Changes In Fluorescence Lifetime Research Articles

Photophysicochemical behaviour of metallophthalocyanines when doped onto silica nanoparticles

Aluminum(III) chloride 2,9(10),16(17),23(24)–tetra–(4–tert–butylphenoxy)phthalocyanine (1), zinc(II) 2,9(10),16(17),23(24)–tetra–(4–tert–butylphenoxy)phthalocyanine (2), zinc(II) 1,8(11),15(18),22(25)–tetra–(4–tert–butylphenoxy) phthalocyanine (3) and zinc(II) 2,9(10),16(17),23(24)–tetra–(4–carboxylphenoxy) phthalocyanine (4) were doped onto silica nanoparticles (SiNPs). There were no significant changes in fluorescence quantum yields and lifetimes of the metallophthalocyanines ( MPcs) when doped onto SiNPs. The triplet quantum yields of the MPcs alone range from 0.22 to 0.85 and in the presence of SiNPs the values range from 0.17 to 0.89. We observed a general decrease of triplet quantum yields of phthalocyanines in the conjugates except for 2 where there was an increase. The values were highly affected by aggregation. Complexes 1 and 3 were highly aggregated when doped onto SiNPs, while 2 and 4 did not show much aggregation.

Hairpin DNA-functionalized gold nanorods for mRNA detection in homogenous solution.

We report a fluorescent probe for mRNA detection. It consists of a gold nanorod (GNR) functionalized with fluorophore-labeled hairpin oligonucleotides (hpDNA) that are complementary to the mRNA of a target gene. This nanoprobe was found to be sensitive to a complementary oligonucleotide, as indicated by significant changes in both fluorescence intensity and lifetime. The influence of the surface density of hpDNA on the performance of this nanoprobe was investigated, suggesting that high hybridization efficiency could be achieved at a relatively low surface loading density of hpDNA. However, steady-state fluorescence spectroscopy revealed better overall performance, in terms of sensitivity and detection range, for nanoprobes with higher hairpin coverage. Time-resolved fluorescence lifetime spectroscopy revealed significant lifetime changes of the fluorophore upon hybridization of hpDNA with targets, providing further insight on the hybridization kinetics of the probe as well as the quenching efficiency of GNRs.

Enhancing Silicon Nanocrystal Photoluminescence through Temperature and Microstructure

Routes to enhancing the photoluminescence (PL) of colloidal silicon nanocrystals (SiNCs) typically focus on changes in surface chemistry and the associated improvements in quantum yield. Here, we report a new more indirect approach that instead exploits the structure of the host matrix. Specifically, we demonstrate that changes in microstructure associated with a thermotropic phase transition in unbound ligand can increase the excitation fluence through scattering, yielding dramatic improvements in PL intensity without any discernible changes in fluorescence lifetime or quantum yield. Using size-purified plasma-synthesized SiNCs prepared as solid and liquid samples, we use experiment and computation to examine both intrinsic size-resolved differences in the temperature-dependent PL and an anomalous contribution linked to matrix microstructure. Beyond revealing a potential new route to improved PL intensity, our results further clarify the role of surface states and the challenges that they present.

Quenching of chlorophyll fluorescence induced by silver nanoparticles

The interaction between chlorophyll (Chl) and silver nanoparticles (AgNPs) was evaluated by analyzing the optical behavior of Chl molecules surrounded by different concentrations of AgNPs (10, 60, and 100nm of diameter). UV–Vis absorption, steady state and time-resolved fluorescence measurements were performed for Chl in the presence and absence of these nanoparticles. AgNPs strongly suppressed the Chl fluorescence intensity at 678nm. The Stern-Volmer constant (KSV) showed that fluorescence suppression is driven by the dynamic quenching process. In particular, KSV was nanoparticle size-dependent with an exponential decrease as a function of the nanoparticle diameter. Finally, changes in the Chl fluorescence lifetime in the presence of nanoparticles demonstrated that the fluorescence quenching may be induced by the excited electron transfer from the Chl molecules to the metal nanoparticles.

In situ kinetics study of the formation of organic nanoparticles by fluorescence lifetime imaging microscopy (FLIM) along a microfluidic device

In this study, a three-dimensional hydrodynamic focusing microfluidic method is presented that allows full control of the nano-precipitation process of adamantyl mesityl BODIPY (4,4-difluoro-3,5-di-(adamantyl)-8-mesityl-4-bora-3a,4a-diaza-s-indacene) (Adambodipy). The precipitation is achieved by combining a central Adambodipy organic flow with a mixture of water and a cationic surfactant, creating a non-solvent precipitation method. The flow and mixing were simulated using COMSOL Multiphysics® 3.4. A good agreement between theory and experiment was obtained for the flow velocity, concentration fields and the subsequent precipitation kinetics. Fluorescence lifetime imaging was used to visualize the precipitation domains following the changes in fluorescence lifetime. The lifetime decreases from 6.1 ns for the molecules down to 0.9 ns for nanoparticles. A principal components analysis of the successive fluorescence decay curves showed that the process could be adequately modeled using three components, which can be attributed to monomers (single molecule), clusters (nuclei) and nanoparticles.

Substrate-based near-infrared imaging sensors enable fluorescence lifetime contrast via built-in dynamic fluorescence quenching elements.

Enzymatic activity sensing in fluorescence lifetime (FLT) mode with "self-quenched" macromolecular near-infrared (NIR) sensors is a highly promising strategy for in vivo imaging of proteolysis. However, the mechanisms of FLT changes in such substrate-based NIR sensors have not yet been studied. We synthesized two types of sensors by linking the near-infrared fluorophore IRDye 800CW to macromolecular graft copolymers of methoxy polyethylene glycol and polylysine (MPEG-gPLL) with varying degrees of MPEGylation and studied their fragmentation induced by trypsin, elastase, plasmin and cathepsins (B,S,L,K). We determined that the efficiency of such NIR sensors in FLT mode depends on sensor composition. While MPEG-gPLL with a high degree of MPEGylation showed rapid (τ1/2=0.1-0.2 min) FLT increase (Δτ=0.25 ns) upon model proteinase-mediated hydrolysis in vivo, lower MPEGylation density resulted in no such FLT increase. Temperature-dependence of fluorescence de-quenching of NIR sensors pointed to a mixed dynamic/static-quenching mode of MPEG-gPLL-linked fluorophores. We further demonstrated that although the bulk of sensor-linked fluorophores were de-quenched due to the elimination of static quenching, proteolysis-mediated deletion of a fraction of short (8-10kD) negatively charged fragments of highly MPEGylated NIR sensor is the most likely event leading to a rapid FLT increase phenomenon in quenched NIR sensors. Therefore, the optimization of "built-in" dynamic quenching elements of macromolecular NIR sensors is a potential avenue for improving their response in FLT mode.

Cellular uptake and intracellular degradation of poly(alkyl cyanoacrylate) nanoparticles.

Background Poly(alkyl cyanoacrylate) (PACA) nanoparticles have shown promise as drug carriers both to solid tumors and across the blood–brain barrier. Efficient drug delivery requires both high cellular uptake of the nanoparticles and release of the drug from the nanoparticles. Release of hydrophobic drugs from PACA nanoparticles is primarily governed by nanoparticle degradation, and this process has been poorly studied at the cellular level. Here we use the hydrophobic model drug Nile Red 668 (NR668) to investigate intracellular degradation of PACA nanoparticles by measuring changes in NR668 fluorescence emission and lifetime, as the spectral properties of NR668 depend on the hydrophobicity of the dye environment. We also assess the potential of poly(butyl cyanoacrylate) (PBCA) and poly(octyl cyanoacrylate) (POCA) nanoparticles for intracellular drug delivery in the prostate cancer cell line PC3 and rat brain endothelial cell line RBE4 and the role of endocytosis pathways in PACA nanoparticle uptake in those cell lines.ResultsFluorescence lifetime imaging, emission spectra analysis and Förster resonance energy transfer indicated that the intracellular degradation was in line with the degradation found by direct methods such as gas chromatography and scanning electron microscopy, showing that PBCA has a faster degradation rate compared to POCA. The combined P(BCA/OCA) nanoparticles had an intermediate degradation rate. The uptake of POCA and PBCA nanoparticles was much higher in RBE4 than in PC3 cells. Endocytosis inhibition studies showed that both clathrin- and caveolin-mediated endocytosis were involved in PACA nanoparticle uptake, and that the former played a predominant role, particularly in PC3 cells.ConclusionsIn the present study, we used three different optical techniques to show that within a 24-hour period PBCA nanoparticles degraded significantly inside cells, releasing their payload into the cytosol, while POCA nanoparticles remained intact. This indicates that it is possible to tune the intracellular drug release rate by choosing appropriate monomers from the PACA family or by using hybrid PACA nanoparticles containing different monomers. In addition, we showed that the uptake of PACA nanoparticles depends not only on the monomer material, but also on the cell type, and that different cell lines can use different internalization pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-015-0156-7) contains supplementary material, which is available to authorized users.

A new turn on coumarin-based fluorescence probe for Ga3 + detection in aqueous solution

The probe CT was synthesized and investigated as a novel label-free chemosensor for Ga3+ detection in water. Probe CT showed remarkable selectivity and sensitivity for Ga3+ in Tris–HCl aqueous buffer solution (pH7.0). The chemosensor responded rapidly to Ga3+ with a 1:1 stoichiometry. Meanwhile, the unapparent changes of fluorescence lifetime decays suggest the turn-on process of probe CT by Ga3+ which appears to be a static mechanism.

Effect of P5 +/Al3 + molar ratio on structure and spectroscopic properties of Nd3 +/Al3 +/P5 + co-doped silica glass

Glasses with compositions of (95.8-x) SiO2–4Al2O3–xP2O5–0.2Nd2O3 (in mol%, x=0–10) were prepared. Structural variation caused by P5+ was investigated by Judd–Ofelt parameters and Raman spectra. Results reveal that Nd3+ preferentially coordinates to PO sites at molar ratio of P5+/Al3+>1 and thus it leads to a sudden change in fluorescence effective linewidth, stimulated emission cross section, fluorescence lifetime and non-radiative transition rate of Nd3+ ions. The fluorescence lifetime changes in the range of 315–379μs. The emission cross section changes in the range of 1.39–1.79×10−20cm2. With the increase of P5+/Al3+ ratio, the fluorescence effective linewidth varies from 45.77 to 37.03nm. The shortest fluorescence lifetime was observed at molar ratio of P5+/Al3+=1. The maximum non-radiative transition rate of 1694/s occurred at molar ratio of P5+/Al3+=1.25. The highest emission cross section of 1.79×10−20cm2 was obtained at the highest P5+/Al3+ molar ratio. Furthermore, structure–property relationship in these glasses was discussed through Judd–Ofelt parameters and luminescent behaviors.

Assessment of Gate Width Size on Lifetime-Based Förster Resonance Energy Transfer Parameter Estimation

Förster Resonance Energy Transfer (FRET) enables the observation of interactions at the nanoscale level through the use of fluorescence optical imaging techniques. In FRET, fluorescence lifetime imaging can be used to quantify the fluorescence lifetime changes of the donor molecule, which are associated with proximity between acceptor and donor molecules. Among the FRET parameters derived from fluorescence lifetime imaging, the percentage of donor that interacts with the acceptor (in proximity) can be estimated via model-based fitting. However, estimation of the lifetime parameters can be affected by the acquisition parameters such as the temporal characteristics of the imaging system. Herein, we investigate the effect of various gate widths on the accuracy of estimation of FRET parameters with focus on the near-infrared spectral window. Experiments were performed in silico, in vitro, and in vivo with gate width sizes ranging from 300 ps to 1000 ps in intervals of 100 ps. For all cases, the FRET parameters were retrieved accurately and the imaging acquisition time was decreased three-fold. These results indicate that increasing the gate width up to 1000 ps still allows for accurate quantification of FRET interactions even in the case of short lifetimes such as those encountered with near-infrared FRET pairs.

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.

Applying fluorescence lifetime imaging microscopy to evaluate the efficacy of anticancer drugs

Fluorescence lifetime imaging microscopy was applied to evaluate the efficacy of anticancer drugs. A decrease in the fluorescence lifetime of the nucleus in apoptotic cancer cells stained by SYTO 13 dye was detected after treatment with antitumor antibiotics such as doxorubicin or epirubicin. It was confirmed that the change in fluorescence lifetime occurred earlier than morphological changes in the cells. We found that the fluorescence lifetime of the nucleus in the cells treated with epirubicin decreased more rapidly than that of the cells treated with doxorubicin. This implies that epirubicin was more efficacious than doxorubicin in the treatment of cancer cells. The change in fluorescence lifetime was, however, not indicated when the cells were treated with cyclophosphamide. The decrease in fluorescence lifetime was associated with the processes involving caspase activation and chromatin condensation. Therefore, this technique would provide useful information about apoptotic cells, particularly in the early stages.

PH sensitivity of FRET reporters based on cyan and yellow fluorescent proteins.

It is generally acknowledged that the popular cyan and yellow fluorescent proteins carried by genetically encoded reporters suffer from strong pH sensitivities close to the physiological pH range. We studied the consequences of these pH responses on the intracellular signals of model Förster resonant energy transfer (FRET) tandems and FRET-based reporters of cAMP-dependent protein kinase activity (AKAR) expressed in the cytosol of living BHK cells, while changing the intracellular pH by means of the nigericin ionophore. Although the simultaneous pH sensitivities of the donor and the acceptor may mask each other in some cases, the magnitude of the perturbations can be very significant, as compared to the functional response of the AKAR biosensor. Replacing the CFP donor by the spectrally identical, but pH-insensitive Aquamarine variant (pK1/2 = 3.3) drastically modifies the biosensor pH response and gives access to the acid transition of the yellow acceptor. We developed a simple model of pH-dependent FRET and used it to describe the expected pH-induced changes in fluorescence lifetime and ratiometric signals. This model qualitatively accounts for most of the observations, but reveals a complex behavior of the cytosolic AKAR biosensor at acid pHs, associated to additional FRET contributions. This study underlines the major and complex impact of pH changes on the signal of FRET reporters in the living cell.

Real-time histology in liver disease using multiphoton microscopy with fluorescence lifetime imaging.

Conventional histology with light microscopy is essential in the diagnosis of most liver diseases. Recently, a concept of real-time histology with optical biopsy has been advocated. In this study, live mice livers (normal, with fibrosis, steatosis, hepatocellular carcinoma and ischemia-reperfusion injury) were imaged by MPM-FLIM for stain-free real-time histology. The acquired MPM-FLIM images were compared with conventional histological images. MPM-FLIM imaged subsurface cellular and subcellular histopathological hallmarks of live liver in mice models at high resolution. Additional information such as distribution of stellate cell associated autofluorescence and fluorescence lifetime changes was also gathered by MPM-FLIM simultaneously, which cannot be obtained from conventional histology. MPM-FLIM could simultaneously image and quantify the cellular morphology and microenvironment of live livers without conventional biopsy or fluorescent dyes. We anticipate that in the near future MPM-FLIM will be evaluated from bench to bedside, leading to real-time histology and dynamic monitoring of human liver diseases.

Correlation of NADH fluorescence lifetime and oxidative phosphorylation metabolism in the osteogenic differentiation of human mesenchymal stem cell.

Reduced nicotinamide adenine dinucleotide (NADH) fluorescence lifetime has been broadly used as a metabolic indicator for stem cell imaging. However, the direct relationship between NADH fluorescence lifetime and metabolic pathway and activity remains to be clarified. In this study, we measured the NADH fluorescence lifetime of human mesenchymal stem cells (hMSCs) as well as the metabolic indictors, such as adenosine triphosphate (ATP) level, oxygen consumption, and lactate release, up to 4 weeks under normal osteogenic differentiation and oxidative phosphorylation-attenuated/inhibited differentiation by oligomycin A (OA) treatment. NADH fluorescence lifetime was positively correlated with oxygen consumption and ATP level during energy transformation from glycolysis to oxidative phosphorylation. Under OA treatment, oxidative phosphorylation was attenuated/inhibited (i.e., oxygen consumption remained the same as controls or lower), cells showed attenuated differentiation under glycolysis, and NADH fluorescence lifetime change was not detected. Increased expression of the overall complex proteins was observed in addition to Complex I. We suggested special caution needs to be exercised while interpreting NADH fluorescence lifetime signal in terms of stemcell differentiation.

Fluorescence axial nanotomography with plasmonics.

We present a novel imaging technique with super-resolution axial sensitivity, exploiting the changes in fluorescence lifetime above a plasmonic substrate. Using conventional confocal fluorescence lifetime imaging, we show that it is possible to deliver down to 6 nm axial position sensitivity of fluorophores in whole biological cell imaging. We employ this technique to map the topography of the cellular membrane, and demonstrate its application in an investigation of receptor-mediated endocytosis in carcinoma cells.

Revealing the Cellular Metabolic State through NADH Autofluorescence Lifetime in Parkinson's Disease

Cellular metabolic imaging of the relative amounts of reduced NADH and the microenvironment of this metabolic electron carrier can be used to noninvasively monitor changes in cell metabolism in various diseases. Moreover, mitochondrial dysfunction, which is directly related to the cellular metabolic state, is considered to be central in developing Parkinson's disease (PD). We applied 2-photon fluorescence lifetime imaging microscopy to determine the fluorescence lifetime as well as the amounts of reduced NADH in PC12 cells (a cell line derived from pheochromocytoma of rat adrenal medulla) treated with Neuronal Growth Factor (NGF) to differentiate into neuronal cells. The neuronal cells were further treated with MPTP to induce PD syndromes and the change in fluorescence lifetime and the amounts of reduced NADH after the treatment were measured. Our preliminary results show a significant increase in the fluorescence lifetime of free as well as bound NADH in the PD induced cells. Moreover, the relative amounts of the ratio of free NADH over bound NADH does not show any significant difference among the control and PD cells. These results may lead to further understanding of the role of cellular metabolism in PD progression.

Eu2+浓度对Sr2Si5N8∶Eu2+红粉热猝灭机制的影响

Eu2+doped Sr2Si5N8 red phosphors with different Eu2 +concentration were synthesized by high-temperature solid state method. The crystal structure,morphology,luminescence and thermal quenching properties were investigated in detail. X-ray diffraction( XRD) patterns show that a pure phase of Sr2Si5N8 phosphor with high crystallinity has been obtained. Photoluminescence( PL)spectra results indicate that there are two different luminescence centers when Eu2 +ions substitute Sr2 +sites. Thermal stability data show that the thermal quenching property is largely dependent on Eu2 +concentration. The changes of relative intensity ratio and fluorescence lifetime were examined for the two-peak emission in Sr2Si5N8∶ Eu2 +. The results reveal that Eu2 +distribution changes with varying Eu2 +concentration,which should be the mainly mechanism of the thermal quenching behavior for Eu2 +doped Sr2Si5N8 phosphors at low dopant levels.

Spectral Properties of Water-Soluble Conjugated Polymer PFP/DNA/Porphyrin Complex

Photodynamic therapy (PDT) is a particular treatment of tumors, which localizes the biological responses by the way that light is applied. In this paper, we have designed a water-soluble system potentially for PDT, which could selectively generate singlet oxygen efficiently under both of one-and two-photon excitation. This complex consists of a cationic water-soluble conjugated polymer PFP, anionic DNA and a cationic porphyrin. The cationic conjugated polymer PFP and porphyrin are connected by anionic DNA through electrostatic interactions. Energy transfer from conjugated polymer PFP (donor) to porphyrin (acceptor) is available since the considerable overlapping between the emission of donor and the absorption of acceptor (S-0 -> S-2). Steady-state spectra and fluorescence lifetime measurements have been performed on the complex with different DNA sequences in order to achieve the energy transfer efficiency. The results of steady-state fluorescence spectra show that there is a remarkable change of fluorescence intensity of the complex with G-quadruplex DNA, which indicates significant energy transfer from the donor to the acceptor. Furthermore, the greatest change of fluorescence lifetime measured by means of time-correlated single photon counting (TCSPC) also proves the highest energy transfer efficiency of the system with G-quadruplex DNA. Singlet oxygen quantum yield can be obtained by the detection of singlet oxygen emission spectra. Porphyrin bound to G-quadruplex DNA has the highest singlet oxygen quantum yield, which suggests this system a possible application in PDT since singlet oxygen could kill tumor cells. Two-photon absorption cross section has also been measured, which reflects the light-harvesting efficiency of the conjugated polymer PFP. The result shows that the conjugated polymer PFP owns large two-photon absorption cross section in the region of 720 similar to 850 nm where the transmission in tissue is higher compared to the visible light. The energy transfer efficiency and singlet oxygen quantum yield are both selective for different DNA sequences. G-quadruplex DNA that is very rich in tumor cells has the highest selectivity, which makes this system a potential application in PDT.

Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor.

The interest in RNA modification enzymes surges due to their involvement in epigenetic phenomena. Here we present a particularly informative approach to investigate the interaction of dye-labeled RNA with modification enzymes. We investigated pseudouridine (Ψ) synthase TruB interacting with an alleged suicide substrate RNA containing 5-fluorouridine (5FU). A longstanding dogma, stipulating formation of a stable covalent complex was challenged by discrepancies between the time scale of complex formation and enzymatic turnover. Instead of classic mutagenesis, we used differentially positioned fluorescent labels to modulate substrate properties in a range of enzymatic conversion between 6% and 99%. Despite this variegation, formation of SDS-stable complexes occurred instantaneously for all 5FU-substrates. Protein binding was investigated by advanced fluorescence spectroscopy allowing unprecedented simultaneous detection of change in fluorescence lifetime, anisotropy decay, as well as emission and excitation maxima. Determination of Kd values showed that introduction of 5FU into the RNA substrate increased protein affinity by 14× at most. Finally, competition experiments demonstrated reversibility of complex formation for 5FU-RNA. Our results lead us to conclude that the hitherto postulated long-term covalent interaction of TruB with 5FU tRNA is based on the interpretation of artifacts. This is likely true for the entire class of pseudouridine synthases.