Project description: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).
Project description:Solid tumours display varied oxygen levels and this characteristic can be exploited to develop new diagnostic tools to determine and exploit these variations. Oxygen is an efficient quencher of emission of many phosphorescent compounds, thus oxygen concentration could in many cases be derived directly from relative emission intensity and lifetime. In this study, we extend our previous work on phosphorescent, low molecular weight platinum(II) complex as an oxygen sensing probe to study the variation in oxygen concentration in a viable multicellular 3D human tumour model. The data shows one of the first examples of non-invasive, real-time oxygen mapping across a melanoma tumour spheroid using one-photon phosphorescence lifetime imaging microscopy (PLIM) and a small molecule oxygen sensitive probe. These measurements were quantitative and enabled real time oxygen mapping with high spatial resolution. This combination presents as a valuable tool for optical detection of both physiological and pathological oxygen levels in a live tissue mass and we suggest has the potential for broader clinical application.
Project description:In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.
Project description:Phosphorescence lifetime imaging microscopy (PLIM) combined with an oxygen (O2)-sensitive luminescent probe allows for high-resolution O2 imaging of living tissues. Herein, we present phosphorescent Ir(III) complexes, (btp)2Ir(acac-DM) (Ir-1) and (btp-OH)3Ir (Ir-2), as useful O2 probes for PLIM measurement. These small-molecule probes were efficiently taken up into cultured cells and accumulated in specific organelles. Their excellent cell-permeable properties allowed for efficient staining of three-dimensional cell spheroids, and thereby phosphorescence lifetime measurements enabled the evaluation of the O2 level and distribution in spheroids, including the detection of alterations in O2 levels by metabolic stimulation with an effector. We took PLIM images of hepatic tissues of living mice by intravenously administrating these probes. The PLIM images clearly visualized the O2 gradient in hepatic lobules with cellular-level resolution, and the O2 levels were derived based on calibration using cultured cells; the phosphorescence lifetime of Ir-1 gave reasonable O2 levels, whereas Ir-2 exhibited much lower O2 levels. Intravenous administration of NH4Cl to mice caused the hepatic tissues to experience hypoxia, presumably due to O2 consumption to produce ATP required for ammonia detoxification, suggesting that the metabolism of the probe molecule might affect liver O2 levels.
Project description:Over the past decade, appreciation of the roles of G-quadruplex (G4) structures in cellular regulation and maintenance has rapidly grown, making the establishment of robust methods to visualize G4s increasingly important. Fluorescent probes are commonly used for G4 detection in vitro; however, achieving sufficient selectivity to detect G4s in a dense and structurally diverse cellular environment is challenging. The use of fluorescent probes for G4 detection is further complicated by variations of probe uptake into cells, which may affect fluorescence intensity independently of G4 abundance. In this work, we report an alternative small-molecule approach to visualize G4s that does not rely on fluorescence intensity switch-on and, thus, does not require the use of molecules with exclusive G4 binding selectivity. Specifically, we have developed a novel thiazole orange derivative, TOR-G4, that exhibits a unique fluorescence lifetime when bound to G4s compared to other structures, allowing G4 binding to be sensitively distinguished from non-G4 binding, independent of the local probe concentration. Furthermore, TOR-G4 primarily colocalizes with RNA in the cytoplasm and nucleoli of cells, making it the first lifetime-based probe validated for exploring the emerging roles of RNA G4s in cellulo.
Project description:Ruthenium dipyridophenazine (dppz) complexes are sensitive luminescent probes for hydrophobic environments. Here, we apply multiple-frequency fluorescence lifetime imaging microscopy (FLIM) to Δ and Λ enantiomers of lipophilic ruthenium dppz complexes in live and fixed cells, and their different lifetime staining patterns are related to conventional intensity-based microscopy. Excited state lifetimes of the enantiomers determined from FLIM measurements correspond well with spectroscopically measured emission decay curves in pure microenvironments of DNA, phospholipid membrane or a model protein. We show that FLIM can be applied to monitor the long-lived excited states of ruthenium complex enantiomers and, combined with confocal microscopy, give new insight into their biomolecular binding and reveal differences in the microenvironment probed by the complexes.
Project description: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.
Project description:Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with fluorescence lifetime imaging microscopy (FLIM) can identify G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.
Project description:Visualization of guanine-rich oligonucleotides that fold into G-quadruplex (G4) helical structures is of great interest in cell biology. There is a large body of evidence that suggests that these noncanonical structures form in vivo and play important biological roles. A promising recent development highlighted fluorescence lifetime imaging microscopy (FLIM) as a robust technique for the direct and quantitative imaging of G4s in live cells. However, this method requires specialized, bespoke synthetic dyes that are not widely available. Herein, we demonstrate that the fluorescence lifetime of commercially available environmentally sensitive dyes Thioflavin T (ThT) and Thiazole Orange (TO) is strongly dependent on the type of DNA topology they bind to, with G4s showing long and distinctive decay times that should allow G4 detection in the biological environment. We applied this observation to visualize G4s in live U2OS cells using FLIM of ThT, upon alteration in G4 levels due to competitive binding or nuclease treatment of cells.
Project description:Time-gated fluorescence lifetime imaging microscopy with the o-BMVC fluorescent probe provides a visualizing method for the study of exogenous G-quadruplexes (G4s) in live cancer cells. Previously, imaging results showed that the parallel G4s are accumulated and that nonparallel G4s are not detected in the lysosomes of CL1-0 live cells. In this work, the detection of the G4 signals from exogenous GTERT-d(FN) G4s in the lysosomes may involve a structural change in live cells from intramolecular nonparallel G4s to intermolecular parallel G4s. Moreover, the detection of the G4 signals in the lysosomes after the 48 h incubation of HT23 G4s with CL1-0 live cells indicates the occurrence of structural conversion from the nonparallel G4s to the parallel G4s of HT23 in the live cells. In addition, the detection of much stronger G4 signals from ss-GTERT-d(FN) than ss-HT23 in the lysosomes of CL1-0 live cells may be explained by the quick formation of the intermolecular parallel G4s of ss-GTERT-d(FN) and the degradation of ss-HT23 before its intramolecular parallel G4 formation. This work provides a new approach to studying G4-lysosome interactions in live cells.