Delayed Fluorescence Signal Research Articles

Pressure-enhanced sensing of tissue oxygenation via endogenous porphyrin: Implications for dynamic visualization of cancer in surgery

Fluorescence guidance is routinely used in surgery to enhance perfusion contrast in multiple types of diseases. Pressure-enhanced sensing of tissue oxygenation (PRESTO) via fluorescence is a technique extensively analyzed here, that uses an FDA-approved human precursor molecule, 5-aminolevulinic acid (ALA), to stimulate a unique delayed fluorescence signal that is representative of tissue hypoxia. The ALA precontrast agent is metabolized in most tissues into a red fluorescent molecule, protoporphyrin IX (PpIX), which has both prompt fluorescence, indicative of the concentration, and a delayed fluorescence, that is amplified in low tissue oxygen situations. Applied pressure from palpation induces transient capillary stasis and a resulting transient PRESTO contrast, dominant when there is near hypoxia. This study examined the kinetics and behavior of this effect in both normal and tumor tissues, with a prolonged high PRESTO contrast (contrast to background of 7.3) across 5 tumor models, due to sluggish capillaries and inhibited vasodynamics. This tissue function imaging approach is a fundamentally unique tool for real-time palpation-induced tissue response in vivo, relevant for chronic hypoxia, such as vascular diseases or oncologic surgery.

Effects of Simulated Acid Rain on Photosynthesis in Pinus massoniana and Cunninghamia lanceolata in Terms of Prompt Fluorescence, Delayed Fluorescence, and Modulated Reflection at 820 nm.

The effects of simulated acid rain (SAR) on the photosynthetic performance of subtropical coniferous species have not been thoroughly investigated. In this study, we treated two coniferous species, Pinus massoniana (PM) and Cunninghamia lanceolata (CL), with four gradients of SAR and then analyzed their photosynthetic activities through measurements of gas exchange, prompt fluorescence (PF), delayed fluorescence (DF), and modulated reflection at 820 nm (MR820). Gas exchange analysis indicated that the decrease in the net photosynthetic rate (Pn) in PM and CL was unrelated to stomatal factors. For the PF transients, SAR induced positive K-band and L-band, a significant reduction in photosynthetic performance index (PIABS), the quantum yield of electron transfer per unit cross-section (ETO/CSm), and maximal photochemical efficiency of photosystem II (Fv/Fm). Analysis of the MR820 kinetics showed that the re-reduction kinetics of PSI reaction center (P700+) and plastocyanin (PC+) became slower and occurred at later times under SAR treatment. For the DF signals, a decrease in the amplitude of the DF induction curve reduced the maximum value of DF (I1). These results suggested that SAR obstructed photosystem II (PSII) donor-side and acceptor-side electron transfer capacity, impaired the connectivity between PSII and PSI, and destroyed the oxygen-evolving complex (OEC). However, PM was better able to withstand SAR stress than CL, likely because of the activation of a protective mechanism.

Understanding delayed fluorescence and triplet decays of Protoporphyrin IX under hypoxic conditions.

Photosensitizers of singlet oxygen exhibit three main types of reverse intersystem-crossing (RISC): thermally activated, triplet-triplet annihilation, and singlet oxygen feedback. RISC can be followed by delayed fluorescence (DF) emission, which can provide important information about the excited state dynamics in the studied system. An excellent model example is a widely used clinical photosensitizer Protoporphyrin IX, which manifests all three mentioned types of RISC and DF. Here, we estimated rate constants of individual RISC and DF processes in Protoporphyrin IX in dimethylformamide, and we showed how these affect triplet decays and DF signals under diverse experimental conditions, such as a varying oxygen concentration or excitation intensity. This provided a basis for a general discussion on guidelines for a more precise analysis of long-lived signals. Furthermore, it has been found that PpIX photoproducts and potential transient excited complexes introduce a new overlapping delayed luminescence spectral band with a distinct lifetime. These findings are important for design of more accurate biological oxygen sensors and assays based on DF and triplet lifetime.

Mitochondrial Oxygen Monitoring During Surgical Repair of Congenital Diaphragmatic Hernia or Esophageal Atresia: A Feasibility Study.

Current monitoring techniques in neonates lack sensitivity for hypoxia at cellular level. The recent introduction of the non-invasive Cellular Oxygen METabolism (COMET) monitor enables measuring in vivo mitochondrial oxygen tension (mitoPO2), based on oxygen-dependent quenching of delayed fluorescence of 5-aminolevulinic acid (ALA)-enhanced protoporphyrin IX. The aim is to determine the feasibility and safety of non-invasive mitoPO2 monitoring in surgical newborns. MitoPO2 measurements were conducted in a tertiary pediatric center during surgical repair of congenital diaphragmatic hernia or esophageal atresia. Intraoperative mitoPO2 monitoring was performed with a COMET monitor in 11 congenital diaphragmatic hernia and four esophageal atresia neonates with the median age at surgery being 2 days (IQR 1.25–5.75). Measurements were done at the skin and oxygen-dependent delayed fluorescence was measurable after at least 4 h application of an ALA plaster. Pathophysiological disturbances led to perturbations in mitoPO2 and were not observed with standard monitoring modalities. The technique did not cause damage to the skin, and seemed safe in this respect in all patients, and in 12 cases intraoperative monitoring was successfully completed. Some external and potentially preventable factors—the measurement site being exposed to the disinfectant chlorohexidine, purple skin marker, or infrared light—seemed responsible for the inability to detect an adequate delayed fluorescence signal. In conclusion, this is the first study showing it is possible to measure mitoPO2 in neonates and that the cutaneous administration of ALA to neonates in the described situation can be safely applied. Preliminary data suggests that mitoPO2 in neonates responds to perturbations in physiological status.

Dimeric conformation sensitive electronic excited states of tetracene congeners and their unconventional non-fluorescent behaviour

Unconventional non-fluorescent J-aggregates of Tetracene (TC) and Naphtho[2,1,8-qra]tetracene (NT) were witnessed and their consequent dramatic quenching was unravelled by a steady state, time-resolved and transient spectroscopy in conjunction with excited state density functional calculations. The TC O-aggregate with slippage angle $$\uptheta = 22.3^{\circ }< 54.7^{\circ }$$ exhibited substantial transition dipole moment (TDM) for both lower (2.79 D) and higher (1.59 D) energy singlet excitations, while, NT formed an ideal J-aggregate (polarization angle, $$\upalpha \sim 0^{\circ })$$ with a predominant TDM to only a lower excitonic state (2.69 D). Subsequently, their unusual quenching was quantified with large drops in the photoluminescence quantum yields (PLQY) from 0.116 to 0.002 upon TC O-aggregation and from 0.478 to 0.038 upon NT J-aggregation. These intense PL drops were systematically investigated for possible occurrence of excimer-like emission quenching and/or photo-degradation of the TC core unit. In view of the TC O-aggregates exhibiting a perfect energetic balance between the singlet (2.34 eV) and triplet (1.28 eV) energies for singlet fission (SF) and a concomitant delayed fluorescence signal, their $$\hbox {S}_{{1}}$$ decay characteristics were attributed to SF followed by an inverse triplet-triplet recombination. In contrast, the energetic imbalance ( $$\hbox {E}(\hbox {S}_{1}) < \hbox {2xE}(\hbox {T}_{1})$$ ) in NT J-aggregates permitted only forward process of SF and the resulting long-lived triplet formation was traced with a positive transient absorption ( $$\hbox {T}_{1} \rightarrow \hbox {T}_{\mathrm{n}})$$ band at 500 nm. Accordingly, the singlet excited state $$(\hbox {S}_{1})$$ dynamics of TC O- and NT J-aggregates, being largely dominated by SF, depicted a depleted $$\hbox {S}_{{1}}$$ population, accounting for the large deviation from aggregation induced enhanced emission, exhibited by classical dye J-aggregates. Unconventional O- and J -aggregates of Tetracene and Naphtho[2,1,8-qra]tetracene showed non-fluorescent behaviour whose singlet excited state (S $$_{1})$$ dynamics was largely governed by fast Singlet Fission decay, accounting for atypical deviation from the well-established aggregation induced enhanced emission from classical J-aggregates.

Room-Temperature Delayed Fluorescence of Gold Nanoclusters in Zinc-Mediated Two-Dimensional Crystalline Assembly.

We report that complexation-reaction-mediated two-dimensional crystalline assembly of gold (Au14) nanoclusters (NCs) exhibits room-temperature delayed fluorescence at 605 nm, with an unprecedented long lifetime of 0.5 ms and an exceptionally high quantum yield of 19.1 ± 0.9%. Interestingly, the as-synthesized Au NCs had a very weak delayed fluorescence signal. The enhancement in delayed fluorescence of Au NCs upon formation of assembly has been attributed to the crystallization-induced structural rigidity, which restricted the nonradiative transitions and enhanced the excited-state lifetime. The attainment of crystalline organization was substantiated by electron diffraction analysis. A possible structure was established based on experimental results and computational optimizations. Atomic force microscopy revealed the formation of multilayered two-dimensional nanosheets with thickness of 2.44 ± 0.48 nm.

Fourier transform of delayed fluorescence as an indicator of herbicide concentration

It is well known that delayed fluorescence (DF) from Photosystem II (PSII) of plant leaves can be potentially used to sense herbicide pollution and evaluate the effect of herbicides on plant leaves. The research of using DF as a measure of herbicides in the literature was mainly conducted in time domain and qualitative correlation was often obtained. Fourier transform is often used to analyze signals. Viewing DF signal in frequency domain through Fourier transform may allow separation of signal components and provide a quantitative method for sensing herbicides. However, there is a lack of an attempt to use Fourier transform of DF as an indicator of herbicide. In this work, the relationship between the Fourier transform of DF and herbicide concentration was theoretically modelled and analyzed, which immediately yielded a quantitative method to measure herbicide concentration in frequency domain. Experiments were performed to validate the developed method.

Drought-induced modifications of photosynthetic electron transport in intact leaves: Analysis and use of neural networks as a tool for a rapid non-invasive estimation

Water deficit is one of the most important environmental factors limiting sustainable crop yields and it requires a reliable tool for fast and precise quantification. In this work we use simultaneously recorded signals of photoinduced prompt fluorescence (PF) and delayed fluorescence (DF) as well as modulated reflection (MR) of light at 820nm for analysis of the changes in the photosynthetic activity in detached bean leaves during drying. Depending on the severity of the water deficit we identify different changes in the primary photosynthetic processes. When the relative water content (RWC) is decreased to 60% there is a parallel decrease in the ratio between the rate of excitation trapping in the Photosystem (PS) II reaction center and the rate of reoxidation of reduced PSII acceptors. A further decrease of RWC to 20% suppresses the electron transfer from the reduced plastoquinone pool to the PSI reaction center. At RWC below values 15%, the reoxidation of the photoreduced primary quinone acceptor of PSII, QA–, is inhibited and at less than 5%, the primary photochemical reactions in PSI and II are inactivated. Using the collected sets of PF, DF and MR signals, we construct and train an artificial neural network, capable of recognizing the RWC in a series of “unknown” samples with a correlation between calculated and gravimetrically determined RWC values of about R2≈0.98. Our results demonstrate that this is a reliable method for determination of RWC in detached leaves and after further development it could be used for quantifying of drought stress of crop plants in situ. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Alterations in delayed and direct phytoplankton fluorescence in response to the diurnal light cycle

Delayed fluorescence (DF) excitation spectrometry was examined as a proxy for phytoplankton activity in comparison to pulse-amplitude-modulated (PAM) fluorometry and dissolved oxygen (DO) evolution. During several day–night cycles, the three target variables were monitored simultaneously, together with pH, temperature and photosynthetically active irradiance in an exponentially growing Chlorella population exposed to natural light conditions. It was found that during a diel cycle prompt and DF signals corresponded to each other and were negatively correlated to the light intensity, with maximum values during night and vice versa. The DF signal showed a strong linear relationship with the quantum yield of photosystem II. Our findings thus suggest that, in addition to the continuous monitoring of active chlorophyll of different taxonomical groups, DF excitation spectrometry also carries the potential to continuously monitor the quantum yields and relative electron transport rates in natural phytoplankton assemblages.

Delayed Chlorophyll Fluorescence as a Monitor for Physiological State of Photosynthetic Apparatus

ABSTRACTIntact plants emit light quanta called delayed fluorescence (DF). DF is result of radiative deactivation of secondary excited chlorophyll molecules in Photosystem II (PS II) antennae complexes. The excitations are produced by backward electron- transfer reactions both in the donor and acceptor sides of PS II. The poly-exponential dark decay of DF in a time interval of tens of nanoseconds to tens of seconds reflects the kinetics of different forward and backward reactions of the photosynthetic electron transfer. The current work reviews the mechanisms of the DF light quanta generation and the methodical approaches that allow us to obtain quantitative information about the photosynthetic machinery state using the DF signal from native objects. We examine an approach for the simultaneous record of DF and prompt chlorophyll fluorescence during the transition of the photosynthetic machinery from dark-adapted to light-adapted state. A new device (Senior PEA) built by Hansatech (King's Lynn, UK) allows us to measure simultaneously the induction transients of prompt chlorophyll fluorescence, DF decaying in a time range 10 μs—240 ms, and the changes in transmission at 820 nm. The comparative analysis of the three types of signals and the application of a model-based description of the processes and reactions that determine the dynamics of the signals during the light-induced transitions (dynamic models and JIP-test) allow us to obtain, from a single few-seconds-long measurement, quantitative information for: a) energetic fluxes and efficiencies at different steps of energy transformation; b) rate constants of electron transfer in and between the two photosystems; c) energization of the thylakoid membrane. This illustrates that DF in combination with other optical and luminescent measurements is a highly informative method for investigation of the physiological state of the photosynthetic apparatus of plants in vivo and in situ, and is an indispensable tool for the purposes of the biophysical phenomics.

Delayed fluorescence as a direct indicator of diurnal variation in quantum and radiant energy utilization efficiencies of phytoplankton

We compared delayed fluorescence (DF) excitation spectrometry with radiocarbon (14C) technique using a monoalgal culture of Chlorella vulgaris grown under natural temperature and irradiance. This was done by monitoring the DF, in parallel to quantum efficiency (QE) and index of radiant energy utilization efficiency (Ψ) as calculated on the basis of carbon uptake measurements by radiocarbon technique. During the diurnal cycle, temperature, irradiance, and chlorophyll (Chl) contents were monitored in the algal culture that was kept in an open transparent plastic tank submerged at the surface of Lake Kinneret, Israel. The DF signal correlated with both the QE (r 2 = 0.869, p<0.01) and Ψ (r 2 = 0.977, p<0.01) during a diurnal cycle. We suggest that, besides the measurement of active Chl and phytoplankton population composition, the DF signal provides additional information on the QE and Ψ in phytoplankton population.

Intramolecular Delayed Fluorescence as a Tool for Imaging Science: Synthesis and Photophysical Properties of a First-Generation Emitter

The synthesis of a molecular dyad comprising two pyrene-based terminals covalently linked via a Hantzsch 1,4-dihydropyridine is described. The dyad is sufficiently flexible to allow the end groups to approach each other in fluid solution, as is evident from the appearance of excimer fluorescence. Transient absorption spectroscopy indicates that both intra- and intermolecular triplet−triplet annihilation (TTA) takes place at modest laser intensities and leads to delayed fluorescence. The spectral distribution of the delayed fluorescence signal matches that of the excimer. The kinetics of TTA and the overall yield of delayed fluorescence are considered in terms of the molecule adopting disparate conformations that interconvert slowly. Consideration is also given to the possible application of such delayed fluorescence in imaging technology.

A Non-invasive and Real-time Monitoring of the Regulation of Photosynthetic Metabolism Biosensor Based on Measurement of Delayed Fluorescence in Vivo

In this paper, a new principle biosensor for non-invasive monitoring of theregulation of photosynthetic metabolism based on quantitative measurement of delayedfluorescence (DF) is developed. The biosensor, which uses light-emitting diode lattice asexcitation light source and a compact Single Photon Counting Module to collect DF signal,is portable and can evaluate plant photosynthesis capacity in vivo. Compared with itsprimary version in our previous report, the biosensor can better control environmentalfactors. Moreover, the improved biosensor can automatically complete the measurements oflight and CO2 response curves of DF intensity. In the experimental study, the testing of theimproved biosensor has been made in soybean (Glycine max Zaoshu No. 18) seedlingstreated with NaHSO3 to induce changes in seedlings growth and photosynthetic metabolism.Contrast evaluations of seedlings photosynthesis were made from measurements of netphotosynthesis rate (Pn) based on consumption of CO2 in tested plants. Current testingresults have demonstrated that the improved biosensor can accurately determine theregulatory effects of NaHSO3 on photosynthetic metabolism. Therefore, the biosensorpresented here could be potential useful for real-time monitoring the regulatory effects ofplant growth regulators (PGRs) and other exogenous chemical factors on plant growth andphotosynthetic metabolism.

Kinetics of delayed chlorophyll a fluorescence registered in milliseconds time range

Delayed fluorescence dark decays in the time interval from 0.35 to 5.5 ms are measured during dark to light adaptation in whole barley leaves and isolated thylakoid membranes, using a disc phosphoroscope. The changes in delayed fluorescence features are compared with variable chlorophyll fluorescence simultaneously registered with the same apparatus as well as in parallel by Handy PEA (Hansatech Instruments Ltd.), and absorbance changes at 820 nm. The registered delayed fluorescence signal is a sum of three components - submillisecond with lifetime of about 0.6 ms, millisecond decayed 2-4 ms and slow component with lifetime > >5.5 ms. The submillisecond delayed fluorescence component is proposed to be a result of radiative charge recombination in Photosystem II reaction centers in the state Z+PQA -QB -, and its lifetime is determined by the rate of electron transfer from QA - to QB -. The millisecond delayed fluorescence component is associated with recombination in Z+PQA -QB = centers with a lifetime determined by the sum of the rate constants of electron transfer from the oxygen-evolving complex to Z+ and of the exchange between the reduced and oxidized plastoquinone pool in the QB-site. On the basis of these assumptions and of the different share of the three components in the integral delayed fluorescence during induction, an attempt has been made to interpret the changes in the delayed fluorescence intensity during the transition of the photosynthetic apparatus from dark to light adapted state.

Rapid Determination of Bitterness in Beer Using Fluorescence Spectroscopy and Chemometrics

Two fluorescence spectroscopic methods with the aim to develop a fast quantitative determination of bitterness in beer were tested. The first method was based on autofluorescence of the diluted and degassed beer samples without any further processing. A total of 21 dark and light beer samples were analyzed and multivariate Partial Least Squares (PLS) regression models to bitterness in form of international bitter units (IBU) were performed. A prediction error in the form of Root Mean Square Error of Cross-Validation (RMSECV) of 2.77 IBU was obtained using six PLS components. Focusing only on the light beer samples the RMSECV was reduced to 1.81 IBU. The second method developed was based on addition of europium to induce delayed fluorescence signals in the beer samples. PLS models yielded an RMSECV of 2.65 IBU for all beers, while a model on the light beer samples gave an RMSECV of 1.75 IBU. The obtained prediction errors were compared to the errors given in the literature for the traditional extraction method of determining IBU.

Characterization of the photosystem II inactivation of heat-stressed barley leaves as monitored by the various parameters of chlorophyll a fluorescence and delayed fluorescence

The effects of elevated temperatures on the photochemical activities of photosystem II (PS II) in situ have been studied for barley leaves using a combination of chlorophyll a fluorescence, delayed fluorescence (DF) in the microsecond range and oxygen evolution rate (OE). Attention is given to the succession of PS II inactivation on donor and acceptor sides and its correlation with reversible and/or irreversible depression of OE. The partial decrease of oxygen evolution at 37.5°C (by 23% compared with the optimal value at 35°C) is found to be mostly reversible and to correlate with a significant increase of the DF signal. The increase of DF indicates that this moderate level of heat stress preferentially induces PS II donor-side inactivation. A nearly irreversible decline of OE occurs at 42.5–45°C. The corresponding decrease of DF signal, by more than 70% if compared with the DF maximum at 40°C, can be explained by the structural changes within PS II which prevent the recombination of separated charges (P680 + Q A − ). Further analysis of chlorophyll a fluorescence supports an idea that this level of heat stress induces significant inactivation of PS II photochemistry on both donor and acceptor sides.

A probe for intracellular concentrations of drugs: Delayed fluorescence from acridine orange

The aim of this work is to develop fluorescent probes that will indicate effective concentrations of therapeutic agents, or endogenous protectors, at important cellular sites. Acridine orange associates with nucleic acids and emits a ‘delayed’ fluorescence signal. This signal is quenched by oxidants such as oxygen, nitroaryl radiosensitizers, adriamycin and mitomycin-c, and reductants such as thiols, ascorbate and other radioprotectors. The quenching of the acridine orange delayed fluorescence reflects the effective concentration of these therapeutically-important oxidants and reductants near DNA. The relative concentration of basic radiosensitizers such as pimonidazole (Ro 03-8799) near the DNA is greater than that of misonidazole. Thiols quench the delayed fluorescence signal according to the degree of ionization of the thiol function; this may model the reactivity of thiols with guanine radical sites in DNA. Ascorbate and aminopyrine do not quench the delayed fluorescence from cells stained with acridine orange as these compounds are taken up by cells very inefficiently.

Changes in phase lags of delayed fluorescence signals due to coherence effects in triplet exciton transport

A generalized master equation approach to triplet exciton motion in molecular solids is used to analyse coherence in the transport via changes in phase lag of periodic delayed fluorescence signals due to triplet-triplet annihilation in crystals under space and time periodic excitation. Coherence effects can lead to increases, as large as = 100%, in the measurable effects due to motion as compared to those for diffusive transport. The method should be considered as the first choice for detection of coherence in triplet transport via delayed fluorescence.

Steady-state delayed flourescence signals in ronchi ruling experiments: theory of coherence effects

Effects of coherence in triplet exciton transport are analyzed in the context of measurements of steady-sdtate delayed fluorescence signals from molecular crystals excited through Ronchi rulings.