Elsevier

Sensors and Actuators B: Chemical

Volume 237, December 2016, Pages 1095-1101
Sensors and Actuators B: Chemical

In-vivo imaging of O2 dynamics on coral surfaces spray-painted with sensor nanoparticles

https://doi.org/10.1016/j.snb.2016.05.147Get rights and content

Highlights

  • Development of a spray-painting method for chemical imaging.

  • O2 sensitive nanoparticle sensors were used to study the surface oxygenation of a coral fragment.

  • O2 production and consumption of the coral were shown with high spatial and temporal resolution.

Abstract

We describe a new method for imaging O2 dynamics on heterogeneous biotic and abiotic surfaces using optical nanosensors. The method can overcome limitations of commonly used optodes or microsensors. To show the potential of the method, the O2 dynamics on the surface of living corals were studied. Small coral fragments were spray-painted with O2 sensor nanoparticles using a conventional airbrush and O2 imaging was done with a simple SLR camera setup. Good coverage of the coral surface was achieved without significantly compromising the coral wellbeing as shown by measuring photosynthetic activity of the photosymbionts at different light levels. The method enabled analysis of spatial heterogeneities in O2 concentration over the coral surface linked to different tissue types. Oxygenic photosynthesis and O2 consumption could be followed at a temporal resolution of <10 s.

Introduction

The concentration of molecular oxygen (O2) is a key environmental parameter in studies of aquatic organisms and ecosystems. The level of O2 is determined by a balance between photosynthetic production, consumption via respiration and other biogeochemical mineralization processes, and the mode of transport between O2 sources and sinks [1], [2], [3]. Aquatic biofilms, sediments, as well as plant and animal tissues exhibit a high cell density that leads to high volumetric rates of O2 turnover. In combination with mass transfer impedance due to the presence of a diffusive boundary layer (DBL) and absence of advective transport [4], such high volumetric O2 production/consumption leads to the establishment of steep concentration gradients between the biomass and the surrounding seawater [5], [6]. Although technically challenging, the characterization and quantification of such gradient microenvironments provides important insights to the regulation of oxic metabolism and photosynthesis including the balance between heterotrophic and autotrophic processes in symbiont-bearing corals, which is strongly affected by the incident light and can lead to shifts between hyperoxic conditions to hypoxia or even anoxia within <10–20 min of darkness [5].

Due to spatial heterogeneity and plasticity in the coral microenvironment, e.g. due to patchy distribution of the photosymbionts, changes in tissue thickness over a complex skeleton topography, tissue contraction/expansion cycles, and local variations in flow and boundary layer characteristics, coral O2 dynamics are complex [7], [8], [9]. Yet such links between structural organization, light and chemical microenvironments provide key insights to the efficiency of the coral symbiosis [10], [11].

Measurements of O2 microenvironments within corals are typically based on electrochemical [5], [12] or optical [13], [14], [15] microsensors. Such measurements can be done with extreme spatial (μm) and temporal (<0.2 s) resolution but are limited to a few spot measurements of local O2 concentration gradients. Due to sample heterogeneity and topography effects, it is thus difficult, if not impossible, to extrapolate microsensor measurements to larger scales. Imaging techniques can partly overcome this limitation when used in combination with optical sensor technology such as luminescent indicators that show reversible quenching of luminescence by O2 [16], [17], [18]. Such O2 imaging typically involves immobilization of the O2 indicators in a sensor film (planar optode) that is positioned relative to the system investigated [17], [19], [20], [21]. Imaging of the O2-dependent change in luminescence can be done via ratiometric luminescence intensity imaging systems [22], [23] or via luminescent lifetime imaging systems that monitor the O2-dependent change in the luminescence decay time [24]. A comparison between these different imaging approaches is given elsewhere [20].

In marine systems, planar optode-based O2 imaging has been applied in a range of different in situ and laboratory studies [25] of e.g. the seagrass rhizosphere [26], sediments [22], [27], microbial mats and biofilms [2], [28], [29], and endolithic communities in coral skeletons [30]. In such studies, it is crucial that the planar sensor film is in close contact to the system investigated. While this works for studying subsurface microenviroments and sample structures that can be pressed up against the planar optode, the lateral O2 distribution over a structurally complex surface topography as found in corals cannot be resolved with planar optodes. There is thus a need for new techniques that can resolve on O2 distribution and dynamics at high spatio-temporal resolution over larger surface areas exhibiting spatial heterogeneity.

A possible solution involves using O2-sensitive magnetic microparticles [31] in combination with life-time based O2 imaging [32]. Using this approach, it was possible to visualize surface O2 dynamics across relatively large (several mm wide) individual coral polyps, albeit with incomplete coverage of the tissue and the need of a strong magnet in the setup in order to keep the particles in place. Furthermore, such sensor microparticles exhibit a relatively slow response to dynamic changes in O2 e. g. upon light-dark shifts. Nanoparticle-based sensor can overcome these problems and enable fast measurement of oxygen dynamics [33], [34], [35].

In this study we present a fast and simple approach to image O2 dynamics on living tissue surfaces. The method is based on spray-painting surfaces with a suspension of O2 sensor nanoparticles and subsequent ratiometric imaging. We present details of sensor calibration and application, and show how this method can discriminate between the O2 microenvironment of different tissue types over a coral colony.

Section snippets

Nanosensor materials

Platinum(II) meso(2,3,4,5,6-pentafluoro)phenyl porphyrin (PtTFPP) was obtained from Frontier Scientific (www.frontiersci.com), and Macrolex® fluorescence yellow 10GN (MY) was obtained from Kremer Pigments (http://kremerpigments.com). The styrene maleic anhydride copolymer (PSMA with 8% MA, Mw: 250,000 g mol−1) XIRAN® was generously provided by Polyscope (http://www.polyscope.eu). Tetrahydrofuran (THF) was obtained from Sigma Aldrich.

Nanosensor preparation

The sensor nanoparticles were prepared according to a previously

Results and discussion

In this study, we evaluate the possibilty of 2D imaging of the O2 distribution over a coral surface. This was done by spraying the coral surface with a thin layer of optical sensor nanoparticles and subsequent ratiometric imaging. We note that this method is intended for laboratory investigations as an addition to currently used microsensor measurements [10], and the coating procedure does not enable in situ application. The method is evaluated by studying dynamics of O2 production and

Conclusion

We present a method for visualizing the distribution of O2 concentration and dynamics over the surface of living coral tissue spray-painted with O2 sensitive nanoparticles and subsequent ratiometric imaging with a simple camera system assembled from commercially available inexpensive components. The coral appeared tolerant to spray-painting and exhibited typical O2 dynamics in light and darkness and we could investigate such dynamics at high spatio-temporal resolution (∼0.1 mm resolution, and < 10 

Funding

The authors declare no competing financial interest.

Contributors

K.K. and M.K. designed the research. K.K., M.K., and S.L.J. conducted experiments. K.K., S.L.J., and M.K. analyzed the data. K.K. wrote the manuscript with editorial help from M.K. All authors have given approval to the final version of the manuscript.

Acknowledgements

This study was supported by grants from the Villum Foundation and the Danish Research Council for Independent Research | Natural Sciences & the Danish Research Council for Independent Research | Technical and Production Sciences. We thank Daniel A. Nielsen and Daniel Wangpraseurt (University of Technology Sydney) for advice on handling the coral samples. Sabrina Kapus is thanked for drawing the TOC picture.

Klaus Koren was born on July 1st 1985. He studied technical Chemistry at the Graz University of Technology, Austria. In 2008 he graduated and continued at this University with his PhD. During his Masters and PhD he worked in the Lab of Prof. Ingo Klimant. At this time he focused on the development of new optical oxygen sensors, indicators and sensor materials. He finished his PhD in 2012. Afterwards he moved towards applying optical sensors in a biological context. In 2014 he joined the lab of

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    Klaus Koren was born on July 1st 1985. He studied technical Chemistry at the Graz University of Technology, Austria. In 2008 he graduated and continued at this University with his PhD. During his Masters and PhD he worked in the Lab of Prof. Ingo Klimant. At this time he focused on the development of new optical oxygen sensors, indicators and sensor materials. He finished his PhD in 2012. Afterwards he moved towards applying optical sensors in a biological context. In 2014 he joined the lab of Prof. Kühl working on development and application of optical sensors in marine environments.

    Sofie L. Jakobsen was born on December 21st 1986 in Denmark. She obtained an education as biomedical laboratory technician in 2010. In 2014 she joined the lab of Prof. Kühl as a technician. She is involved in different projects ranging from high resolution structure analysis (SEM and TEM) to culturing of marine species.

    Michael Kühl was born on June 16, 1964. He received his M.Sc. in biology (1988) and Ph.D. in microbiology (1992) from the University of Aarhus, Aarhus (Denmark). From 1992–1998 he established and headed the microsensor research group at the Max-Planck-Institute for Marine Microbiology, Bremen (Germany) developing electrochemical and fiber-optic microsensors and advanced imaging techniques for environmental analysis. Since 1998 he has continued this research at the Marine Biological Section, Department of Biology, University of Copenhagen (Denmark) where he is full professor in microbial ecology and heads the Microenvironmental Ecology research group. He is a member of the Royal Danish Academy of Sciences and Letters, associate editor of Marine Biology, Aquatic Microbiology, Environmental Biology, and Faculty of 1000/Environmental Microbiology.

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