Time-resolved pH imaging in marine sediments with a luminescent planar optode

Abstract

A laboratory-based optical pH sensor for 2-dimensional pH imaging at benthic interfaces is presented. The sensor consists of a single-layer hydrogel matrix embedding the fluorescent pH indicator 2′, 7′-dihexyl-5(6)-Noctadecyl-carboxamidofluorescein ethyl ester (DHFAE) and the phosphorescent ruthenium(II)-Tris-4,7-diphenyl-1,10-phenanthroline incorporated in nanoparticles, serving as an inert reference standard. The measuring principle is based on time domain dual-lifetime referencing (t-DLR). The fluorophore/phosphor couple is simultaneously excited by a green LED (λ max 530 nm) pulsed in the microsecond range, and the sensor emission is recorded by a fast-gateable CCD camera. For each pH image, intensity images in 2 time windows (1 during and 1 after the excitation phase) are taken. The ratio of these 2 images is proportional to the pH of the sample and not affected by the overall signal intensity. The sensor has a dynamic range suitable for marine conditions (pH 7.3 to 9.3) with an apparent pK a of 8.3. The sensor was long-term stable (months) when kept in darkness and had a response time of < 200 s when going from pH 8.3 to 7.6. Light proved to have a negative effect on the sensor performance due to photobleaching of the pH indicator, resulting in a negative drift in the signal ratio at higher pH after prolonged light exposure. The spatial resolution (83 by 83 μm/pixel) of the sensor was capable of resolving smallscale spatial variability in the pH at a heterogeneous sediment-water interface, and time series of calibrated pH images also expressed a marked temporal variability in the pH distribution across this interface. Hotspots with intensified microbial activity were observed, and pH minima along burrow walls of polychaetes indicated elevated diagenetic activity in these zones. Individually extracted profiles from the pH images agreed well with independently measured pH microelectrode profiles, confirming the robustness of the approach.

A simple and inexpensive high resolution color ratiometric planar optode imaging approach: application to oxygen and pH sensing.

“…The ratio between the blue and the red image show a typical sigmoid relationship for pH calibration curves (Hulth et al 2002;Zhu et al 2005;Stahl et al 2006) (Fig. 6B).…”

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“…6B). The pK a value of 7.47 is slightly higher than those reported for other HPTSbased sensors (Zhu et al 2005;Hulth et al 2002) but lower than pH sensors based on the carboxyfluorescein derivative, 2′,7′-dihexyl-5(6)-N-octadecyl-carboxamidofluorescein ethyl ester (DHFAE) (Schröder et al 2005;Stahl et al 2006).…”

“…For future, in situ calibration values of pK a , R0, a, and b have to be determined before deployment. Due to the ion permeable hydrogel matrix, the pH sensor is affected by salinity (ionic strength) as with other described pH sensors (Zhu et al 2005;Stahl et al 2006). This should be taken into account when calibrating the sensor.…”

“…Recently, measurements in sea-ice have also provided promising results for resolving physical and biologically induced O 2 dynamics (Rysgaard et al 2008). Although studies on O 2 dominate the planar optode literature, recent advances also allow two-dimensional studies of pH (Hulth et al 2002;Zhu et al 2005;Stahl et al 2006), pCO 2 (Zhu et al 2006;Schröder et al 2007) and ammonia (Strömberg and Hulth 2005) in benthic substrates. There is a broad interest in applying planar optodes to benthic communities but the requirements of relatively complicated measuring systems has limited the number of users.…”

“…In the t-DLR system, the intensity of the indicator is referenced to the luminescent lifetime of a reference dye. The t-DLR has been used for pH imaging in marine systems (Schröder et al 2005) and for planar optode studies of marine sediments (Stahl et al 2006).…”