Fluorophores In Solution Research Articles

Unraveling solvent effects on the electronic absorption spectra of TRITC fluorophore in solution: a theoretical TD-DFT/PCM study

In this paper the equilibrium between the two ground state forms of TRITC in solvents of different polarity has been studied by means of state of the art DFT calculations using the polarizable continuum model (PCM) for including bulk solvent effects. TD-DFT/PCM calculations have been used to calculate vertical excitation energies and the UV-Vis spectra of TRITC has been interpreted in terms of the equilibrium between different forms, which is modulated by the microenvironment embedding the molecule. We show that the most intense absorption bands are sensitive to solute-solvent interactions and provide a distinctive signature of specific interactions. The good agreement with experimental data makes our approach suitable to study and monitor modifications of the chemical environment of fluorophores in different solutions as well as in interaction with biological systems and nanoparticles.

Nano-Devices for Probing Single Molecules

By confining molecules to 2-dimensional sheets or zero-dimensional wells, with nanometer-scale dimensions, one can observe individual fluorescently labeled molecules for long times, at high concentrations, and without surface attachment. We present two simple nano-devices that achieve these goals. We use these devices to obtain previously inaccessible information on molecular size, composition, and dynamics.2-dimensional confinement is achieved near the point of contact between a convex lens and a planar coverslip. The lens-coverslip spacing varies smoothly from zero to many microns as the radial distance from the point of contact is increased. Commercial fused silica optics have surface roughness of approximately 1 nm, so one can select a vertical confinement with nanometer accuracy simply by imaging at a given radius from the point of contact. The lens-coverslip system allows: a) fluorescence imaging of immobilized single molecules in the presence of a micromolar concentration of diffusing fluorophores; b) long-time observations of freely diffusing single molecules in dilute solution, which further allows determination of diffusion coefficients, brightness, and spectral dynamics molecule-by-molecule; and c) direct mechanical measurement of the size distribution in a population of fluorescently labeled molecules.Zero-dimensional confinement is achieved in nanometer-scale wells in a fused silica coverslip. A solution of fluorophores is washed over the wells, and then the bulk solution is replaced with a fluorinated oil. At most one molecule, or molecular complex, is immobilized in a nanoscale water droplet in each well. As with the lens-coverslip system, the dimple machine allows long-time observations of individual molecules, without surface attachment and in the presence of a high fluorescence background. By counting the number of photobleaching steps in each of several thousand chambers, we determine the distribution of stoichiometries in multimeric complexes.

H-Type Dimer Formation of Fluorophores: A Mechanism for Activatable, in Vivo Optical Molecular Imaging

In vivo molecular imaging with target-specific activatable "smart" probes, which yield fluorescence only at the intended target, enables sensitive and specific cancer detection. Dimerization and fluorescence quenching has been shown to occur in concentrated aqueous solutions of various fluorophores. Here, we hypothesized that fluorophore dimerization and quenching after conjugation to targeting proteins can occur at low concentration. This dimerization can be exploited as a mechanism for fluorescence activation. Rhodamine derivatives were conjugated to avidin and trastuzumab, which target D-galactose receptor and HER2/neu antigen, respectively. After conjugation, a large proportion of R6G and TAMRA formed H-type dimers, even at low concentrations, but could be fully dequenched upon dissociation of the dimers to monomers. To demonstrate the fluorescence activation effect during in vivo fluorescence endoscopic molecular imaging, a highly quenched probe, avidin-TAMRA, or a minimally quenched probe, avidin-Alexa488, was administered into mice with ovarian metastases to the peritoneum. The tumors were clearly visualized with avidin-TAMRA, with low background fluorescence; in contrast, the background fluorescence was high for avidin-Alexa488. Thus, H-dimer formation as a mechanism of fluorescence quenching could be used to develop fluorescence activatable probes for in vivo molecular imaging.

Fluorescence detection of DNA using an amorphous silicon p-i-n photodiode

A microdevice based on a thin-film hydrogenated amorphous silicon (a-Si:H) n-i-p photodiode is used to quantify the density of DNA oligonucleotides labeled with a fluorophore in solution, adsorbed, and hybridized on the device surface. Excitation of single-stranded DNA molecules tagged with the fluorophore Alexa Fluor 430 with near UV/blue light results in the emission of visible light at 539 nm. The emitted light produces an electrical signal in the photodetector, allowing the optoelectronic detection and quantification of the DNA molecules. An integrated high-pass filter cuts the excitation light while allowing the collection of the emission light by the photodiode. The detection limit of the present device is of the order of 5 nM in solution. A surface density of the order of 2 pmol/cm2 was measured for DNA targets hybridized to immobilized DNA probes. The sensitivity of the current device is limited by the efficiency of the filtering of the excitation light. The detection concept explored can enable on-chip electronic data acquisition in lab-on-a-chip devices and microarrays.

Characterization of long pathlength capillary waveguides for evanescent fluorescence sensing applications

Abstract The optical properties of a novel fused silica fiber-optic capillary (FOCap) waveguide for fluorescence chemical sensing are evaluated. Evanescent fluorescence from samples contained in the FOCap is measured by coupling the FOCap to a light source and a fluorescence spectrophotometer. The FOCap has negligible excitation light loss over long lengths (tested to 20 m). Evanescent fluorescence is linear in FOCaps up to 20 m long when filled with a fluorophore solution and up to 15 m when a fluorophore is covalently attached to the inner surface. Evanescent fluorescence measurements in 50, 150, and 250 μm inner diameter FOCaps indicate that greater sensitivity is achieved with thinner-walled capillaries which have more internal reflections. The ability of the waveguide to function as an indirect chemical sensor for nitroaromatic compounds is demonstrated via fluorescence quenching of a pyrene-labeled FOCap.

Nitrogen Gas Purging for the Deoxygenation of Polyaromatic Hydrocarbon Solutions in Cyclohexane for Routine Fluorescence Analysis

During routine fluorescence analysis, the presence of dissolved oxygen in solutions can result in the dynamic quenching of a fluorophore's emission through collisional deexcitation of the fluorophore's excited state. In order to avoid this type of fluorescence quenching, dissolved oxygen is often removed from solutions by an inert gas purging procedure. However, the details and quantification of this purging process are often limited in fluorescence studies. In this work, standard 10 mm x 10 mm fluorescence cuvettes are filled with polyaromatic hydrocarbon (PAH) solutions in cyclohexane and purged using nitrogen gas, and the experimental purging parameters (nitrogen flow rate, amount of volatile solvent loss, and rate of oxygen removal) are measured and analyzed. For experimental conditions similar to those used in this study, we are able to provide useful guidelines for the deoxygenation of solutions, specifically the purge times required for solutions of fluorophores with various fluorescence lifetimes. Enhancement factors, or F(0)/F values (the ratio of fluorescence intensity of a completely deoxygenated solution to the fluorescence intensity of an aerated solution), for chrysene, phenanthrene, naphthalene, and pyrene solutions in cyclohexane were found to be 3.61 +/- 0.02, 4.17 +/- 0.02, 7.63 +/- 0.07, and 21.81 +/- 0.35, respectively.

Fluorescence Detection of DNA Hybridization Using an Integrated Thin-Film Amorphous Silicon n-i-p Photodiode

ABSTRACTThis paper presents the fluorescence detection of DNA hybridization with a surface immobilized probe using an hydrogenated amorphous silicon photosensor. This sensor integrates a SiO2 layer for DNA probe immobilization, a p-i-n amorphous silicon (a-Si:H) photodiode for fluorescence detection and a fluorescence filter of amorphous silicon carbon (a-SiC:H) to cut the excitation light. With this integrated photosensor system, a five order of magnitude difference was obtained in the signal measured at the emission wavelength and that measured at the excitation wavelength for the same incident photon flux. The fluorophore Alexa Fluor 430 was used to label the DNA target molecules and a laser at 405 nm and a photon flux of 5.7×1016 cm−2.s−1 was used as the excitation light source. The detection limit achieved for fluorophores in solution in contact with the device and for fluorophores immobilized on the device surface is 5×10−9 M and 0.4 pmol/cm2, respectively. The fluorescence detection of the DNA target hybridization with a covalently or electrostatically immobilized probe was successfully detected at a surface density of ∼3 pmol/cm2.

Intracellular Zinc Elevation Measured with a “Calcium‐Specific” indicator: A Question on Calcium Signals

Much of our current evidence concerning of the role of calcium (Ca2+) or Ca2+ signaling comes from fluorescent studies with so-called “calcium indicators.” However, many of those same Ca2+ indicators have a higher affinity for divalent cation zinc (Zn2+) than for Ca2+. Here, we investigated metal-ion responses by screening nominally Ca2+-selective fluorophores in solutions containing various concentrations of Zn2+. Ca2+-sensitive dyes such as Calcium Green-1, Fura-2, and Fluo-3 showed a wide range of interaction with Zn2+, even responding to Zn2+ in the presence of high concentrations of Ca2+, demonstrating that these fluorescent Ca2+ indicators can be used effectively to measure dynamic changes of cytosolic Zn2+in situ. We then investigated the rising Zn2+ transients in neurons during ischemia simulated by oxygen and glucose deprivation (OGD) which caused a marked rise in intracellular zinc. Next we used the calcium probe, Calcium Green-1, to determine whether calcium, like zinc, also rises intracellularly. The Calcium Green-1 fluorescence did in fact rise dramatically, but chelation of zinc (and not Ca2+) by TPEN dramatically reversed that increased fluorescence, suggesting that rising zinc, not rising Ca2+, is the primary source of the increasing calcium-green fluorescence. Finally, zinc chelation gave a substantial reduction in the number of “dying” neurons, and also reduced thapsigargin induced “Ca2+” transient. Therefore, re-evaluation of the role of intracellular calcium in neuronal injury is warranted. Supports: the American Heart Association (Y.V.L) and the National Institutes of Health (Y.V.L)

Intensity Calibration and Shading Correction for Fluorescence Microscopes

Standardization in image cytometry involves intensity calibration and shading correction. This unit presents a method using concentrated solutions of fluorophores. A drop of highly concentrated dye solution placed between a slide and a coverslip produces a spatially uniform fluorescent sample with reproducible quantum yield and resistance to photobleaching. The technique has a number of practical features that make it inexpensive, reproducible, and straightforward. Descriptions are given for both wide-field and confocal scanning fluorescence microscopy.

Determining zinc with commonly used calcium and zinc fluorescent indicators, a question on calcium signals

Investigations into the roles of Ca 2+ and Zn 2+ in cell biology have been facilitated by the development of sensitive fluorometric probes that have enabled the measurement of Ca 2+ or Zn 2+ in both extracellular and intracellular environments. It is critical to be aware of the specificity and relative selectivity of a probe for the targeted ion. Here, we investigated metal-ion responses by screening nominally Zn 2+- or Ca 2+-selective fluorophores in solutions containing various concentrations of Ca 2+, as a potential interferent for Zn 2+, or Zn 2+, as a potential interferent for Ca 2+. The results suggested that Zn 2+-sensitive dyes were more specific for their targeted ion than dyes that targeted Ca 2+. Ca 2+-sensitive dyes such as Calcium Green-1, Fura-2, and Fluo-3 showed a wide range of interaction with Zn 2+, even responding to Zn 2+ in the presence of high concentrations of Ca 2+. We demonstrate that these Ca 2+ indicators can effectively measure dynamic changes of cytosolic Zn 2+. Our results appeal for a new generation of Ca 2+ fluorophores that are more specific for Ca 2+ over Zn 2+. One implication of these results is that data obtained using Ca 2+-sensitive dyes may need to be re-examined to determine if results previously attributed to Ca 2+ could, in part, be due to Zn 2+.

Influence of Fluorescence Anisotropy on Fluorescence Intensity and Lifetime Measurement: Theory, Simulations and Experiments

The significance of fluorescence anisotropy in fluorescence intensity and lifetime measurements, and erroneous measurements and interpretations resulting from its disregard, are thoroughly discussed, formulated and quantified. In all fluorescence-related measurements--including excitation and emission spectra, relative fluorescence intensity (FI), fluorescenc life time (FLT), fluorescence resonance energy transfer (FRET), etc., with the exception of fluorescence polarization and anisotropy--it is generally true that the higher the fluorescence anisotropy, the greater the distortion of fluorescence measurements. Quantifiable distortions occur when fluorescence measurements are conducted without considering the influence of fluorescence anisotropy. Here, this influence is described by numerous newly developed mathematical expressions which are simulated and experimentally confirmed utilizing single and binary fluorescent solutions of fluorophores with different spectroscopic characteristics. A marked agreement is shown between the theory and experimental data, clearly indicating the legitimacy of the physical suppositions and the mathematical expressions presented in this paper. Practical and instructive implications are discussed. The following findings are of special applicative importance: 1) the existence of an infinite number of couples of Magic Angles; 2) the deviation between two equally fluorescing particles having different fluorescence anisotropies; 3) the relation between the detected fluorescence intensity and anisotropy when measured under various setups of emission and excitation polarizers; 4) the dependence of the artificial normalized steady-state weight of a single-exponentially decaying fluorophore on its fluorescence anisotropy.

Temporally and spectrally resolved sampling imaging with a specially designed streak camera

We present a novel sampling imaging technique capable of performing simultaneous two-dimensional measurements of the temporal and spectral characteristics of light-emission processes by use of a specially designed streak camera. A proof-of-principle experiment was performed with a homemade multifocal multiphoton fluorescence microscope. The system was calibrated with a Fabry-Perot etalon and a standard fluorophore solution (rhodamine 6G in ethanol) and was shown to have temporal and spectral resolution of 6.5 ps and 3 nm, respectively, as well as high accuracy and reproducibility in lifetime and spectrum measurement. Temporally and spectrally resolved images of 4 x 4 foci on the sample can be obtained with a snapshot.

Broad Spectral Domain Fluorescence Wavelength Modulation of Visible and Near-Infrared Emissive Polymersomes

Incorporation of an extended family of multi[(porphinato)zinc(II)] (PZn)-based supermolecular fluorophores into the lamellar membranes of polymersomes (50 nm to 50 mum diameter polymer vesicles) gives rise to electrooptically diverse nano-to-micron (meso) scale soft materials. Studies that examine homogeneous suspensions of 100 nm diameter emissive polymersomes demonstrate fluorescence energy modulation over a broad spectral domain of the visible and near-infrared (600-900 nm). These polymersomal structures highlight that the nature of intermembranous polymer-to-fluorophore contacts depends on the position and identity of the porphyrins' phenyl ring substituents. Emissive polymersomes are shown to possess reduced spectral heterogeneity with respect to the established optical signatures of these PZn-based supermolecular fluorophores in solution; additionally, selection of fluorophore ancillary substituents predictably controls the nature of polymer-emitter noncovalent interactions to provide an important additional mechanism to further modulate the fluorescence band maxima of these meso-scale emissive vesicles.

Photophysical study of a conjugated–non-conjugated PPV-type electroluminescent copolymer

This paper reports the photo- and electroluminescence studies of emitting species of one of the first reported blue emitters, the conjugated–non-conjugated multi-block copolymer, poly[1,8-octanedioxy-2,6-dimethoxy-1,4-phenylene-1,2-ethenylene-1,4-phenylene-1,2-ethenylene-3,5-dimethoxy-1,4-phenylene]. Because the conjugation length of the emissive center is very well defined (two and half phenylene–vinylene units) the differences found between the fluorescence profile of the fluorophore in solution, at several concentrations and that in the solid state allowed us to conclude that in solid the emission comes from associated forms, such as ground-state dimers and/or excimers. Time-resolved fluorescence in a nanosecond time scale recorded at ϖ em=24,096 cm −1 showed a monoexponential decay of 1.5 ns, which is compatible with rigid forms of stilbene derivatives.

Optical Properties of Alexa™ 488 and Cy™5 Immobilized on a Glass Surface

The absorption and emission spectra were measured for Cy5 and Alexa 488 fluorophores confined on a glass surface. The data were obtained using fluorometry and spectroscopic ellipsometry. Red shifts of the surface-immobilized fluorophore absorption spectra relative to the fluorophore spectra in aqueous solution were observed using both methods. We interpret these red shifts in terms of a change in the polarizability and polarity of the effective solvent. A formula is given that can be used to estimate expected shifts in absorption and emission maxima for surface-immobilized fluorophores. Spectroscopic ellipsometry measurements provide identification of the fluorophores confined on a glass surface. These results suggest that the design of microarray detection systems should be based on the optical properties of fluorophores attached to the surface and not on the optical properties of fluorophores in solution.

Heterocyclic quinol-type fluorophores. Synthesis of novel imidazoanthraquinol derivatives and their photophysical properties in benzene and in the crystalline state

An isomeric pair of novel imidazoanthraquinol fluorophores 2 and 3 exhibiting two tautomeric forms (A and B) that are possible for the imidazole ring have been developed. In order to control the tautomeric form to improve the fluorescence properties, the N-butylated imidazole derivatives 6 and 7 have further been synthesized. The fluorescence intensity of the quinols was in the order 3 ≪ 6 < 7 < 2 in benzene, which is quite different from the order 3 ≪ 2 ≪ 6 < 7 in the crystalline state. To investigate the effect of N-alkylation of imidazole ring on the photophysical properties of the quinol fluorophores in solution and in the crystalline state, we have performed semi-empirical molecular orbital calculations (AM1 and INDO/S) and X-ray crystallographic analysis. On the basis of the results of calculations and the X-ray crystal structures, the N-alkylation effects on the chemical structure of the quinol fluorophores and on their solid-state photophysical properties are discussed.

Perylene Bisimide Based Macrocycles: Effective Probes for the Assessment of Conformational Effects on Optical Properties

Conformation-dependent tuning of color: Starting from aryloxy-substituted perylene bisimides two isomeric diagonally (A) and laterally (B) bridged macrocycles were synthesized (see picture) for three different bridge lengths. These isomers comprise different conformational features and can, therefore, be used as molecular probes to deduce the conformational influence on the optical properties of this important class of fluorophores in solution.

Absolute Optical Cross Section of Individual Fluorescent Molecules

Besides having revealed transitions masked in the bulk, optical spectroscopy of single molecules has enabled the observation of elementary molecular quantum events, such as the absorption and emission of a photon. Single photon transitions are quantified by a cross section that can be regarded as the photon capture area of a molecule. Therefore, absolute optical cross sections can readily be measured in the bulk from the decrease (by absorption) or the gain (by stimulated emission) of the number of photons in a beam. Unfortunately, this method is not applicable to individual molecules, because the resulting change in intensity is negligible. If the fluorescence quantum efficiency Qem is known, the molecular absorption cross section can be calculated from the fluorescence emission. However, this method re-introduces the hallmark of ensemble averaging, because Qem is known only from the bulk. As a result, the absolute photon capture area of a specific molecule has remained elusive. Here we report on the measurement of absolute cross sections of stimulated emission at room temperature. No a priori information about the individual molecule is needed. In addition, we demonstrate the instant control of a molecule.s excited state by light and thus, as a matter of fact, also the viability of pump–probe experiments with individual molecules at room temperature. Molecular optical absorption usually takes place from the vibrationally relaxed ground state S0 to a hot Franck–Condon state S1* of the first excited singlet state (Figure 1a). After vibrational relaxation, which occurs on a (sub)picosecond time scale, organic fluorophores emit within a few nanoseconds with a probability given by the fluorescence quantum yield. Alternatively, the S1!S0 transition can be enforced by light, that is by stimulated emission. Stimulated emission has been employed to probe the temporal evolution of the excited state in a pump–probe fashion, as well as to alter the fluorescence lifetime and anisotropy of organic fluorophores in solution. Furthermore, stimulated emission depletion (STED) of the excited state has been introduced in fluorescence microscopy to break the diffraction resolution barrier, but has recently also been used to measure cross sections in the bulk. We attained single molecule sensitivity with a scanning confocal setup fed by two synchronized pulse trains: a first pulse at a wavelength of l= 565 nm for excitation was followed by a red-shifted pulse at l= 778 nm for STED, both adapted to the spectrum (Figure 1b) of the xanthene dye JA 26 (1). Efficient STED demands a STED pulse duration t

Cumulant Analysis in Fluorescence Fluctuation Spectroscopy

A novel technique for the analysis of fluorescence fluctuation experiments is introduced. Fluorescence cumulant analysis (FCA) exploits the factorial cumulants of the photon counts and resolves heterogeneous samples based on differences in brightness. A simple analytical model connects the cumulants of the photon counts with the brightness ɛ and the number of molecules N in the optical observation volume for each fluorescent species. To provide the tools for a rigorous error analysis of FCA, expressions for the variance of factorial cumulants are developed and tested. We compare theory with experiment by analyzing dye mixtures and simple fluorophore solutions with FCA. A comparison of FCA with photon-counting histogram (PCH) analysis, a related technique, shows that both methods give identical results within experimental uncertainty. Both FCA and PCH are restricted to data sampling times that are short compared to the diffusion time of molecules through the observation volume of the instrument. But FCA theory, in contrast to PCH, can be extended to treat arbitrary sampling times. Here, we derive analytical expressions for the second factorial cumulant as a function of the sampling time and demonstrate that the theory successfully models fluorescence fluctuation data.

Fluorophore Conformation in Green Fluorescent Protein: A Quantum Mechanics/Molecular Mechanics Study

AbstractGreen Fluorescent Protein (GFP) is a widely used fluorescent marker exhibiting two excitation peaks – a strong peak at 398 nm and a second at 475 nm, with the fluorescence at ca. 510 nm. Its molecular structure consists of a β-barrel composed of 11 β-strands and a central helix containing the fluorophore. Two different forms of the fluorophore – a protonated/neutral fluorophore and a de-protonated/anionic fluorophore – are thought to be responsible for the two distinct spectroscopic states. Notably, the isolated fluorophore in solution is efficiently quenched. Conformational flexibility within the protein cavity is an implicitly important factor that governs the photochemistry of GFP. However, the literature contains accounts of studies that reach conflicting conclusions, claiming that either the fluorophore's barrier to internal rotation is negligibly small or that the protein cavity is not complementary to a planar fluorophore. In this work, we calculate the torsional potential of one of the two exocyclic bonds that connect the two rings in the fluorophore, taking into account its immediate environment by applying a quantum mechanics/molecular mechanics method, with the ultimate aim of evaluating the protein-environment effects on the fluorescence.