Use Of Fluorophores Research Articles

Surface Plasmon-Coupled Emission of Rhodamine 110 Aggregates in a Silica Nanolayer.

First analysis of strong directional surface plasmon-coupled emission (SPCE) of ground-state formed intermolecular aggregates of Rhodamine 110 (R110) in silica nanofilms deposited on silver nanolayers is reported. Until now, the processes of energy transport and its trapping due to aggregate formation have not been studied in the presence of SPCE. A new approach to multicomponent systems with weakly and strongly fluorescent centers making use of fluorophore-surface plasmon interaction is presented. The analysis is based on comparison of experimental free-space emission spectra (F-SE), experimental SPCE with theoretical surface plasmon resonance spectra (SPR). It is shown that, due to the dispersion of SPCE, the detection of weak aggregate emission is straightforward if only the monomers and aggregates fluorescence spectra are somewhat spectrally shifted. SPCE studies confirmed the formation of weakly fluorescent higher order aggregates of R110 in silica films. The results indicate that the increase of energy transfer from monomers to aggregates is due to fluorophore-plasmon interaction.

Spurious Observation of the Marcus Inverted Region in Bimolecular Photoinduced Electron Transfer

The effect of viscosity on the bimolecular electron transfer quenching of a series of coumarins by N,N-dimethylaniline was investigated using steady-state and time-resolved fluorescence spectroscopy. The data reveal that the static and transient stages of the quenching become dominant as viscosity increases. When extracting the quenching rate constants using a simple Stern-Volmer analysis, a decrease of the rate constant with increasing driving force is observed above ~2 cP. However, this apparent Marcus inverted region, already reported several times with the same system in micelles and room temperature ionic liquids, totally vanishes when analyzing the data with a model accounting for the static and transient stages of the quenching. It appears that the apparent Marcus inverted region arises from the neglect of these quenching regimes together with the use of fluorophores with different excited-state lifetimes.

Fluorescence Microscopy Light Sources

Fluorescence microscopy techniques are now prevalent throughout the life sciences and many of the physical sciences. These techniques are often dependent on white light sources that have evolved from the more traditional mercury arc lamp to metal halide sources to the more recent light emitting diodes (LEDs). The newer light sources show more uniform power across the visible light spectrum, allowing for the use of fluorophores and fluorescent proteins outside of the peak wavelengths associated with the more traditional light sources.

CLASI-FISH: Principles of combinatorial labeling and spectral imaging

Although the number of phylotypes present in a microbial community may number in the hundreds or more, until recently, fluorescence in situ hybridization has been used to label, at most, only a handful of different phylotypes in a single sample. We recently developed a technique, CLASI-FISH for combinatorial labeling and spectral imaging – fluorescence in situ hybridization, to greatly expand the number of distinguishable taxa in a single FISH experiment. The CLASI technique involves labeling microbes of interest with combinations of probes coupled with spectral imaging to allow the use of fluorophores with highly overlapping excitation and emission spectra. Here, we present the basic principles and theory of CLASI-FISH along with some guidelines for performing CLASI-FISH experiments. We further include a protocol for creating fluorescence spectral reference standards, a vital component of successful CLASI-FISH.

Fluorescence-based sensing with optical nanowires: a generalized model and experimental validation

A model for the fluorescence sensing properties of small-core high-refractive-index fibers (optical nanowires) is developed and compared quantitatively with experiment. For the first time, higher-order modes and loss factors relevant to optical nanowires are included, which allows the model to be compared effectively with experiment via the use of fluorophore filled suspended optical nanowires. Numerical results show that high-index materials are beneficial for fluorescence-based sensing. However, both numerical and experimental results show that the fluorescence signal is relatively insensitive to core size, except for low concentration sensing where nanoscale fiber cores are advantageous due to the increased evanescent field power.

Using fluorescence spectroscopy EEM to evaluate the efficiency of organic matter removal during coagulation–flocculation of a tropical surface water (Agbo reservoir)

This study evaluate the use of fluorophores A, C and T fluorescence intensities to access the coagulation efficiency for removing dissolved organic carbon (DOC) in the raw water from Agbo reservoir in Ivory Coast. A coagulation–flocculation was conducted with aluminium sulphate as coagulant and DOC residual and fluorescence intensities were acquired. The consistency of fluorescence data was evaluated to ensure that no inner-effect, quenching or enhanced intensities affect the data. Fluorescence-inferred DOC removal was then calculated in percentage terms from the decrease in organic matter fluorescence intensity for each peak between raw and clarified water and correlated with measured DOC removal. The results indicate a high significant correlation between measured DOC removal and fluorescence-inferred DOC removal calculated for peak A ( R 2 = 0.91), peak C ( R 2 = 0.89), peak T ( R 2 = 0.92) indicating a strong linear relationship between DOC removal and fluorescence intensities. Furthermore, tryptophan-like (peak T) was found to be the least eliminated and thus, may be considered as an indicator of DOC residual after coagulation–flocculation process. This result shows that fluorescence spectroscopy offers a robust analytical technique to be used to evaluate DOM removal efficiency in water treatment.

Analysis of Protein–Protein Interactions by Using Droplet‐Based Microfluidics

Every little drop: The K(D) values of angiogenin (ANG) interactions as shown by FRET analysis of thousands of pL-sized droplets agree with data from bulk-fluorescence polarization measurements. Importantly, the use of fluorophores does not affect the activity of ANG or the binding of anti-ANG antibodies to ANG. Such an experimental platform could be applied to the high-throughput analysis of protein-protein interactions.

Towards non-blinking colloidal quantum dots

At a single-molecule level, fluorophore emission intensity fluctuates between bright and dark states. These fluctuations, known as blinking, limit the use of fluorophores in single-molecule experiments. The dark-state duration shows a universal heavy-tailed power-law distribution characterized by the occurrence of long non-emissive periods. Here we have synthesized novel CdSe-CdS core-shell quantum dots with thick crystalline shells, 68% of which do not blink when observed individually at 33 Hz for 5 min. We have established a direct correlation between shell thickness and blinking occurrences. Importantly, the statistics of dark periods that appear at high acquisition rates (1 kHz) are not heavy tailed, in striking contrast with previous observations. Blinking statistics are thus not as universal as thought so far. We anticipate that our results will help to better understand the physico-chemistry of single-fluorophore emission and rationalize the design of other fluorophores that do not blink.

Fluorescent stains for quantification of DNA by confocal laser scanning microscopy in 3-D

Confocal microscopy requires the use of fluorophores to visualize structures of interest within a specimen. To perform reliable measurements of the intensity of fluorescence, the stain should be specific, penetrate well into tissue sections, and bind stoichiometrically. Furthermore, emission must be linear with respect to DNA content and brightness, and fluorescence should be stable. Confocal microscopy is used to determine DNA ploidy and to analyze texture of nuclei, which is accomplished in three dimensions, because nuclei can be measured within the original tissue context. For this purpose the sample must be stained with a DNA binding fluorophore with the properties described above. Stains with different properties have been developed for different applications. We review here the advantages and disadvantages of these different stains for analyzing DNA ploidy and nuclear texture using three-dimensional microscopy. We conclude that SYBR green I and TO-PRO-3 are the most suitable stains for this purpose at present.

Single-Molecule Studies of Enhanced Fluorescence on Silver Island Films

We investigated enhanced fluorescence of Cyanine 5 dyesnear silver island films (SIFs) by the use of single moleculespectroscopic method. We found that, on average, the Cy5molecules are 18-fold brighter on SIFs than on a glasssurface and that single-molecule lifetimes are 10-foldshorter on SIFs as compared to glass. We observed Cy5molecules until photobleaching occurred and found that themolecules on the SIF emitted 20-fold more photons ascompared to those on glass prior to photobleaching. Theseresults demonstrate that the use of fluorophore–metalinteractions can increase the brightness and photostabilityof fluorophores for single-molecule detection.During the past decade, there has been considerableprogress in the development of fluorescence probes. Manyprobes, such as the cyanine dyes, have high extinctioncoeffiecients and high quantum yields. Hence, it is unlikelythat the future probe development will result in dramaticincreases in detectability for small organic fluorphores. Forthis reason, there is an interest in quantum dots andpolymeric probes. In the past years we have taken adifferent approach to create improved probes, this being theuse of metallic nanoparticles [1–3]. Ensemble experimentshaverevealedthatproximitytosilverparticlescouldincreasethe intensity and photostability of fluorphores [4–9]. Thiseffect is due to the through-space near-field interactionbetween the fluorphore and surface plasmons on the metals.Thus, the use of metallic nanostructure to enhancefluorescence has great potential for applications in thefields of medical diagnostics and biotechnology.The fluorescence spectral properties of fluorphores areespecially important for sensitive detection. The photo-stability of the fluorphore resolves the time it can beobserved prior to photobleaching. Hence, it is of interest todetermine if the improved spectral properties observed atthe ensemble level extends to the single-molecule level.The interactions of fluorophores with metallic particleshave been studied theoretically and empirically at thesingle-molecule level in recent years [10–13]. In thesereports, the major concern was the dependence of theemission rate on the distance between the dye layer and themetallic surface. The emission can be quenched due toradiation energy transfer to the metal as molecules adsorbeddirectly on the surface [12, 13]. Another consideration is theenhanced fluorescence due to increased electromagneticfield of surface plasmon and/or enhanced quantum yield[10, 11]. Therefore, we examined the widely used fluores-cent probe Cy5 near SIFs. These films consist of sub-wavelength-size silver particles on a glass surface [4, 5].Our single-molecule experiments of this system reveal boththe spectral changes due to the metal particles and theheterogeniety due to a range of fluorophore–metaldistances.For these studies, the Cy5 molecules were spin-coatedonto the SIFs from an aqueous solution containing 0.5%polyvinyl alcohol (PVA). Initially, we examined theensemble emission spectra in which the Cy5 concentrationis 20-fold higher than for the single-molecule experiments(Figure 1). Both Cy5 spectra exhibit emission maxima near680 nm. A significant enhancement in fluorescenceemission is clearly evident on silvered surfaces. Thepresence of silver nanostructure did not distort the Cy5spectrum. Additionally, the SIF sample without Cy5 did notcontribute significant background. This initial ensembleresult indicates that the Cy5 dyes deposited on SIF display

Fluorescence-based detection of thiols in vitro and in vivo using dithiol probes

Thiols play a central role in maintaining biological homeostasis. Their levels can change dramatically in response to oxidative stress associated with toxic insults, bacterial infection, and disease. Therefore, a reagent that can monitor thiol levels both in vitro and in vivo would be useful for assays and as a biomarker. Such a reagent should (i) be selective for thiols, (ii) be able to penetrate cell walls, and (iii) have a low reduction potential so as not to create oxidative stress in a cell. We have developed such a fluorescent reagent (DSSA) based on a dithiol linker: (i) the use of a dithiol linker makes it selective for thiols; (ii) the use of fluorophores that populate neutral states at physiological pH improves cell wall penetration; and (iii) because of the reagent’s low reduction potential (−0.60 V), it will not stress cells oxidatively. For example, 5 μM of reagent is responsive to changes in glutathione levels in the physiologically relevant range of 1 to 10 mM, yet this would oxidize less than 1% of cellular glutathione. In Escherichia coli, decreased thiol levels were detected in cells deficient in glutathione synthesis. In zebrafish embryos, the DSSA reagent permitted detection of unusually high thiol levels in the zebrafish chorion.

Development and Characterization of Green Fluorescent Protein Mutants with Altered Lifetimes

Multiple-probe fluorescence imaging applications demand an ever-increasing number of resolvable probes, and the use of fluorophores with resolvable fluorescence lifetimes can help meet this demand. Green fluorescent protein (GFP) and its variants have been widely used in spectrally resolved multiprobe imaging, but as yet, there has not been a systematic set of mutants generated with resolvable lifetimes. Therefore, to generate such mutants, we have utilized error-prone PCR and fluorescence lifetime imaging to screen for mutants of UV-excited green fluorescent protein (GFPuv) that exhibit altered fluorescence decay lifetimes. This has resulted in the isolation of GFPuv mutants displaying at least three distinctly different lifetimes in the range of 1.9-2.8 ns. Mutation of Y145 to either histidine or cysteine was found to shift the fluorescence lifetime of GFPuv from 3.03 +/- 0.03 to 2.78 +/- 0.05 ns for the Y145H mutant and to 2.74 +/- 0.05 ns for Y145C. Some of the shorter-lifetime mutants exhibited excitation peaks that were red-shifted relative to their maximal absorption, indicating that the mutations allowed the adoption of additional conformations relative to wtGFPuv. The utility of these mutants for applications in simultaneous imaging and quantification is shown by the ability to quantify the composition of binary mixtures in time-resolved images using a single detector channel. The application of the screening method for generating lifetime mutants of other fluorescent proteins is also discussed.

Criteria for quantitative PLIF experiments using high-power lasers

In order to interpret concentration measurements using planar laser-induced fluorescence (PLIF), it is necessary to understand the systematic and statistical errors that accompany those measurements. High-power lasers provide both increased signal-to-noise ratios (desirable) and intensity-induced deviations from linearity (undesirable). This paper reviews and argues for modification of current criteria for use of fluorophores for quantitative PLIF imaging, and makes use of a modified new protocol to qualify the fluorophore Rhodamine-WT for use in quantitative PLIF imaging. The key element is the decision to operate the imaging experiment under optically thin conditions.

Enhanced photostability of ICG in close proximity to gold colloids

Photobleaching of fluorophores frequently limits their detectability or observation time. We examined Indocyanine green (ICG) which is widely used in medical testing and is highly unstable. We showed that spatial localization of ICG near metallic gold colloids resulted in increased photostability. This suggests the use of fluorophore–metal conjugates in situations adversely affected by photobleaching.

High resolution detection of fluorescently labeled microorganisms in environmental samples using time-resolved fluorescence microscopy

The high level of discrimination offered by fluorescence microscopy has led to its widespread use for the analysis of individual microbial cells. The major limitation of fluorescence microscopy in microbial ecology is that many types of environmental samples contain autofluorescent material that can obscure emission from a fluorescent label. Time-resolved fluorescence microscopy (TRFM) is a technique that greatly reduces background autofluorescence whilst maintaining signal strength of the fluorescent target. TRFM differs from fluorescent microscopy in the use of fluorophores that are characterized by long-lived luminescence. Samples are briefly illuminated to excite fluorescence then capture of luminescence is delayed for a time interval sufficient to ensure autofluorescence has largely faded. TRFM has not been extensively used in microbiology because of the limitations and cost of available time-resolved microscopes and the lack of suitable long-lived fluorescent labels. Here we describe modification of a commercial fluorescence microscope for time-resolved operation through the addition of an image-intensified camera and low cost flashlamp. The TRFM was used in combination with a novel immunofluorophore for the specific detection of Giardia cysts in a water sample containing large amounts of autofluorescent material. A 60-mus gate delay between excitation and detection resulted in a 30-fold increase in contrast of labeled parasites compared to conventional immunostaining. To our knowledge, this is the first report of the use of TRFM for the detection of microorganisms in environmental samples.

High resolution detection of fluorescently labeled microorganisms in environmental samples using time-resolved fluorescence microscopy

The high level of discrimination offered by fluorescence microscopy has led to its widespread use for the analysis of individual microbial cells. The major limitation of fluorescence microscopy in microbial ecology is that many types of environmental samples contain autofluorescent material that can obscure emission from a fluorescent label. Time-resolved fluorescence microscopy (TRFM) is a technique that greatly reduces background autofluorescence whilst maintaining signal strength of the fluorescent target. TRFM differs from fluorescent microscopy in the use of fluorophores that are characterized by long-lived luminescence. Samples are briefly illuminated to excite fluorescence then capture of luminescence is delayed for a time interval sufficient to ensure autofluorescence has largely faded. TRFM has not been extensively used in microbiology because of the limitations and cost of available time-resolved microscopes and the lack of suitable long-lived fluorescent labels. Here we describe modification of a commercial fluorescence microscope for time-resolved operation through the addition of an image-intensified camera and low cost flashlamp. The TRFM was used in combination with a novel immunofluorophore for the specific detection of Giardia cysts in a water sample containing large amounts of autofluorescent material. A 60-μs gate delay between excitation and detection resulted in a 30-fold increase in contrast of labeled parasites compared to conventional immunostaining. To our knowledge, this is the first report of the use of TRFM for the detection of microorganisms in environmental samples.

Preparation of Na,K-ATPase Specifically Modified on the Anti-fluorescein Antibody-Inaccessible Site by Fluorescein 5′-Isothiocyanate

Specific labeling is required for energy transfer measurements and to avoid artifacts in the use of fluorophores as reporter groups. Therefore, a method for specific modification by one of the most popular reagents for P-type ATPases (fluorescein 5′-isothiocyanate) has been developed. Sulfhydryl reagents protected against modification of cysteine residues, and treatment with dithiothreitol eliminated a slow doubling of the fluorescence of conventionally modified Na,K-ATPase upon dilution that is attributed to disappearance of self-energy transfer. Removal of nonspecifically bound fluorescein was also confirmed by titration of the modified Na,K-ATPase with anti-fluorescein antibody and by time resolution of the fluorescence change when the modified enzyme was mixed with Na+ in a stopped-flow instrument. The only fluorescence change when specifically modified Na,K-ATPase was mixed with Na+ was the signal from fluorescein at the antibody-inaccessible, substrate-protectable site that reports the conformational change in unphosphorylated enzyme. The magnitude of the fluorescence change reporting the conformational change increased from between 8 and 12% to between 25 and 30% without affecting the kinetic constants estimated from titrations with Na+ and K+. The method should be generally applicable to the preparation of specifically labeled P-type pumps for use in kinetic and equilibrium titrations or energy transfer measurements.

Fluorescence spectroscopy of single biomolecules.

Recent advances in single-molecule detection and single-molecule spectroscopy at room temperature by laser-induced fluorescence offer new tools for the study of individual macromolecules under physiological conditions. These tools relay conformational states, conformational dynamics, and activity of single biological molecules to physical observables, unmasked by ensemble averaging. Distributions and time trajectories of these observables can therefore be measured during a reaction without the impossible need to synchronize all the molecules in the ensemble. The progress in applying these tools to biological studies with the use of fluorophores that are site-specifically attached to macromolecules is reviewed.

Micro-CT imaging of structure-to-function relationship of bone microstructure and associated vascular involvement

We are exploring methods of quantitating the 3D microstructure of bone in a way that will provide quantitative information about the functional status of the bone. The basic strategy is to image the spatial distribution of a selected, local, marker of function (e.g., material properties or new bone formation) and relate this to the simultaneously imaged 3D anatomic microstructure. Many of these approaches are extensions of well-established 2D imaging techniques (e.g., use of fluorophores and autoradiography) to 3D micro-CT. Local stresses throughout the microstructure can be estimated from the 3D geometry (and change in that geometry in response to stress applied to the outside of the bones) and correlated to the local function. In addition to study of bone, we are also exploring calcification of arterial walls, both within the bone and outside the bone, such as coronary arteries. Arterial calcification in ovariectomised rats has been observed.

Use of platinum coproporphyrin and delayed luminescence imaging to extend the number of targets FISH karyotyping.

Combinatorial use of fluorophores in multicolor fluorescence in situ hybridization (FISH) allows for the recognition of all human chromosomes. Here we introduce the concept of the use of delayed luminescence labels such as phosphorescent platinum coproporphyrins (PtCP) to extend the number of simultaneously detectable targets in multicolor FISH karyotyping. PtCP-conjugated antibodies were used in combination with conventional FISH labels such as cascade blue, fluorescein, lissamine rhodamine, Cy5, and Cy7. Probe sets for all human chromosomes were generated and labeled with these dyes in a combinatorial approach. Delayed luminescence of PtCP was accomplished using a standard fluorescence microscope in which a specially constructed module for visualization of delayed luminescence was incorporated. The module consists of a minichopper incorporated in the standard block that holds the shutter and diaphragm, and a FLC polarizing shutter mounted in a filter holder at the emission side. Multicolor FISH staining was applied to normal metaphase chromosomes and to chromosomes generated from cultured JVM-2 cells with known translocations. Multicolor FISH images (conventional and delayed) were registered using a slow-scan CCD camera. Recognition of all 24 chromosomes was feasible, since the delayed PtCP fluorescence (lifetime, 90 micros) could be easily distinguished from the conventional promptly fluorescing dyes. We discuss possibilities for extending the number of targets far beyond the 24 demonstrated so far.