Choice of medium affects PBMC quantification, cell size, and downstream respiratory analysis.

High-resolution respirometry (HRR) can assess peripheral blood mononuclear cell (PBMC) bioenergetics, but no standardized medium for PBMC preparation and HRR analysis exist. Here, we study the effect of four different media (MiR05, PBS, RPMI, Plasmax) on the count, size, and HRR (Oxygraph-O2k) of intact PBMCs. Remarkably, the cell count was 21 % higher when PBMCs were resuspended in MiR05 than in PBS or Plasmax, causing O2 flux underestimation during HRR due to inherent adjustments. Moreover, smaller cell size and cell aggregation was observed in MiR05. Based on our findings, we propose that Plasmax, PBS or RPMI is more suitable than MiR05 for HRR of intact PBMCs. We provide oxygen solubility factors for Plasmax and PBS and encourage further optimization of a standardized HRR protocol for intact PBMCs.

Multiparameter Sensing of Oxygen and pH at Biological Interfaces via Hyperspectral Imaging of Luminescent Sensor Nanoparticles.

Chemical dynamics in biological samples are seldom stand-alone processes but represent the outcome of complicated cascades of interlinked reaction chains. In order to understand these processes and how they correlate, it is important to monitor several parameters simultaneously at high spatial and temporal resolution. Hyperspectral imaging is a promising tool for this, as it provides broad-range spectral information in each pixel, enabling the use of multiple luminescent indicator dyes, while simultaneously providing information on sample structures and optical properties. In this study, we first characterized pH- and O2-sensitive indicator dyes incorporated in different polymer matrices as optical sensor nanoparticles to provide a library for (hyperspectral) chemical imaging. We then demonstrate the successful combination of a pH-sensitive indicator dye (HPTS(DHA)3), an O2-sensitive indicator dye (PtTPTBPF), and two reference dyes (perylene and TFPP), incorporated in polymer nanoparticles for multiparameter chemical imaging of complex natural samples such as green algal biofilms (Chlorella sorokiniana) and seagrass leaves (Zostera marina) with high background fluorescence. We discuss the system-specific challenges and limitations of our approach and further optimization possibilities. Our study illustrates how multiparameter chemical imaging with hyperspectral read-out can now be applied on natural samples, enabling the alignment of several chemical parameters to sample structures.

Seagrass-mediated rhizosphere redox gradients are linked with ammonium accumulation driven by diazotrophs.

Seagrasses can enhance nutrient mobilization in their rhizosphere via complex interactions with sediment redox conditions and microbial populations. Yet, limited knowledge exists on how seagrass-derived rhizosphere dynamics affect nitrogen cycling. Using optode and gel-sampler-based chemical imaging, we show that radial O2 loss (ROL) from rhizomes and roots leads to the formation of redox gradients around below-ground tissues of seagrass (Zostera marina), which are co-localized with regions of high ammonium concentrations in the rhizosphere. Combining such chemical imaging with fine-scale sampling for microbial community and gene expression analyses indicated that multiple biogeochemical pathways and microbial players can lead to high ammonium concentration within the oxidized regions of the seagrass rhizosphere. Symbiotic N2-fixing bacteria (Bradyrhizobium) were particularly abundant and expressed the diazotroph functional marker gene nifH in Z. marina rhizosphere areas with high ammonium concentrations. Such an association between Z. marina and Bradyrhizobium can facilitate ammonium mobilization, the preferred nitrogen source for seagrasses, enhancing seagrass productivity within nitrogen-limited environments. ROL also caused strong gradients of sulfide at anoxic/oxic interfaces in rhizosphere areas, where we found enhanced nifH transcription by sulfate-reducing bacteria. Furthermore, we found a high abundance of methylotrophic and sulfide-oxidizing bacteria in rhizosphere areas, where O2 was released from seagrass rhizomes and roots. These bacteria could play a beneficial role for the plants in terms of their methane and sulfide oxidation, as well as their formation of growth factors and phytohormones. ROL from below-ground tissues of seagrass, thus, seems crucial for ammonium production in the rhizosphere via stimulation of multiple diazotrophic associations. IMPORTANCE: Seagrasses are important marine habitats providing several ecosystem services in coastal waters worldwide, such as enhancing marine biodiversity and mitigating climate change through efficient carbon sequestration. Notably, the fitness of seagrasses is affected by plant-microbe interactions. However, these microscale interactions are challenging to study and large knowledge gaps prevail. Our study shows that redox microgradients in the rhizosphere of seagrass select for a unique microbial community that can enhance the ammonium availability for seagrass. We provide first experimental evidence that Rhizobia, including the symbiotic N2-fixing bacteria Bradyrhizobium, can contribute to the bacterial ammonium production in the seagrass rhizosphere. The release of O2 from rhizomes and roots also caused gradients of sulfide in rhizosphere areas with enhanced nifH transcription by sulfate-reducing bacteria. O2 release from seagrass root systems thus seems crucial for ammonium production in the rhizosphere via stimulation of multiple diazotrophic associations.

Fine-scale mapping of physicochemical and microbial landscapes of the coral skeleton.

The coral skeleton harbours a diverse community of bacteria and microeukaryotes exposed to light, O2 and pH gradients, but how such physicochemical gradients affect the coral skeleton microbiome remains unclear. In this study, we employed chemical imaging of O2 and pH, hyperspectral reflectance imaging and spatially resolved taxonomic and inferred functional microbiome characterization to explore links between the skeleton microenvironment and microbiome in the reef-building corals Porites lutea and Paragoniastrea benhami. The physicochemical environment was more stable in the deep skeleton, and the diversity and evenness of the bacterial community increased with skeletal depth, suggesting that the microbiome was stratified along the physicochemical gradients. The bulk of the coral skeleton was in a low O2 habitat, whereas pH varied from pH 6-9 with depth. Physicochemical gradients of O2 and pH of the coral skeleton explained the ß-diversity of the bacterial communities, and skeletal layers that showed O2 peaks had a higher relative abundance of endolithic algae, reflecting a link between the abiotic environment and the microbiome composition. Our study links the physicochemical, microbial and functional landscapes of the coral skeleton and provides new insights into the involvement of skeletal microbes in the coral holobiont metabolism.

Think outside the box: 3D bioprinting concepts for biotechnological applications - recent developments and future perspectives.

3D bioprinting - the fabrication of geometrically complex 3D structures from biocompatible materials containing living cells using additive manufacturing technologies - is a rapidly developing research field with a broad range of potential applications in fundamental research, regenerative medicine and industry. Currently, research into 3D bioprinting is mostly focused on new therapeutic concepts for the treatment of injured or degenerative tissue by fabrication of functional tissue equivalents or disease models, utilizing mammalian cells. However, 3D bioprinting also has an enormous potential in biotechnology. Due to the defined spatial arrangement of biologically active (non-mammalian) cells in a biomaterial matrix, reaction compartments can be designed according to specific needs, or co-cultures of different cell types can be realized in a highly organized manner to exploit cell-cell interactions. Thus, 3D bioprinting technology can enable new biotechnological concepts, for example, by implementing perfusion systems while protecting shear sensitive cells or performing cascaded bioreactions. Here, we review the use of 3D bioprinting to manufacture defined 3D microenvironments for biotechnological applications using bacteria, fungi, microalgae, plant cells and co-cultures of different cell types. We discuss recent approaches to apply 3D bioprinting in biotechnological applications and - as it is a particular challenge - concepts for the real-time monitoring of the physiological state, growth and metabolic activity of the embedded cells in 3D bioprinted constructs. With these insights, we outline new applications of 3D bioprinting in biotechnology, engineered living materials and space research.

Hyperspectral Luminescence Imaging in Combination with Signal Deconvolution Enables Reliable Multi-Indicator-Based Chemical Sensing.

Although real-time monitoring of individual analytes using reversible optical chemical sensors (optodes) is well established, it remains a challenge in optical sensing to monitor multiple analyte concentrations simultaneously. Here, we present a novel sensing approach using hyperspectral imaging in combination with signal deconvolution of overlapping emission spectra of multiple luminescent indicator dyes, which facilitates multi-indicator-based chemical imaging. The deconvolution algorithm uses a linear combination model to describe the superimposed sensor signals and employs a sequential least-squares fit to determine the percent contribution of the individual indicator dyes to the total measured signal. As a proof of concept, we used the algorithm to analyze the measured response of an O2 sensor composed of red-emitting Pd(II)/Pt(II) porphyrins and NIR-emitting Pd(II)/Pt(II) benzoporphyrins with different sensitivities. This facilitated chemical imaging of O2 over a wide dynamic range (0-950 hPa) with a hyperspectral camera system (470-900 nm). The applicability of the novel method was demonstrated by imaging the O2 distribution in the heterogeneous microenvironment around the roots of the aquatic plant Littorella uniflora. The presented approach of combining hyperspectral sensing with signal deconvolution is flexible and can easily be adapted for use of various multi-indicator- or even multianalyte-based optical sensors with different spectral characteristics, enabling high-resolution simultaneous imaging of multiple analytes.

Substantial near-infrared radiation-driven photosynthesis of chlorophyll f-containing cyanobacteria in a natural habitat.

Far-red absorbing chlorophylls are constitutively present as chlorophyll (Chl) d in the cyanobacterium Acaryochloris marina, or dynamically expressed by synthesis of Chl f, red-shifted phycobiliproteins and minor amounts of Chl d via far-red light photoacclimation in a range of cyanobacteria, which enables them to use near-infrared-radiation (NIR) for oxygenic photosynthesis. While the biochemistry and molecular physiology of Chl f-containing cyanobacteria has been unraveled in culture studies, their ecological significance remains unexplored and no data on their in situ activity exist. With a novel combination of hyperspectral imaging, confocal laser scanning microscopy, and nanoparticle-based O2 imaging, we demonstrate substantial NIR-driven oxygenic photosynthesis by endolithic, Chl f-containing cyanobacteria within natural beachrock biofilms that are widespread on (sub)tropical coastlines. This indicates an important role of NIR-driven oxygenic photosynthesis in primary production of endolithic and other shaded habitats.

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

We describe a method to image dissolved oxygen (O2), in 2D at high spatial (< 50-100 µm) and temporal (< 10 s) resolution. The method employs O2 sensitive luminescent sensor foils (planar optodes) in combination with a specialized camera system for imaging luminescence lifetime in the frequency-domain. Planar optodes are prepared by dissolving the O2-sensitive indicator dye in a polymer and spreading the mixture on a solid support in a defined thickness via knife coating. After evaporation of the solvent, the planar optode is placed in close contact with the sample of interest - here demonstrated with the roots of the aquatic plant Littorella uniflora. The O2 concentration-dependent change in the luminescence lifetime of the indicator dye within the planar optode is imaged via the backside of the transparent carrier foil and aquarium wall using a special camera. This camera measures the luminescence lifetime (µs) via a shift in phase angle between a modulated excitation signal and emission signal. This method is superior to luminescence intensity imaging methods, as the signal is independent of the dye concentration or intensity of the excitation source, and solely relies on the luminescence decay time, which is an intrinsically referenced parameter. Consequently, an additional reference dye or other means of referencing are not needed. We demonstrate the use of the system for macroscopic O2 imaging of plant rhizospheres, but the camera system can also easily be coupled to a microscope.

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

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 O2 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.

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

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.

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

There is a strong need for techniques that can quantify the important reactive oxygen species hydrogen peroxide (H2O2) in complex media and in vivo. We combined chemiluminescence-based H2O2 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 H2O2 concentration and dynamics utilizing the chemiluminescent reaction of H2O2 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 H2O2 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 H2O2 quantification was used in different biological systems ranging from marine biology, using the model organism Exaiptasia pallida (light stress induced H2O2 release up to ~ 2.7 µM), over biomedical applications quantifying enzyme dynamics (glucose oxidase in a glucose solution producing up to ~ 60 µM H2O2 and the subsequent addition of catalase to monitor the H2O2 degradation process) and the ability of bacteria to modify their direct environment by regulating H2O2 concentrations in their surrounding media. This was shown by the bacteria Pseudomonas aeruginosa degrading ~ 18 µM background H2O2 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.

Seagrass-Mediated Phosphorus and Iron Solubilization in Tropical Sediments.

Tropical seagrasses are nutrient-limited owing to the strong phosphorus fixation capacity of carbonate-rich sediments, yet they form densely vegetated, multispecies meadows in oligotrophic tropical waters. Using a novel combination of high-resolution, two-dimensional chemical imaging of O2, pH, iron, sulfide, calcium, and phosphorus, we found that tropical seagrasses are able to mobilize the essential nutrients iron and phosphorus in their rhizosphere via multiple biogeochemical pathways. We show that tropical seagrasses mobilize phosphorus and iron within their rhizosphere via plant-induced local acidification, leading to dissolution of carbonates and release of phosphate, and via local stimulation of microbial sulfide production, causing reduction of insoluble Fe(III) oxyhydroxides to dissolved Fe(II) with concomitant phosphate release into the rhizosphere porewater. These nutrient mobilization mechanisms have a direct link to seagrass-derived radial O2 loss and secretion of dissolved organic carbon from the below-ground tissue into the rhizosphere. Our demonstration of seagrass-derived rhizospheric phosphorus and iron mobilization explains why seagrasses are widely distributed in oligotrophic tropical waters.