Luminescence Lifetime Imaging of O<sub>2</sub> with a Frequency-Domain-Based Camera System

We describe a method to image dissolved oxygen (O2), in 2D at high spatial (&#60; 50-100 &#181;m) and temporal (&#60; 10 s) resolution. The method employs ...

Aarhus University

There is a strong need for techniques that can quantify the important reactive oxygen species hydrogen peroxide (HO) in complex media and in vivo. We combined chemiluminescence-based HO measurements on a commercially available flow injection analysis (FIA) system with sampling of the analyte using microdialysis probes (MDPs), typically used for measurements in tissue. This allows minimally invasive, quantitative measurements of extracellular HO concentration and dynamics utilizing the chemiluminescent reaction of HO with acridinium ester. By coupling MDPs to the FIA system, measurements are no longer limited to filtered, liquid samples with low viscosity, as sampling via a MDP is based on a dynamic exchange through a permeable membrane with a specific cut-off. This allows continuous monitoring of dynamic changes in HO concentrations, alleviates potential pH effects on the measurements, and allows for flexible application in different media and systems. We give a detailed description of the novel experimental setup and its measuring characteristics along with examples of application in different media and organisms to highlight its broad applicability, but also to discuss current limitations and challenges. The combined FIA-MDP approach for HO quantification was used in different biological systems ranging from marine biology, using the model organism Exaiptasia pallida (light stress induced HO release up to ~ 2.7 µM), over biomedical applications quantifying enzyme dynamics (glucose oxidase in a glucose solution producing up to ~ 60 µM HO and the subsequent addition of catalase to monitor the HO degradation process) and the ability of bacteria to modify their direct environment by regulating HO concentrations in their surrounding media. This was shown by the bacteria Pseudomonas aeruginosa degrading ~ 18 µM background HO in LB-broth. We also discuss advantages and current limitations of the FIA-MDP system, including a discussion of potential cross-sensitivity and interfering chemical species.

Extracellular hydrogen peroxide measurements using a flow injection system in combination with microdialysis probes - Potential and challenges.

The composition and development of naturally occurring microbial communities are defined by a complex interplay between the community and the surrounding environment and by interactions between community members. Intriguingly, these interactions can in some cases cause synergies, where the community is able to outperform its single-species constituents. However, the underlying mechanisms driving community interactions are often unknown and difficult to identify due to high community complexity. Here, we show how opposite pH drift induced by specific community members leads to pH stabilization of the microenvironment, acting as a positive interspecies interaction, driving  community synergy in a model consortium of four coisolated soil bacteria, , , , and  We use microsensor pH measurements to show how individual species change the local pH microenvironment and how cocultivation leads to a stabilized pH regime over time. Specifically,  acid production from  and alkali production primarily from  led to an overall pH stabilization of the local environment over time, which in turn resulted in enhanced community growth. This specific type of interspecies interaction was found to be highly dependent on medium type and concentration; however, similar pH drift from the individual species could be observed across medium variants. Understanding interspecies interactions in bacterial communities is important for unraveling species dynamics in naturally occurring communities. These dynamics are fundamental for identifying evolutionary drivers and for the development of efficient biotechnological industry applications. Recently, pH interplay among community members has been identified as a factor affecting community development, and pH stabilization has been demonstrated to result in enhanced community growth. The use of model communities in which the effect of changing pH level can be attributed to specific species contributes to the investigation of community developmental drivers. This contributes to assessment of the extent of emergent behavior and members' contributions to community development. Here, we show that pH stabilization of the microenvironment  in a synthetic coisolated model community results in synergistic growth. This observation adds to the growing diversity of community interactions leading to enhanced community growth and hints toward pH as a strong driver for community development in diverse environments.

 Community Synergy between Bacterial Soil Isolates Can Be Facilitated by pH Stabilization of the Environment.

Most aquatic systems rely on a multitude of biogeochemical processes that are coupled with each other in a complex and dynamic manner. To understand such processes, minimally invasive analytical tools are required that allow continuous, real-time measurements of individual reactions in these complex systems. Optical chemical sensors can be used in the form of fiber-optic sensors, planar sensors, or as micro- and nanoparticles (MPs and NPs). All have their specific merits, but only the latter allow for visualization and quantification of chemical gradients over 3D structures. This review (with 147 references) summarizes recent developments mainly in the field of optical NP sensors relevant for chemical imaging in aquatic science. The review encompasses methods for signal read-out and imaging, preparation of NPs and MPs, and an overview of relevant MP/NP-based sensors. Additionally, examples of MP/NP-based sensors in aquatic systems such as corals, plant tissue, biofilms, sediments and water-sediment interfaces, marine snow and in 3D bioprinting are given. We also address current challenges and future perspectives of NP-based sensing in aquatic systems in a concluding section. Graphical abstract ᅟ.

Nanoparticle- and microparticle-based luminescence imaging of chemical species and temperature in aquatic systems: a review.

Luminescence lifetime based imaging is still the most reliable method for generating chemical images using chemical sensor technology. However, only few commercial systems are available that enable imaging lifetimes within the relevant nanosecond to microsecond range. In this technical note we compare the performance of an older time-domain (TD) based camera system with a frequency-domain (FD) based camera system regarding their measuring characteristics and applicability for O and pH imaging in environmental samples and with different indicator dye systems emitting in the visible and near-infrared part of the spectrum. We conclude that the newly introduced FD imaging system delivers comparable if not better results than its predecessor, now enabling robust and simple chemical imaging based on FD luminescence lifetime measurements.

Luminescence Lifetime Imaging of Chemical Sensors-A Comparison between Time-Domain and Frequency-Domain Based Camera Systems.

Cable bacteria belonging to the family Desulfobulbaceae couple sulfide oxidation and oxygen reduction by long-distance electron transfer over centimeter distances in marine and freshwater sediments. In such habitats, aquatic plants can release oxygen into the rhizosphere. Hence, the rhizosphere constitutes an ideal habitat for cable bacteria, which have been reported on seagrass roots recently. Here, we employ experimental approaches to investigate activity, abundance, and spatial orientation of cable bacteria next to the roots of the freshwater plant Littorella uniflora. Fluorescence in situ hybridization (FISH), in combination with oxygen-sensitive planar optodes, demonstrated that cable bacteria densities are enriched at the oxic-anoxic transition zone next to roots compared to the bulk sediment in the same depth. Scanning electron microscopy showed cable bacteria along root hairs. Electric potential measurements showed a lateral electric field over centimeters from the roots, indicating cable bacteria activity. In addition, FISH revealed that cable bacteria were present in the rhizosphere of Oryza sativa (rice), Lobelia cardinalis and Salicornia europaea. Hence, the interaction of cable bacteria with aquatic plants of different growth forms and habitats indicates that the plant root-cable bacteria interaction might be a common property of aquatic plant rhizospheres.

The rhizosphere of aquatic plants is a habitat for cable bacteria.

Coastal eutrophication is a growing problem worldwide, leading to increased epiphyte overgrowth of seagrass leaves. Yet little is known about how epiphytes affect key biogeochemical conditions and processes in the seagrass phyllosphere. We used electrochemical microsensors to measure microgradients of O , pH, and CO at the bare and epiphyte-covered leaf surface of seagrass (Zostera marina L.) to determine effects of epiphytes on the leaf chemical microenvironment. Epiphytes result in extreme daily fluctuations in pH, O , and inorganic carbon concentrations at the seagrass leaf surface severely hampering the plant's performance. In light, leaf epiphyte biofilms and their diffusive boundary layer lead to strong basification, markedly reducing the CO and HCO availability at the leaf surface, leading to reduced photosynthetic efficiency as a result of carbon limitation and enhanced photorespiration. With epiphytes, leaf surface pH increased to >10, thereby exceeding final pH levels (~9.62) and CO compensation points for active photosynthesis. In darkness, epiphyte biofilms resulted in increased CO and hypoxia at the leaf surface. Epiphytes can lead to severe carbon limitation in seagrasses owing to strong phyllosphere basification leading to CO depletion and costly, yet limiting, HCO utilization, increasing the risk of plant starvation.

Strong leaf surface basification and CO limitation of seagrass induced by epiphytic biofilm microenvironments.

Microbially induced CaCO precipitation (MICP) can give concrete self-healing properties. MICP agents are typically bacterial endospores which are coated into shelled granules, infused into expanded clay, or embedded into superabsorbent polymer (SAP). When small cracks appear in the cured concrete, the encapsulation is broken and the metabolic CO production from the germinated bacteria causes healing of the cracks by precipitation of CaCO. Such systems are being tested empirically at large scales, but survival of endospores through preparation and application, as well as germination and growth kinetics of the germinated vegetative cells, remains poorly resolved. We encapsulated endospores of Bacillus subtilis and Bacillus alkalinitrilicus in crosslinked acrylamide-based SAP and quantified their germination, growth, and, in the case of B. alkalinitrilicus, CaCO precipitation potential. The endospores survived crosslinking and desiccation inside the polymer matrix. Microcalorimetry and microscopy showed that ~ 80% of the encapsulated endospores of both strains readily germinated after rehydration of freeze-dried SAP. Germinated cells grew into dense colonies of cells inside the SAP, and those of B. alkalinitrilicus calcified with up to 0.3 g CaCO produced per g desiccated SAP when incubated aerobically. Measurements by planar optodes indicated that the precipitation rates were inherently oxygen limited due to diffusional constraints, rather than limited by electron donor or Ca availability. Such oxygen limitation will limit MICP in all water-saturated and oxygen-dependent systems, and MICP agents based on anaerobic bacteria, e.g., nitrate reducers, should be developed to broaden the applicability of bioactive self-healing concretes to wet and waterlogged environments.

Constraints on CaCO precipitation in superabsorbent polymer by aerobic bacteria.

Leakage from cementitious structures with a retaining function can have devastating environmental consequences. Leaks can originate from cracks within the hardened cementitious material that is supposed to seal the structure off from the surrounding environment. Bioactive self-healing concretes containing bacteria capable of microbially inducing CaCO precipitation have been suggested to mitigate the healing of such cracks before leaking occurs. An important parameter determining the biocompatibility of concretes and cements is the pH environment. Therefore, a novel ratiometric pH optode imaging system based on an inexpensive single-lens reflex (SLR) camera was used to characterize the pH of porewater within cracks of submerged hydrated oil and gas well cement. This enabled the imaging of pH with a spatial distribution in high resolution (50 μm per pixel) and a gradient of 1.4 pH units per 1 mm. The effect of fly ash substitution and hydration time on the pH of the cement surface was evaluated by this approach. The results show that pH is significantly reduced from pH >11 to below 10 with increasing fly ash content as well as hydration time. The assessment of bioactivity in the cement was evaluated by introducing superabsorbent polymers with encapsulated  endospores into the cracks. The bacterial activity was measured using oxygen optodes, which showed the highest bacterial activity with increasing amounts of fly ash substitution in the cement, correlating with the decrease in the pH. Overall, our results demonstrate that the pH of well cements can be reliably measured and modified to sustain the microbial activity.

Klaus Koren

Aarhus University Centre for Water Technology,
Section for Microbiology,
Department of Bioscience,
Aarhus University Centre for Water Technology, Section for Microbiology, Department of Bioscience

Aarhus University

Luminescence Lifetime Imaging of O₂ with a Frequency-Domain-Based Camera System

Klaus Koren

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Aarhus University Centre for Water Technology,Section for Microbiology,Department of Bioscience,Aarhus University Centre for Water Technology, Section for Microbiology, Department of Bioscience

Aarhus University

Luminescence Lifetime Imaging of O2 with a Frequency-Domain-Based Camera System

Aarhus University Centre for Water Technology,
Section for Microbiology,
Department of Bioscience,
Aarhus University Centre for Water Technology, Section for Microbiology, Department of Bioscience

Luminescence Lifetime Imaging of O₂ with a Frequency-Domain-Based Camera System