Recent theoretical studies highlight how nonclassical photon correlations in entangled photon pairs can selectively address nonlinear optical pathways. However, the resulting signals are typically too weak for practical time-resolved experiments. Here, we propose two-dimensional (2D) time-resolved fluorescence spectroscopy that exploits these correlations and operates with current single-photon detectors. The method provides two advantages over conventional 2D electronic spectroscopy: (i) It yields 2D spectra without phase-stable multipulse control, relying instead on heralded twin-photon correlations, and (ii) it simplifies spectra by isolating the contribution that is spectroscopically equivalent to stimulated emission, thereby suppressing ground-state bleaching and excited-state absorption. Numerical calculations for a natural pigment-protein complex-inspired trimer show that this pathway selectivity enables the extraction of rich information on energy transfer dynamics. These results indicate a feasible route to real-time observation of molecular dynamics using entangled photon pairs.

Despite the growing interest in deep eutectic solvents (DESs), studies involving excited-state photochemical dynamics in such media are very scarce. Herein, we investigated the excited-state proton transfer (ESPT) of a photoacid (HPTS) in betaine (BET)-based DESs with systematically varied hydrogen-bond donors (HBD): ethylene glycol (EG), 1,2-propanediol (PD), and glycerol (Gly), using steady-state and time-resolved fluorescence spectroscopy. The results confirm strong ESPT in these DESs; the time-resolved fluorescence displays multiexponential decay behavior, and the initial proton-transfer and dissociation times are 0.17, 0.43, and 0.63 ns, and 0.51, 0.78, and 0.93 ns, respectively, in BET-EG, BET-PD, and BET-Gly systems. Time-resolved emission spectra show decay of the protonated state and growth of the deprotonated state without an iso-emissive point, accompanied by a continuous red shift in both emissions, indicating that ESPT and solvation occur on comparable time scales. The solvation dynamics of both states of the photoacid exhibit biphasic relaxation, with components of a few hundred picoseconds and a few nanoseconds. Similar solvation time scales, 0.10-0.30 ns and 0.94-2.36 ns for the nonproton-transferring analogue, and 0.10-0.20 ns and 0.40-0.80 ns for a standard solute (C153) demonstrate that ESPT in these DESs is governed by solvent reorganization. Temperature-dependent measurements reveal concurrent acceleration of both solvation and ESPT rate, with the strongest temperature sensitivity in BET-Gly and the weakest in BET-EG. These findings demonstrate that the photochemical reactivity of HPTS is regulated by the solvation-shell dynamics, and that tuning the HBD component of a DES provides a rational strategy for modulating proton transport in these novel media.

Time-resolved and steady-state measurements of proton transfer reactions for the two main types of neutral photoacids, the R*NH2 and R*OH photoacids, are compared and analyzed through free-energy correlations. We show that significant similarities in the proton transfer mechanisms exist in the proton dissociation of the two types of photoacids and that the observed differences in the free-energy correlations are likely to be caused by differences in the local reorganization energy emerging from the solvent. While both the R*NH2 and R*OH photoacids may release the proton to the solvent, R*NH2 photoacids are too weak to do so noticeably within the short excited-state lifetime of the photo-excited photoacids. For this reason, we constructed for R*NH2 photoacids a free-energy correlation between the proton transfer rate of the NH2 group and a series of strong Brønsted-base proton-acceptors R*NH2 + B ⇄ R*NH- + BH+ using steady-state and time-resolved fluorescence spectroscopies. We discuss how this correlation compares with the free-energy correlation of a series of R*OH photoacids of various strengths, which proton-dissociate in water according to R*OH ⇄ R*O- + H+ or proton-dissociate to a strong base like the proton transfer reactions we study with the R*NH2 photoacids.

Hesperetin-conjugated silver nanoparticles (HSP-AgNPs) were prepared using hesperetin as a reducing and stabilizing agent. UV-Vis and FTIR spectroscopy, dynamic light scattering, XPS, XRD and HRTEM were used to confirm the formation of functionalized nanoparticles. HSP-AgNPs displayed notable sensitivity and selectivity in the detection of Fe3+ in water. The probe also exhibited strong anti-interference performance against other metal ions. A detection limit (LOD) of 0.41 µM suggested prospective application of HSP-AgNPs in environmental sensing of Fe3+. Further, to explore the potential of HSP-AgNPs in biosensing of Fe3+, the biodistribution of nanoparticles was checked by studying their interaction with bovine serum albumin (BSA), a model carrier protein. Interactions between BSA and HSP-AgNPs were explored using UV-Vis, steady-state, time-resolved and synchronous fluorescence spectroscopy. HSP-AgNPs caused static quenching of tryptophan fluorescence of BSA. The thermodynamic parameters of binding (ΔH and ΔS) suggested the predominant involvement of hydrophobic interactions between BSA and nanoparticles.

Multiphoton imaging coupled with steady-state and time-resolved fluorescence spectroscopy reveals protein-bound FAD in femtosecond-laser-injured regenerating muscle.

Molecular choreography in crowded environments: Insights from solvation dynamics.

Utilization of minor red-shifted chlorophyll a for oxygenic photosynthesis under far-red light in green algae.

Solvation dynamics play a central role in shaping nucleic acid structure, flexibility, and recognition, yet their molecular origins remain poorly understood for RNA, whose diverse architectures and intrinsic conformational plasticity far exceed those of DNA. Here, we present the atomistic, microsecond-scale computational dissection of solvation dynamics in two structurally homologous but dynamically distinct viral RNAs-BIV TAR and HIV-2 TAR-in both apo and peptide-bound states, mimicking the salt concentration of the experimental time-resolved fluorescent spectroscopic measurements. By combining high-temporal-resolution solvation time correlation functions with detailed energy decomposition analyses, we uncover how water, ions, and RNA motions cooperatively shape relaxation across ultrafast to nanosecond timescales. Our results reveal that, unlike DNA, where slow components primarily reflect long-lived hydration and ion condensation, RNA can generate slow solvation decay either through long-lived hydration or through its own internal conformational fluctuations, such as involving spontaneous base-flipping events. Peptide binding modulates this conformational landscape in strikingly system-specific ways: BIV TAR RNA undergoes classical fluctuation quenching, where TAT binding suppresses RNA motions but shifts relaxation more toward solvent-RNA correlation, while HIV-2 TAR RNA exhibits a non-classical redistribution of solvent-ion-peptide correlations stemming from its weaker and more dynamic binding interface with TAT. The dominant slow decay in HIV-2 apo TAR maps directly onto an allosteric communication channel previously identified from structural analyses, demonstrating that solvation responses can sensitively report on RNA allostery. Together, this study bridges the experimental observations of time-resolved fluorescence spectroscopy with mechanistic molecular insights, establishes solvation dynamics as a powerful probe of RNA conformational energetics, and highlights how subtle differences in RNA-protein recognition can imprint distinct signatures on hydration and ion reorganization.

Protein aggregation into amyloid fibrils underlies numerous human diseases, yet the most widely used fluorescent probe, Thioflavin T (ThT), offers an incomplete picture of the process and fails to detect certain fibril structures. Here, we introduce and characterize the photophysical properties of DANIR-2b(2OH), a water-soluble push-pull dye that overcomes these limitations. It successfully binds early prefibrillar aggregates and small fibrils of the human Islet Amyloid Polypeptide that elude detection by ThT, which we confirm by time-resolved cryo-electron microscopy of aliquots taken during the kinetic assays. We further demonstrate that DANIR-2b(2OH) can also track the aggregation of other amyloid proteins, such as insulin and Aβ1-42. The protein-dye interaction was characterized via steady-state and time-resolved fluorescent spectroscopy. DANIR-2b(2OH) features environment-sensitive emission, high photostability, and a straightforward synthesis. Critically, it provides a substantially lower noise level in standard plate-reader assays, allowing the tracking of aggregation processes that are not visible in standard ThT measurements. This establishes DANIR-2b(2OH) as a highly sensitive and broadly applicable probe for real-time amyloid aggregation measurements and imaging.

Effectively separating critical materials, including intra-lanthanide separations, is crucial for meeting growing application demands. Liquid-liquid extraction (LLE) is the industry standard for lanthanide separations, where the selectivity can depend on small changes in metal coordination. In this work, we investigate representative lanthanide Eu coordination with a neutral malonamide extractant in an imidazolium bistriflimide ionic liquid (IL) solvent. Through systematic titrations of the water and extractant and under extraction conditions, we observe surprising cooperative Eu solvation with the IL anion and extractant. Time-resolved fluorescence spectroscopy measurements show strong extractant coordination in water-saturated IL. Lifetime measurements show no water coordination, and extended X-ray fine structure spectroscopy data provide a coordination number of 10. Molecular dynamics simulations confirm this coordination number and reveal IL anion coordination in the final Eu complex, even though it is ordinarily a significantly weaker ligand compared to water. The lack of water in the final extracted complex and IL anion coordination potentially explain the increased extraction in LLE systems using ILs, as evidenced by higher distribution ratios for cation exchange extraction, despite the energetic cost of cation transfer to the aqueous phase. These results highlight the opportunities for tuning metal coordination to drive extraction in unique solvent systems.

Circulating estradiol is predominantly protein-bound, with human serum albumin (HSA) serving as its major carrier. While traditionally considered a carrier with low affinity and readily reversible binding at a single site, the molecular details and kinetics of estradiol-HSA interactions remain incompletely understood. We employed equilibrium dialysis, steady-state and time-resolved fluorescence spectroscopy to characterize estradiol-HSA interactions. Surface plasmon resonance (SPR) was used to elucidate the kinetics of estradiol's association and dissociation with HSA. Structural and energetic features of binding were investigated using molecular docking and structure network analyses. Binding isotherms generated using equilibrium dialysis, steady-state and time-resolved fluorescence spectroscopy revealed nonlinear asymmetric binding with apparent Kd that varied as a function of estradiol and HSA concentrations, inconsistent with canonical model of low-affinity, single-site interaction characterized by a fixed Kd. Kinetic analyses by SPR revealed initial rapid association dynamics followed by a slower second phase. Molecular modeling identified a high-affinity estradiol-binding pocket in Sudlow's Site I and two additional low-affinity sites within a highly interconnected hub of structural blocks. Spatially coordinated conformational rearrangements accompanying estradiol partitioning into the high-affinity pocket of Sudlow's Site I and two additional moderate-affinity sites suggest an allosterically-coupled binding architecture that enables albumin to actively regulate estradiol bioavailability across a broad, physiologically relevant concentration range. Estradiol's binding to HSA is a dynamic, multi-equilibrium process driven by ligand-induced conformational rearrangements within HSA; the binding data are inconsistent with canonical model of estradiol-HSA interaction with 1:1 stoichiometry and a fixed Kd.

In recent decades, the extractant N,N,N',N'-tetraoctyldiglycolamide (TODGA) has garnered significant attention for its strong selectivity across the lanthanide (Ln) series. This selectivity has been connected to outer-sphere interactions, particularly hydrogen bonding between TODGA, outer-sphere counteranions, and coextracted acid and water molecules. The addition of phase modifiers to the organic phase that help avoid phase splitting, such as linear alcohols, can also influence the Ln distribution ratios. However, the mechanism by which alcohols affect Ln extraction and their impact on Ln selectivity remain poorly understood. This study investigates the extraction of Ln by TODGA in mixtures of alcohol and n-dodecane to explore how the alkyl structure of the alcohol influences Ln extraction and speciation in both HNO3 and HCl systems. Our findings demonstrate that incorporating alcohols into n-dodecane organic phases can significantly enhance Ln selectivity, with an order-of-magnitude increase in separation factors for many systems. Using multiple spectroscopic techniques, such as ultraviolet-visible (UV-Vis)-NIR, time-resolved fluorescence spectroscopy (TRFS), extended X-ray absorption fine structure (EXAFS), and NMR, we confirm that alcohols do not alter the inner-sphere coordination of Ln, preserving the 1:3 [Ln(TODGA)3]3+ complex, but instead associate in the outer sphere. This demonstrates a means beyond inner-sphere coordination to control and significantly enhance Ln selectivity with phase modifiers.

In most organisms, ATP synthesis is powered by the proton motive force (pmf) and catalyzed by ATP synthase. While the chemiosmotic theory originally proposed a "delocalized coupling" between proton pumps and consumers, growing evidence implicates the membrane in mediating localized proton transfer (PT). To directly track ultrafast PT at the membrane surface as a function of ATP synthase activity, we developed an in vitro system. We tethered a light-activated excited-state photoacid to the bilayer of unilamellar vesicles to confine PT to the membrane interface and co-reconstituted a thermophilic Bacillus TFOF1 ATP synthase. Using steady-state and time-resolved fluorescence spectroscopy, we quantified PT and lateral proton diffusion from the anchored photoacid under conditions with non-ATP- and ATP-producing enzymes. Our results show that the membrane accepts protons at its interface and that PT is enhanced only when ATP synthase is active. A comparison with soluble photoacid positioned near the membrane shows that protons consumed by ATP synthase do not equilibrate with the bulk aqueous phase. Instead, they are transferred directly along the two-dimensional membrane interface to the enzyme. This localized coupling can explain how ATP synthesis can proceed even when the apparent bulk pmf seems insufficient. Our results refine the proton translocation during ATP synthesis by revealing that the membrane itself is an active participant in PT, thereby strengthening the case for localized proton coupling in bioenergetics.

Accurate identification and mechanism of breast invasive ductal carcinoma based on combining steady-state and time-resolved label-free fluorescence spectroscopy.

Fluorosolvatochromism and dual emission of D-π-A+ thiophene based derivatives: An approach to white light emitters (II).

Gold nanospheres (AuNSs) functionalized with DNA are powerful tools for studying nanoscale biomolecular interactions through fluorescence modulation. Understanding how DNA conformation influences fluorescence is essential for advancing biosensor design. We examine how DNA strand length and surface loading govern the fluorescence behavior of Cy5-labeled single-stranded DNA (polyA-Cy5) bound to AuNSs and how these properties change upon hybridization with complementary polyT strands. PolyA-Cy5 strands of different bases were conjugated to AuNSs and analyzed using steady-state and time-resolved fluorescence spectroscopy before and after polyT hybridization. Surface attachment induced strong fluorescence quenching, with intensity varying with strand length due to differences in DNA conformation. Short strands remained rigid and upright, whereas longer strands adopted more flexible geometries. Upon hybridization, longer duplexes exhibited fluorescence enhancement attributed to increased fluorophore-metal spacing as dsDNA becomes more upright. Lifetime measurements supported these conformational changes and suggest a Förster resonance energy transfer (FRET)-based quenching mechanism. Experiments in fetal bovine serum (FBS) confirmed that hybridization-induced enhancement persists in biologically relevant media. The results reveal strand-length and density-dependent conformational dynamics of DNA on AuNSs and establish a robust fluorescence-based method for probing nanoscale assembly. The nanosystem can also be tracked intracellularly, as demonstrated by its detectable signal in fluorescence imaging.

Human serum albumin (HSA) is a key transport protein whose ability to bind multiple endogenous and exogenous ligands is governed by site heterogeneity and long-range conformational coupling; however, the mechanisms underlying ligand redistribution among binding sites, particularly in nanoparticulate HSA, remain poorly understood. To address this, we systematically examined the binding of ANS, DAUDA, palmitic acid (PA), and the anticancer lipopeptide PA-EQRPR to monomeric HSA (mHSA) and HSA nanoparticles (HSA-NPs) using steady-state and time-resolved fluorescence spectroscopy complemented by molecular modeling. In mHSA, three spectroscopically distinct binding species were resolved, with fluorescence lifetimes of ∼22.7, 14.5, and 1.6 ns and dissociation constants of 0.33, 9.0, and 3.3 μM, revealing multiple binding environments with distinct affinities and dynamics. Competitive binding experiments demonstrated cooperative PA binding and showed that PA-EQRPR not only displaces ANS or DAUDA but also promotes their redistribution to alternative, more hydrophobic sites, consistent with ligand-induced allosteric site–site communication. Lifetime-resolved analysis of DAUDA further revealed that PA stabilizes long-lived, high-affinity binding states, while PA-EQRPR shifts ligand populations toward deeper hydrophobic environments, enhancing fluorescence. HSA-NPs prepared using ethanol or acetone exhibited markedly different binding behaviors from mHSA, highlighting the impact of protein organization on ligand accessibility. Ethanol-induced HSA-NPs favored long-lifetime, hydrophobic binding species, whereas acetone-induced particles showed reduced site heterogeneity. Docking and molecular dynamics simulations revealed ligand-driven conformational rearrangements that reshape HSA’s hydrophobicity landscape. Together, these findings introduce an allosteric population-shift framework that rationalizes multisite ligand binding and redistribution in both monomeric and nanoparticulate HSA.

Collagen autofluorescence provides valuable intrinsic contrast for assessing tissue structure, composition, and pathology. However, a comprehensive understanding of the fluorescence properties across different collagen types remains limited. This knowledge gap may limit the development of advanced label-free fluorescence spectroscopy and imaging techniques for specific tissue characterization and diagnostic applications. This study aims to comprehensively characterize the fluorescence intensity excitation-emission matrices (I-EEMs) and time-resolved excitation-emission matrices (TR-EEMs) of collagen standards from Types I, II, III, IV, and V obtained from various organ sources under both dry and hydrated conditions, to identify optimal excitation-emission parameters for each collagen type discrimination, and to establish a reference dataset that supports future research in label-free tissue characterization. We employed a time-resolved fluorescence spectroscopy system equipped with an optical parametric oscillator laser (excitation: 200-2000 nm, pulse width: 30 ps) as an excitation source to generate I-EEMs and TR-EEMs of human and bovine collagen Types I-V. The fluorescence light was obtained by a multichannel plate photomultiplier tube through a monochromator (spectral range: 200-1000 nm). Measurements were conducted using collagen standards, under both dry and hydrated states. Additionally, photobleaching effects were assessed to ensure the reliability and reproducibility of fluorescence data. Each collagen type exhibited distinct I-EEM and TR-EEM signatures, with fluorescence lifetimes ranging from 2.5 ns (Type III, bovine skin) to 5.3 ns (Types II and V). Fibrillar collagens (Types I and V) displayed broader I-EEMs, whereas basement membrane collagen (Type IV) showed the narrowest spectral distribution. Organ-source-dependent variations were evident within the same collagen type. Type I collagen from human placenta exhibited an inverse lifetime-emission wavelength relationship compared to bovine sources. Hydration consistently red-shifted emission peaks into the 395-420 nm range and reduced fluorescence lifetimes across all collagen types (e.g., Type I bovine Achilles tendon: 3.2-5.0 ns dry vs. 3.0-4.5 ns hydrated). Despite excitation wavelength- and fluence-dependent photobleaching of fluorescence intensity, fluorescence lifetimes remained relatively stable, confirming the robustness of lifetime-based measurements. This study establishes a comprehensive reference dataset for the fluorescence properties of collagen Types I-V and demonstrates the potential of combined I-EEMs and TR-EEMs analysis for tissue characterization. The results highlight species-, organ-, type-, and environment-specific optical fingerprints of similar collagens, which must be considered before implementing more in-depth studies on how the optical properties of collagen change in different medical applications.

Quantitative determination of submerged oil concentrations by time-resolved fluorescence spectroscopy: A comparison of multi-way calibration strategies.

ABSTRACT Radiative lifetimes of 18 even levels of Ho ii in the energy range between 28692.100 and 44997.566 cm−1 and those of seven levels of Ho iii from 31313.51 to 62804.69 cm−1 were measured by the time-resolved laser-induced fluorescence spectroscopy in laser-induced plasma. To our knowledge, 13 levels of Ho ii and seven levels of Ho iii were measured for the first time. By combining the experimental lifetimes and theoretical branching fractions, the transition probabilities and oscillator strengths for two lines in Ho ii and 30 lines in Ho iii were determined.