Time-resolved Total Internal Reflection Fluorescence Research Articles

Adsorption and Aggregation at Silica/Methanol Interfaces: The Role of Solute Structure

Second harmonic generation (SHG) and time-resolved, total internal reflection fluorescence (TR-TIRF) spectroscopy were used to examine the adsorption, solvation, and aggregation of a coumarin solute, Coumarin 152 (C152), at the silica/methanol interface. Experiments were performed as a function of bulk C152 concentration with SHG data providing information about relative surface coverage and ground state solvation environment. TR-TIRF data measured emission lifetimes of C152 adsorbed to the silica/methanol interface. SHG spectra show strong resonance enhancement at 365 nm, a result that is blue-shifted considerably from C152’s electronic excitation of ∼400 nm in bulk methanol. Given C152’s solvatochromic behavior, this observation implies that C152 adsorbed to the silica/methanol interface experiences a local dielectric environment that is significantly less polar than in bulk methanol. TR-TIRF decays at sub-micromolar bulk concentrations were fit to two lifetimes: one assigned to C152 emission in bulk methanol (0.9 ns) and a longer lifetime assigned to contributions from adsorbed C152 (∼4 ns). The longer lifetime is similar to C152 in alkanes, a result that is consistent with SHG data. Isothermal data from SHG experiments show unusual behavior as bulk C152 concentration increases. Instead of approaching an asymptotic limit signifying monolayer coverage, the SHG response rises at the lowest C152 concentrations and then decreases dramatically, suggesting the onset of aggregate formation. Changes in the TR-TIRF emission behavior of C152 at higher C152 bulk concentrations support this hypothesis. These findings are interpreted in terms of C152’s ability to self-associate, and the energetics of dimer formation are explored using ab initio calculations and polarizable continuum models.

Single-Molecule Imaging of a Fluorescent Unnatural Amino Acid Incorporated Into Nicotinic Receptors

We report on the first, to our knowledge, successful detection of a fluorescent unnatural amino acid (fUAA), Lys(BODIPYFL), incorporated into a membrane protein (the muscle nicotinic acetylcholine receptor, nAChR) in a living cell. Xenopus oocytes were injected with a frameshift-suppressor tRNA, amino-acylated with Lys(BODIPYFL) and nAChR ( α/ β19′GGGU/ γ/ δ) mRNAs. We measured fluorescence from oocytes expressing nAChR β19′Lys(BODIPYFL), using time-resolved total internal reflection fluorescence microscopy. Under conditions of relatively low receptor density (<0.1 receptors/ μm 2), we observed puncta with diffraction-limited profiles that were consistent with the point-spread function of our microscope. Furthermore, diffraction-limited puncta displayed step decreases in fluorescence intensity, consistent with single-molecule photobleaching. The puncta densities agreed with macroscopic ACh-induced current densities, showing that the fUAA was incorporated, and that receptors were functional. Dose-response relations for the nAChR β19′Lys(BODIPYFL) receptors were similar to those for wild-type receptors. We also studied nAChR β19′Lys(BODIPYFL) receptors labeled with α-bungarotoxin monoconjugated with Alexa488 ( αBtxAlexa488). The nAChR has two αBtx binding sites, and puncta containing the Lys(BODIPYFL) labeled with αBtxAlexa488 yielded the expected three discrete photobleaching steps. We also performed positive control experiments with a nAChR containing enhanced green fluorescent protein in the γ-subunit M3-M4 loop, which confirmed our nAChR β19′Lys(BODIPYFL) measurements. Thus, we report on the cell-based single-molecule detection of nAChR β19′Lys(BODIPYFL).

Photoinduced Redox Cycle of Riboflavin at a Water/Oil Interface

A photoinduced redox reaction cycle of Riboflavin (RF) at a water/CCl4 interface was studied directly by means of both steady-state and time-resolved total internal reflection (TIR) fluorescence spectroscopies. The TIR fluorescence spectrum of RF observed at the water/CCl4 interface with the maximum wavelength of 517 nm was assigned to the pi-pi* transition from the excited singlet-state of the isoalloxazine chromophore in RF. Upon prolonged laser irradiation (400 nm) in the presence of N,N-dioctadecyl-[1,3,5]triazine-2,4,6-triamine (DTT) as a guest for RF in the CCl4 phase, on the other hand, a new TIR fluorescence band appeared at around 480 nm. Furthermore, the fluorescence intensity at around 480 nm increased in the presence of acetic acid in the water phase. Detailed studies demonstrated that the new fluorescence band should be ascribed to 1,5-dihydoroflavin (RFH2). The present results indicated that RFH2 was produced through the photoreaction of the RF-DTT hydrogen-bonded complex formed at the water/CCl4 interface, whose reaction mechanisms were discussed on the basis of the results observed by fluorescence spectra/dynamics measurements under the TIR conditions as well as by transient absorption spectroscopy.

Time-resolved total internal reflection fluorescence study on hybridization of complementary single-stranded DNAs at a water/oil interface.

Hybridization of complementary single-stranded DNAs (ssDNA) at a water/CCl4 interface was studied on the basis of picosecond total internal reflection fluorescence spectroscopy. Complementary ssDNAs dissolved in water were shown to produce the relevant double-stranded DNA (dsDNA) at a water/CCl4 interface in the presence of octadecylamine (ODA) in the oil phase, while hybridization between ssDNAs did not proceed in the water phase, as demonstrated by the fluorescence dynamics of ethidium bromide as a probe for the DNA structure. The structures of dsDNA and the roles of ODA in hybridization of ssDNA at the interface were discussed.

Direct observation of molecular recognition mediated by triple hydrogen bonds at a water/oil interface: time-resolved total internal reflection fluorometry study.

Molecular recognition mediated by hydrogen-bonding interactions at a water/CCl(4) interface was investigated directly by means of time-resolved total internal reflection (TIR) fluorescence spectroscopy. The TIR fluorescence decay profile of riboflavin (RF) in the absence of a guest in the CCl(4) phase was fitted satisfactorily by a single-exponential function. In the presence of N,N-dioctadecyl-[1,3,5]triazine-2,4,6-triamine (DTT) as a guest in the CCl(4) phase, on the other hand, the fluorescence decay profiles were best fitted by double-exponential functions with the relevant amplitude (A(i)) being varied with the concentration of DTT. Furthermore, the rotational reorientation time of RF at the interface determined by fluorescence dynamic anisotropy was 210 ps in the absence of DTT, while fast (160-220 ps) and slow (670-750 ps) rotational reorientation times were observed in the presence of DTT. This slow rotational reorientation time was shown to ascribe to that of the RF-DDT complex formed at the water/CCl(4) interface. These results indicate that molecular recognition mediated by complementary hydrogen bonding takes place effectively at the water/CCl(4) interface, which was observed directly by both fluorescence dynamics and fluorescence dynamic anisotropy measurements under the TIR conditions.

Solvation Dynamics of Fluorophores with an Anthroyloxy Group at the Heptane/Water Interface as Studied by Time-Resolved Total Internal Reflection Fluorescence Spectroscopy

Solvent relaxation processes of 12-(9-anthroyloxy) stearic acid (12-AS) and 4-(9-anthroyloxy) butanoic acid (4-ABA) at the heptane/water interface are examined by time-resolved total internal reflection fluorescence spectroscopy. In a bulk heptane solution containing 3 M ethanol, both 12-AS and 4-ABA show time-dependent fluorescence spectral shifts, and the decay profiles can be analyzed by the two-state kinetics model. The results obtained for the binary mixture of heptane and ethanol can be ascribed to a local enrichment of ethanol molecules around fluorophores, that is, preferential solvation. A similar result is obtained for 12-AS but not for 4-ABA at the heptane/water interface, indicating that the preferential solvation occurs for 12-AS at the heptane/water interface. Since 4-ABA does not show a time-dependent spectral shift at the interface, the fluorophore of 4-ABA is surrounded by more water molecules, by which quenching of fluoresence was accelerated, than 12-AS at the heptane/water interface, and it is located more closely to the water phase than 12-AS.

Excitation Energy Transfer from Sulforhodamine 101 to Acid Blue 1 at a Liquid/Liquid Interface: Experimental Approach To Estimate Interfacial Roughness

Dynamic fluorescence anisotropy of Sulforhodamine 101 (SR101) and excitation energy transfer from SR101 to Acid Blue 1 (AB1) at water/carbon tetrachloride (CCl4) and water/1,2-dichloroethane (DCE) interfaces were studied by using time-resolved total-internal-reflection (TIR) fluorometry. A magic-angle dependence of the TIR fluorescence dynamic anisotropy revealed that rotational reorientation of SR101 at the water/CCl4 interface was restricted in the two-dimensional plane of the interface, while that at the water/DCE interface took place rather freely, similar to reorientation in an isotropic medium. Furthermore, the structural dimension derived from the excitation energy transfer dynamics measurements at the water/CCl4 or water/DCE interface was 1.93 or 2.48, respectively. These results indicated that the water/CCl4 interface was sharp with respect to the molecular size of SR101 (∼10 A), while the water/DCE interface was relatively rough compared to the water/CCl4 interface. Structural dimension analysis...

Time-resolved total internal reflection fluorometry of ternary europium(III) complexes formed at the liquid/liquid interface

The formation of ternary europium(III) complexes with 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione (Htta) and 4,7-diphenyl-1,10-phenanthroline disulfonate (bps) formed at the toluene/water interface was studied by time-resolved total internal reflection fluorescence spectrometry. The fluorescence decay profile of the interfacial complexes was fitted to the double-exponential decay function with the lifetimes of 22±8 and 203±34 µs, which proved the existence of two different species. Dependence of the fluorescence intensity of each species on the ligand concentrations suggested the formation of Eu(tta)2bps- (long lifetime) and Eu2(tta)2(bps)2 (short lifetime) at the interface. The anomalously short lifetime of the binuclear complex was discussed in terms of a ligand–metal charge transfer state.

Total Internal Reflection Fluorescence Dynamic Anisotropy of Sulforhodamine 101 at a Liquid/Liquid Interface: Rotational Reorientation Times and Interfacial Structures

The dynamic anisotropy of Sulforhodamine 101 (SR101) at water/phthalate ester (PE, bis(2-ethylhexyl) phthalate, di-n-heptyl phthalate, di-n-butyl phthalate, or di-n-ethyl phthalate) interfaces was studied by using time-resolved total internal reflection (TIR) fluorometry. A magic-angle dependence of the TIR fluorescence dynamics revealed that rotational reorientation of SR101 at the water/PE interface was restricted in the X−Y plane (in-plane) of the interface. The results indicated that the interface was sharp with respect to the molecular size of SR101 (∼10 A). In-plane rotational reorientation of SR101 at the interface showed two time constants (τrot). The fast component (τ1rot) was similar to that in water irrespective of the nature of PE, while the slow one (τ2rot) was affected by the viscosity of PE but not directly by the macroscopic viscosity. The two rotational reorientation times of SR101 characteristic to the water/PE interface were explained in terms of different adsorption modes of the dye on...

Microenvironments of 8-anilino-1-naphthalenesulfonate at the heptane-water interface: time-resolved total internal reflection fluorescence spectroscopy

Picosecond time-resolved total internal reflection fluorescence spectroscopy was applied to study the microenvironments of 8-anilino-1-naphthalenesulfonate molecules at the heptane-water interface. By analyzing the fluorescence decay profiles, it was found that the fluorophores in the vicinity of the interface are present in two different solvated structures. One of them is located up to a few nanometers from the interface, and the other is located in a relatively wide range toward the aqueous phase. The former solvated structure is the cause of a long fluorescence lifetime which reflects the low polarity or the extremely high viscosity of the microenvironments.

Excimer formation of pyrene in a solid/polymer solution interface layer. A time-resolved total internal reflection fluorescence study

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTExcimer formation of pyrene in a solid/polymer solution interface layer. A time-resolved total internal reflection fluorescence studySanyo Hamai, Naoto Tamai, and Hiroshi MasuharaCite this: J. Phys. Chem. 1995, 99, 14, 4980–4985Publication Date (Print):April 1, 1995Publication History Published online1 May 2002Published inissue 1 April 1995https://doi.org/10.1021/j100014a018RIGHTS & PERMISSIONSArticle Views192Altmetric-Citations14LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts

Salt effects on a proton transfer reaction of excited 1-naphthol in a solid/liquid interface layer: A picosecond time-resolved total internal reflection fluorescence study

Analyses of the excited-state proton transfer reaction of 1-naphthol in sapphire/water interface layers have been performed using fluorescence decay data obtained by a picosecond time-resolved total internal reflection fluorescence spectroscopy. The rate constant of the excited-state proton transfer reaction, k 1, of 1-naphthol in an interface layer was reduced as compared to that in a bulk aqueous solution. Addition of NaCl to aqueous solution of 1-naphthol resulted in a decrease in k 1 for both in the interface layer and in the bulk solution, suggesting that a proton acceptor from excited 1-naphthol is not a single water molecule but a water cluster and that water clusters in an interface layer are also destroyed by NaCl.

Aggregation of pyrene in poly(alkyl methacrylate) films revealed by time-resolved total internal reflection fluorescence spectroscopy

Fluorescence spectra and decay curves of pyrene in poly(alkyl methacrylate) (methyl, n-propyl, isobutyl and cyclohexyl) films cast on sapphire substrates were measured under total internal reflection and normal excitation conditions, and were compared with each other. On the basis of the intensity ratio of the excimer to the monomer fluorescence and the fluorescence decay curve under the normal condition, the degree of pyrene aggregation in these films is discussed in terms of the solubility parameter, chain flexibility and hydrophobicity of the host polymers. Both the difference in the degree of pyrene aggregation and the difference in micropolarity around pyrene molecules between the vicinity of the sapphire/polymer interface and the bulk were found to be quite common for these polymer films cast on sapphire plates, and are discussed in terms of the interaction between substrate surface and polymers. The effect of tacticity of poly(methyl methacrylate) on the degree of the pyrene aggregation and the effect of labelling with pyrene were also investigated.

Effects of the solid/liquid interface on the excimer formation of pyrene in toluene containing poly (methyl methacrylate). A time-resolved total internal reflection fluorescence study

By means of time-resolved total internal reflection (TIR) fluorescence measurements, rise and decay profiles of the pyrene excimer fluorescence were examined in toluene containing 3.3×10 −4 mol dm −3 of poly(methyl methacrylate) (PMMA, MW = 120000). In addition to a reduction of the fluorescence intensity of the pyrene excimer relative to the monomer, the rise and decay rates of the excimer fluorescence were decelerated in a sapphire/toluene interface layer of a thickness less than 60 nm. These findings were interpreted by an increase in the viscosity of the interface layer including PMMA adsorbed on a sapphire surface.

Inhomogeneous Aggregation of a Merocyanine Dye at the Solid/Liquid Interface Layer. A Picosecond Time-Resolved Total Internal Reflection Fluorescence Study

Abstract Fluorescence spectra and their rise and decay dynamics of a merocyanine dye in the interface layer have been studied as a function of penetration depth of the evanescent wave intensity by total internal reflection fluorescence spectroscopy. The aggregation of the dye was enhanced in the interface layer of up to ≈ 100 nm thickness compared to the bulk solution.

Excited-state proton transfer of 1-naphthol in liquid—solid interface layers. Picosecond time-resolved total internal reflection fluorescence study

Picosecond time-resolved total internal reflection fluorescence spectroscopy was applied to analyze the proton transfer reaction of 1-naphthol in water—sapphire interface layers. The rate constant of the proton transfer reaction from excited neutral species became slow in the interface layer as compared with that in the bulk aqueous solution. The anomaly observed up to the penetration depth of 100 nm was interpreted in terms of rotational fluctuations of water aggregates in the interface layer.

Solvation dynamics of a coumarin dye at liquid—solid interface layer. Picosecond total internal reflection fluorescence spectroscopic study

Fluorescence rise and decay curves of coumarin 460 in 1-butanol/sapphire interface layer were measured by time-resolved total internal reflection fluorescence spectroscopy. The fast rise and decay components due to the solvent relaxation became slow in the interface layer, and the anomaly was observed up to the penetration depth of 100 nm. The result was interpreted in terms of the longitudinal relaxation of 1-butanol affected by hydrogen bonding interactions between the hydrophilic sapphire surface and 1-butanol.

Interfacial Characteristics of Poly(methyl methacrylate) Film: Aggregation of Pyrene and Micropolarity Revealed by Time-Resolved Total Internal Reflection Fluorescence Spectroscopy

Interfacial Characteristics of Poly(methyl methacrylate) Film: Aggregation of Pyrene and Micropolarity Revealed by Time-Resolved Total Internal Reflection Fluorescence Spectroscopy

パルスレーザーを用いた有機薄膜の計測評価と反応

Principle of laser oscillation and a variety of laser application were interpreted, and recent advances in laser chemistry were described. For organic thin films, time-resolved total internal reflection fluorescence spectroscopy is very fruitful, since it provides novel information on relaxation dynamics of electronic excitation energy, photochemical reaction, dopant concentration, and so on near the surface. Using fluorescence microscope this methodology was developed to be a dynamic fluorescence microprobe. Intense pulsed laser excitation of organic films loads to an efficient ablation. Molecular and electronic aspect of this phenomenon were studied by examining polymer films doped with π-electronic molecules whose photophysical and photochemical properties are well know.

Dynamic Fluorescence Microprobe Method Utilizing Total Internal Reflection Phenomenon

Abstract An optical set-up for time-resolved total internal reflection fluorescence spectroscopy under a microscope was developed and its performance was examined by using specially prepared polymer films. This microspectroscopic method provides ps dyamic information for a microvolume of 0.1 μm (depth)×5 μm (diameter).