In the ideal case, a fluorescent sensor responds reversibly and fast (within a few seconds to few minutes) to changing analyte concentrations, which enables the visualization of the distribution of an analyte in a sample with a high spatial and temporal resolution. In fact, some sensors are capable of responding in the microsecond range, particularly in the case of oxygen- or temperature-sensitive probes used for instationary pressure and temperature measurements on surfaces. As the excitation of the fluorophores and fluorescence imaging can be performed in a remote way, optrodes or nanoprobes can be applied in a nondestructive way with a low impact on the sample. This paves the way for tracking chemical species in living systems.
Planar optrodes are useful tools in marine research, particularly in marine microbiology. The acidification of sea water due to the global increase in atmospheric CO
2 is a major problem for calcifying organisms such as shellfish and corals at the sea bottom. Seasonal changes in oxygen concentration and pH are further objects of systematic scientific studies. Oxygen penetration into sediments and its dynamics or oxygen consumption due to the degradation of organic matter can be also monitored with fluorescence imaging methods
[1].
2. Planar Optrodes
Imaging methods are mainly applied at sediment–water interfaces. Changes in the chemical composition in the depth of sediments are considered as relatively steady. However, the activities of benthic organisms can generate heterogeneities and complex 3D transport and reaction patterns over millimeter to meter scales in surficial sediments. The state of the art in 2D imaging techniques using planar optrodes to investigate biogeochemical processes in heterogeneous sediments and soils was reviewed by Li et al.
[9]. Scholz et al. reviewed different methods for mapping chemical gradients around seagrass roots, including planar-optrode-based techniques for O
2 and pH imaging
[10]. Optrodes together with other techniques for sensing pCO2 in seawater were summarized 2016 by Clarke et al.
[11].
2.1. Oxygen Sensors
Studying the impact of temperature and irradiance on benthic microalgal communities is one specific aspect of major interest. For this purpose, the effects of temperature and light on oxygen production and photosynthesis were studied by the 2D imaging of O
2 and recording chlorophyll A fluorescence
[12]. It was shown that irradiance increased pore-water O
2 concentration, sediment net O
2 production, and gross photosynthesis. Increasing temperatures stimulated the consumption of O
2 more than photosynthesis. Thus, the community becomes more heterotrophic at elevated temperatures. The authors concluded that the imaging approach demonstrates a great potential for studying environmental effects on photosynthetic activity and O
2 turnover in complex phototrophic benthic communities. The optrode was prepared from an iridium(III) complex (Ir(CS)
2acac) with polystyrene as a polymer binder. The sensors were integrated in a flume with two fiber optic faceplates as windows containing sediments and a read out with a CCD camera.
PtTFPP combined with a coumarin dye Macrolex
® fluorescent yellow 10GN as an antenna was dissolved in a polystyrene matrix, and the resulting oxygen sensor membrane was coated onto glass inserts. These were fitted into the front window of an incubation box to study the spatiotemporal dynamics of the reaction of manure solids with soil. Phosphorus release, oxygen consumption, and greenhouse gas emissions were recorded after the addition of organic fertilizers to soil
[13]. Planar optrodes of the same composition were utilized to map the O
2 micro-distribution around
Vallisneria spiralis roots. The availability of O
2 in the sediment–root interface is critical for the survival of macrophytes. Long-term imaging results were gathered during a 36-day period
[14]. The O
2 dynamics under different environmental conditions were also mapped in the rhizosphere of
L. hexandra. The results obtained with planar optrodes were compared to DGT-LA-ICP-MS (laser ablation inductively coupled plasma mass spectrometry) to investigate the mobilization mechanisms of trace metals triggered by radial oxygen loss and rhizosphere acidification. It was demonstrated that, with an increasing light intensity, air humidity, or atmospheric CO
2 concentrations, the O
2 concentrations and oxidized areas in the rhizosphere were significantly reduced
[15]. Li et al. investigated the O
2 distribution and dynamics in the rhizosphere of
Phragmites australis, and their impact on nutrient removal in sediments
[16].
The results from chemical imaging were compared with nitrogen porewater measurements to assess the extent of plant-induced changes in soil redox dynamics. These determine the spatio-temporal patterns in porewater O
2 and pH, which influence nitrogen biogeochemical cycling during dosed applications of nitrogen-rich artificial wastewater. Imaging using planar optrodes revealed O
2 fluxes to anoxic sediments via radial oxygen loss from
Typha latifolia roots
[17].
Commercial oxygen optrodes were used to study the spatial organization of bacterial populations. This is of importance, because the activity of microbes in soil is spatially inhomogenous, which influences biogeochemical processes. The sensors were placed in pore networks in soil and at a peripheral port. The generation of oxygen gradients was verified by the means of fluorescence imaging using the VisiSens system
[18].
2.2. Sensors for pH
A sensor system for 2D pH imaging in alkaline sediments and water was presented by Han et al. They prepared a dual luminophore sensor with the pH sensitive dye chlorophenyliminopropenyl aniline, which can be excited at 550 nm and emits red light with a maximum of 590 nm, and Macrolex
® fluorescence yellow 10 GN (emission at 502 nm) as a reference, embedded in PVC
[19]. Referenced imaging was performed with an RGB camera. The dynamic range of the sensor was between pH 7.5 and 10.5. Images were recorded in natural freshwater sediments and water associated with the photosynthesis of
Vallisneria spiral species.
Chemical imaging techniques can be also used to study dynamic pH and CO
2 distributions in the area of plant roots. These are the main drivers of processes in the rhizosphere. Planar optrodes were used in different set ups to monitor plant-root–soil interactions. Continuous and real-time measurements of the pH and CO
2 dynamics around roots and nodules of different plants were carried out. Again, the commercially available VisiSens system was used (see above) in combination with commercially available planar optodes for pH and CO
2 [20].
2.3. Multiple Sensor Systems
A multilayer planar optrode system for oxygen and pH imaging was built by embedding Platinum(II)octaethylporphyrin as an oxygen indicator and quantum dots as a reference in polystyrene, with a second layer consisting of the pH indicator 5-hexadecanoylamino-fluorescein in a Hydromed D4 matrix. An optical isolation layer containing carbon black in Hydromed D4 was coated on the top. All indicator and reference dyes could be excited by a 405 nm LED, while their emissions matched the red, green, and blue channels of a 3CCD camera. The set up was used to analyze the pH and oxygen dynamics of the seawater under the influence of rain drops
[21].
The spatial O
2 and CO
2 distribution around the roots of Lobelia under light and dark conditions was studied by integrating the commercially available VisiSens sensor foils for both parameters inside the walls of a rhizobox
[22]. Koop-Jakobsen et al. used the same system for taking high-resolution 2D-images of the O
2, pH, and CO
2 distributions around roots of the intertidal salt-marsh plant
Spartina anglica during alternating light–dark cycles by ratiometric fluorescence imaging. They demonstrated that the roots affected O
2, pH, and CO
2 dynamics, resulting in distinct gradients of these parameters in the rhizosphere
[23]. The sensor foils were placed in direct contact with the roots and sediment.
The most important chemical parameters associated with emissions from soil, NH
3, O
2, and pH microenvironments were analyzed with a triple optrode approach. A newly developed optrode for NH
3 with a limit of detection of 2.11 ppm and a large dynamic range up was fabricated by the immobilization of the fluorescent pH indicator Oxazine 170 perchlorate and an inert reference dye (Macrolex yellow) in a polyurethane hydrogel
[24]. The NH
3 optrode can be excited with a 470 nm LED, and fluorescence images were obtained using a 530 nm longpass filter. All three optrodes were integrated into an experimental set up within a thin soil layer which was sandwiched between two glass plates equipped with optrodes. Two NH
3 optrodes were placed on one side and the O
2 and pH optrodes on the other side, with a read out with ratiometric fluorescence imaging.
A new dual sensor system for the simultaneous imaging of O
2 and pH with low cross-talk was developed by Moßhammer et al. They combined an O
2-sensitive europium complex
[25] with a near-infrared emitting pH indicator based on BODIPY and an inert reference coumarin dye for ratiometric imaging. The sensor foil also contained diamant powder as a signal enhancer. The sensors were calibrated in transparent flow chambers and tested in photosynthetic microbial mats
[26].
The simultaneous sensing of pH and O
2 was used for the evaluation of bioactive cement
[27]. It contains bacteria which are capable of inducing CaCO
3 precipitation for the self-healing of cracks in concrete structures before leaking occurs. The pH is an important parameter determining the biocompatibility of concretes and cements. A ratiometric imaging system using pH-sensitive optrodes was developed to characterize the pH of porewater within the cracks of submerged hydrated oil and gas well cement. Again, the ratiometric optode for oxygen sensing consisted of PtTFPP and Macrolex fluorescence yellow as a reference dye, dispersed in polystyrene. The pH-sensitive optrode was composed of an aza-BODIPY dye with NIR emission and a high pK
a suited for measurements in highly alkaline concrete, and a green-emitting coumarin derivative as a reference dye in Hydromed D4 polymer. The authors showed that the pH was significantly reduced from pH > 11 to below 10 with an increasing fly ash content as well as hydration time. The bacterial activity was measured using the oxygen optrodes.
Similar to medical applications, the use of planar optrodes is restricted to very specific problems and conditions, e.g., for measurements in sediments or in the rhizosphere. Imaging systems have been designed that can be used on-site in marine environments or under laboratory conditions integrated in small basins or incubation boxes.
3. Micro- and Nanoparticles
Koren et al. developed an approach to overcome the typical limitations of optrodes
[28]. They studied the O
2 dynamics on the surface of living corals by sensor nanoparticles that were spray-painted on the corals by a conventional airbrush. The nanosensors consisted of PtTFPP in a styrene-maleic anhydride copolymer. The dye Macrolex
® fluorescence yellow 10GN was integrated as a reference. Imaging was performed with a ratiometric RGB camera set up, using the red channel for oxygen sensing and the green channel as a reference. The same type of particles was applied to visualize the O
2 dynamics around the roots of seagrasses. In the experimental set up, the below-ground tissue of the seagrass was embedded in an artificial sediment containing the O
2-sensitive nanoparticles. Images were recorded with a digital SLR camera and a 405 nm multichip LED, which was mounted perpendicular to the transparent chamber wall
[29].
The spatio-temporal distribution and dynamics of O
2 at biologically active surfaces with complex surface topography were quantified with oxygen-sensitive magnetic microparticles with a diameter of around 100 µm. These were prepared from a styrene-maleic acid anhydride copolymer containing the NIR-emitting luminophore platinum (II) meso-tetra(4-fluorophenyl)-tetrabenzoporphyrin
[30]. The particles were magnetized by the addition of lipophilic magnetite nanoparticles and titanium dioxide nanoparticles. The particles were distributed across the surface tissue of the scleractinian coral Caulastrea furcata and fixed with a magnet. Their luminescence response was recorded with a fluorescence lifetime imaging system. Thus, the lateral surface heterogeneity of the O
2 microenvironment across coral polyps exposed to flow could be mapped. Studying the steady-state O
2 concentrations under constant light and O
2 dynamics during experimental light–dark shifts enabled the identification of zones of different photosynthetic activities.