Oliver Kohls

A modular luminescence lifetime imaging system for mapping oxygen distribution in biological samples

We developed a new modular luminescence lifetime imaging system (MOLLI), that enables the imaging of luminescence lifetimes in the range of 1 ms to 1 s. The system can easily be adapted to different experimental applications. The central parts of the system are a recently released CCD-camera with a fast electronic shutter and gated LED (light emitting diode) or Xe excitation light sources. A personal computer controls the gating and image acquisition via a pulse delay generator. Here we present the new imaging system and give examples of its performance when used for measuring two-dimensional oxygen distributions with planar optodes. Furthermore, future applications of the system in biology are discussed.

HETEROGENEITY OF OXYGEN PRODUCTION AND CONSUMPTION IN A PHOTOSYNTHETIC MICROBIAL MAT AS STUDIED BY PLANAR OPTODES

By applying planar optodes and imaging techniques to a benthic photosynthetic mat, we demonstrated an extensive vertical and horizontal variation in O2 concentrations, O2 consumption, and O2 production. In light, the oxic zone could be divided into three horizons: 1) an upper zone dominated by diatoms that had a moderate net O2 production, 2) another zone dominated by Microcoleus‐like cyanobacteria with a high net O2 production, and 3) a lower zone with disintegrating microalgae and cyanobacteria with a high O2 consumption rate. From the O2 images, the net O2 production/consumption was calculated at a spatial resolution of 130 μM. This allowed us to identify microsites with high rates of O2 turnover within the photic zone. Sites with high net O2 consumption (>1.5 nmol·cm−3·s−1) were typically situated next to sites with a relatively high net production (>2 nmol·cm−3·s−1), revealing a mosaic in which the highest O2 consumption sites were surrounded by the highest O2 production sites. This suggested a tight spatial coupling between production and consumption of O2 within the photic zone. Light stimulated the O2 consumption within the photic zone. At irradiances above 400 μmol photons·m−2·s−1, the stimulated O2 production was almost completely balanced by enhanced O2 consumption at microsites exhibiting net consumption of O2 even at maximum irradiance (578 μmol photons·m−2·s−1). Our observations strongly supported the idea that light‐stimulated respiration was caused by stimulated heterotrophic activity fueled by organic carbon leakage from the phototrophs. Despite microsites with high net O2 consumption, anoxic microniches were not encountered in the investigated mat. Images of gross photosynthetic rates also revealed an extensive horizontal variation in gross rates, with microsites of low or no photosynthesis within the otherwise photic zone. Calculations based on the obtained images revealed that at maximum light (578 μmol photons·m−2·s−1), 90% of the O2 produced was consumed within the photic zone. The presented data demonstrate the great potential offered by planar optode for studies of benthic photosynthetic communities.

Calcite dissolution driven by benthic mineralization in the deep-sea: in situ measurements of Ca2+, pH, pCO2 and O2

Abstract In situ measured microprofiles of Ca 2+ , pCO 2 , pH and O 2 were performed to quantify the CaCO 3 dissolution and organic matter mineralization in marine sediments in the eastern South Atlantic. A numerical model simulating the organic matter decay with oxygen was used to estimate the calcite dissolution rate. From the oxygen microprofiles measured at four stations along a 1300-m isobath of the eastern African margin and one in front of the river Niger at a water depth of 2200 m the diffusive oxygen uptake (DOU) and oxygen penetration depth (OPD) was calculated. DOU rates were in the range of 0.3 to 3 mmol m −2 d −1 and showed a decrease with increasing water depth, corresponding to an increase in OPD. The calculated amount of degradated organic matter is in the range of 1 to 8.5 gC m −2 a −1 . The metabolic CO 2 , released from mineralization of the organic matter drives calcite dissolution in these sediments overlain by calcite-supersaturated water. Fluxes across the sediment water interface calculated from the in situ Ca 2+ microprofiles were 0.6 mmol m −2 d −1 for two stations at a water depth of 1300 m. The ratio of calcite dissolution flux and organic C degradation is 0.53 and 0.97, respectively. The microprofiles indicate that CO 2 produced within the upper oxic sediment layer dissolves up to 85% of the calcite rain to the seafloor. Modeling our O 2 , pH and Ca 2+ profiles from one station predicted a calcite dissolution rate constant for this calcite-poor site of 1000 mol kgw −1 a −1 (mol per kg water and year), which equals 95% d −1 . This rate constant is at the upper end of reported in situ values.

Setup of a fiber optical oxygen multisensor-system and its applications in biotechnology

Abstract The development of a simple and very easy-to-use fiber optical oxygen multisensor measuring system is described. This fiber optical chemosensor (optode) is based on the fluorescence quenching of a special fluorophor by oxygen. Standard applications of the well-established amperometric oxygen sensor can be carried out with this optical sensor. Beyond that, many special applications for oxygen measurement become possible (O.S. Wolfbeis, Fiber Optic Chemical Sensors and Biosensors, vols. I and II, CRC Press, Boca Raton, USA, 1991), which cannot be performed by sensors of the Clark (L.C. Clark, Electrochemical device for chemical analysis, US Patent 2913386, 1956) type. In comparison to electrochemical sensors fiber optical chemosensors have several advantages, which are illustrated in this paper. Details of the sensor chemistry, instrumental system and the oxygen sensitivity are presented. The application in a precultivation processes, in Ca-alginate beads and layers with immobilized cells in packed-bed-reactors are presented. Diffusion coefficients were calculated from the experiments.

Adaptation, test and in situ measurements with O2 microopt(r)odes on benthic landers

Oxygen microopt(r)odes have recently been introduced as an alternative to microelectrodes in the field of aquatic biology. We here describe adaptation, test results and first in situ measurements made with O2 microopt(r)odes on deep-sea benthic landers. This includes a detailed description of the sensors, the mechanical mounting, and the necessary measuring system. Hydrostatic pressure effects on the sensors and the optical penetrators are evaluated and discussed. Further, in situ micoopt(r)ode data obtained by a profiling lander (Profilur) and a benthic chamber lander (Elinor) are presented, discussed and compared to measurements obtained simultaneously by Clark type O2 microelectrodes. The obtained data demonstrated that opt(r)odes are a realistic and good alternative to electrodes for landers and other measuring platforms during deep-sea deployments.

Development and comparison of pH microoptodes for use in marine systems

Traditionally microscale measurements of pH are based on potentiometric measurements with a pH glass microelectrode. The preparation of these electrodes is, however, very time consuming. We developed pH micro-optodes for use in seawater in the range of pH 7 - 9. The optodes are based on immobilized acid-base indicators, which change their color and/or fluorescence properties as a function of the pH. Various dyes were immobilized directly on the tip of a tapered optical fiber by different techniques. We then investigated these pH optodes with respect to response time, mechanical stability and calibration characteristics. Dependent on the optical properties of the indicator material we used different light emitting diodes (LEDs) as the light sources and either a photodiode or a photomultiplier as detector.

Characterization and application of temperature micro-optodes for use in aquatic biology

Benthic aquatic environments like biofilms or sediments are often investigation by measuring profiles of chemical or physical parameters at a high spatial resolution (< 50 micrometers ). This is necessary to understand e.g. transport processes and the biogeochemistry of the sediment water interface. A variety of electrochemical and optical microsensors has been developed and used for this purpose. In most of these applications the temperature of the investigated biofilms or sediments is assumed to be constant. However measurements with thermocouples of an appr. diameter of 300 micrometers have shown that this is not always the case for illuminated shallow water sediments and biofilms. We developed new microoptodes for measuring temperature distributions at a high spatial (< 50 micrometers ) and thermal (< 0.2 degree(s)C) resolution in aquatic systems. The new sensors are based on a fluorophore that is well known for its application in oxygen sensing-Ruthenium(II)- tris-1,10-phenantroline. Demas et al. (1992) discussed the possible use of highly luminescent transition metal complexes as temperature indicators. We have approached this idea from our experiences with ruthenium complexes as oxygen indicators. The first realized sensor consists of a closed microcapillary filled with an indicator solution and in inserted tapered optical fiber. The principle uses the temperature dependence of the fluorescence lifetime in the solution. To keep the solution oxygen free an oxygen scavenger is added to it. The change of the lifetime is detected by a special measuring device that uses a phase modulation technique.

Micro-optodes: the role of fibre tip geometry for sensor performance

Established sensors for fine scale measurements in natural environments are based on electrochemical measuring principles for e. g. oxygen and pH. The preparation of such electrochemical sensors is, however, a time consuming process.

Thionine as an indicator for use as a hydrogen sulfide optode

The amount of dissolved hydrogen sulfide is an important parameter in many environmental applications. Conventional methods for H2S detection depend on iodometric titration or spectroscopic measurements. Unfortunately these methods are not applicable for direct measurements in natural systems. A recently described method for the on-line detection of H2S is based on quenching of fluorescence of thioneine. The reaction between H2S and thioneine was described as reversible photo-reduction. This reaction was tested in order to design an optical microsensor for the measurement of H2S in sediments and other biological systems. We immobilized thioneine in several matrices and investigated these materials with respect to response time, mechanical stability, the influence of the excitation light and the reversibility. The sensing materials were deposited on the tip of optical fibers. The measuring system for the excitation and detection of the fluorescence consisted of a yellow light emitting diode, a fiber-optic coupler and a photomultiplier. The excitation light was intensity modulated to enable measurements in ambient light. Our results indicate that the thioneine based reaction scheme for H2S detection is not very suitable for use in a H2S optode due to lack of reversibility, long response times, and the need for regeneration of the sensor chemistry.

MICROX II: a new generation of portable measuring systems for micro-optodes

Sediments, microbial mats, biofilms and other microbial communities are characterized by steep gradients of physical and chemical parameters. Fibre optical microsensors, microoptodes, that we developed over the last three years have become powerful tools to investigate and measure these parameters with a sufficient spatial resolution and with a minor disturbance of the micro-environment in natural systems.