Time-resolved Fluorescence Measurements Research Articles

Time-resolved observation of DHR123 nano-clay radio-fluorogenic gel dosimeters by photoluminescence-detected pulse radiolysis

The importance of real-time dose evaluation has increased for recent advanced radiotherapy. However, conventional methods for real-time dosimetry using gel dosimeters face challenges owing to the delayed dose response caused by the slow completion of radiation-induced chemical reactions. In this study, a novel technique called photoluminescence-detected pulse radiolysis (PLPR) was developed, and its potential to allow real-time dose measurements using nano-clay radio-fluorogenic gel (NC-RFG) dosimeters was investigated. PLPR is a time-resolved observation method, and enables time-resolved fluorescence measurement. NC-RFG dosimeters were prepared, typically consisting of 100 μM dihydrorhodamine 123 (DHR123) and 2.0 wt.% nano-clay, along with catalytic and dissolving additives. We successfully achieved time-resolved observation of the increase in fluorescence intensity upon irradiation of the dosimeter. Dose evaluation was possible at 1 s after irradiation. The dose-rate effect was not observed for the deoxygenated dosimeter, but was observed for the aerated dosimeter. Besides the dose-rate effect, linear dose responses were obtained for both conditions. Furthermore, we made a novel observation of a decay in the fluorescence intensity over time in the early stages which named fluorescence secondary loss (FSL) and elucidated the conditions under which this phenomenon occurs.

Investigation of H-Bonding and pH on the Fluorescence Spectral Behavior of Valsartan.

The photophysical properties of valsartan (VAL), a potent phenyl tetrazole derivative sartan, were investigated. Valsartan has absorption bands at 230nm and 255nm and a fluorescence band at about [Formula: see text] = 346nm in butanol which is red shifted depending on the H-bonding capability of the solvent. The role of H-bonding in the excited state was approved through the linear correlation of the emission energy of VAL with Camlet-Taft acidity and basicity parameters, α and β, of polar protic solvents. The position and intensity of fluorescence emission bands of VAL are found to be pH dependent, shifting from 425nm at pH 2 to 375nm at pH 4 with enhancement of intermolecular H-bonding and fluorescence intensity depletion beyond pH 5 with formation of tetrazole anion. The results were supported by time-resolved fluorescence measurements which indicated the presence of different species with different lifetimes in the excited state depending on solution pH value. Computational results based on time dependent density functional methods (TDDFT) show that the tetrazole moiety is involved in the [Formula: see text] absorption transitions, while natural bond analysis (NBO) shows that VAL adopts a dimer conformation in water because of effective intermolecular H-bonding.

Optical Characterization of Fluorescent Chitosan-Based Carbon Dots Embedded in Aqueous Natural Dye

(1) Background: This work evaluated the optical characterization of aqueous fluorescent chitosan-based carbon dots (or carbon nanoparticles CNPs) embedded in natural dye for potential functional packaging applications. Chitosan-based materials are nontoxic, biodegradable, biocompatible, bactericidal, and produced from renewable polymer sources. Anthocyanins are pigments of different colors with a large range of potential applications, such as in bioindicators and biomonitoring; (2) Methods: The CNPs were synthetized in aqueous solutions using chitosan as a carbon source. The natural dye was extracted from the leaves of Tradescantia pallida Purpurea in aqueous solutions. The fluorescence quantum efficiency (η) and fluorescence lifetime (τ) were determined using the mode-mismatched pump–probe thermal lens (TL) technique and time-resolved fluorescence lifetimes (TRFL) measurements, respectively; (3) Results: The η and τ were measured for CNPs embedded in natural dye solution at different concentrations (5.2, 12.09, and 21.57 mass percentage composition). The η and τ photophysical parameters obtained for CNPs embedded in natural dye were compared with those of other CNPs synthesized using different carbon sources, such as leaves, seeds, and protein; (4) Conclusions: Fluorescence spectra and time-resolved fluorescence measurements corroborate the TL results, and relatively high values of η were obtained for the CNP synthesized and embedded in natural dye.

A comprehensive investigation of the nano-[Cu2-(DIP)2-EA] effects on HSA through spectroscopic procedures and computer simulations

In this research, the toxicity of nano-[Cu2-(DIP)2-EA], a metal nano-complex consisting of ellagic acid and bathophenanthroline ligands, on human serum albumin (HSA) at a protein level was investigated. Molecular docking simulations and spectral analyses were conducted in a simulated physiological environment at pH 7.4 to explore the interaction of nano-[Cu2-(DIP)2-EA] with HSA. The results represented an increase in albumin absorption upon exposure to nano-[Cu2-(DIP)2-EA], demonstrating significant interaction between the two compounds. Steady-state and time-resolved fluorescence measurements pointed out that nano-[Cu2-(DIP)2-EA] induced static quenching of the albumin's intrinsic fluorescence with a high binding affinity of approximately 106 mol/L in a 1:1 interaction ratio. The thermodynamic variables clarified that binding of nano-[Cu2-(DIP)2-EA] to albumin occurs spontaneously and primarily driven by van der Waals interactions and H-bonds. The results of the computer simulations and the binding displacement experiments utilizing the site markers warfarin and ibuprofen revealed that nano-[Cu2-(DIP)2-EA] binds to site I within the subdomain IIA of albumin. Circular dichroism analysis elaborated that nano-[Cu2-(DIP)2-EA] slightly perturbed the microenvironment around of tryptophan residues and diminished the α-helix structure stability to a negligible amount.

Monitoring cancer treatments in melanoma cells using a fluorescence lifetime nanoprobe based on a CdSe/ZnS quantum dot functionalized with a peptide containing D-penicillamine and histidine

Anticancer therapies with cisplatin and volasertib (BI-6727) were monitored by fluorescence lifetime imaging microscopy (FLIM) in live SK-Mel-2 melanoma cells. A CdSe/ZnS quantum dot functionalized with a peptide containing D-penicillamine and histidine (CdSe/ZnS-PH) was used as intracellular pH fluorescent probe. A faster cytosol acidification was observed for cells treated with cisplatin when compared to volasertib. The first changes in the intracellular pH were found after 2 hours of treatment with cisplatin and 8 hours with volasertib. Additionally, the relationship between cytosol acidification and apoptosis was investigated using an innovative methodology based on time-resolved fluorescence measurements. Similar low percentages of apoptotic cells were observed after short incubation periods (2 – 8 hours) with both drugs. In contrast, late apoptosis and death were found for a large fraction of cells during 24-hour incubation with cisplatin but not volasertib. Thus, the early acidification observed in cisplatin treatment could accelerate apoptosis and cell death. Despite volasertib treatment shows slower mechanism of action than cisplatin, similar inhibitory effects were found for both drugs at longer incubation periods (72 hours). This study proves the potential of CdSe/ZnS-PH nanoparticle as a fluorescence lifetime probe in the study of the mechanism of action of antitumor drugs.

Dependence of photophysical properties on stereoisomers: The case of nonsymmetric 2-aryl(benz)imidazole-annulated derivatives

Nonsymmetric 2-aryl (benz)imidazole derivatives were previously synthesized via C-H/N-H annulation of imidazole derivatives from lapachol moieties with internal alkynes as an efficient tool to increase the π-system of lapimidazole derivatives. To investigate their photophysical properties, time-resolved and steady-state fluorescence measurements were carried out on dropcast film, powder, and diluted solution. Despite having the same number of single and double bonds, slight differences in their structures lead to distinct photophysical properties. The time-correlated single-photon counting measurements also demonstrated that the aspects of the temporal decay curves are dependent on the treatment of the samples during the measurements. The decay curves were relatively well fitted using an alternative method based on the exponentially modified Gaussian function, giving rise to a more complete set of fitting parameters. Finally, theoretical calculations corroborate that small structural changes in molecules can significantly alter their conformational dynamics, electronic spectra, and overall electronic behavior and stability.

Green synthesis of zinc ferrite nanoparticles from Nyctanthes arbor-tristis: unveiling larvicidal potential, protein binding affinity and photocatalytic activities.

Environmental pollution, being a major concern worldwide, needs a unique and ecofriendly solution. To answer this, researchers are aiming in utilizing plant extracts for the synthesis of nanoparticles. These NPs synthesized using plant extracts provide a potential, environmentally benign technique for biological and photocatalytic applications. Especially, plant leaf extracts have been safe, inexpensive, and eco-friendly materials for the production of nanoparticles in a greener way. In this work, zinc ferrite nanoparticles (ZnFe2O4 NPs) were prepared using Nyctanthes arbor-tristis leaf extract by hydrothermal method, and its biological and photocatalytic properties were assessed. The synthesized ZnFe2O4 NPs were characterized using powder X-ray diffraction (XRD), scanning electron microscopy (SEM), and Fourier-transform infrared spectroscopy (FT-IR). X-ray diffraction confirmed the arrangement of the fcc crystal structure of the nanoparticles and that some organic substances were encapsulated within the zinc ferrite. According to the SEM analysis, the resulting nanoparticles got agglomerated and spherical in shape. The ZnFe2O4 nanoparticles are in their pure form, and all of their elemental compositions were shown by the energy-dispersive X-ray analysis (EDAX) spectrum. The FTIR results revealed that the produced nanoparticles contained distinctive functional groups. Fluorescence spectroscopy was used to examine the binding affinities between bovine serum albumin (BSA) and ZnFe2O4 nanoparticles in terms of protein binding, stability, and conformation. The interaction between BSA and ZnFe2O4 NPs was examined using steady-state and time-resolved fluorescence measurements, and it was evident that static quenching occurred. The ability of ZnFe2O4 nanoparticles to kill Culex quinquefasciatus (C. quinquefasciatus) larvae was evaluated. The synthesized NPs demonstrated a noteworthy toxic effect against the fourth instar larvae of C. quinquefasciatus with LC50 values of 43.529µg/mL and LC90 values of 276.867µg/mL. This study revealed the toxicity of green synthesized ZnFe2O4 NPs on mosquito larvae, proving that these NPs are good and effective larvicides. Furthermore, the ZnFe2O4 NPs were utilized for dye degradation of methylene blue under visible light treatment and achieved 99.5% degradation.

Anion Effect on the Excited-State Intramolecular Proton Transfer of 4'-N,N-Diethylamino-3-hydroxyflavone in Ionic Liquids.

The excited-state intramolecular proton transfer (ESIPT) reaction of 4'-N,N,-diethylamino-3-hydroxyflavone (C2HF) was studied using time-resolved fluorescence measurements in ionic liquids (ILs) of various anions with a fixed cation (1-ethyl-3-methylimidazolium [C2mim]+). C2HF showed an ESIPT reaction from the normal excited state (N*; keto form) to the tautomer excited state (T*; enol form) where both states are emissive. The ESIPT rate and yield were obtained by analyzing the time-resolved fluorescence spectra measured using the optical Kerr gate method. Both the ESIPT rate and yield decreased with increasing hydrogen-bond accepting ability of the anion. According to density functional theory calculations, the complex formation energy between C2HF and the anion became significantly negative with increasing the hydrogen-bond accepting ability of anion. The pseudoequilibrium constant between N* and T* ([T*]/[N*]) in the electronic excited state decreased with increasing hydrogen-bond accepting ability of the anion, while it increased with increasing the alkyl-chain length of alkyl sulfonate. The excitation wavelength dependence of the ESIPT rate and yield was studied for C2HF in [C2mim][C6H13SO3]. The ESIPT yield decreased by nearly a factor of 2 with increasing excitation wavelength from 360 to 425 nm, although the change in the ESIPT rate was small. The solvation heterogeneity due to the alkyl chain in the anion was considered to be the reason for the excitation wavelength dependence.

L-cysteine modified N-doped carbon quantum dots derived from peach palm (Bactris gasipaes) peels for detection of mercury ions

Establishing an effective strategy for Hg2+ determination with good sensitivity and high selectivity is of particular concern. In this work, N-doped carbon quantum dots (NCQDs) were obtained for the first time from peels of peach palm fruit (Bactris gasipaes) through microwave-assisted one-pot synthesis. Surface NCQDs was modified with l-cysteine (NCQDs/CYS) by a chemical reaction (known as amide coupling) between the nitrogen-containing groups (amine group) of l-cysteine ligand and the carboxyl groups of NCQDs. The chemical bonding was examined by Fourier Transform Infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). NCQDs/CYS nanoparticles exhibited a quantum yield of fluorescence (ΦFL) of 25.4%, high solubility in water, and a surface with thiol groups from l-cysteine. Mercury ions induced quenching of fluorescence of NCQDs/CYS at low concentrations (Limit of Detection, LOD = 0.19 μM) with great sensitivity. A significant preference for Hg2+ ions was found because of the high affinity between the thiolate groups of NCQDs/CYS and the analyte. Time-resolved fluorescence measurements were done to study the way that led to fluorescence quenching when Hg2+ ions were present, evidencing a static quenching fluorescence mechanism. The new NCQDs/CYS material could be applied to accurately measure the concentration of Hg2+ ions in water samples in a fast and cost-effective way.

Non-covalent self-assembly of substituted phthalocyanine with C60, C70, and 9-phenylanthracene: Spectroscopic insights and DFT calculations

Self-assembly of non-covalent donor-acceptor systems based on (octakis-3,5-di-tert-butylphenoxy)phthalocyanine (H2Pc(3,5-tBuPhO)8) and С60, С70 or 9-phenylanthracene (PhAn) is observed and described using the UV–vis and steady-state/time-resolved fluorescence methods. The 1:1 stoichiometry of the H2Pc(3,5-tBuPhO)8 - С60/С70/PhAn supramolecule was established by Job's method. The fluorescence quenching of phthalocyanine in the mentioned systems in toluene, indicating close bonding between a donor and an acceptor, was observed. The bonding constants are 1.2 × 104 M−1, 2.4 × 104 M−1, and 3.9 × 102 M−1 for the C60, C70, and PhAn derivatives, respectively. Using time-resolved fluorescence measurements, the static mechanism of quenching was established. The H2Pc(3,5-tBuPhO)8 fluorescence lifetimes in the absence and presence of С60/С70/PhAn, the charge separation rate constant (kCS) and the charge-separated state quantum yield (ΦCS) of donor-acceptor systems were calculated. (С70)H2Pc(3,5-tBuPhO)8 displays the highest kCS and ΦCS in good agreement with its stability parameter. Quantum-chemical calculations of the molecular spatial/electronic structure and the HOMO/LUMO composition that were revealed by DFT and TD DFT methods point to the H2Pc(3,5-tBuPhO)8 → fullerenes redistribution of the electron density and to the opposite one in the PhAn complex.

A Coronin 1-Derived Peptide Inhibits Membrane Fusion by Modulating Membrane Organization and Dynamics.

Membrane fusion is considered the first step in the entry of enveloped viruses into the host cell. Several targeted strategies have been implemented to block viral entry by limiting the fusion protein to form a six-helix bundle, which is a prerequisite for fusion. Nonetheless, the development of broad-spectrum fusion inhibitors is essential to combat emerging and re-emerging viral infections. TG-23, a coronin 1, a tryptophan-aspartate-rich phagosomal protein-derived peptide, demonstrated inhibition of fusion between small unilamellar vesicles (SUVs) by modulating the membrane's physical properties. However, its inhibitory efficacy reduces with an increasing concentration of membrane cholesterol. The present work aims to develop a fusion inhibitor whose efficacy would be unaltered in the presence of membrane cholesterol. A stretch of the tryptophan-aspartic acid-containing peptide with a similar secondary structure and hydrophobicity profile of TG-23 from coronin 1 was synthesized, and its ability to inhibit SUV-SUV fusion with varying concentrations of membrane cholesterol was evaluated. Our results demonstrate that the GG-21 peptide inhibits fusion irrespective of the cholesterol content of the membrane. We have further evaluated the peptide-induced change in the membrane organization and dynamics utilizing arrays of steady-state and time-resolved fluorescence measurements and correlated these results with their effect on fusion. Interestingly, GG-21 displays inhibitory efficacy in a wide variety of lipid compositions despite having a secondary structure and physical properties similar to those of TG-23. Overall, our results advocate that the secondary structure and physical properties of the peptide may not be sufficient to predict its inhibitory efficacy.

Near-infrared two-photon absorption and excited state dynamics of a fluorescent diarylethene derivative

Near-infrared two-photon absorption and excited state dynamics of a fluorescent diarylethene (fDAE) derivative were investigated by time-resolved absorption and fluorescence spectroscopies. Prescreening with quantum chemical calculation predicted that a derivative with methylthienyl groups (mt-fDAE) in the closed-ring isomer has a two-photon absorption cross-section larger than 1000 GM, which was experimentally verified by Z-scan measurements and excitation power dependence in transient absorption. Comparison of transient absorption spectra under one-photon and simultaneous two-photon excitation conditions revealed that the closed-ring isomer of mt-fDAE populated into higher excited states deactivates following three pathways on a timescale of ca. 200 fs: (i) the cycloreversion reaction more efficient than that by the one-photon process, (ii) internal conversion into the S1 state, and (iii) relaxation into a lower state (S1’ state) different from the S1 state. Time-resolved fluorescence measurements demonstrated that this S1’ state is relaxed to the S1 state with the large emission probability. These findings obtained in the present work contribute to extension of the ON–OFF switching capability of fDAE to the biological window and application to super-resolution fluorescence imaging in a two-photon manner.Graphical

Inhibition of amyloid fibrillation of single amino acid tryptophan by tannic acid: An insight into targeted therapy of hypertryptophanemia disorder

Amyloid fibrillation involving specific amino acids is a key factor contributing to the development of various neurodegenerative disorders, which remain being investigated. A promising therapeutic approach for treatment involves the use of small molecules to inhibit this amyloid fibrillation. In this study, we present experimental evidence demonstrating the efficacy of tannic acid (TA) as a potent inhibitor of fibrillation, particularly in the case of the single amino acid tryptophan (Trp), which is associated with hypertryptophanemia disorder. To investigate the kinetics of fibril formation and its suppression by TA, we have employed a combination of steady-state and time-resolved fluorescence measurements. These techniques have allowed us to monitor changes in the intrinsic fluorescence of Trp and an amyloid-specific fluorophore over time. To visualize the effects of TA on Trp fibrils, we have employed various imaging techniques such as, atomic force microscopy, transmission electron microscopy, and field emission scanning electron microscopy. These imaging techniques have vividly captured the TA-induced disruption of long Trp fibrils. Dynamic light scattering measurements have complemented these microscopic observations, further supporting the notion that TA effectively hinders the formation and stability of Trp fibrils. Additionally, we have determined the binding constant and calculated changes in the free energy associated with Trp-TA interactions through temperature-dependent spectroscopic measurements. These thermodynamic analyses provide valuable insights into the underlying mechanisms of TA-induced disaggregation of larger Trp fibrils.

Kinetics of pair-induced quenching in holmium-doped optical fibers

We present a study of the dynamics of the rapid energy transfer upconversion that occurs within pairs or clusters of holmium ions, commonly referred to as pair-induced quenching. The lifetime of excited holmium ion pairs is determined to be around 0.4 µs from time-resolved fluorescence measurements. We develop a rate equation model that incorporates this lifetime and evaluate numerically and experimentally the consequences this finite intra-cluster upconversion rate has on the performance of a nanosecond-pulsed Ho-doped fiber amplifier. We demonstrate a significant improvement in efficiency by utilizing pulsed pumping on a timescale shorter than the measured excited ion pair lifetime.

Fluorescent Sensor Based on 1H-Pyrazolo[3,4-b]quinoline Derivative for Detecting Zn2+ Cations.

The photophysical and sensory properties of the donor-acceptor pyrazoloquinoline derivative (PQPc) were investigated using absorption, steady-state, and time-resolved fluorescence measurements. The compound synthesized from commercial, readily available substrates exhibited absorptions in the UV-Vis range, with a maximum of the longwave band around 390 nm. The maximum fluorescence was around 460-480 nm, depending on the solvent. The quantum yield was between 12.87% (for n-hexane) and 0.75% (for acetonitrile) and decreased with increasing solvent polarity. The PET mechanism was implicated as the cause of fluorescence quenching. Divalent ions such as Zn2+, Pb2+, Cd2+, Ca2+, Mg2+, Co2+, Ni2+, and Cu2+ were introduced to study the fluorescent response of PQPc. A 13-times increase in fluorescence quantum yield was observed after the addition of Zn2+ ions. Detailed research was carried out for the PQPc-Zn2+ system in order to check the possibility of analytical applications of PQPc as a fluorescent sensor. A detection limit of Zn2+ was set at the value level 1.93 × 10-7 M. PQPc-Zn2+ complexes had a stoichiometry of 1:1 with a binding constant of 859 M-1. Biological studies showed that the sensor was localized in cells near the membrane and cytoplasm and may be used to detect zinc ions in eukaryotic cells.

The decay curves of luminescence from Eu2+ in β-SiAlON are effectively analyzed using the general-order kinetics formula

Defects in phosphors affect not only luminescence intensity but also emission peak width, decay time, and afterglow. The green phosphor β-SiAlON:Eu2+ exhibits the green emission of Eu2+ at 520 nm and the blue emission of nitrogen vacancies at 460 nm in time-resolved fluorescence measurements. The decay time of the intrinsic Eu2+ transition is 0.7 μs, but afterglow is detected from 50 μs to 0.01 s. This afterglow decay curve is the same for the green emission of Eu2+ and the blue emission of nitrogen vacancies, suggesting that the defect levels of the nitrogen vacancies affect the Eu2+ transition. The afterglow decay curves were analyzed using the formula of the general-order kinetics, 1+t/τB−n , where n is the decay power and τB is the decay time. This equation is generally used when analyzing afterglow on the order of seconds to hours but has not been examined systematically applied in samples with different concentrations of Eu2+ and temperatures on the order of nanoseconds to milliseconds. The decay power n is approximately 1 for all Eu2+ concentrations (x = 0.001–0.3) and undoped β-SiAlON. The decay time τB is correlated with the density of the nitrogen vacancies determined by electron spin resonance. Furthermore, the value of n is approximately 1 for 50 μs to 0.01 s and 0.3 for 1–1000 s. Thus, the luminescence mechanism of Eu2+ can be discussed by comparing n and τB obtained from the decay curves. In addition, several different Eu2+-doped phosphors, namely SrAl2O4:Eu2+, Dy3+, CaAlSiN3:Eu2+, and CaS:Eu2+, Tm3+, are studied.

Time-resolved fluorescence measurements of excitonic dimer-bearing oligonucleotides

Time-resolved fluorescence measurements of excitonic dimer-bearing oligonucleotides

Energy transfer from phycobilisomes to photosystem I at room temperature.

The phycobilisomes function as the primary light-harvesting antennae in cyanobacteria and red algae, effectively harvesting and transferring excitation energy to both photosystems. Here we investigate the direct energy transfer route from the phycobilisomes to photosystem I at room temperature in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that lacks photosystem II. The excitation dynamics are studied by picosecond time-resolved fluorescence measurements in combination with global and target analysis. Global analysis revealed several fast equilibration time scales and a decay of the equilibrated system with a time constant of ≈220 ps. From simultaneous target analysis of measurements with two different excitations of 400 nm (chlorophyll a) and 580 nm (phycobilisomes) a transfer rate of 42 ns-1 from the terminal emitter of the phycobilisome to photosystem I was estimated.

Chitosan polymers randomly labeled with pyrene: synthesis, photophysics, and selective quenching by metal cations and anions

The synthesis together with a comprehensive electronic spectral and photophysical study of two chitosan polymers randomly labeled with different degrees of pyrene were here undertaken. The polymers, denoted as ChiNPy-A and ChiNPy-B, have different degrees of substitution with pyrene (%DSPy), with values of 0.5 and 4.5, respectively. The resulting polymers consist in four distinct units, including the deacetylated and acetylated units and the grafted imine and amine pyrene units, obtained by a Schiff base formation reaction and subsequent reduction, respectively. Time-resolved fluorescence measurements in aqueous solution revealed triple exponential decay fits that characterizes the fluorescence properties of the amine- and imine-pyrene-linked units, as well as self-quenching of pyrene units. The ChiNPy polymer showed fluorescence quenching of its pyrene amine units, with lead (II) and mercury (II) exhibiting the highest degree of fluorescence quenching. From Stern-Volmer type plots, two distinct populations of fluorophores were observed. These findings emphasize the potential of chitosan pyrene-labeled polymers as sensitive fluorescent probes, particularly for detecting metal cations.

Anomalous proton transfer of a photoacid HPTS in nonaqueous reverse micelles.

The proton transfer reaction is one of the fundamental chemical reactions where the reaction dynamics strongly depend on solvent properties such as acidity or basicity. A photoacid 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) shows a sharp decrease of pKa (7.7 → 0.5) upon photoexcitation, and the excited-state proton transfer (ESPT) occurs with ultrafast time constants of 2.5 and 89 ps in bulk aqueous solution. However, the two-step proton transfers via the contact ion pair formation and the proton diffusion are strongly limited inside the nanopools of reverse micelles (RMs). The confinement in small RMs strongly impeded the proton transfer reactions. In this work, we report the ESPT of HPTS confined in methanol-in-oil RMs by steady-state and time-resolved electronic spectroscopy. Interestingly, HPTS shows substantial deprotonation in the excited state only in small RMs, while the ESPT of HPTS does not occur in bulk methanol solution due to the low basicity of aliphatic alcohols. The kinetic analysis of time-resolved fluorescence and transient absorption measurements will compare the proton transfer dynamics of HPTS in the water-in-oil and methanol-in-oil RMs. The ESPT of photoacids, especially in the nonaqueous RMs, can be crucial in understanding many important chemical reactions involving proton transfer in the confined environments of cells and membranes.