Time-resolved Fluorescence Data Research Articles

Dual excitation spectral autofluorescence lifetime and reflectance imaging for fast macroscopic characterization of tissues.

Advancements in optical imaging techniques have revolutionized the field of biomedical research, allowing for the comprehensive characterization of tissues and their underlying biological processes. Yet, there is still a lack of tools to provide quantitative and objective characterization of tissues that can aid clinical assessment in vivo to enhance diagnostic and therapeutic interventions. Here, we present a clinically viable fiber-based imaging system combining time-resolved spectrofluorimetry and reflectance spectroscopy to achieve fast multiparametric macroscopic characterization of tissues. An essential feature of the setup is its ability to perform dual wavelength excitation in combination with recording time-resolved fluorescence data in several spectral intervals. Initial validation of this bimodal system was carried out in freshly resected human colorectal cancer specimens, where we demonstrated the ability of the system to differentiate normal from malignant tissues based on their autofluorescence and reflectance properties. To further highlight the complementarity of autofluorescence and reflectance measurements and demonstrate viability in a clinically relevant scenario, we also collected in vivo data from the skin of a volunteer. Altogether, integration of these modalities in a single platform can offer multidimensional characterization of tissues, thus facilitating a deeper understanding of biological processes and potentially advancing diagnostic and therapeutic approaches in various medical applications.

Analysis of individual nanoscale block copolymer vesicles by atomic force microscopy combined with time-resolved fluorescence microscopy

Abstract We report on the analysis of individual dye loaded block copolymer (BCP) vesicles via a combination of confocal laser scanning microscopy (CSLM)/fluorescence lifetime imaging microscopy (FLIM) on the one hand and atomic force microscopy (AFM) on the other hand. AFM measurements on ATTO 647N-loaded poly(styrene-block-poly(acrylic acid)) (PS115-b-PAA15) vesicles were carried out with high spatial resolution and afforded morphology and dimensions of BCP vesicles on solid substrates. By contrast the CSLM and FLIM data are diffraction limited, but from the time resolved fluorescence data information on the local vicinity of the reporter dye can be extracted. In the combined experiment individual dye-loaded vesicles and vesicle aggregates were discerned, analyzed metrologically by AFM and in more detail by CSLM and FLIM. On the basis of FLIM data the partitioning of the reporter dye was analyzed. The dye resides preferentially in the hydrophilic corona inside the vesicles. The dye concentration in the polymersome was about 90 times higher than in the solution used for encapsulation. These results underline that the combination of AFM with sensitive optical techniques, especially FLIM, is a promising approach for obtaining a deeper understanding of molecular interactions and nanoenvironments in supramolecular macromolecular structures and beyond.

Cation Exchange during the Synthesis of Colloidal Type-II ZnSe-Dot/CdS-Rod Nanocrystals.

Cation exchange is known to occur during the synthesis of colloidal semiconductor heteronanoparticles, affecting their band gap and thus altering their optoelectronic properties. It is often neglected, especially when anisotropic heterostructures are discussed. We present a study on the role of cation exchange inevitably occurring during the growth of anisotropic dot-in-rod structures consisting of a spherical ZnSe core enclosed by a rod-shaped CdS shell. The material combination exhibits a type-II band alignment. Two reactions are compared: the shell-growth reaction of CdS on ZnSe and an exchange-only reaction of ZnSe cores to CdSe. Transmission electron microscopy and a comprehensive set of optical spectroscopy data, including linear and time-resolved absorption and fluorescence data, prove that cation exchange from ZnSe to CdSe is the dominant process in the initial stages of the shell-growth reaction. The degree of cation exchange before significant shell growth starts was determined to be about 50%, highlighting the importance of cation exchange during the heteronanostructure growth.

Fluorescence probing and molecular docking analysis of the binding interaction of bovine serum albumin (BSA) with the polarity probe AICCN.

In this work, the fluorescent probe 2-amino-4-(1H-indol-3-yl)-4H-chromene-3-carbonitrile (AICCN) has been used to evaluate its potential as a prospective polarity probe. From detailed fluorescence studies of the probe, it could be shown that AICCN can indeed function as an effective polarity probe. The calculated dipole moments of AICCN in both the ground state and excited state in various solvents lend support to the steady state fluorescence results. It was also shown that AICCN can be used to probe the micropolarity of micelles and can be used successfully for the determination of CMC of the surfactants. The binding process of the probe AICCN to BSA has been followed by plotting the binding isotherms and Scatchard Plots. The time-resolved fluorescence data indicate that the preferred binding site of AICCN in BSA lies close to the buried Trp residue Trp-213 in Domain II. This contention is further supported by the molecular docking studies. The interaction study of the probe AICCN with proteins is relevant for future use of AICCN as a hydrophobic drug. Information was also obtained about the effect of probe binding on the serum albumin structure, which may be correlated to its physiological activity. Thus, the probe AICCN can serve not only as a good reporter of polarity of the microenvironment in biological systems but also as an efficient fluorophore to monitor conformational changes in proteins in future.

Multisite phosphorylation and binding alter conformational dynamics of the 4E-BP2 protein

Intrinsically disordered proteins (IDPs) play critical roles in regulatory protein interactions, but detailed structural/dynamic characterization of their ensembles remain challenging, both in isolation and when they form dynamic “fuzzy” complexes. Such is the case for mRNA cap-dependent translation initiation, which is regulated by the interaction of the predominantly folded eukaryotic initiation factor 4E (eIF4E) with the intrinsically disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. Single-molecule Förster resonance energy transfer showed that the conformational changes of 4E-BP2 induced by binding to eIF4E are non-uniform along the sequence; while a central region containing both motifs that bind to eIF4E expands and becomes stiffer, the C-terminal region is less affected. Fluorescence anisotropy decay revealed a non-uniform segmental flexibility around six different labeling sites along the chain. Dynamic quenching of these fluorescent probes by intrinsic aromatic residues measured via fluorescence correlation spectroscopy report on transient intra- and inter-molecular contacts on nanosecond-to-microsecond timescales. Upon hyperphosphorylation, which induces folding of ∼40 residues in 4E-BP2, the quenching rates decreased at most labeling sites. The chain dynamics around sites in the C-terminal region far away from the two binding motifs significantly increased upon binding to eIF4E, suggesting that this region is also involved in the highly dynamic 4E-BP2:eIF4E complex. Our time-resolved fluorescence data paint a sequence-level rigidity map of three states of 4E-BP2 differing in phosphorylation or binding status and distinguish regions that form contacts with eIF4E. This study adds complementary structural and dynamics information to recent studies of 4E-BP2, and it constitutes an important step toward a mechanistic understanding of this important IDP via integrative modeling.

Assembly of Bleomycin Saccharide-Decorated Spherical Nucleic Acids.

Glyco-decorated spherical nucleic acids (SNAs) may be attractive delivery vehicles, emphasizing the sugar-specific effect on the outer sphere of the construct and at the same time hiding unfavorable distribution properties of the loaded oligonucleotides. As examples of such nanoparticles, tripodal sugar constituents of bleomycin were synthesized and conjugated with a fluorescence-labeled antisense oligonucleotide (AONARV7). Successive copper(I)-catalyzed azide-alkyne and strain-promoted alkyne-nitrone cycloadditions (SPANC) were utilized for the synthesis. Then, the glyco-AONARV7 conjugates were hybridized with complementary strands of a C60-based molecular spherical nucleic acid (i.e., a hybridization-mediated carrier). The formation and stability of these assembled glyco-decorated SNAs were evaluated by polyacrylamide gel electrophoresis (PAGE), UV melting profile analysis, and time-resolved fluorescence spectroscopy. Association constants were extracted from time-resolved fluorescence data. Preliminary cellular uptake experiments of the glyco-AONARV7 conjugates (120 nM solutions) and of the corresponding glyco-decorated SNAs (10 nM solutions) with human prostate cancer cells (PC3) showed an efficient uptake in each case. A marked variation in intracellular distribution was observed.

Multi responsive chemosensor for the determination of metal ions (Co2+, Cu2+, and Zn2+ ions)

A multi-responsive Schiff-based chemosensor 3-[1-(benzylimino)ethyl]-2-hydroxy-6-methyl-4H-pyran-4-one (DBA) was synthesized and characterized. Single probe (DBA) exhibited sensing behaviour towards Co2+, Cu2+, and Zn2+ ions. The binding approach of DBA towards Co2+, Cu2+, and Zn2+ ions was calculated on basis of jobs plot (1:1) and which was further confirmed on basis of the mass spectral technique. The detection limit of DBA towards Co2+, Cu2+ and Zn2+ ions was found to be 6.82 × 10-7 M, 9.74 × 10-8 M, and 1.14 × 10-7 M respectively. DFT computational studies also validated the results obtained from absorption and emission studies. Time-resolved fluorescence data suggested that the copper complex was more stable as compared to the cobalt and zinc complex. The stability of complex formed by DBA with Co2+, Cu2+, and Zn2+ ions were chequered by time-reliant studies. The synthesized Schiff base DBA can detect Co2+, Cu2+, and Zn2+ ions over a wide pH range, and its practical applicability was effectively executed in environmental water samples.

Optical, Electrochemical, Third-Order Nonlinear Optical Investigations of 3,4,5-Trimethoxy Phenyl Substituted Non-Aqueous Phthalocyanines.

A new series of non-aqueous phthalocyanines having 3,4,5-trimethoxy phenyl group at peripheral positions in which the central cavity possessing Cu(II), Zn(II), and without metals has been synthesized, and its absorption, fluorescence (steady-state and excited state lifetimes), electrochemical, and third-order nonlinear optical (NLO) properties were evaluated. Absorption studies data suggest that all three phthalocyanines obey Beer–Lambert’s law, and the redox properties indicate that both oxidation and reduction reactions are macrocyclic centered. The singlet quantum yields were measured in different solvents and were found to be in the range of 0.2–0.5 in the case of free-base, whereas it was in the range of 0.1–0.5 in zinc derivative, while the time-resolved fluorescence data revealed lifetimes of typically a few ns. The third-order NLO properties were investigated using the Z-scan technique with kilohertz (for retrieving true electronic nonlinearities) and megahertz repetition rate femtosecond pulses at 800 nm. Intensity-dependent Z-scan studies revealed robust NLO coefficients for solutions and thin films (two-photon absorption cross-sections of 9,300–57,000 GM) of these molecules suggesting a strong potential for optical switching, imaging, and optical limiting applications.

Multivariate Curve Resolution Slicing of Multiexponential Time-Resolved Spectroscopy Fluorescence Data.

Time-resolved fluorescence spectroscopy (TRFS), i.e., measurement of fluorescence decay curves for different excitation and/or emission wavelengths, provides specific and sensitive local information on molecules and on their environment. However, TRFS relies on multiexponential data fitting to derive fluorescence lifetimes from the measured decay curves and the time resolution of the technique is limited by the instrumental response function (IRF). We propose here a multivariate curve resolution (MCR) approach based on data slicing to perform tailored and fit-free analysis of multiexponential fluorescence decay curves. MCR slicing, taking as a basic framework the multivariate curve resolution-alternating least-squares (MCR-ALS) soft-modeling algorithm, relies on a hybrid bilinear/trilinear data decomposition. A key feature of the method is that it enables the recovery of individual components characterized by decay profiles that are only partially describable by monoexponential functions. For TRFS data, not only pure multiexponential tail information but also shorter time delay information can be decomposed, where the signal deviates from the ideal exponential behavior due to the limited time resolution. The accuracy of the proposed approach is validated by analyzing mixtures of three commercial dyes and characterizing the mixture composition, lifetimes, and associated contributions, even in situations where only ternary mixture samples are available. MCR slicing is also applied to the analysis of TRFS data obtained on a photoswitchable fluorescent protein (rsEGFP2). Three fluorescence lifetimes are extracted, along with the profile of the IRF, highlighting that decomposition of complex systems, for which individual isomers are characterized by different exponential decays, can also be achieved.

Machine-learning-assisted electron-spin readout of nitrogen-vacancy center in diamond

Machine learning is a powerful tool in finding hidden data patterns for quantum information processing. Here, we introduce this method into the optical readout of electron-spin states in diamond via single-photon collection and demonstrate improved readout precision at room temperature. The traditional method of summing photon counts in a time gate loses all the timing information crudely. We find that changing the gate width can only optimize the contrast or the state variance, not both. In comparison, machine learning adaptively learns from time-resolved fluorescence data and offers the optimal data processing model that elaborately weights each time bin to maximize the extracted information. It is shown that our method can repair the processing result from imperfect data, reducing 7% in spin readout error while optimizing the contrast. Note that these improvements only involve recording photon time traces and consume no additional experimental time, and they are, thus, robust and free. Our machine learning method implies a wide range of applications in the precision measurement and optical detection of states.

Macroscopic fluorescence lifetime topography enhanced via spatial frequency domain imaging.

We report on a macroscopic fluorescence lifetime imaging (MFLI) topography computational framework based around machine learning with the main goal of retrieving the depth of fluorescent inclusions deeply seated in bio-tissues. This approach leverages the depth-resolved information inherent to time-resolved fluorescence data sets coupled with the retrieval of in situ optical properties as obtained via spatial frequency domain imaging (SFDI). Specifically, a Siamese network architecture is proposed with optical properties (OPs) and time-resolved fluorescence decays as input followed by simultaneous retrieval of lifetime maps and depth profiles. We validate our approach using comprehensive in silico data sets as well as with a phantom experiment. Overall, our results demonstrate that our approach can retrieve the depth of fluorescence inclusions, especially when coupled with optical properties estimation, with high accuracy. We expect the presented computational approach to find great utility in applications such as optical-guided surgery.

Total Emission Time Resolved Decay: a Method for Measurement and Resolution of Broad-Band Emission.

This article reports a time-resolved fluorescence data acquisition technique termed as "Total Emission Time Resolved Decay" (TETRD). TETRD is recorded by using zero-order diffraction of emission grating in TCSPC instrument. TETRD decay curve has entire wavelength dependent decay information buried in it. Cut-off filters are used to avoid scattering contamination. Two existing approaches are used for analysing the interconnected TETRD data. (i) First, global analysis: for discretely decaying multiple components, TETRD dataset is analyzed using global analysis. The normalized pre-exponentials (αi) and relative amplitudes (fi) recovered from global analysis reflect the individual component emission more faithfully and resembles with steady-state spectral data as well. (ii) Second, stretched exponential fitting (StrEF): for continuous lifetime distribution systems, StrEF (I(t) = I0 exp[-(t/τ)1/h]) has been used to analyse TETRD data. The average lifetime (τ) of StrEF matches well with the average lifetime of multi-exponential fitting, the heterogeneity factor (h) of StrEF is an additional parameter, which informs about local heterogeneity in the system. It is shown that the lifetimes obtained with TETRD matches well with the lifetimes obtained using conventional time resolved emission spectra (TRES). TETRD holds advantage in rapid data acquisition and facilitates inclusion of another variable (like concentration, solvent composition, pH, excitation wavelength etc.) into experimental design. Further, with the use of an appropriate data analysis tool, the multi-component decay profiles can be resolved conveniently.

Multispectral LIF-Based Standoff Detection System for the Classification of CBE Hazards by Spectral and Temporal Features.

Laser-induced fluorescence (LIF) is a well-established technique for monitoring chemical processes and for the standoff detection of biological substances because of its simple technical implementation and high sensitivity. Frequently, standoff LIF spectra from large molecules and bio-agents are only slightly structured and a gain of deeper information, such as classification, let alone identification, might become challenging. Improving the LIF technology by recording spectral and additionally time-resolved fluorescence emission, a significant gain of information can be achieved. This work presents results from a LIF based detection system and an analysis of the influence of time-resolved data on the classification accuracy. A multi-wavelength sub-nanosecond laser source is used to acquire spectral and time-resolved data from a standoff distance of 3.5 m. The data set contains data from seven different bacterial species and six types of oil. Classification is performed with a decision tree algorithm separately for spectral data, time-resolved data and the combination of both. The first findings show a valuable contribution of time-resolved fluorescence data to the classification of the investigated chemical and biological agents to their species level. Temporal and spectral data have been proven as partly complementary. The classification accuracy is increased from 86% for spectral data only to more than 92%.

Tryptanthrin from indigo: Synthesis, excited state deactivation routes and efficient singlet oxygen sensitization

The microwave-assisted synthesis of tryptanthrin from indigo in mild oxidation conditions, and a comprehensive study of the excited state properties of this compound in a variety of solvents with different polarity and viscosity values at room and low temperatures are reported. In contrast with indigo, emission of the triplet state of tryptanthrin is observed with a very efficient singlet oxygen sensitization quantum yield, indicating that the triplet state is efficiently populated. From time-resolved fluorescence and femtosecond transient absorption data, further supported with time-dependent density functional theory (TDDFT) calculations, two species, with S1 states with locally excited (LE) of π,π* nature and a charge transfer (CT) of n,π* characteristics, originated from an initially populated Frank-Condon S2 state (π,π*), are observed. The two electronically independent species are energetically nearly degenerate and inter-conversion is predicted (and rate constants determined) to occur between LE (S1) and CT (S1) species. Due to the low value of the fluorescence quantum yield (~10−3) and high triplet state yield (ϕT≥ϕΔ), the high stability of this compound is associated to the high efficiency of the radiationless deactivation processes which involve the formation of the CT state which efficiently converts, through S1 ~~> Tn intersystem crossing, to the T1 triplet state.

Proton Abstraction Mediates Interactions between the Super Photobase FR0-SB and Surrounding Alcohol Solvent.

We report on the motional and proton transfer dynamics of the super photobase FR0-SB in the series of normal alcohols C1 (methanol) through C8 (n-octanol) and ethylene glycol. Steady-state and time-resolved fluorescence data reveal that the proton abstraction dynamics of excited FR0-SB depend on the identity of the solvent and that the transfer of the proton from solvent to FR0-SB*, forming FR0-HSB+*, fundamentally alters the nature of interactions between the excited molecule and its surroundings. In its unprotonated state, solvent interactions with FR0-SB* are consistent with slip limit behavior, and in its protonated form, intermolecular interactions are consistent with a much stronger interaction of FR0-HSB+* with the deprotonated solvent RO-. We understand the excited-state population dynamics in the context of a kinetic model involving a transition state wherein FR0-HSB+* is still bound to the negatively charged alkoxide, prior to solvation of the two charged species. Data acquired in ethylene glycol confirm the hypothesis that the rotational diffusion dynamics of FR0-SB* are largely mediated by solvent viscosity while proton transfer dynamics are mediated by the lifetime of the transition state. Taken collectively, our results demonstrate that FR0-SB* extracts solvent protons efficiently and in a predictable manner, consistent with a ca. 3-fold increase in dipole moment upon photoexcitation as determined by ab initio calculations based on the equation-of-motion coupled-cluster theory.

Systematically controlled fluorescence resonance energy transfer from cadmium telluride quantum dots to rhodamine 101 dye: steady-state versus time-resolved measurements

This article highlights the studies on precise measurements of energy transfer efficiency in a steady-state method by adopting sample preparation methods. Steady-state and time-resolved fluorescence measurements were employed to evaluate the cadmium telluride (CdTe) quantum dot (QD) photoluminescence quenching induced by the proximal rhodamine 101 dye (Rh 101) molecule via fluorescence resonance energy transfer (FRET). Further, the evaluation of steady-state measurements in comparison with time-resolved measurements was carried out. The FRET parameters, such as spectral overlap, Forster distance, and intermolecular distance for each donor–acceptor pair, were determined and variation of these parameters with respect to the size of the QD is discussed. Further, efforts to understand the optical and carrier relaxation dynamics of CdTe QDs are made using steady-state and time-resolved fluorescence data.

Influence of 1,3,5-triazine Core and Electron Donor Group in Photophysical Properties of BODIPY Dyes.

The relationship between the number of BODIPY in a compound and the increase on its fluorescence has been established such as an aggregation induced by multiple BODIPY. We aimed to determine the influence of an electron donor substituent in the BODIPY-triazine system. In this sense, as a first step, we collected data such as photophysical characteristics about BODIPY without substituent and meso-triazine-BODIPY system. Then, three more meso-triazine-BODIPY were synthetized by Lyndsey method. In addition, absorption and emission spectra, fluorescence quantum yields and time-resolved fluorescence data were obtained. Furthermore, solvatochromism was determined by solvent descriptors and photophysical parameters. Finally, the results showed that the triazine core stabilized the system and we observed that the number of BODIPY increased fluorescence mainly in polar solvents. While electron donation maintained the conjugation that reduced the influence of the solvent on the photophysical characteristics.

Mapping the excitation energy migration pathways in phycobilisomes from the cyanobacterium Acaryochloris marina

In this study, we use ultrafast time-resolved absorption and fluorescence spectroscopies to examine A. marina phycobilisomes isolated from cells grown under light of different intensities and spectral regimes. Investigations were performed at room temperature and at 77 K. The study demonstrates that if complexes are stabilized by high phosphate (900 mM) buffer, there are no differences between them in temporal and spectral properties of fluorescence. However, when the complexes are allowed to disassemble into trimers in low phosphate (50 mM) buffer, differences are clearly observed. The fluorescence properties of intact or disassembled phycobilisomes from cells grown in low intensity white light are unresponsive to variation in phosphate concentration. This antenna complex was further studied in detail with application of femtosecond time-resolved absorption at room temperature. Combined spectroscopic and kinetic analysis of time-resolved fluorescence and absorption data of this antenna allowed us to identify spectrally different forms of phycocyanobilins and to propose a simplified model of how they could be distributed within the phycobilisome structure.

Decay- and evolution-associated spectra of time-resolved fluorescence of LHCII aggregates

Non-photochemical quenching (NPQ) is responsible for the protection of the photosynthetic apparatus of plants from photodamage at high-light conditions. It is commonly agreed that NPQ takes place in the major light-harvesting complexes (LHCII), however, its exact mechanisms are still under debate. Valuable information about its molecular nature can be provided by measuring time-resolved fluorescence (TRF) spectra of LHCII complexes and their aggregates. Previously [Chmeliov et al., Nat. Plants 2, 16045 (2016)], we analysed the corresponding TRF spectra using the multivariate curve resolution method and proposed a three-state model to describe the spectroscopic data. Usually, such data is described in terms of global analysis resulting in decay or evolution-associated spectra. In this work, we apply such analysis to the TRF data of LHCII aggregates and show that, although mathematically feasible, it cannot be directly related to the physical kinetic model. Nevertheless, a careful examination supplemented with additional spectroscopic information still results in the same three-state model proposed before.

A comprehensive photophysical and NMR investigation on the interaction of a 4-methylumbelliferone derivative and cucurbit[7]uril

The host-guest interaction of a 7-hydroxycoumarin (3-[2-(diethylamino)ethyl]-7-hydroxy-4-methylcoumarin, 3ED4MU) with cucurbit[7]uril, CB7, was investigated in aqueous solution. From the steady-state fluorescence Job plot and 1H NMR spectroscopy the formation of a 1:1 inclusion complex, with a binding constant of 1.75 × 105 M−1 was obtained. Time-resolved fluorescence data in water, DMSO and dioxane for 3ED4MU and for umbelliferone (UM, here taken has the model compound) show that the decays are double or triple-exponentials mirroring the presence of excited neutral (N*), anionic (A*), tautomeric (T*) and zwitterionic (T*′) species (the latter only present in 3ED4MU). In the time-resolved experiments, the interaction of 3ED4MU with CB7 is mirrored by an increase of the pre-exponential factor associated to the zwitterionic species (thus giving support for the stabilization of this species when interacting with CB7) and a dramatic change in the pKa* value (from ~0.5 to ~8), associated to a displacement of the water environment probed by 3ED4MU.