Phosphorescence Lifetime Imaging Research Articles

Biocompatible Re-Containing Block Copolymers for Intracellular pH Mapping in the PLIM Mode.

Monitoring of intracellular pH is of great importance since deviation of this parameter from the "normal" magnitudes can be considered as an indicator of various pathologies. Thus, the development of new efficient and biocompatible sensors suitable for application in biological systems and capable of quantitative pH estimation remains an urgent chemical task. Herein, we report the synthesis of a series of phosphorescent rhenium [Re(NN)(CO)2(PR3)2]+ complexes based on the NN diimine ligands containing pH-responsive carboxylic groups and styrene-containing phosphine ligands. The complexes, which display the highest pH sensitivity, were copolymerized with polyvinylpyrrolidone using the RAFT protocol to impart water solubility and to protect the chromophores from interaction with molecular oxygen. The resulting copolymers show an emission lifetime response onto pH variations in the physiological range. Cellular experiments with Chinese hamster ovary cells (CHO-K1) reveal easy internalization of the probes in cell culture and an approximately uniform distribution in cells, with some preference for location in acidic compartments (late endosomes and lysosomes). Using nigericin to homogenize intra- and extracellular pH, we built a calibration of lifetime versus pH in live CHO-K1 cells. Analysis of the phosphorescence lifetime imaging microscopy (PLIM) data confirms the applicability of the obtained sensors for monitoring the intracellular pH in cell cultures.

Ultraphotostable Phosphorescent Nanosensors for Sensing the Lysosomal pH at the Single-Cell Level over Long Durations.

Ultraphotostable phosphorescent nanosensors have been designed for continuously sensing the lysosome pH over a long duration. The nanosensors exhibited excellent photostability, high accuracy, and capability to measure pH values during cell proliferation for up to 7 days. By arranging a metal-ligand complex of long phosphorescence lifetime and pH indicator in silica nanoparticles, we discover efficient Förster resonance energy transfer, which converts the pH-responsive UV-vis absorption signal of the pH indicator into a phosphorescent signal. Both the phosphorescent intensity and lifetime change at different pH values, and intracellular pH values can be accurately measured by our custom-built rapid phosphorescent lifetime imaging microscopy. The excellent photostability, high accuracy, and good biocompatibility prove that these nanosensors are a useful tool for tracing the fluctuation of pH values during endocytosis. The methodology can be easily adapted to design new nanosensors with different pKa or for different kinds of intracellular ions, as there are hundreds of pH and ion indicators readily available.

A Mitochondria-Targeted Nitric Oxide Probe for Multimodality Imaging of Macrophage Immune Responses.

Nitric oxide (NO) is a crucial signal molecule closely linked to the biological immune response, especially in macrophage polarization. When activated, macrophages enter a pro-inflammatory state and produce NO, a marker for the M1 phenotype. In contrast, the anti-inflammatory M2 phenotype does not produce NO. We developed a mitochondria-targeted two-photon iridium-based complex (Ir-ImNO) probe that can detect endogenous NO and monitor macrophages' different immune response states using various imaging techniques, such as one- and two-photon phosphorescence imaging and phosphorescence lifetime imaging. Ir-ImNO was used to monitor the immune activation of macrophages in mice. This technology aims to provide a clear and comprehensive visualization of macrophage immune responses.

Biocompatible Phosphorescent Ir(III) Complexes for Hypoxia Sensing in PLIM Mode

AbstractIn this work, two new biscyclometalated Ir(III) complexes with diimine ligands were synthesized. The compounds were characterized by 1D and 2D NMR spectroscopy and HR ESI mass spectrometry. All complexes exhibit efficient phosphorescence with pronounced sensitivity to the presence of oxygen. The photophysical properties of the obtained compounds, including absorption, emission spectra, lifetimes and quantum yields of phosphorescence were measured in various aqueous and model biological media with variations in pH, temperature and at various O2 concentrations. The complexes exhibit a very good sensory response to the changes in oxygen concentration, resulting in an increase in quantum yields and lifetimes by 2.8–5.5 times upon oxygen removal from aerated aqueous solutions. Biological tests with CHO‐K1 cell line have shown that these compounds display low toxicity, rapid internalization into cells and localization predominantly in lysosomes. For the most promising complex the phosphorescence lifetime imaging experiments were conducted, revealing that this sensor markedly changes the phosphorescence lifetime values in cells from 1.8 to 4.1 μs upon transition from normoxia to simulated hypoxia. The obtained results indicate that this type of iridium chromophores can be effectively used for assessing the oxygen status of living objects by using time‐resolved luminescent microscopy.

Monitoring the Mitochondrial Viscosity Changes During Cuproptosis with Iridium(III) Complex Probe via In Situ Phosphorescence Lifetime Imaging.

Cuproptosis is a novel copper-dependent form of programmed cell death, displaying important regulatory functions in many human diseases, including cancer. However, the relationship between the changes in mitochondrial viscosity, a key factor associated with cellular malfunction, and cuproptosis is still unclear. Herein, we prepared a phosphorescent iridium (Ir) complex probe for precisely monitoring the changes of mitochondrial viscosity during cuprotosis via phosphorescence lifetime imaging. The Ir complex probe possessed microsecond lifetimes (up to 1 μs), which could be easily distinguished from cellular autofluorescence to improve the imaging contrast and sensitivity. Benefiting from the long phosphorescence lifetime, excellent viscosity selectivity, and mitochondrial targeting abilities, the Ir complex probe could monitor the increase in the mitochondrial viscosity during cuproptosis (from 46.8 to 68.9 cP) in a quantitative manner. Moreover, through in situ fluorescence imaging, the Ir complex probe successfully monitored the increase in viscosity in zebrafish treated with lipopolysaccharides or elescolomol-Cu2+, which were well-known cuproptosis inducers. We anticipate that this new Ir complex probe will be a useful tool for in-depth understanding of the biological effects of mitochondrial viscosity during cuproptosis.

Color‐Tunable Luminescence of Eu‐Doped LaF3 Particles Sensitized by d–f Energy Transfer from a Two‐Photon Absorbing Ir(III) Complex

AbstractThe surface of Eu0.3La0.7F3 submicron particles is functionalized with a blue‐emitting Ir(III) complex (1) specifically designed to coordinate lanthanide(III) ions efficiently through a carboxylic unit. In this Eu0.3La0.7F3@1 composite, the Ir(III) complexes are randomly coordinated to either superficial La(III) ions or Eu(III) ions (Ir‐Lasurface and Ir‐Eusurface pairs, respectively) and its color emission, a balance between blue (Ir‐based) and red (Eu‐based) light, can be tuned as a function of the excitation wavelength. Irradiation at the maximum of the excitation band in 1 promotes blue emission from Ir‐Lasurface pairs and red emission from Eusurface ions sensitized by Ir → Eusurface energy transfer (EnT). At λexc = 396 nm (maximum of the Eu(III) 5L6 ← 7F0 absorption band) the red emission from inner Eu(III) ions becomes dominant. Excitation of 1 can also be achieved by two photon‐absorption (TPA) since this complex has a moderate cross‐section of σ2 = 9.4 ± 1.0 GM at 780 nm (σ2 = 5.8 ± 0.6 GM at 800 nm). Phosphorescence lifetime imaging (PLIM) allows the emission of individual particles to be visualized and the Ir‐, Eusurface‐, and Eucore‐based emissions can be distinguished due to the significant differences in their respective emission lifetimes.

Effects of Photodynamic Therapy on Tumor Metabolism and Oxygenation Revealed by Fluorescence and Phosphorescence Lifetime Imaging.

This work was aimed at the complex analysis of the metabolic and oxygen statuses of tumors in vivo after photodynamic therapy (PDT). Studies were conducted on mouse tumor model using two types of photosensitizers-chlorin e6-based drug Photoditazine predominantly targeted to the vasculature and genetically encoded photosensitizer KillerRed targeted to the chromatin. Metabolism of tumor cells was assessed by the fluorescence lifetime of the metabolic redox-cofactor NAD(P)H, using fluorescence lifetime imaging. Oxygen content was assessed using phosphorescence lifetime macro-imaging with an oxygen-sensitive probe. For visualization of the perfused microvasculature, an optical coherence tomography-based angiography was used. It was found that PDT induces different alterations in cellular metabolism, depending on the degree of oxygen depletion. Moderate decrease in oxygen in the case of KillerRed was accompanied by an increase in the fraction of free NAD(P)H, an indicator of glycolytic switch, early after the treatment. Severe hypoxia after PDT with Photoditazine resulted from a vascular shutdown yielded in a persistent increase in protein-bound (mitochondrial) fraction of NAD(P)H. These findings improve our understanding of physiological mechanisms of PDT in cellular and vascular modes and can be useful to develop new approaches to monitoring its efficacy.

Bromine indirubin FLIM/PLIM sensors to measure oxygen in normoxic and hypoxic PDT conditions

BackgroundThe induction of phototoxicity during photodynamic therapy (PDT) is dependent on oxygen availability. For this reason, the development of sensors to measure oxygen and oxygen consumption is extremely important. ApproachIn this project we have used Fluorescence Lifetime imaging (FLIM) and Phosphorescence Lifetime Imaging/ delayed Fluorescence Lifetime Imaging (PLIM/dFLIM) to investigate the ability of bromine indirubin derivatives as oxygen sensors. ResultsThe oxygen sensitivity of bromine indirubins was detected through PLIM/dFLIM. Moreover, we have observed, by measuring nicotinamide adenine dinucleotide (NADH) FLIM, that bromine indirubin has a significant impact on cellular metabolism by shifting the SCC-4 Cells metabolism from oxidative phosphorylation (OXPHOS) to glycolysis. ConclusionsIn conclusion, this study successfully achieves its goals and provides important insights into the use of indirubin as a potential oxygen consumption sensor with the capability to identify and differentiate between normoxic and hypoxic regions within the cells.

Oxygen Assessment in Tumors In Vivo Using Phosphorescence Lifetime Imaging Microscopy.

The oxygen level in a tumor is a crucial factor for its development and response to therapies. Phosphorescence lifetime imaging (PLIM) with the use of phosphorescent oxygen probes is a highly sensitive, noninvasive optical technique for the assessment of molecular oxygen in living cells and tissues. Here, we present a protocol for microscopic mapping of oxygen distribution in a mouse tumor model in vivo. We demonstrate that PLIM microscopy, in combination with an Ir(III)-based probe, enables visualization of cellular-level heterogeneity of tumor oxygenation.

Maximum-A-Posteriori Estimation of Retinal Oxygen Tension Based on Photon Counting Statistics.

The regularization of retinal oxygen tension estimation was previously proposed with an assumption that phosphorescence intensity images are corrupted by additive Gaussian noise. Based on this assumption, a regularized least-squares estimate has been shown to be better than a conventional least-squares estimation. However, this assumption is inconsistent with the acquisition process of phosphorescence intensity images acquired using an intensified charge-coupled device camera. Almost the entire acquisition process is governed by the natural aspects of photons. Therefore, a method based on photon counting statistics is more appropriate. In this study, we propose a regularized oxygen tension estimation method based on photon counting statistics and a phosphorescence lifetime imaging model.

Monitoring the Intracellular pH and Metabolic State of Cancer Cells in Response to Chemotherapy Using a Combination of Phosphorescence Lifetime Imaging Microscopy and Fluorescence Lifetime Imaging Microscopy.

The extracellular matrix (ECM), in which collagen is the most abundant protein, impacts many aspects of tumor physiology, including cellular metabolism and intracellular pH (pHi), as well as the efficacy of chemotherapy. Meanwhile, the role of collagen in differential cell responses to treatment within heterogeneous tumor environments remains poorly investigated. In the present study, we simultaneously monitored the changes in pHi and metabolism in living colorectal cancer cells in vitro upon treatment with a chemotherapeutic combination, FOLFOX (5-fluorouracil, oxaliplatin and leucovorin). The pHi was followed using the new pH-sensitive probe BC-Ga-Ir, working in the mode of phosphorescence lifetime imaging (PLIM), and metabolism was assessed from the autofluorescence of the metabolic cofactor NAD(P)H using fluorescence lifetime imaging (FLIM) with a two-photon laser scanning microscope. To model the ECM, 3D collagen-based hydrogels were used, and comparisons with conventional monolayer cells were made. It was found that FOLFOX treatment caused an early temporal intracellular acidification (reduction in pHi), followed by a shift to more alkaline values, and changed cellular metabolism to a more oxidative state. The presence of unstructured collagen markedly reduced the cytotoxic effects of FOLFOX, and delayed and diminished the pHi and metabolic responses. These results support the observation that collagen is a factor in the heterogeneous response of cancer cells to chemotherapy and a powerful regulator of their metabolic behavior.

Analysis of tumour oxygenation in model animals on a phosphorescence lifetime based macro-imager

Monitoring of tissue O2 is essential for cancer development and treatment, as hypoxic tumour regions develop resistance to radio- and chemotherapy. We describe a minimally invasive technique for the monitoring of tissue oxygenation in developing grafted tumours, which uses the new phosphorescence lifetime based Tpx3Cam imager. CT26 cells stained with a near-infrared emitting nanoparticulate O2 probe NanO2-IR were injected into mice to produce grafted tumours with characteristic phosphorescence. The tumours were allowed to develop for 3, 7, 10 and 17 days, with O2 imaging experiments performed on live and euthanised animals at different time points. Despite a marked trend towards decreased O2 in dead animals, their tumour areas produced phosphorescence lifetime values between 44 and 47 µs, which corresponded to hypoxic tissue with 5–20 μM O2. After the O2 imaging in animals, confocal Phosphorescence Lifetime Imaging Microscopy was conducted to examine the distribution of NanO2-IR probe in the tumours, which were excised, fixed and sliced for the purpose. The probe remained visible as bright and discrete ‘islands’ embedded in the tumour tissue until day 17 of tumour growth. Overall, this O2 macro-imaging method using NanO2-IR holds promise for long-term studies with grafted tumours in live animal models, providing quantitative 2D mapping of tissue O2.

A Mitochondria-Localized Iridium(III) Complex for Simultaneous Two-Photon Phosphorescence Lifetime Imaging of Downstream Products N2O3 and ONOO- of Endogenous Nitric Oxide.

Nitric oxide (NO) serves as a ubiquitous and fundamental signaling molecule involved in intricate effects on both physiological and pathological processes. NO, biosynthesized by nitric oxide synthase (NOS) or generated from nitrite, can form nitrosation reagent N2O3 (4NO + O2 = 2N2O3) through its oxidation or quickly produce peroxynitrite anion ONOO- (NO + •O2- = ONOO-) by reacting with superoxide anion (•O2-). However, most of the existing luminescent probes for NO just focus on specificity and utilize only a single signal to distinguish products N2O3 or ONOO-. In most of the present work, they differentiate one product from another simply by fluorescence signal or fluorescence intensity, which is not enough to distinguish accurately the behavior of NO in living cells. Herein, a new mitochondria-targeted and two-photon near-infrared (NIR) phosphorescent iridium(III) complex, known as Ir-NBD, has been designed for accurate detection and simultaneous imaging of two downstream products of endogenous NO, i.e., N2O3 and ONOO-. Ir-NBD exhibits a rapid response to N2O3 and ONOO- in enhanced phosphorescence intensity, increased phosphorescence lifetime, and an exceptionally high two-photon cross-section, reaching values of 78 and 85 GM, respectively, after the reaction. Furthermore, we employed multiple imaging methods, phosphorescence intensity imaging, and phosphorescence lifetime imaging together to image even distinguish N2O3 and ONOO- by probe Ir-NBD. Thus, coupled with its excellent photometrics, Ir-NBD enabled the detection of the basal level of intracellular NO accurately by responding to N2O3 and ONOO- in the lipopolysaccharide-stimulated macrophage model in virtue of fluorescence signal and phosphorescence lifetime imaging, revealing precisely the endogenous mitochondrial NO distribution during inflammation in a cell environment.

Structural Elucidation of Some Coordination Compounds With Reference Phosphorescent Metal Composite as Theranostic Anticancer Agents

This paper reveals that the phosphorescent metal composite are a new kind of multifunctional antitumor compounds that can integrate imaging and antitumor functions in a single molecule. In this minireview, we summarize the recent research progress in this field, concentrating on the theranostic applications of phosphorescent iridium(III), ruthenium(II) and rhenium(I) composite. The infinitesimal design that affords these composite with tumour- or subcellular organelle-targeting properties is elucidated. The potential of these composite to induce and monitor the dynamic behavior of subcellular organelles and the changes in microenvironment during the process of therapy is demonstrated. Moreover, the potential and advantages of applying new technologies, such as super-resolution imaging and phosphorescence lifetime imaging, are also described. Finally, the challenges faced in the development of novel theranostic metallo-anticancer composite for possible clinical translation are proposed.

Platinum(II)-Based Optical Probes for Imaging Quadruplex DNA Structures via Phosphorescence Lifetime Imaging Microscopy.

G-quadruplex DNA is a non-canonical structure that forms in guanine-rich regions of the genome. There is increasing evidence showing that G-quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. Herein we report on a series of new cyclometallated platinum(II) complexes which, upon binding to G-quadruplex DNA, display an increase in their phosphorescence, acting as switch-on probes. More importantly, upon binding to G-quadruplexes they display a selective and distinct lengthening of their emission lifetime. We show that this effect can be used to selectively visualise these structures in cells using Phosphorescence Lifetime Imaging Microscopy (PLIM).

Platinum(II)-Based Optical Probes for Imaging Quadruplex DNA Structures via Phosphorescence Lifetime Imaging Microscopy.

G-quadruplex DNA is a non-canonical structure that forms in guanine-rich regions of the genome. There is increasing evidence showing that G-quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. Herein we report on a series of new cyclometallated platinum(II) complexes which, upon binding to G-quadruplex DNA, display an increase in their phosphorescence, acting as switch-on probes. More importantly, upon binding to G-quadruplexes they display a selective and distinct lengthening of their emission lifetime. We show that this effect can be used to selectively visualise these structures in cells using Phosphorescence Lifetime Imaging Microscopy (PLIM).

Three-dimensional oxygen concentration monitoring in hydrogels using low-cost phosphorescence lifetime imaging for tissue engineering.

Oxygen concentration measurement in 3D hydrogels is vital in 3D cell culture and tissue engineering. However, standard 3D imaging systems capable of measuring oxygen concentration with adequate precision are based on advanced microscopy platforms, which are not accessible in many laboratories due to the system's complexity and the high price. In this work, we present a fast and low-cost phosphorescence lifetime imaging design for measuring the lifetime of oxygen-quenched phosphorescence emission with 0.25 µs temporal precision and sub-millimeter spatial resolution in 3D. By combining light-sheet illumination and the frequency-domain lifetime measurement using a commercial rolling-shutter CMOS camera in the structure of a conventional optical microscope, this design is highly customizable to accommodate application-specific research needs while also being low-cost as compared to advanced instruments. As a demonstration, we made a fluidic device with a gas-permeable film to create an artificial oxygen gradient in the hydrogel sample. Dye-embedded beads were distributed in the hydrogel to conduct continuous emission lifetime monitoring when nitrogen was pumped through the fluidic channel and changed oxygen distribution in the sample. The dynamics of the changes in lifetime co-related with their location in the gel of size 0.5 mm×1.5 mm×700 µm demonstrate the ability of this design to measure the oxygen concentration stably and precisely in 3D samples.

Phosphorescent O2-Probes Based on Ir(III) Complexes for Bioimaging Applications

The design, synthesis, and investigation of new molecular oxygen probes for bioimaging, based on phosphorescent transition metal complexes are among the topical problems of modern chemistry and advanced bioimaging. Three new iridium [Ir(N^C)2(N^N)]+ complexes with cyclometallating 4-(pyridin-2-yl)-benzoic acid derivatives and different di-imine chelate ligands have been synthesized and characterized by mass spectrometry and NMR spectroscopy. The periphery of these complexes is decorated with three relatively small “double-tail” oligo(ethylene glycol) fragments. All these complexes exhibit phosphorescence; their photophysical properties have been thoroughly studied, and quantum chemical calculations of their photophysical properties were also performed. It turned out that the changes in the nature of the di-imine ligand greatly affected the character of the electronic transitions responsible for their emission. Two complexes in this series show the desired photophysical characteristics; they demonstrate appreciable quantum yield (14–15% in degassed aqueous solutions) and a strong response to the changes in oxygen concentration, ca. three-fold increase in emission intensity, and an excited state lifetime upon deaeration of the aqueous solution. The study of their photophysical properties in model biological systems (buffer solutions containing fetal bovine serum—FBS) and cytotoxicity assays (MTT) showed that these complexes satisfy the requirements for application in bioimaging experiments. It was found that these molecular probes are internalized into cultured cancer cells and localized mainly in mitochondria and lysosomes. Phosphorescent lifetime imaging (PLIM) experiments showed that under hypoxic conditions in cells, a 1.5-fold increase in the excitation state lifetime was observed compared to aerated cells, suggesting the applicability of these complexes for the analysis of hypoxia in biological objects.

An Optochemical Oxygen Scavenger Enabling Spatiotemporal Control of Hypoxia.

We present an optochemical O2 scavenging system that enables precise spatiotemporal control of the level of hypoxia in living cells simply by adjusting the light intensity in the illuminated region. The system employs rhodamine containing a selenium or tellurium atom as an optochemical oxygen scavenger that rapidly consumes O2 by photochemical reaction with glutathione as a coreductant upon visible light irradiation (560-590 nm) and has a rapid response time, within a few minutes. The glutathione-consuming quantum yields of the system were calculated as about 5 %. The spatiotemporal O2 consuming in cultured cells was visualized with a hypoxia-responsive fluorescence probe, MAR. Phosphorescence lifetime imaging was applied to confirmed that different light intensities could generate different levels of hypoxia. To illustrate the potential utility of this system for hypoxia research, we show that it can spatiotemporally control calcium ion (Ca2+ ) influx into HEK293T cells expressing the hypoxia-responsive Ca2+ channel TRPA1.

An Optochemical Oxygen Scavenger Enabling Spatiotemporal Control of Hypoxia

AbstractWe present an optochemical O2 scavenging system that enables precise spatiotemporal control of the level of hypoxia in living cells simply by adjusting the light intensity in the illuminated region. The system employs rhodamine containing a selenium or tellurium atom as an optochemical oxygen scavenger that rapidly consumes O2 by photochemical reaction with glutathione as a coreductant upon visible light irradiation (560–590 nm) and has a rapid response time, within a few minutes. The glutathione‐consuming quantum yields of the system were calculated as about 5 %. The spatiotemporal O2 consuming in cultured cells was visualized with a hypoxia‐responsive fluorescence probe, MAR. Phosphorescence lifetime imaging was applied to confirmed that different light intensities could generate different levels of hypoxia. To illustrate the potential utility of this system for hypoxia research, we show that it can spatiotemporally control calcium ion (Ca2+) influx into HEK293T cells expressing the hypoxia‐responsive Ca2+ channel TRPA1.