Novel Approach for Lifetime-Proportional Luminescence Imaging Using Frame Straddling.

Optode-based chemical imaging is a rapidly evolving field that has substantially enhanced our understanding of the role of microenvironments and chemical gradients in biogeochemistry, microbial ecology, and biomedical sciences. Progress in sensor chemistry has resulted in a broadened spectrum of analytes, alongside enhancements in sensor performance (e.g.

SlipOChip - single-cell respiration under tuneable environments.

In disciplines like toxicology and pharmacology, oxygen (O) respiration is a universal metric for evaluating the effects of chemicals across various model systems, including mammalian and microalgal cells. However, for these cells the common practice is to segregate populations into control and exposure groups, which assumes direct equivalence in their responses and does not take into account heterogeneity among individual cells. This lack of resolution impedes our ability to precisely investigate differences among experimental groups with small or limited sample sizes.

Microbial dietary preference and interactions affect the export of lipids to the deep ocean.

Lipids comprise a significant fraction of sinking organic matter in the ocean and play a crucial role in the carbon cycle. Despite this, our understanding of the processes that control lipid degradation is limited. We combined nanolipidomics and imaging to study the bacterial degradation of diverse algal lipid droplets and found that bacteria isolated from marine particles exhibited distinct dietary preferences, ranging from selective to promiscuous degraders.

Size-related variability of oxygen consumption rates in individual human hepatic cells.

Accurate descriptions of the variability in single-cell oxygen consumption and its size-dependency are key to establishing more robust tissue models. By combining microfabricated devices with multiparameter identification algorithms, we demonstrate that single human hepatocytes exhibit an oxygen level-dependent consumption rate and that their maximal oxygen consumption rate is significantly lower than that of typical hepatic cell cultures. Moreover, we found that clusters of two or more cells competing for a limited oxygen supply reduced their maximal consumption rate, highlighting their ability to adapt to local resource availability and the presence of nearby cells.

Exploring BPA alternatives - Environmental levels and toxicity review.

Bisphenol A alternatives are manufactured as potentially less harmful substitutes of bisphenol A (BPA) that offer similar functionality. These alternatives are already in the market, entering the environment and thus raising ecological concerns. However, it can be expected that levels of BPA alternatives will dominate in the future, they are limited information on their environmental safety.

PACMan: A software package for automated single-cell chlorophyll fluorometry.

Microalgae, small photosynthetic unicells, are of great interest to ecology, ecotoxicology and biotechnology and there is a growing need to investigate the ability of cells to photosynthesize under variable conditions. Current strategies involve hand-operated pulse-amplitude-modulated (PAM) chlorophyll fluorimeters, which can provide detailed insights into the photophysiology of entire populations- or individual cells of microalgae but are typically limited in their throughput. To increase the throughput of a commercially available MICROSCOPY-PAM system, we present the PAM Automation Control Manager ('PACMan'), an open-source Python software package that automates image acquisition, microscopy stage control and the triggering of external hardware components.

Simultaneous visualization of flow fields and oxygen concentrations to unravel transport and metabolic processes in biological systems.

From individual cells to whole organisms, O transport unfolds across micrometer- to millimeter-length scales and can change within milliseconds in response to fluid flows and organismal behavior. The spatiotemporal complexity of these processes makes the accurate assessment of O dynamics via currently available methods difficult or unreliable. Here, we present "sensPIV," a method to simultaneously measure O concentrations and flow fields.

Photophysiological response of Symbiodiniaceae single cells to temperature stress.

Photosynthetic dinoflagellates in the family Symbiodiniaceae engage in symbiosis with scleractinian corals. As coral 'bleaching' is partly governed by the thermal sensitivity of different Symbiodiniaceae lineages, numerous studies have investigated their temperature sensitivity. However, the systematic identification of single-cells with increased temperature resistance among these dinoflagellates has remained inaccessible, mostly due to a lack of technologies operating at the microscale.

Possible Functional Roles of Patellamides in the Ascidian- Symbiosis.

Patellamides are highly bioactive compounds found along with other cyanobactins in the symbiosis between didemnid ascidians and the enigmatic cyanobacterium . The biosynthetic pathway of patellamide synthesis is well understood, the relevant operons have been identified in the genome and genes involved in patellamide synthesis are among the most highly transcribed cyanobacterial genes in hospite. However, a more detailed study of the in vivo dynamics of patellamides and their function in the ascidian- symbiosis is complicated by the fact that remains uncultivated despite numerous attempts since its discovery in 1975.

Using High-Sensitivity Lipidomics To Assess Microscale Heterogeneity in Oceanic Sinking Particles and Single Phytoplankton Cells.

Sinking particulate organic matter (POM) is a primary component of the ocean's biological carbon pump that is responsible for carbon export from the surface to the deep sea. Lipids derived from plankton comprise a significant fraction of sinking POM. Our understanding of planktonic lipid biosynthesis and the subsequent degradation of lipids in sinking POM is based on the analysis of bulk samples that combine many millions of plankton cells or dozens of sinking particles, which averages out natural heterogeneity.

A microscopy-compatible temperature regulation system for single-cell phenotype analysis - demonstrated by thermoresponse mapping of microalgae.

This work describes a programmable heat-stage compatible with in situ microscopy for the accurate provision of spatiotemporally defined temperatures to different microfluidic devices. The heat-stage comprises an array of integrated thin-film Joule heaters and resistance temperature detectors (RTDs). External programming of the heat-stage is provided by a custom software program connected to temperature controllers and heater-sensor pairs.

Bistability in oxidative stress response determines the migration behavior of phytoplankton in turbulence.

Turbulence is an important determinant of phytoplankton physiology, often leading to cell stress and damage. Turbulence affects phytoplankton migration both by transporting cells and by triggering switches in migratory behavior, whereby vertically migrating cells can actively invert their direction of migration upon exposure to turbulent cues. However, a mechanistic link between single-cell physiology and vertical migration of phytoplankton in turbulence is currently missing.

Optofluidic Raman-activated cell sorting for targeted genome retrieval or cultivation of microbial cells with specific functions.

Stable isotope labeling of microbial taxa of interest and their sorting provide an efficient and direct way to answer the question "who does what?" in complex microbial communities when coupled with fluorescence in situ hybridization or downstream 'omics' analyses. We have developed a platform for automated Raman-based sorting in which optical tweezers and microfluidics are used to sort individual cells of interest from microbial communities on the basis of their Raman spectra. This sorting of cells and their downstream DNA analysis, such as by mini-metagenomics or single-cell genomics, or cultivation permits a direct link to be made between the metabolic roles and the genomes of microbial cells within complex microbial communities, as well as targeted isolation of novel microbes with a specific physiology of interest.

PhenoChip: A single-cell phenomic platform for high-throughput photophysiological analyses of microalgae.

Photosynthetic microorganisms are key players in aquatic ecosystems with strong potential for bioenergy production, yet their systematic selection at the single-cell level for improved productivity or stress resilience ("phenotyping") has remained largely inaccessible. To facilitate the phenotyping of microalgae and cyanobacteria, we developed "PhenoChip," a platform for the multiparametric photophysiological characterization and selection of unicellular phenotypes under user-controlled physicochemical conditions. We used PhenoChip to expose single cells of the coral symbiont to thermal and chemical treatments and monitor single-cell photophysiology via chlorophyll fluorometry.

Life in the dark: far-red absorbing cyanobacteria extend photic zones deep into terrestrial caves.

Chlorophyll (Chl) f and d are the most recently discovered chlorophylls, enabling cyanobacteria to harvest near-infrared radiation (NIR) at 700-780 nm for oxygenic photosynthesis. Little is known about the occurrence of these pigments in terrestrial habitats. Here, we provide first details on spectral photon irradiance within the photic zones of four terrestrial cave systems in concert with a detailed investigation of photopigmentation, light reflectance and microbial community composition.

A Robust Proton Flux (pHlux) Assay for Studying the Function and Inhibition of the Influenza A M2 Proton Channel.

The M2 protein is an important target for drugs in the fight against the influenza virus. Because of the emergence of resistance against antivirals directed toward the M2 proton channel, the search for new drugs against resistant M2 variants is of high importance. Robust and sensitive assays for testing potential drug compounds on different M2 variants are valuable tools in this search for new inhibitors.

In situ metabolomic- and transcriptomic-profiling of the host-associated cyanobacteria Prochloron and Acaryochloris marina.

The tropical ascidian Lissoclinum patella hosts two enigmatic cyanobacteria: (1) the photoendosymbiont Prochloron spp., a producer of valuable bioactive compounds and (2) the chlorophyll-d containing Acaryochloris spp., residing in the near-infrared enriched underside of the animal.

Chlorophyll f-driven photosynthesis in a cavernous cyanobacterium.

Chlorophyll (Chl) f is the most recently discovered chlorophyll and has only been found in cyanobacteria from wet environments. Although its structure and biophysical properties are resolved, the importance of Chl f as an accessory pigment in photosynthesis remains unresolved. We found Chl f in a cyanobacterium enriched from a cavernous environment and report the first example of Chl f-supported oxygenic photosynthesis in cyanobacteria from such habitats.

Rapid TaqMan-based quantification of chlorophyll d-containing cyanobacteria in the genus Acaryochloris.

Reports of the chlorophyll (Chl) d-containing cyanobacterium Acaryochloris have accumulated since its initial discovery in 1996. The majority of this evidence is based on amplification of the gene coding for the 16S rRNA, and due to the wide geographical distribution of these sequences, a global distribution of Acaryochloris species was suggested. Here, we present a rapid, reliable, and cost-effective TaqMan-based quantitative PCR (qPCR) assay that was developed for the specific detection of Acaryochloris species in complex environmental samples.

Reactive oxygen production induced by near-infrared radiation in three strains of the Chl d-containing cyanobacterium Acaryochloris marina.

Cyanobacteria in the genus Acaryochloris have largely exchanged Chl a with Chl d, enabling them to harvest near-infrared-radiation (NIR) for oxygenic photosynthesis, a biochemical pathway prone to generate reactive oxygen species (ROS). In this study, ROS production under different light conditions was quantified in three Acaryochloris strains (MBIC11017, HICR111A and the novel strain CRS) using a real-time ethylene detector in conjunction with addition of 2-keto-4-thiomethylbutyric acid, a substrate that is converted to ethylene when reacting with certain types of ROS. In all strains, NIR was found to generate less ROS than visible light (VIS).

Microenvironmental Ecology of the Chlorophyll b-Containing Symbiotic Cyanobacterium Prochloron in the Didemnid Ascidian Lissoclinum patella.

The discovery of the cyanobacterium Prochloron was the first finding of a bacterial oxyphototroph with chlorophyll (Chl) b, in addition to Chl a. It was first described as Prochloron didemni but a number of clades have since been described. Prochloron is a conspicuously large (7-25 μm) unicellular cyanobacterium living in a symbiotic relationship, primarily with (sub-) tropical didemnid ascidians; it has resisted numerous cultivation attempts and appears truly obligatory symbiotic.

Biofilm growth and near-infrared radiation-driven photosynthesis of the chlorophyll d-containing cyanobacterium Acaryochloris marina.

The cyanobacterium Acaryochloris marina is the only known phototroph harboring chlorophyll (Chl) d. It is easy to cultivate it in a planktonic growth mode, and A. marina cultures have been subject to detailed biochemical and biophysical characterization.

Microbial diversity of biofilm communities in microniches associated with the didemnid ascidian Lissoclinum patella.

We assessed the microbial diversity and microenvironmental niche characteristics in the didemnid ascidian Lissoclinum patella using 16S rRNA gene sequencing, microsensor and imaging techniques. L. patella harbors three distinct microbial communities spatially separated by few millimeters of tunic tissue: (i) a biofilm on its upper surface exposed to high irradiance and O(2) levels, (ii) a cloacal cavity dominated by the prochlorophyte Prochloron spp.