose signal was collected by a data-acquisition card (National Instruments) for PC data acquisition. Calibrations of the sensors were conducted at the experimental temperatures and salinities. Air-saturated water or water saturated with nitrogen gas as well as anoxic sediment parts, if present in the experiments, served for the determination of air saturation and zero oxygen calibration values, respectively. The sensors were attached to a micro-manipulator mounted on a motorised linear stage (VT-80, Micos) and placed above the sample, enabling reproducible positioning (precision +/- 1 µm) of the sensor tip.

Studied characteristics and experimental designs Light-guidance effect (“spatial cross-talk” effect)

Planar oxygen optodes glued onto the aquaria walls were calibrated prior to the measurements and the lifetime values of τ0 and τ100 corresponding to the 0% and 100% air saturation, respectively, were determined. The aquaria were subsequently filled with the intertidal mudflat sediment (German Wadden Sea) and a microbial mat (Jonkers et al. 2003). Oxygen images were calculated from the lifetime image measured by the MOLLI system using the calibration values τ0 and τ100.

Steady state 2D oxygen distributions and steep vertical oxygen gradients were used to determine the light guidance effects. Oxygen steady state in the microbial mats was achieved by illumination with a halogen lamp (~ 800 µmol photons m-2 s-1). In the aquarium filled with mudflat sediment, steep oxygen gradients developed as a result of oxygen consumption by the sediment and continuous bubbling of the overlying water with air. The aquaria were filled with the sediment so that the sediment depth was the same in all aquaria.

To test the light-guidance effect at different experimental conditions, different transparent materials were used as walls in the aquaria. These included polymethylmethacrylate (PMMA, Malon, custom-made, product nr. 16) and polycarbonate (PC), both ~ 7 mm thick, as well as quartz glass (Schott) of different thicknesses (2, 3, 5 and 10 mm). The surfaces of the PMMA and PC walls were coated with a special, scratch- resistant material (available as Axxis, Cadillac-Plastics or Lexan, Röhm or Makrolon, Bayer).

Signal-to-Noise ratio

The signal-to-noise ratio and accuracy of the planar optode were determined by repeated oxygen measurements (every 5 s) conducted in small flow-through aquaria, filled either with air saturated water or anoxic sediment. The oxygen concentrations were simultaneously measured with microelectrodes.

Temporal resolution

The temporal resolution was quantified by comparing parallel planar-optode and microelectrode measurements in oxygen-consuming intertidal permeable sediments and oxygen-producing/consuming Chiprana microbial mats. Oxygen concentrations in the permeable sediment were manipulated by starting and stopping the percolation with aerated water (flow-through method, as described by Polerecky et al. (2005)). Rapid

Long-term stability and robustness of semi-transparent planar optodes

Calibration characteristics of the semi-transparent planar optodes, i.e., the dynamic range and luminescence intensity, were determined before, during and after extensive studies conducted in a wave tank (Precht et al. 2004). In these experiments, propagating sediment ripples were frequently created, resulting in the movement of sand grains along the surface of the optodes. Additionally, square sampling cores equipped with semi- transparent optodes, which were extensively applied in the flow-through measurements (Polerecky et al., 2005), were used to assess the robustness and durability of the optodes.