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Assessment of hypoxia, nutrients, metabolite gradients, and other hallmarks of the tumor microenvironment within 3D multicellular spheroid and organoid models represents a challenging analytical task. Here, we report red/near-infrared (NIR) emitting cell staining with O2-sensitive nanoparticles, which enable measurements of spheroid oxygenation on a conventional fluorescence microscope. Nanosensor probes, termed “MMIR” (multimodal infrared), incorporate an NIR O2-sensitive metalloporphyrin (PtTPTBPF) and deep red aza-BODIPY reference dyes within a biocompatible polymer shell, allowing for oxygen gradient quantification via fluorescence ratio and phosphorescence lifetime readouts. We optimized staining techniques and evaluated the nanosensor probe characteristics and cytotoxicity. Subsequently, we applied nanosensors to the live spheroid models based on HCT116, DPSCs, and SKOV3 cells, at rest, and treated with drugs affecting cell respiration. We found that the growth medium viscosity, spheroid size, and formation method influenced spheroid oxygenation. Some spheroids produced from HCT116 and dental pulp stem cells exhibited “inverted” oxygenation gradients, with higher core oxygen levels than the periphery. This contrasted with the frequently encountered “normal” gradient of hypoxia toward the core caused by diffusion. Further microscopy analysis of spheroids with an “inverted” gradient demonstrated metabolic stratification of cells within spheroids: thus, autofluorescence FLIM of NAD(P)H indicated the formation of a glycolytic core and localization of OxPhos-active cells at the periphery. Collectively, we demonstrate a strong potential of NIR-emitting ratiometric nanosensors for advanced microscopy studies targeting live and quantitative real-time monitoring of cell metabolism and hypoxia in complex 3D tissue models.

Transient early endosome (EE)-mitochondria interactions can mediate mitochondrial iron translocation, but the associated mechanisms are still elusive. We showed that Divalent Metal Transporter 1 (DMT1) sustains mitochondrial iron translocation via EE-mitochondria interactions in triple-negative MDA-MB-231, but not in luminal A T47D breast cancer cells. DMT1 silencing increases labile iron pool (LIP) levels and activates PINK1/Parkin-dependent mitophagy in MDA-MB-231 cells. Mitochondrial bioenergetics and the iron-associated protein profile were altered by DMT1 silencing and rescued by DMT1 re-expression. Transcriptomic profiles upon DMT1 silencing are strikingly different between 2D and 3D culture conditions, suggesting that the environment context is crucial for the DMT1 knockout phenotype observed in MDA-MB-231 cells. Lastly, in vivo lung metastasis assay revealed that DMT1 silencing promoted the outgrowth of lung metastatic nodules in both human and murine models of triple-negative breast cancer cells. These findings reveal a DMT1‐dependent pathway connecting EE-mitochondria interactions to mitochondrial iron translocation and metastatic fitness of breast cancer cells.

Bacterial metabolisms using electron acceptors other than oxygen (e.g., methanogenesis and fermentation) largely contribute to element cycling and natural contaminant attenuation/mobilization, even in well-oxygenated porous environments, such as shallow aquifers. This paradox is commonly explained by the occurrence of small-scale anoxic microenvironments generated by the coupling of bacterial respiration and dissolved oxygen (O2) transport by pore water. Such microenvironments allow facultative anaerobic bacteria to proliferate in oxic environments. Microenvironment dynamics are still poorly understood due to the challenge of directly observing biomass and O2distributions at the microscale within an opaque sediment matrix. To overcome these limitations, we integrated a microfluidic device with transparent O2planar optical sensors to measure the temporal behavior of dissolved O2concentrations and biomass distributions with time-lapse videomicroscopy. Our results reveal that bacterial colony morphology, which is highly variable in flowing porous systems, controls the formation of anoxic microenvironments. We rationalize our observations through a colony-scale Damköhler number comparing dissolved O2diffusion and a bacterial O2uptake rate. Our Damköhler number enables us to predict the pore space fraction occupied by anoxic microenvironments in our system for a given bacterial organization.

Aprotic alkali metal–O2batteries face two major obstacles to their chemistry occurring efficiently, the insulating nature of the formed alkali superoxides/peroxides and parasitic reactions that are caused by the highly reactive singlet oxygen (1O2). Redox mediators are recognized to be key for improving rechargeability. However, it is unclear how they affect 1O2formation, which hinders strategies for their improvement. Here we clarify the mechanism of mediated peroxide and superoxide oxidation and thus explain how redox mediators either enhance or suppress 1O2formation. We show that charging commences with peroxide oxidation to a superoxide intermediate and that redox potentials above ~3.5 V versus Li/Li+drive 1O2evolution from superoxide oxidation, while disproportionation always generates some 1O2. We find that 1O2suppression requires oxidation to be faster than the generation of 1O2from disproportionation. Oxidation rates decrease with growing driving force following Marcus inverted-region behaviour, establishing a region of maximum rate.

Metallocorroles involving 5d transition metals are currently of interest as near-IR phosphors and as photosensitizers for oxygen sensing and photodynamic therapy. Their syntheses, however, are often bedeviled by capricious and low-yielding protocols. Against this backdrop, we describe rhenium-imido corroles, a new class of 5d metallocorroles, synthesized simply and in respectable (∼30%) yields via the interaction of a free-base corrole, Re2(CO)10, K2CO3, and aniline in 1,2,4-trichlorobenzene at ∼190 °C in a sealed vial under strict anaerobic conditions. The generality of the method was shown by the synthesis of six derivatives, including those derived from meso-tris(pentafluorophenyl)corrole, H3[TPFPC], and five different meso-tris(p-X-phenyl)corroles, H3[TpXPC], where X = CF3, F, H, CH3, OCH3. Single-crystal X-ray structures obtained for two of the complexes, Re[TpFPC](NPh) and Re[TpCF3PC](NPh), revealed relatively unstrained equatorial Re–N distances of ∼2.00 Å, a ∼ 0.7-Å displacement of the Re from the mean plane of the corrole nitrogens, and an Re–Nimidodistance of ∼1.72 Å. Details of the corrole skeletal bond distances, diamagnetic 1H NMR spectra, relatively substituent-independent Soret maxima, and electrochemical HOMO–LUMO gaps of ∼2.2 V all indicated an innocent corrole macrocycle. Surprisingly, unlike several other classes of 5d metallocorroles, the Re-imido complexes proved nonemissive in solution at room temperature and also failed to sensitize singlet oxygen formation, indicating rapid radiationless deactivation of the triplet state, presumably via the rapidly rotating axial phenyl group. By analogy with other metal-oxo and -imido corroles, we remain hopeful that the Re-imido group will prove amenable to further elaboration and thereby contribute to the development of a somewhat challenging area of coordination chemistry.

A glucose optode measuring the internal oxygen gradient is presented. The multilayer biosensor is composed of (i) analyte-impermeable transparent support, (ii) first oxygen-sensing layer combined with an enzymatic layer, (iii) diffusion barrier, and (iv) second oxygen-sensing layer. To make this design suitable for measurement in subcutaneous tissue, a pair of NIR phosphorescent indicators with very different spectral properties is chosen. Combination of a conventional Pt(II) tetrabenzoporphyrin dye (absorption and emission maxima at 617 and 772 nm, respectively) used in the first layer and a new intramolecularly bridged Pt(II) complex (absorption and emission maxima at 673 and 872 nm, respectively) in the second layer enables efficient separation of both emission signals. This specially designed dye class is accessible via Scholl-reaction from tetraphenyltetrabenzoporphyrin complexes. For the first time, the new optode allows simultaneous glucose and oxygen measurement in a single spot and therefore accurate compensation of oxygen heterogeneities resulting from fluctuations in the tissue. The presented material covers the dynamic ranges from 0 to 150 hPa O2and from 0 to 360 mg/dL (20 mM) glucose (at 37 °C).

Optical temperature sensors based on self-referenced readout schemes such as the emission ratio and the decay time are crucial for a wide range of applications, with the former often preferred due to simplicity of instrumentation. This work describes a new group of dually emitting dyes, platinum(II) pincer complexes, that can be used directly for ratiometric temperature sensing without an additional reference material. They consist of Pt(II) metal center surrounded by a pyridinedipyrrolide ligand (PDP) and a terminal ligand (benzonitrile, pyridine, 1-butylimidazol or carbon monoxide). Upon excitation with blue light, these complexes exhibit green to orange emission, with quantum yields in anoxic toluene at 25 °C ranging from 13% to 86% and decay times spanning from 8.5 to 97 μs. The emission is attributed to simultaneous thermally activated delayed fluorescence (TADF) and phosphorescence processes on the basis of photophysical investigations and DFT calculations. Rather uniquely, simple manipulations in substituents of the PDP ligand and alteration of the terminal ligand allow fine-tuning of the ratio between TADF and phosphorescence from almost 100% TADF emission (Pt(MesPDPC6F5(BN)) to over 80% of phosphorescence (Pt(PhPDPPh(BuIm)). Apart from ratiometric capabilities, the complexes also are useful as decay time-based temperature indicators with temperature coefficients exceeding 1.5% K–1in most cases. Immobilization of the dyes into oxygen-impermeable polyacrylonitrile produces temperature sensing materials that can be read out with an ordinary RGB camera or a smartphone. In addition, Pt(PhPDPPh)Pycan be incorporated into biocompatible RL100 nanoparticles suitable for cellular nanothermometry, as we demonstrate with temperature measurements in multicellular colon cancer spheroids.

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 O2and 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.

Novel fluorescent diazaoxatriangulenium (DAOTA) pH indicators for lifetime-based self-referenced pH sensing are reported. The DAOTA dyes were decorated with phenolic-receptor groups inducing fluorescence quenching via a photoinduced-electron-transfer mechanism. Electron-withdrawing chlorine substituents ensure response in the most relevant pH range (apparent pKa′values of ∼5 and 7.5 for the p,p-dichlorophenol- and p-chlorophenol-substituted dyes, respectively). The dyes feature long fluorescence lifetimes (17–20 ns), high quantum yields (∼60%), and high photostabilities. Planar optodes are prepared upon immobilization of the dyes into polyurethane hydrogel D4. Apart from the response in the fluorescence intensity, the optodes show pH-dependent lifetime behavior, which makes them suitable for studying 2D pH distributions with the help of fluorescence-lifetime-imaging techniques. The lifetime response is particularly pronounced for the sensors with high dye concentrations (0.5–1 wt % with respect to the polymer) and is attributed to the efficient homo-FRET mechanism.

A series of bis(phenylacetylido)(porphyrinato) E(IV) (E=Si, Ge, Sn) derivatives were obtained by reacting lithium phenylacetylide with the corresponding bis(chlorido)(porphyrinato) E(IV) precursors. Crystal structure determinations demonstrated that the trans-coordinated acetylide ligands deviate from their expected ideal geometry. Density functional theory calculations and comparison of the packing of the molecules among the homologous series of Si(IV), Ge(IV) and Sn(IV) revealed causes for the deviation from the simple valence shell electron pair repulsion model. Fluorescence and phosphorescence of these organometallic group 14 derivatives were studied and the results are compared to the literature-known properties of the related bis(chlorido)(porphyrinato) E(IV) compounds (E=Si, Ge, Sn). The first crystal structure of a porphyrinato Ge(IV) compound with σ-bonded acetylide ligands is reported.

We report a family of Pt and Pd benzoporphyrin dyes with versatile photophysical properties and easy access from cheap and abundant chemicals. Attaching 4 or 8 alkylsulfone groups onto a meso-tetraphenyltetrabenzoporphyrin (TPTBP) macrocylcle renders the dyes highly soluble in organic solvents, photostable, and electron-deficient with the redox potential raised up to 0.65 V versus the parent porphyrin. The new dyes intensively absorb in the blue (Soret band, 440–480 nm) and in the red (Q-band, 620–650 nm) parts of the electromagnetic spectrum and show bright phosphorescence at room-temperature in the NIR with quantum yields up to 30% in solution. The small singlet–triplet energy gap yields unusually efficient thermally activated delayed fluorescence (TADF) at elevated temperatures in solution and in polymeric matrices with quantum yields as high as 27% at 120 °C, which is remarkable for benzoporphyrins. Apart from oxygen sensing, these properties enable unprecedented simultaneous, self-referenced oxygen and temperature sensing with a single indicator dye: whereas oxygen can be determined either via the decay time of phosphorescence or TADF, the temperature is accessed via the ratio of the two emissions. Moreover, the dyes are efficient sensitizers for triplet–triplet annihilation (TTA)-based upconversion making possible longer sensitization wavelength than the conventional benzoporphyrin complexes. The Pt-octa-sulfone dye also features interesting semireversible transformation in basic media, which generates new NIR absorbing species.

A new optical carbon dioxide sensor for environmental monitoring is presented. It combines a robust and long-term stable sensing material with a compact read-out device. The sensing material relies on a NIR pH indicator immobilized into ethyl cellulose along with a quaternary ammonium base. The perfluorinated polymer Hyflon AD 60 used as a protection layer significantly enhances the long-term and mechanical stability of the sensor foils, as well as the robustness against poisoning gases, e.g.hydrogen sulfide. The sensor can be stored under ambient conditions for more than six weeks, whereas sensors covered with silicone rubber deteriorate within one week under the same conditions. The complete sensor device is applicable after a three-point (re)calibration without a preconditioning step. The carbon dioxide production and consumption of the water plant Egeria densawas measured in the laboratory. Furthermore, results of profiling carbon dioxide measurements during a research cruise on the Baltic Sea at water depths up to 225 m are presented.

High brightness, chemical and photostability, tunable characteristics, and spectral and surface properties are important attributes for nanoparticle probes designed for live cell imaging. We describe a class of nanoparticles for high-resolution imaging of O2that consists of a substituted conjugated polymer (polyfluorene or poly(fluorene-alt-benzothiadiazole)) acting as a FRET antenna and a fluorescent reference with covalently bound phosphorescent metalloporphyrin (PtTFPP, PtTPTBPF). The nanoparticles prepared from such copolymers by precipitation method display stability, enhanced (>5–10 times) brightness under one- and two-photon excitation, compatibility with ratiometric and lifetime-based imaging modes, and low toxicity for cells. Their cell-staining properties can be modulated with positively and negatively charged groups grafted to the backbone. The “zwitter-ionic” nanoparticles show high cell-staining efficiency, while their cell entry mechanisms differ for the different 3D models. When injected in the bloodstream, the cationic and anionic nanoparticles show similar distribution in vivo. These features and tunable properties make the conjugated polymer based phosphorescent nanoparticles a versatile tool for quantitative O2imaging with a broad range of cell and 3D tissue models.

Monitoring cell and tissue oxygenation is important for the analysis of cell development and differentiation, mitochondrial function, and common (patho)physiological conditions such as ischemia, cancer, neurodegenerative disorders. A number of materials for sensing cellular oxygen (O2) by optical means have been described in recent years, but the diverse range of biological models and measurement tasks demands more versatile, flexible, and simple O2sensors. A new cell‐penetrating phosphorescent nanosensor material called MM2 probe is presented. In it, the highly photostable phosphorescent reporter dye Pt(II)‐5,10,15,20‐tetrakis‐(2,3,4,5,6‐pentafluorophenyl)‐porphyrin (PtTFPP; emission at 650 nm) and poly(9,9‐dioctylfluorene) (PFO) fluorophore act as Förster resonance energy transfer (FRET) donor and two‐photon antennae are embedded in cationic hydrogel nanoparticles. Such probe formulation provides efficient delivery into the cell and subsequent sensing and high‐resolution imaging of cellular O2in different detection modalities, including ratiometric intensity and phosphorescence lifetime‐based sensing under one‐photon and two photon excitation. MM2 probe combines high brightness, photo‐ and chemical stability, low toxicity, and ease of fabrication and use. Its versatility and analytical performance are demonstrated in physiological experiments with adherent cells and neurospheres representing 2D and 3D respiring objects and detection on time‐resolved fluorescent readers, confocal and multiphoton microscopes, and customized microsecond fluorescence/phosphorescence lifetime imaging microscopy (FLIM) systems.

A novel concept for designing optical oxygen sensing materials is reported. Oxygen‐sensitive anti‐Stokes emission is generated via triplet–triplet annihilation‐based upconversion and serves as an analytical parameter. Porous glass beads are used to incorporate the “sensing chemistry” including a sensitizer and an annihilator dissolved in a high boiling solvent. The beads are dispersed in silicone rubber or Teflon AF to produce solid state optodes. Inexpensive low power light sources (LEDs) are used for the excitation. The upconverted emission shows unmatched sensitivity both for the luminescence decay time and for the luminescence intensity. The latter features unusual quadratic Stern‐Volmer plots. Much lower sensitivity of the residual NIR luminescence of the sensitizer allows determination of pO2in the broad dynamic range from trace oxygen quantities to ≈40 kPa. Interrogation of the sensors in frequency domain is demonstrated. Influence of the excitation light power on the calibration, temperature effects, dynamic response to altering pO2, and photostability of the sensing materials are also investigated.

A new intracellular O2(icO2) sensing probe is presented, which comprises a nanoparticle (NP) formulation of a cationic polymer Eudragit RL-100 and a hydrophobic phosphorescent dye Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP). Using the time-resolved fluorescence (TR-F) plate reader set-up, cell loading was investigated in detail, particularly the effects of probe concentration, loading time, serum content in the medium, cell type, density, etc. The use of a fluorescent analogue of the probe in conjunction with confocal microscopy and flow cytometry analysis, revealed that cellular uptake of the NPs is driven by nonspecific energy-dependent endocytosis and that the probe localizes inside the cell close to the nucleus. Probe calibration in biological environment was performed, which allowed conversion of measured phosphorescence lifetime signals into icO2concentration (μM). Its analytical performance in icO2sensing experiments was demonstrated by monitoring metabolic responses of mouse embryonic fibroblast cells under ambient and hypoxic macroenvironment. The NP probe was seen to generate stable and reproducible signals in different types of mammalian cells and robust responses to their metabolic stimulation, thus allowing accurate quantitative analysis. High brightness and photostability allow its use in screening experiments with cell populations on a commercial TR-F reader, and for single cell analysis on a fluorescent microscope.

Synthesis and characterization of four iridium(III)–octaethylporphyrins and a π‐extended iridium(III)–benzoporphyrin are presented. Strong room‐temperature phosphorescence was observed for all of the complexes with quantum yields of up to 30 %. Axial ligands were introduced to tune the photophysical properties and the solubility. Complexes bearing lipophilic ligands such as pyridine or N‐(n‐butyl)imidazole were incorporated into polystyrene to obtain optical oxygen sensors. Covalent coupling of the dye is possible by introduction of ligands with binding domains (1‐imidazoleacetic acid). This enabled preparation of a water‐soluble oxygen probe (by staining bovine serum albumin) and a trace oxygen sensor (by coupling to amino‐modified silica gel).

Magnetic optical sensor particles with multifunctional cores and shells are synthesized via a facile nanoprecipitation method and the subsequent modification of the particle shell. The hydrophobic particle core includes optical oxygen indicators, a light harvesting system, photosensitizers, and magnetic nanoparticles. Further functionalities are introduced by modifying the shell with enzymes, antibodies, multiple layers of polyelectrolytes, stimuli‐responsive polymers, and luminescent indicator dyes. The hydrodynamic diameter is tunable by varying different precipitation parameters.

A new thermographic phosphor based on chromium(III)-doped yttrium aluminum borate (YAB) is obtained as single crystals by high temperature flux growth and as a microcrystalline powder via solution combustion synthesis. The phosphor is excitable both in the blue (λmax422 nm) and in the red part of the spectrum (λmax600 nm) and shows bright NIR emission. The brightness of the phosphor is comparable to that of a well-known lamp phosphor Mn(IV)-doped magnesium fluorogermanate. At ambient temperatures, the Cr(III)-doped YAB shows high temperature dependence of the luminescence decay time, which approaches 1% per deg. The material shows no decrease in luminescence intensity at higher temperatures. The new phosphor is particularly promising for applications in temperature-compensated optical chemosensors (including those based on NIR-emitting indicators) and in pressure-sensitive paints.

A new class of oxygen indicators is described. Platinum(II) and palladium(II) complexes of azatetrabenzoporphyrins occupy an intermediate position between tetrabenzoporphyrins and phthalocyanines and combine features of both. The new dyes are excitable in the red part of the spectrum and possess strong room-temperature NIR phosphorescence. Other features include excellent spectral compatibility with the red laser diodes and 632.8 nm line of He−Ne laser, excellent photostability, and significantly shorter decay times than for the respective meso-tetraphenyltetrabenzoporphyrins. Applicability of the complexes for optical oxygen sensing is demonstrated.

A sensitive and broad‐ranged optical thermometer, based on the thermally activated delayed fluorescence of fullerene C70, is presented. It consists of C70molecularly dispersed in a polymer film. Several polymer matrices were investigated. In the absence of oxygen the fluorescence intensity increases markedly with temperature. At 25 °C the fluorescence intensity of C70increases maximally by a factor of between 17 and 22, depending on the polymer, whereas at 100 °C the fluorescence intensity can be 79 times higher. In the absence of oxygen and for temperatures above 20 °C, the red fluorescence of C70in the films is so intense that it is easily perceived by the naked eye. For the systems studied, the fluorescence intensity is very sensitive to temperature. This results in a working range from −80 to at least 140 °C in the case of C70in poly(tert‐butyl methacrylate) (PtBMA). Perylene was incorporated into the film as an internal reference in order to enable ratiometric measurements. The sensitivity of the lifetime of the delayed fluorescence to temperature is also high and results in an even wider working range. The performance of the C70/PtBMA film was measured against a well‐known optical temperature probe, [Ru(phen)3] (phen=phenanthroline). The results show that the C70/PtBMA film is a very good system for optical temperature‐sensing over a wide range of temperatures, outperforming known standards.

Simultaneous fluorometric sensing of two analytes becomes possible using a modified dual lifetime referencing (m-DLR) method. In this scheme, two luminescent indicators are needed that have overlapping absorption and emission spectra but largely different decay times. They are excited by a single light source, and both emissions are measured simultaneously. In the frequency domain m-DLR method, the phase of the short-lived fluorescence of a first indicator is referenced against that of the long-lived luminescence of the second indicator. The analytical information is obtained by measurement of the phase shifts at two modulation frequencies. The method is demonstrated to work for the case of dually sensing oxygen and carbon dioxide. It benefits from simple instrumentation and optical setup. The approach is perceived to be of wide applicability. Examples include (1) analysis of two luminescent analytes, (2) analytical determinations that make use of two probes, and (3) sensing of two species such as carbon dioxide and oxygen (as demonstrated here), or oxygen and chlorophyll, provided the luminophores meet the condition of having largely different decay times and overlapping absorption and emission spectra.