In disciplines like toxicology and pharmacology, oxygen (O2) 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. To overcome this barrier, we introduce SlipO2Chip, an innovative glass microfluidic platform for precisely quantifying single-cell O2 respiration in the coordinated absence and presence of chemical solutes. SlipO2Chip comprises a wet-etched fused silica channel plate on the top and a dry-etched borosilicate microwell plate at the bottom. The microwells are coated with Pt(II) meso-tetra(pentafluorophenyl)porphine (PtTFPP), an O2 sensing optode material and an O2-independent reference dye. A custom 3D-printed holder facilitates the controlled horizontal movement ('slipping') of the channel plate over the microwell plate, thereby establishing or disrupting the fluid path over microwells. Collectively, these design elements enable the immobilization of single-cells in microwells, their exposure to controlled fluid flows, the coordinated opening and closing of microwells and repeated measurements of single-cell O2 respiration. Uniquely, by sequentially executing opening and closing it becomes possible to measure single-cell respiration prior to and after exposure to chemical solutes. In a proof-of-concept application, we utilized SlipO2Chip to measure the impact of increasing exposures of the marine bacterial signal 2-heptyl-4-quinolone (HHQ) on the dark respiration of the diatom Ditylum brightwellii at single-cell resolution. Results revealed a concentration-dependent decrease in per-cell O2 dark respiration, with a maximum reduction of 40.2% observed at HHQ concentrations exceeding 35.5 μM, and a half-maximal effective concentration (EC50) of 5.8 μM, consistent with that obtained via conventional bulk respiration methods. The ability of SlipO2Chip to sequentially assess the effects of chemical substances on single-cell O2 metabolism is advantageous for research where sample volumes are limited, such as clinical biopsies, studies involving rare microbial isolates, and toxicological studies aiming to address exposure effects while accounting for cell-to-cell variability.

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. We used our approach to characterize the covariance of size and oxygen consumption rate within a cell population, showing that size matters, since oxygen metabolism covaries lognormally with cell size. Our study paves the way for linking the metabolic activity of single human hepatocytes to their tissue- or organ-level metabolism and describing its size-related variability through scaling laws.

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. PACMan comes with a user-friendly graphical user interface and is released together with a stand-alone tool (PAMalysis) for the automated calculation of per-cell maximum quantum efficiencies (= Fv /Fm ). Using these two software packages, we successfully tracked the photophysiology of >1000 individual cells of green algae (Chlamydomonas reinhardtii) and dinoflagellates (genus Symbiodiniaceae) within custom-made microfluidic devices. Compared to the manual operation of MICROSCOPY-PAM systems, this represents a 10-fold increase in throughput. During experiments, PACMan coordinated the movement of the microscope stage and triggered the MICROSCOPY-PAM system to repeatedly capture high-quality image data across multiple positions. Finally, we analyzed single-cell Fv /Fm with the manufacturer-supplied software and PAMalysis, demonstrating a median difference <0.5% between both methods. We foresee that PACMan, and its auxiliary software package will help increase the experimental throughput in a range of microalgae studies currently relying on hand-operated MICROSCOPY-PAM technologies.

We combined microfabricated devices with multiparameter identification algorithms to probe the variability in size-dependent oxygen consumption parameters of single human hepatic cells. We demonstrate that single cells exhibit an oxygen-dependent metabolic rate, typical of Michaelis-Menten kinetics, and that their maximal oxygen consumption is significantly lower than that of monolayers or 3D hepatic cell aggregates. Notably, we found that clusters of two or more cells competing for a limited oxygen supply reduced their maximal single-cell consumption rate, highlighting their ability to adapt to local resource availability and the presence of nearby cells. Next, we used our high-throughput approach to characterize the covariance of size and oxygen consumption within a cell population.The results show that cooperative behaviour emerges in cell clusters, and that single-cell size and metabolism can be described by a lognormal joint probability density. Our study thus serves as a foundation to connect the metabolic activity of single human hepatocytes to their tissue-or organ-level metabolism as well as describe its size-related variability through scaling laws.### Competing Interest StatementThe authors have declared no competing interest.

Transport of oxygen (O2) is essential for life on Earth as it sustains aerobic organisms respiring O2. Biological respiration leads to the local depletion of O2 while diffusive and advective transport processes replenish the O2 required for proper biological functioning. From individual cells to whole organisms, O2 transport unfolds across micrometer to millimeter length scales and can change within milliseconds in response to hydrodynamic flows and organismal behavior. The spatio-temporal complexity of these processes makes the accurate assessment of O2 fluxes via currently available methods difficult, laborious or unreliable. Here, we combined microscale ratiometric and lifetime-based O2 imaging methods with particle velocimetry to determine O2 concentrations and flow fields simultaneously. This new method - ‘sensPIV‘ - now enables non-invasive measurements of O2 exchange fluxes across a wide-range of spatial scales at unprecedented accuracy and speed. We document three proof-of-concept applications that highlight the power of sensPIV to measure O2 fluxes within i) microfluidic devices, ii) sinking model particles and iii) complex colony-forming corals. SensPIV can now quantitatively link O2 usage to transport processes across a wide range of life-science and engineering applications.

From individual cells to whole organisms, O2 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 O2 dynamics via currently available methods difficult or unreliable. Here, we present "sensPIV," a method to simultaneously measure O2 concentrations and flow fields. By tracking O2-sensitive microparticles in flow using imaging technologies that allow for instantaneous referencing, we measured O2 transport within (1) microfluidic devices, (2) sinking model aggregates, and (3) complex colony-forming corals. Through the use of sensPIV, we find that corals use ciliary movement to link zones of photosynthetic O2 production to zones of O2 consumption. SensPIV can potentially be extendable to study flow-organism interactions across many life-science and engineering applications.