Controllable metal-enhanced fluorescence in organized films and colloidal system

In recent years, considerable efforts have been devoted to better understand the unique emission properties of fluorophores enhanced by the localized surface plasmon resonance of metal nanoparticles (NPs), due to the widespread applications of fluorescence techniques. It is demonstrated by experiment and theoretical calculation that the enhancement efficiency strongly depends on the morphology of the metal NPs, the spectral overlap between metal and fluorophores, the separation distance between them, and other factors. Among these aspects to be considered are suitable spacer material and assembling methods to control the spatial arrangement of plasmonic NPs and fluorophore with proper optical properties and interactions. In this contribution, we provide a brief overview on recent progress of metal-enhanced fluorescence in organized films and colloidal systems.     

Excitation volumetric effects (EVE) in metal-enhanced fluorescence

Metal-Enhanced Fluorescence (MEF) effects from different density silver island films (SiFs) and the effects of far-field excitation irradiance on the observed enhancement of fluorescence were studied. It is shown that MEF non-linearly depends on silver nanoparticle (NP) size/density, reaching a maximum value for SiFs made at a deposition time (DT) of ～5 minutes, i.e. just before SiFs become continuous. Numerical simulations of the silver-islands growing on glass revealed that the near-field magnitude depends non-linearly on size and interparticle distance exhibiting dramatic enhancement at ～10 nm distance between the NPs. In addition, a remarkable effect of modulation in MEF efficiency by far-field excitation irradiance has been observed, which can be correlated well with numerical simulations that show an excitation power volume dependence. The near-field volume changes non-linearly with far-field power. This unique observation has profound implications in MEF, which has rapidly emerged as a powerful tool in the biosciences and ultimately allows for tunable fluorescence enhancement factors.     

Distance-dependent metal-enhanced fluorescence from Langmuir-Blodgett monolayers of alkyl-NBD derivatives on silver island films

We described the effect of fluorophore distance from the silver island films (SIFs) on the metal-enhanced fluorescence (MEF) from two newly developed long-chain nitrobenzoxadiazole derivatives (NBD-C16 and NBD-C18). The well-established Langmuir-Blodgett technique is used to deposit the fluorophores at defined distances from the SIFs surface, and an inert amphiphilic stearic acid is used to control the distance. NBD probes deposited directly on the SIFs surface show the highest metal-enhanced fluorescence of approximately 32-fold, and both of the probes that were studied show a consistent decrease in metal-enhanced fluorescence when increasing the distance from the fluorophore to the SIFs surface. The lowest fluorescence enhancement of approximately 4-fold is observed for the probes located 90 nm from the SIFs surface. Additionally, we also have noticed the shortest fluorescence lifetimes for the NBD probes deposited directly onto the SIFs surface, and the lifetimes are consistently increased when increasing the distances between the fluorophore and SIFs surfaces. These contrasting spectral changes, enhanced fluorescence, and decreased fluorescence lifetimes are in accordance with an increase in the rate of radiative decay for fluorophores near the silver particles. The present study provides significant information on the effect of fluorophore distance on the metal-enhanced fluorescence phenomenon.     

Self-duplex formation of an A(py)-substituted oligodeoxyadenylate and its unique fluorescence

Unexpected homoadenine self-duplexes are formed when pyrene units are bound covalently to the deoxyadenosine bases at specific distances (1,4 relationships). This discovery illustrates how small-molecule pyrene intercalators can be used to drive unknown nucleic acid assembly with a concomitant change in fluorescence. When a pair of pyrene fluorophore units is located within an oligodeoxyadenylate chain, the system can display three different colors (reddish-orange, green, or blue) depending on the relative location of the fluorophores. A unique fluorescence signal, a reddish band peaking at 580 nm, appears when the oligomers possess more than two spacers between the pyrene fluorophores(1,4 relationships). Several spectroscopic experiments, for example, recording variable-concentration spectra, CD, UV, melting temperature, and gel electropherogram, indicate that this new reddish band came from an intermolecular homoadenine self-duplex. Time-resolved fluorescence measurements using both TCSPC and upconversion methods indicate that this unique fluorescence has a long lifetime.     

Ultraviolet laser induced fluorescence of human aorta

Laser induced fluorescence from normal human aorta is studied with u.v. excitations of 305 to 310 nm, observing emission from 320 to 500 nm. In this region LIF lineshapes are strongly dependent on the excitation wavelength, suggesting that at least two fluorophores are being observed. The short wavelength fluorophore, peaking at 34Onm, is identified as tryptophan, while the longer wavelength fluorophore, peaking at 387 nm, is associated with collagen and elastin. In addition, fluorescence time decays of each component are measured with a time correlated photon counting system. A four-exponential fit of each decay is necessary to extract fluorescence lifetimes, which range from 33 ps to 8.6 ns.     

Fluorescence resonance energy transfer by quencher adsorption into hydrogels containing fluorophores

We synthesized anionic hydrogels containing fluorophores and investigated the adsorption of a cationic quencher having an amino group into hydrogels by fluorescence resonance energy transfer (FRET). FRET from the fluorophore to the quencher in hydrogels was examined by fluorescence intensity and fluorescence decay using a fluorescence spectrophotometer and femtosecond laser spectroscopy. The fluorescence intensity of the fluorophore‐containing hydrogels decreased rapidly with increasing amounts of adsorbed cationic quencher. The fluorescence emission of the fluorophore in the quencher‐adsorbed hydrogels containing fluorophores decayed more rapidly than that of the original hydrogels. The aforementioned result indicates that the fluorescence of the fluorophore‐containing hydrogels is quenched due to FRET from the fluorophore to the quencher as the cationic quenchers can approach the fluorophores in hydrogels by electrostatic interactions. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 3245–3252, 2006     

Specific and stable fluorescence labeling of histidine-tagged proteins for dissecting multi-protein complex formation

Labeling of proteins with fluorescent dyes offers powerful means for monitoring protein interactions in vitro and in live cells. Only a few techniques for noncovalent fluorescence labeling with well-defined localization of the attached dye are currently available. Here, we present an efficient method for site-specific and stable noncovalent fluorescence labeling of histidine-tagged proteins. Different fluorophores were conjugated to a chemical recognition unit bearing three NTA moieties (tris-NTA). In contrast to the transient binding of conventional mono-NTA, the multivalent interaction of tris-NTA conjugated fluorophores with oligohistidine-tagged proteins resulted in complex lifetimes of more than an hour. The high selectivity of tris-NTA toward cumulated histidines enabled selective labeling of proteins in cell lysates and on the surface of live cells. Fluorescence labeling by tris-NTA conjugates was applied for the analysis of a ternary protein complex in solution and on surfaces. Formation of the complex and its stoichiometry was studied by analytical size exclusion chromatography and fluorescence quenching. The individual interactions were dissected on solid supports by using simultaneous mass-sensitive and multicolor fluorescence detection. Using these techniques, formation of a 1:1:1 stoichiometry by independent interactions of the receptor subunits with the ligand was shown. The incorporation of transition metal ions into the labeled proteins upon labeling with tris-NTA fluorophore conjugates provided an additional sensitive spectroscopic reporter for detecting and monitoring protein-protein interactions in real time. A broad application of these fluorescence conjugates for protein interaction analysis can be envisaged.     

Sulforhodamine Adsorbed Langmuir-Blodgett Layers on Silver Island Films: Effect of Probe Distance on the Metal-Enhanced Fluorescence

Metal-Enhanced Fluorescence (MEF) has become an important method in biomedical sensing. In this paper, we present the distance-dependent MEF of sulforhodamine B (SRB) monolayer on silver island films (SIFs). SRB is electrostatically incorporated into the Langmuir-Blodgett (LB) layers of octadecylamine (ODA) deposited on glass and SIFs substrates. The distances between SRB and SIFs or glass surfaces are controlled by depositing a varied number of inert stearic acid (SA) spacer layers. SRB is incorporated into positively charged LB layers of ODA by immersing the ODA deposited substrates into aqueous solution of SRB. Dye incorporated ODA layers with 10 nm separation distance from the SIFs surface show maximum metal-enhanced fluorescence intensity; ~7-fold increase in intensity as compared to that from the glass surface. The corresponding enhancement factor is reduced with increasing or decreasing the probe distance from the SIFs surface. Additionally, SRB on SIF surfaces show reduced lifetimes. We observed the shortest lifetime from the SRB with 5 nm distance from the SIF surfaces and the lifetime increased consistently with increasing the distances between the fluorophore and the SIFs surface. These observed spectral changes, increase in fluorescence intensity and decreased fluorescence lifetimes, are in accordance with the expected effects due to near-field interactions between the silver nanoparticles and fluorophores. We have also analyzed the complex fluorescence heterogeneous decays on metallic nanostructured surfaces using continuous distributions of decay times. The decay-time distributions appear to be sensitive to the distance between the metal and fluorophore and represent the underlying heterogeneity of the samples. The present systematic study provides significant information on the effect of fluorophore distance on the metal-enhanced fluorescence phenomenon.     

Fast and stable photochromic oxazines for fluorescence switching

The stringent limitations imposed by diffraction on the spatial resolution of fluorescence microscopes demand the identification of viable strategies to switch fluorescence under optical control. In this context, the photoinduced and reversible transformations of photochromic compounds are particularly valuable. In fact, these molecules can be engineered to regulate the emission intensities of complementary fluorophores in response to optical stimulations. On the basis of this general design logic, we assembled a functional molecular construct consisting of a borondipyrromethene fluorophore and a nitrospiropyran photochrome and demonstrated that the emission of the former can be modulated with the interconversion of the latter. This fluorophore-photochrome dyad, however, has a slow switching speed and poor fatigue resistance. To improve both parameters, we developed a new family of photochromic switches based on the photoinduced opening and thermal closing of an oxazine ring. These compounds switch back and forth between ring-closed and -open isomers on nanosecond-microsecond timescales and tolerate thousands of switching cycles with no sign of degradation. In addition, the attachment of appropriate chromophoric fragments to their switchable oxazine ring can be exploited to either deactivate or activate fluorescence reversibly in response to illumination with a pair of exciting beams. Specifically, we assembled three dyads, each based on either a borondipyrromethene or a coumarin fluorophore and an oxazine photochrome, and modulated their fluorescence in a few microseconds with outstanding fatigue resistance. The unique photochemical and photophysical properties of our fluorophore-photochrome dyads can facilitate the development of switchable fluorophores for superresolution imaging and, ultimately, provide valuable molecular probes for the visualization of biological samples on the nanometer level.     

Multivariate curve resolution analysis excitation-emission matrices of fluorescence of humic substances

Excitation-emission matrices (EEM) of fluorescence of aqueous solutions of humic substances (HS), and sets of EEM acquired as function of the HS concentration, were analysed by multivariate curve resolution alternating least squares (MCR-ALS). Three types of HS samples were studied: one commercial humic acid; two samples of fulvic acid (FA) extracted from a pinewood soil; two samples of FA extracted from recycled wastes. The fluorescence measurements were carried out at HS concentration between 5 and 100 mg/L and at pH 6. The application of MCR-ALS algorithm on each individual EEM, as well as on column-wise augmented matrices, allows the identification of three major fluorophores in all HS samples analysed. The emission and excitation spectra of these fluorophores were recovered and are characteristic of each sample. Moreover, the variation of the fluorescence intensities of each fluorophore with HS concentration shows deviations from linearity at HS concentration higher than 30 mg/L, depending on the fluorophore and/or sample. This behaviour reveals the existence of inner filter effects that affect the proportionally between the fluorescent signal and concentration but do not provoke measurable distortions on the fluorescence spectra of the detected fluorophores.     

Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy

Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1-20 ms), and demonstrate its generation via both one- and multistep excitation processes. Apart from reflecting a major pathway for photobleaching, our results also suggest the exploitation of the photoinduced ionization and the subsequent reduction by antioxidants for biomolecular monitoring purposes and as a possible switching mechanism with applications in high-resolution microscopy.     

Water-source characterization and classification with fluorescence EEM spectroscopy: PARAFAC analysis

Water-quality protection and environmental forensics require rapid water monitoring and source identification. In this paper, parallel factor analysis (PARAFAC) of fluorescence excitation-emission matrix spectra (EEMS) was used to characterize and classify water samples from landfills, wastewater treatment plants, lakes, and rivers. The study showed that the optimal number of components was four to represent the data set. The fluorescence fingerprints for water samples from different sources were sufficiently different, so qualitative water classification could be achieved. Specifically, Component 1 was the major fluorescing centre in river waters, with characteristics consistent with humic-like fluorophores; Component 2 was the dominant fluorophore in the treated wastewaters; Component 3 was the characteristic fluorophore in landfill leachates; and Components 1, 3, and 4 existed in lake waters at comparable weight, among which Component 4 may be considered as a protein- or amino acid-like fluorophore.     

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.     

Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probes

Many genomics assays use profluorescent oligonucleotide probes that are covalently labeled at the 5' end with a fluorophore and at the 3' end with a quencher. It is generally accepted that quenching in such probes without a stem structure occurs through F?rster resonance energy transfer (FRET or FET) and that the fluorophore and quencher should be chosen to maximize their spectral overlap. We have studied two dual-labeled probes with two different fluorophores, the same sequence and quencher, and with no stem structure: 5'Cy3.5-beta-actin-3'BHQ1 and 5'FAM-beta-actin-3'BHQ1. Analysis of their absorption spectra, relative fluorescence quantum yields, and fluorescence lifetimes shows that static quenching occurs in both of these dual-labeled probes and that it is the dominant quenching mechanism in the Cy3.5-BHQ1 probe. Absorption spectra are consistent with the formation of an excitonic dimer, an intramolecular heterodimer between the Cy3.5 fluorophore and the BHQ1 quencher.     

A fluorescence ratiometric sensor for hypochlorite based on a novel dual-fluorophore response approach

A fluorescence ratiometric sensor for OCl⁻ has been developed based on a novel dual fluorophore response approach. The sensor molecule contains a coumarin fluorophore and a rhodamine fluorophore, and the two fluorophores are directly linked to an OCl⁻ recognition group. The structure of the sensor was characterized by ESI-MS, NMR, and X-ray crystallographic analysis. Upon treatment with OCl⁻, both fluorophores in the sensor responded simultaneously at two separate optical windows, with large enhancement of the fluorescence ratio (I₅₇₈/I₅₀₁) from 0.01 to 39.55. The fluorescence ratios for the sensor showed a good linearity with the concentration of OCl⁻ in the range of 0.2–40 μM and the detection limits is 0.024 μM (S N⁻¹ = 3). Investigation of reaction products indicated that the sensor reaction with OCl⁻ produced two new fluorescent molecules, which were responsible for the fluorescence changes in two optical windows. In addition, the sensor showed high selectivity to OCl⁻ over other reactive oxygen species, reactive nitrogen species, cations, and anions. The sensor has also been successfully applied to detection of OCl⁻ in natural water samples with satisfactory recovery.     

Optical modulation and selective recovery of Cy5 fluorescence

Fluorescence modulation offers the opportunity to detect low-concentration fluorophore signals within high background. Applicable from the single-molecule to bulk levels, we demonstrate long-wavelength optical depopulation of dark states that otherwise limit Cy5 fluorescence intensity. By modulated excitation of a long-wavelength Cy5 transient absorption, we dynamically modulate Cy5 emission. The frequency dependence enables specification of the dark-state timescales enabling optical-demodulation-based signal recovery from high background. These dual-laser illumination schemes for high-sensitivity fluorescence-signal recovery easily improve signal-to-noise ratios by well over an order of magnitude, largely by discrimination against background. Previously limited to very specialized dyes, our utilization of long-lived dark states in Cy5 enables selective detection of this very common single-molecule and bulk fluorophore. Although, in principle, the "dark state" can arise from any photoinduced process, we demonstrate that cis-trans photoisomerization, with its unique transient absorption and lifetime enables this sensitivity boosting, long-wavelength modulation to occur in Cy5. Such studies underscore the need for transient absorption studies on common fluorophores to extend the impact of fluorescence modulation for high-sensitivity fluorescence imaging in a much wider array of applications.     

Angular-dependent metal-enhanced fluorescence from silver colloid-deposited films: opportunity for angular-ratiometric surface assays

We describe an exciting opportunity for Metal-Enhanced Fluorescence (MEF)-based surface assays using an angular-ratiometric approach to the observed enhanced emission from fluorophores in close proximity to silver colloids deposited on glass substrates. This approach utilizes the radiationless energy transfer (coupling) between the excited states of the fluorophore and the induced surface plasmons of the silver colloids, and the subsequent angular-dependent fluorescence emission from the fluorophore-silver colloid system. Since MEF is related to surface plasmons' ability to scatter light, angular-dependent light scattering from three different silvered surfaces and glass substrates were investigated using two common excitation angles, 45 and 90 degrees . The scattered light from silvered surfaces with a high loading was observed at wider angles on both sides of the glass substrates, while forward scattering (from the back of the glass) was dominant for the silvered surfaces with low loading, as explained by both Mie and Rayleigh theories. When silver colloids were placed between the fluorophore and glass interface, the coupled fluorescence emission through the higher refractive index glass (and in air), increased in an angular-dependent fashion, following closely the angular-dependent light scattering pattern of the silver colloids themselves. Similar observations for fluorescence emission from fluorophores deposited onto glass surfaces alone were made, but at much narrower angles on both sides of the fluorophore-glass interface and were simply explained by Lambert's cosine law. As the loading of silver on glass was increased, the enhanced fluorescence emission was observed at wider angles (towards 0 and 180 degrees ) at both sides of the silvered surfaces. Glass surfaces without silver colloids were used as control samples to demonstrate the benefits of MEF for enhancing fluorescence signatures in an elegant, angular-dependent fashion. Finally, the utility of the angular-dependent MEF phenomenon for intensity-based angular-ratiometric surface assays is demonstrated.     

Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

Modern fluorescence microscopy applications go along with increasing demands for the employed fluorescent dyes. In this work, we compared antifading formulae utilizing a recently developed reducing and oxidizing system (ROXS) with commercial antifading agents. To systematically test fluorophore performance in fluorescence imaging of biological samples, we carried out photobleaching experiments using fixed cells labeled with various commonly used organic dyes, such as Alexa 488, Alexa 594, Alexa 647, Cy3B, ATTO 550, and ATTO 647N. Quantitative evaluation of (i) photostability, (ii) brightness, and (iii) storage stability of fluorophores in samples mounted in different antifades (AFs) reveal optimal combinations of dyes and AFs. Based on these results we provide guidance on which AF should preferably be used with a specific dye. Finally, we studied the antifading mechanisms of the commercial AFs using single-molecule spectroscopy and reveal that these empirically selected AFs exhibit similar properties to ROXS AFs.     

Local heterogeneity for a Eu3+-doped glass evidenced by time-resolved fluorescence spectroscopy coupled to scanning near-field optical microscopy

Time-resolved fluorescence spectroscopy (TRFS) was applied to an aluminate glass sample doped with Eu3+ cation as a fluorescent probe of the chemical environment and local symmetry. Conventional far-field experiments revealed the presence of two different phases: an amorphous phase featured by a highly disordered environment surrounding the Eu3+ cation and a more ordered polycrystalline phase that exhibits a significant increase in the Eu3+ fluorescence decay time compared to that of the amorphous phase. Near-field fluorescence spectra and decay kinetics were recorded in the frontier region between the two phases using a home-built scanning near-field optical microscope. SNOM-TRFS experiments confirmed the presence of local heterogeneities in this part of the glass at a sub-micrometric spatial scale. Polycrystalline sites featured an important shear-force interaction with the probing fiber optic tip, a longer fluorescence decay time, and a higher Stark splitting of the 5D0 --> 7FJ (J = 1-4) electronic transitions of the Eu3+ cations.     

Deconvolution of fluorescence spectra: contribution to the structural analysis of complex molecules

Fluorescence spectroscopy is a sensitive analytical tool in the studies of both simple and complex molecular structures. In complex molecules, however, determining the number and position of components may give a specific insight into the structure, complementary to the other analytical techniques. We applied log–normal model to analyze fluorescence of simple monofluorophore molecule. In order to analyze spectra where both fluorophores and Raman emission bands were present, we developed a method obtained by combination of the symmetric, Gaussian, for Raman and asymmetric, log–normal model, for fluorescence, applicable to the molecules of different complexity. Technically, for each sample we varied excitation wavelength with 5 nm step and recorded the corresponding emission spectra. They were subsequently used for component analysis. Position of each component was plotted against the excitation wavelength. Applying this approach we could identify minimal number of components having stable positions, while their approximate probability density (APD) in a spectral series was correlated with the probable number of fluorophores in the molecule. The method was tested on molecules containing different number of fluorophores: monomers involved in the synthesis of plant polymer lignin—coniferyl alcohol (one fluorophore), ferulic acid (two fluorophores) and on lignin model compound produced from these monomers (many fluorophores). All investigated species belong to benzene-substituted class of compounds, and it is reasonable to assume that they have similar fluorescence band contour. We also report the results of environmental scanning electron microscopy (ESEM) studies showing multilayered dehydrogenative polymer (DHP) structure, in order to show complexity of the polymer. Our results present complementarity of these two approaches in the structural studies of the lignin model compound.