O2 Probes Research Articles

Ratiometric fluorescence detection of O2•− based on dual-emission schiff base polymer/rhodamine-B nanocomposites

A novel ratiometric fluorescent sensor for superoxide radical (O2•−) detection was developed based on Schiff base polymer (SBP)/rhodamine-B (SBP/RDB) nanocomposites. The SBP/RDB was investigated by atomic force microscopy, scanning electron microscopy, transmission electron microscopy, X-ray powder diffraction, Fourier transform infrared spectroscopy, N2 adsorption/desorption isotherms and fluorescence spectroscopy, etc. The results showed the SBP was two-dimensional (2D) crystalline nanosheet with the thickness of 0.5 nm. RDB could be self-assembled on each of 2D SBP surface to form SBP/RDB due to the hydrogen bond between N atoms of SBP and –COOH of RDB as well as π-π interaction. The SBP/RDB exhibited the characteristic fluorescence emission of both SBP at 485 nm and RDB at 585 nm under an excitation of 270 nm. The 2D SBP was not only acted as template to assemble RDB, but also used as reference fluorescence for O2•− detection. The SBP/RDB fluorescence probe for O2•− detection demonstrated high sensitivity and selectivity with good linear range (11.1 ± 0.76 nM–8.0 ± 0.36 μM) and low detection limit (3.7 ± 0.42 nM). Because of the low cost and simple operation, the work sheds some new light to construct ratiometric fluorescent sensors based on SBP to detect O2•− in drinking water and human blood serum for commercial applications.

91 - Unveil mitochondrial superoxide anion transport pathway in ischemia/reperfusion injury of the mouse liver via two-photon fluorescence imaging

Monitor dynamic changes in mitochondrial and cytoplasmic superoxide anion (O2•−) levels can unveil O2•− signalling pathway of hepatic ischemia/reperfusion (IR) injury and find relative therapeutic target. However, reversible and ratio two-photon fluorescence probe for mitochondrial O2•− hasn’t been presented. Therefore, we developed a reversible and ratio two-photon fluorescence probe for mitochondrial O2•−. With the aid of a cytoplasm O2•− probe and two-photon fluorescent microscopy, the crosstalk between mitochondrial O2•− and cytoplasmic O2•− has been explored. We found that mitochondrial O2•− transported to cytoplasm via VDAC channel in hepatic ischemia/reperfused mice. Furthermore, blocking VDAC channel and increasing mitochondrial Mn-SOD may reduce the risk of IR injury death. Our work uncovers mitochondria O2•− possible transport pathway and molecular mechanism of IR injury, also provides a new therapeutic strategy for IR injury.

A platinum(II)-acetylide-based conjugated polyelectrolyte for hypoxia imaging via ratiometric and time-resolved luminescence microscopy

A platinum(II)-acetylide-based conjugated polyelectrolyte has been designed and synthesized by using polyfluorenes as an O2-insensitive fluorophore and Pt(II) complex as an O2-sensitive phosphor, which can generate conjugated polyelectrolyte nanoparticle (CPE-nanoparticle) in the aqueous solution owing to their amphiphilic structures. The CPE-nanoparticle displays good sensitivity to O2 concentration and can detect oxygen levels reversibly. The intracellular ratiometric O2 sensing performance of the CPE-nanoparticle has been demonstrated by the remarkable variation in the Igreen/Iblue ratio values (0.18–0.85) in HeLa cells under different O2 levels. Furthermore, O2 level detection was carried out through time-resolved luminescence imaging (TRLI) to demonstrate the accuracy of the probe based on the CPE-nanoparticle. The CPE-nanoparticle shows a high phosphorescence quantum yield (19.98%) and oxygen quenching efficiency (0.975), which are superior to the existing O2 probe. The CPE-nanoparticle has been successfully applied in photoluminescence lifetime imaging and time-gated luminescence imaging for monitoring intracellular O2 levels.

Hypothermia Decreases O2 Cost for Ex Vivo Contraction in Mouse Skeletal Muscle.

Evidence suggests that the energy efficiency of key ATPases involved in skeletal muscle contractile activity is improved in a hypothermic condition. However, it is unclear how a decrease in temperature affects skeletal muscle O2 consumption (mVO2) induced by muscle contraction. Isolated mouse extensor digitorum longus (EDL) muscles were incubated in a temperature-controlled (37°C or 25°C) bath that included an O2 probe. EDL muscles from one limb were subjected to the measurement of resting mVO2, and the contralateral EDL muscles were used for the measurement of mVO2 with electrically stimulated contraction. For the resting protocol, muscles were suspended at resting tension for 15 min with continuous O2 recordings. For the contraction protocol, EDL muscles underwent 10 electrically stimulated isometric contractions with continuous O2 recordings for 15 min. The rate of O2 disappearance was quantified as micromoles of O2 per minute and normalized to the wet weight of the muscle. Resting mVO2 was greater at 37°C than at 25°C, consistent with the idea that lower temperature reduces basal metabolic rate. Electrically stimulated contraction robustly increased mVO2 at both 37°C and 25°C, which was sustained for ~3 min postcontraction. During that period, mVO2 was elevated approximately fivefold at both 37°C and 25°C. Greater contraction-induced mVO2 at 37°C compared with 25°C occurred despite lower force generated at 37°C than at 25°C. Together, O2 cost for muscle contraction (force-time integral per O2 consumed) was greater at 37°C than at 25°C. Levels of high-energy phosphates were consistent with greater energy demand at 37°C compared with 25°C. In conclusion, these results indicate that muscle contraction that occurs at subnormal temperature requires less O2 than at 37°C.

Tandem Payne/Dakin Reaction: A New Strategy for Hydrogen Peroxide Detection and Molecular Imaging.

Hydrogen peroxide (H2 O2 ) has been recognized as one of the most significant ROS (reactive oxygen species) in human health and disease. Because of the intrinsic attributes of H2 O2 -such as its low reactivity under physiological pH-it is exceedingly challenging to develop small-molecule fluorescent probes with high selectivity and sensitivity for visualization of H2 O2 in an intricate biological milieu. To address this gap, a rationally designed tandem Payne/Dakin reaction is reported that is specific to molecular recognition of H2 O2 . New H2 O2 probes based on this unique chemical strategy can be easily synthesized by a general coupling reaction, and the practical applicability of those probes has been confirmed by the visualization of endogenously produced H2 O2 in living cells. In particular, starvation-induced H2 O2 production in mouse macrophages has been detected by the novel probe in both confocal imaging and flow cytometry. This tandem Payne/Dakin reaction provides a basis for developing more sophisticated molecular tools to interrogate H2 O2 functions in biological phenomena.

Design of a phosphinate-based bioluminescent probe for superoxide radical anion imaging in living cells.

Superoxide radical anion (O2 ˙- ) as an important member of reactive oxygen species (ROS) plays a vital role both in physiology and pathology. Herein we designed and synthesized a novel phosphinate-based bioluminescence probe for O2 ˙- detection in living cells, which exhibited good sensitivity for capturing O2 ˙- at the nanomole level and high selectivity against other ROS. The probe was further found to be of low toxicity for living cells and was then successfully employed for sensing endogenous O2 ˙- by using phorbol-12-myristate-13-acetate (PMA) as a traditional O2 ˙- stimulator in Huh7 cells. Moreover, the increasing production and use of nanoparticles, has given rise to many concerns and debates among the public and scientific authorities regarding their safety and final fate in biological systems. Herein it was found that mondisperse polystyrene particles could stimulate O2 ˙- generation in Huh7 cells. Overall, the probe was demonstrated to have a great potential as a novel bioluminescent sensor for detecting O2 ˙- in living cells. To our knowledge, this is the first small-molecule phosphinate-based bioluminescence probe that will open up great opportunities for unlocking the mystery of O2 ˙- in human health and disease.

Pentameric PdAu and PdPt nanoparticles on the MgO(1 0 0) surface and their CO and O2 adsorption properties

The surface mode of the Birmingham Cluster Genetic Algorithm (S-BCGA), which performs an unbiased global optimisation search for clusters adsorbed on a surface, has been employed for the global optimisation of noble metal pentamers on an MgO(1 0 0) substrate. The effect of element identity and alloying in surface-bound neutral subnanometre particles is calculated by energetic analysis of all compositions of supported 5-atom PdAu and PdPt clusters. Our results show that the binding strengths of the component elements to the surface are in the order Pt > Pd > Au. In addition, alloying Pd with Au and Pt is favorable for this size since excess energy calculations show a preference for bimetallic clusters for both cases. Furthermore, the electronic behaviour, which is intermediate between molecular systems and bulk metals allows tuning of the characteristics of particles in the subnanometre size range. The adsorption of CO and O2 probe molecules are also modelled and it is found that CO and O2 adsorption leads to a weakening of the cluster–surface interaction.

A Breath of Fresh Air: A Genetically Encoded O2 Probe for Direct Mapping and Quantification of Oxygenation Levels in Cells via Fluorescence Lifetime Imaging

Molecular oxygen is an important reporter of metabolic and physiological status at the cellular and tissue level, and its concentration can be used for the evaluation of diseases of various nature (e.g.: cancer, coronary artery disease). The development of accurate and quantitative methods to measure O2 concentration in living cells, tissues and organisms has remained challenging and is subject of intense research. We developed a protein-based, fluorescent oxygen sensor that can be expressed directly in cells to monitor oxygen levels in the cytoplasmic environment. We fused Myoglobin (Mb), a physiological oxygen carrier, with mCherry, a fluorescent protein, to build a fluorescence resonance energy transfer (FRET) pair where Myoglobin acts as a variable dark acceptor. The changes in the spectral properties of Myoglobin upon oxygen binding translate into changes of the FRET-depleted emission intensity of mCherry, and this effect is best detected by monitoring the fluorescence lifetime of the probe. We present in this work the expression and characterization of the Mb-mCherry system in vitro to establish a calibration curve of its response to [O2] and other relevant effectors. Further, by using Fluorescence Lifetime Imaging Microscopy (FLIM), we show that we are able to map the oxygenation level of the probe ex vivo in cells, providing a quantitative description. A genetically encoded oxygen probe based on fluorescence addresses the most common issues with currently available methods to monitor O2 levels in living organisms: data collection speed, spatial resolution and localization within cells and tissues. Customization of the response can be achieved by exploiting the mutations of Mb to tune O2 binding affinity, or the response to external modulators (e.g.: lactate) present in the cell.

A two-photon fluorescent probe for endogenous superoxide anion radical detection and imaging in living cells and tissues

Superoxide anion radical (O2−), the “primary” reactive oxygen species (ROS) in living systems, is linked to a variety of physiological and pathological processes. Therefore, developing an effective strategy to monitor the fluctuation of O2− in biological systems is of great importance. This paper describes a new turn-on two-photon fluorescent probe for endogenous O2− detection and imaging, which was rationally designed and synthesized via a non-redox strategy. In the presence of O2−, the probe exhibited notable fluorescence enhancement (∼235-fold) with a low detection limit down to 1nM, indicating a high signal-to-background ratio and excellent sensitivity. In addition, short response time, good biocompatibility, low cytotoxicity, long-term stability against light illumination, specificity to O2− over general reductants, and pH stability were demonstrated, indicating that the requirements for cellular O2− determination are met. Furthermore, the probe was successfully applied in two-photon fluorescence imaging of endogenous O2− in living cells and tissues and showed high imaging resolution and a deep-tissue imaging depth of ∼150μm, illustrating the promising potential for practical applications in complex biosystems and providing a valuable theoretical basis and technical support for the study of physiological and pathological functions of O2−.

Remote Terpyridine Integrated NHC-IrIII Luminophores as Potential Dual-Emissive Ratiometric O2 Probes.

A new class of bi-luminophoric dyad has been designed, consisting of an oxygen-sensitive phosphorescent NHC-IrIII center with a remotely integrated oxygen-insensitive fluorescent terpyridine unit. The new terpyridine flurophore-integrated NHC-IrIII molecule was demonstrated as a potential ratiometric O2 probe with built-in internal reference, exhibiting tunable dual-emissive features, as well as highly linear and reversible O2 -response behavior.

Probing whole‐stream metabolism: influence of spatial heterogeneity on rate estimates

Summary Whole‐stream metabolism has been estimated by measuring in‐stream oxygen (O2) concentrations since the method was introduced over 50 years ago. However, the influence of measurement location and estimation method on metabolism rates is understudied. We examined how the placement of O2 probes (i.e. depth, separation from the thalweg), differences in methodology (1‐station, 2‐station, area‐weighted) and reach lengths influenced estimated rates of whole‐stream metabolism in a tallgrass prairie watershed. Metabolism estimates made in the thalweg differed from estimates made in backwaters due to disconnection in flow, and estimates made in deep pools differed from surface estimates due to thermal stratification (temporary flow disconnection). The 1‐station respiration estimates differed from short 2‐station reach‐scale estimates (c. 20 m) but were more similar to larger 2‐station reach‐scale estimates (c. 100 m). In contrast, the 1‐station gross primary production was most similar to the short 2‐station reaches occurring immediately upstream and became less similar at longer 2‐station reach lengths. The different estimation methodologies (1‐station, 2‐station, area‐weighted) accounting for the longest reach scale did not result in different metabolism rates. The temporary phenomena of thermal stratification of stream pools during a warm day, which disconnected pool bottoms from the surface waters, likely affected not only the pool estimates but also estimates made in the downstream thalweg (i.e. an O2 deficit accrued from respiration during the day in the bottom of the pool abruptly moved downstream during mixing). Oxygen probe placement mattered and affected rate estimates according to habitat type and reach length (i.e. scale) due to the influence of small‐scale heterogeneity on community respiration. Selection of reach length can be critical for studies depending on whether local heterogeneity is of interest or should be averaged. We conclude that the intuitive use of thalwegs and reaches that are at least 10 times the stream width are likely appropriate for whole‐stream metabolism estimates, although the exact reach length necessary and potential stream‐specific characteristics, such as stratified pools, need to be carefully considered in probe placement. We encourage other studies to report the placement characteristics of O2 probes in streams as well as consider the potential confounding factor of local habitat heterogeneity.

443 - The Measurement of Cellular Bioenergetics at Defined Steady-State Oxygen Concentrations: A Novel Microplate Based Method

Although often overlooked, most cell-based in vitro assays are conducted under hyperoxic conditions, while the impact of oxygen concentration ([O2]) on experimental outcome is typically ignored. It is known however that for cells cultured at lower than ambient [O2], there exists a potential for higher [O2] to significantly impact cellular bioenergetics and, by extension, data output. While there is a growing appreciation of this deficiency, technical limitations have prevented broad uptake of [O2]-informed in vitro assay design. These limitations have been ameliorated by recent the advances in both instrumentation and assay technologies: integrated atmospheric control units (ACUs) now facilitate measurement at defined [O2] while advanced phosphorescent intracellular O2 and fluorescent extracellular pH probes, allowing simultaneous multiparametric measurements of cellular oxygenation, electron transport chain activity and glycolytic flux. Described herein is a high-throughput method for measuring all three key metabolic parameters under defined [O2] thereby further addressing such technical limitations. The method utilizes open-flow respirometry, a technique that allows for the measurement of cellular respiration at steady state (e.g. O2 supply = O2 demand). Preliminary data on respiration rate and media acidification at predefined [O2] is presented, facilitating a more detailed interrogatioen for the effect of [O2] on the balance between oxidative phosphorylation and glycolytic activity. This is further delineated using known metabolic effectors including the mitochondrial modulators, oligomycin, antimycin A and FCCP and the CPT-1 inhibitor Etomoxir. The method therefore complements existing technologies, and as a cost-effective, efficient means to measure cellular bioenergetics at an [O2] most relevant to a given cell type or experimental model.

Reactive Oxygen Species and Oxidative Stress in Obesity-Recent Findings and Empirical Approaches.

High levels of reactive oxygen species (ROS) are intricately linked to obesity and associated pathologies, notably insulin resistance and type 2 diabetes. However, ROS are also thought to be important in intracellular signaling, which may paradoxically be required for insulin sensitivity. Many theories have been developed to explain this apparent paradox, which have broadened our understanding of these important small molecules. While many sites for intracellular ROS production have been described, mitochondrial generated ROS remain a major contributor in most cell types. Mitochondrial ROS generation is controlled by a number of factors described in this review. Moreover, these studies have established both a demand for novel sensitive approaches to measure ROS, as well as a need to standardize and review their suitability for different applications. To properly assess levels of ROS and mitochondrial ROS in the development of obesity and its complications, a growing number of tools have been developed. This paper reviews many of the common methods for the investigation of ROS in mitochondria, cell, animal, and human models. Available approaches can be generally divided into those that measure ROS-induced damage (e.g., DNA, lipid, and protein damage); those that measure antioxidant levels and redox ratios; and those that use novel biosensors and probes for a more direct measure of different forms of ROS (e.g., 2',7'-di-chlorofluorescein (DCF), dihydroethidium (DHE) and its mitochondrial targeted form (MitoSOX), Amplex Red, roGFP, HyPer, mt-cpYFP, ratiometric H2 O2 probes, and their derivatives). Moreover, this review provides caveats and strengths for the use of these techniques in different models. Advances in these techniques will undoubtedly advance the understanding of ROS in obesity and may help resolve unanswered questions in the field.

Simultaneous Near-Infrared and Two-Photon In Vivo Imaging of H2 O2 Using a Ratiometric Fluorescent Probe based on the Unique Oxidative Rearrangement of Oxonium.

A new ratiometric fluorescent H2 O2 probe, benzopyrylium-coumarin (BC), is designed by using an oxonium moiety as the unique H2 O2 response site. The BC probe exhibits an extremely large emission shift of 221 nm in response to H2 O2 , and is successfully applied for the simultaneous near-infrared and two-photon imaging of H2 O2 in living cells, mouse-liver tissues, and zebrafish.

Influence of loading rate and modes on infiltration of treated wastewater in soil-based constructed wetland

ABSTRACTOver the last 10 years soil-based constructed wetlands for discharge of treated wastewater (TWW) are commonly presented as a valuable option to provide tertiary treatment. The uncomplete knowledge in soil modifications and a lack of clear design practices laid the foundation of this work. The aim of this study was to determine optimal hydraulic loads and to observe the main critical parameters affecting treating performances and hydraulic loads acceptance. For this purpose, a soil rich in clay and backfill was chosen to perform column infiltration tests with TWW. Two loading rates and two loading modes were compared to study the influence of an intermittent feeding. Inlet and outlet waters were periodically analysed and columns were instrumented with balances, tensiometers, O2 and temperature probes. Soil physico-chemical characteristics were also taken into account to better understand the modification of the soil. One of the main expectations of tertiary treatment is to improve phosphate removal. A particular attention was thus given to phosphorus retention. The interest of an intermittent feeding in presence of a soil with high clay content was showed. This study highlighted that an intermittent feeding could make possible the use of a clay-rich soil for water infiltration.

Fluorescence Measurement of Pericellular Oxygen

Under normal physiological conditions, the mitochondrial electron transport chain consumes cellular/mitochondrial oxygen (O2) to generate a proton motive force (ΔΨm) which powers ATP synthesis. Thus, many cellular processes that depend on [ATP] are either directly or indirectly regulated by the availability of O2 to the cells. Restriction of tissue blood flow disrupts the O2 supply to cause ischemia, and is detrimental to virtually all cell types. Nevertheless, the cellular, organellar, and molecular processes that enable cells to survive episodes of ischemia remain largely unknown. This is due in part to our inability to control and to accurately measure local pericellular O2, particularly during high-resolution single cell measurements. Here we present a fluorescent O2-probe comprising silica-supported O2 micro sensors integrated in an O2-permeable silicon matrix. A micro-layer (20-50 μm) of the probe serves as a substrate beneath adherent single cells. This enables us to measure the pericellular partial pressure of O2 (ppO2) without disrupting the flow of extracellular solutions. The O2 probe responds rapidly to abrupt changes of ppO2 at rates approximately 100 times faster than Clark-type microelectrodes. Since the ppO2 microsensors and the cell are spatially resolved, calibrated changes of pericellular ppO2 and intracellular events can be imaged simultaneously with high spatiotemporal resolution. We have used this technique to measure the pericellular ppO2 of cardiomyocytes, which are extremely vulnerable to O2 deprivation, owing to their heavy reliance on mitochondrial oxidative phosphorylation to generate ATP. During time-lapse experiments, the ΔΨm in different subcellular regions is monitored. Dynamic cytosolic [Ca2+] levels are also measured during electrically stimulated cell contractions and their sensitivity to O2 is tested. This new probe provides a direct means of measuring pericellular ppO2 in real-time, and thus opens new avenues to investigate the physiological and pathophysiological responses to hypoxia at the single-cell level.

Metallochelate Coupling of Phosphorescent Pt-Porphyrins to Peptides, Proteins, and Self-Assembling Protein Nanoparticles.

Specific and reversible metallochelate coupling via nitrilotriacetate (NTA) moiety is widely used for immobilization, purification, and labeling of oligo(histidine)-tagged proteins. Here, we evaluated this strategy to label various peptides and proteins with phosphorescent Pt-porphyrin derivatives bearing NTA group(s). Zn(2+) complexes were shown to have minimal effect on the photophysics of the porphyrin moiety, allowing quenched-phosphorescence sensing of O2. We complexed the PtTFPP-NTA conjugate with His-containing peptide that can facilitate intracellular loading, and observed efficient accumulation and phosphorescent staining of MEF cells. The more hydrophilic PtCP-NTA conjugate was also seen to form stable complexes with larger polypeptide constructs based on fluorescent proteins, and with subunits of protein nanoparticles, which retained their ability to self-assemble. Testing in phosphorescence lifetime based O2 sensing assays on a fluorescence reader and PLIM microscope revealed that phosphorescent metallochelate complexes perform similarly to the existing O2 probes. Thus, metallochelate coupling allows simple preparation of different types of biomaterials labeled with phosphorescent Pt-porphyrins.

Mitochondria Targetable Time-Gated Luminescence Probe for Singlet Oxygen Based on a β-Diketonate-Europium Complex.

Singlet oxygen ((1)O2) plays a key role in the photodynamic therapy (PDT) technique of neoplastic diseases. In this work, by using a 9,10-dimethyl-2-anthryl-containing β-diketone, 1,1,1,2,2-pentafluoro-5-(9',10'-dimethyl-2'-anthryl)-3,5-pentanedione (Hpfdap), as a (1)O2-recognition ligand, a novel β-diketonate-europium(III) complex that can act as a luminescence probe for (1)O2, [Eu(pfdap)3(tpy)] (tpy = 2,2',2″-terpyridine), has been designed and synthesized for the time-gated luminescence detection of (1)O2 in living cells. The complex is weakly luminescent due to the quenching effect of 9,10-dimethyl-2-anthryl groups. After reaction with (1)O2, accompanied by the formation of endoperoxides of 9,10-dimethyl-2-anthryl groups, the luminescence quenching disappears, so that the long-lived luminescence of the europium(III) complex is switched on. The complex showed highly selective luminescence response to (1)O2 with a remarkable luminescence enhancement. Combined with the time-gated luminescence imaging technique, the complex was successfully used as a luminescent probe for the monitoring of the time-dependent generation of (1)O2 in 5-aminolevulinic acid (a PDT drug) loaded HepG2 cells during the photodynamic process. In addition, by coloading the complex and a mitochondrial indicator, Mito-Tracker Green, into HepG2 cells, the specific localization of [Eu(pfdap)3(tpy)] molecules in mitochondria of HepG2 cells was demonstrated by confocal fluorescence imaging measurements.

Embryo oxygenation in pipefish brood pouches: novel insights

The pipefish brood pouch presents a unique mode of parental care that enables males to protect, osmoregulate, nourish and oxygenate the developing young. Using a very fine O2 probe, we assessed the extent to which males of the broad-nosed pipefish (Syngnathus typhle) oxygenate the developing embryos and are able to maintain pouch fluid O2 levels when brooding in normoxia (100% O2 saturation) and hypoxia (40% O2 saturation) for 24 days. In both treatments, pouch fluid O2 saturation levels were lower compared with the surrounding water and decreased throughout the brooding period, reflecting greater offspring demand for O2 during development and/or decreasing paternal ability to provide O2 to the embryos. Male condition (hepatosomatic index) was negatively affected by hypoxia. Larger males had higher pouch fluid O2 saturation levels compared with smaller males, and levels were higher in the bottom section of the pouch compared with other sections. Embryo size was positively correlated with O2 availability, irrespective of their position in the pouch. Two important conclusions can be drawn from our findings. First, our results highlight a potential limitation to brooding within the pouch and dismiss the notion of closed brood pouches as well-oxygenated structures promoting the evolution of larger eggs in syngnathids. Second, we provide direct evidence that paternal care improves with male size in this species. This finding offers an explanation for the documented strong female preference for larger partners because, in terms of oxygenation, the brood pouch can restrict embryo growth.

Luminescence Lifetime Imaging Microscopy For Analysis Of Oxygen In The Cancer Cell

Molecular oxygen (O2), the key component of aerobic metabolism, plays important roles in normal and cancer cells and tissues. A real‐time and quantitative O2 imaging in 3D cell models would allow to discriminate complex effects of hypoxia on cell function in complex (heterogeneous) cell populations at subcellular resolution. Here, we used luminescence lifetime imaging (FLIM, PLIM) to develop and evaluate phosphorescent O2 probes with 3D spheroid and multi‐cellular aggregate models. We tested rat embryonic neurosphere culture with cationic nanoparticle O2 probes (MM2, NanO2) and developed O2 imaging protocol, multiplexed with analysis of distribution of specific protein markers such as HIF‐2α. To address the problem of efficient in‐depth staining, we designed two novel intracellular O2 probes: small molecule Pt‐Glc and nanoparticle PA2. Both probes displayed high analytical performance (brightness, photostability, O2 calibration) and were evaluated with rat pheochromocytoma PC12 and human colon cancer HCT116 cell aggregates. Resting cells cultured in 3D aggregates of >50 μm size displayed significant deoxygenation even under ambient atmospheric O2 levels. Still, they showed high viability and experienced different levels of oxygenation, upon treatment with mitochondrial uncouplers, inhibitors or pharmacological drugs. This data confirms the importance of probing of cellular O2 for experiments with 3D cell models. When combined with other live cell imaging dyes, such as markers of necrosis and apoptosis, the method allows for multi‐parameter quantitative assessment of cell physiology within relevant 3D micro‐environment.Supported by Science Foundation Ireland (SFI) grant 13/SIRG/2144.