LIGHT QUENCHING OF FLUORESCENCE: A NEW METHOD TO CONTROL THE EXCITED STATE LIFETIME AND ORIENTATION OF FLUOROPHORES

Abstract Experimental studies have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modem high-repetition rate lasers, a phenomenon we call “light quenching.” In this overview article, we describe the possible effects of light quenching on the steady-state and time-resolved intensity and anisotropy of fluorophores. One can imagine two classes of experiments. Light quenching can occur within the single excitation pulse, or light quenching can be accomplished with a second time-delayed quenching pulse. The extent of light quenching depends on the amplitude of the emission spectrum at the quenching wavelength. Different effects are expected for light quenching by a single laser beam (within a single laser pulse) or for a time-delayed quenching pulse. Depending upon the polarization of the light quenching beam, light quenching can decrease or increase the anisotropy. Remarkably, the light quenching can break the usual z-axis symmetry of the excited state population, and the measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. The polarization can increase to unity under selected conditions. Quenching with time-delayed light pulses can result in step changes in the intensity or anisotropy, which is predicted to result in oscillations in the frequency-domain intensity and anisotropy decays. These predicted effects of light quenching, including oscillations in the frequency-domain data, were demonstrated to occur using selected fluorophores. The increasing availability and use of pulsed laser sources requires consideration of the possible effects of light quenching and offers the opportunity for a new class of two-pulse or multiple-pulse time-resolved experiments where the sample is prepared by the excitation pulse and subsequent quenching pulses to modify the excited state population, followed by time- or frequency-domain measurement of the optically prepared excited fluorophores.     

High-resolution colocalization of single molecules within the resolution gap of far-field microscopy.

To obtain detailed information about the three-dimensional (3D) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 630/650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 630/650) that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.     

Aggregation of Poly(γ‐benzyl L‐glutamate)s Followed by Time‐Resolved Emission Anisotropy

Syntheses of poly(γ‐benzyl L ‐glutamate)s (PBLGs) labeled with various fluorophores (tryptophan, dansyl, and anthracene) having different molecular weights are reported. Association of PBLG chains was studied by time‐resolved emission anisotropy in the solvents supporting the aggregation process (1,4‐dioxane and tetrahydrofuran) and in N,N‐dimethylformamide, where the aggregates were not formed. The influence of molecular weight and polymer concentration on PBLG association was studied as well. The limiting emission anisotropy (r_∞) and rotational correlation times (ϕ) were determined. The chain relaxation dynamics were compared with the fluorescence lifetimes of the fluorophores and spectroscopically suitable labels were selected. Tryptophan was found to be an inconvenient fluorophore for the association study of PBLGs because of its short excited‐state lifetime. Dansyl and anthracene fluorophores, however, proved to be suitable labels for the chain dynamics study of PBLGs in solution. The mobilities of PBLG chains in 1,4‐dioxane were slower than those in tetrahydrofuran and N,N‐dimethylformamide because of PBLG association in this solvent.     

A novel two-fluorophore approach to ratiometric sensing of Zn(2+)

We present a small molecule ratiometric Zn2+-sensing system based on two fluorophores excited by visible light, a Zn2+-insensitive reporter fluorophore, coumarin 343, and a Zn2+-sensitive fluorescein-based compound, ZPA-1. The two fluorophores are linked by an ester to give Coumazin-1, a membrane-permeable, essentially nonfluorescent compound. Upon exposure to esterases, Coumazin-1 is hydrolyzed to its constituent fluorophores. Measurement of the ratio of coumarin emission at 488 nm (lambdaexc = 445 nm) and comparison with ZPA-1 emission at 534 nm (lambdaexc = 505 nm) affords information about the amount of sensor present as well as the amount of Zn2+ present. A generally applicable synthetic route to amide-functionalized ZP1 sensors is also described. The Zn2+-sensing properties of one member of this class are similar to those of the parent ZP1 sensor, with slightly tighter binding and lower background signal.     

Fluorescence resonance energy transfer in polydiacetylene liposomes

Conjugated polydiacetylene (PDA) possessing stimuli-responsive properties has been intensively investigated for developing efficient sensors. We report here fluorescence resonance energy transfer (FRET) in liposomes synthesized using different molar ratios of dansyl-tagged diacetylene and diacetylene-carboxylic acid monomers. Photopolymerization of diacetylene resulted in cross-linked PDA liposomes. We used steady-state electronic absorption, emission, and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). We found that the monomer ratio of acceptor to donor ( R ad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. For R ad = 10 000, the acceptor emission intensity was amplified by more than 18 times when the liposome solution was heated from 298 to 338 K. A decrease in R ad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to insertion of dansyl in the bilayer of the liposomes, which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. Furthermore, receptor tagged onto PDA liposomes can interact with ligands present on proteins, enzymes, and cells, which will produce emission sensing signal. Therefore, using the present approach, there exist opportunities for designing FRET-based highly sensitive and selective chemical and biochemical sensors.     

Selective Monitoring of Stern-Volmer Quenching of Chiral Molecules Via Fluorescence Detected Circular Dichroism

Abstract

The use of fluorescence detected circular dichroism (FDCD) for monitoring the quenching of chiral molecules is described. This technique combines the specificity for chiral centers of circular dichroism (CD), the sensitivity and selectivity of the fluorescence measurement with the additional chemical information provided by fluorescence quenching. Spectra are acquired using a multidimensional FDCD spectrometer which obtains a FDCD matrix of spectral data as a function of multiple excitation wavelengths (CD information) and multiple fluorescence emission wavelengths. This combination of FDCD and fluorescence quenching produces a very selective monitor for chiral fluorophores.     

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.     

Dual‐Polarization Imaging of a Dual‐Fluorophore Ion Sensor: A Single‐Molecule Study

The fluorescence emission of the dual‐fluorophore Ca²⁺ ion sensor molecule, calcium‐green 2 (CG‐2), has been characterized using dual‐polarization imaging at the single‐molecule level. By comparing the fluorescence intensity of individual CG‐2 molecules in two mutually orthogonal polarization image channels, information about the relative orientation of the two constituent fluorophores in the molecule is obtained. Experimental results from polarization measurements are compared with those predicted from a geometric model based on coupled‐fluorophores that are randomly distributed in space. The results confirm previous optical spectroscopy‐based predictions of the orientation of CG‐2′s fluorophores, and the general applications of this dual‐polarization imaging approach for characterizing the optical properties of molecules containing multiple fluorophores is discussed.     

Chemoselective Alteration of Fluorophore Scaffolds as a Strategy for the Development of Ratiometric Chemodosimeters

Ratiometric sensors generally couple binding events or chemical reactions at a distal site to changes in the fluorescence of a core fluorophore scaffold. However, such approaches are often hindered by spectral overlap of the product and reactant species. We provide a strategy to design ratiometric sensors that display dramatic spectral shifts by leveraging the chemoselective reactivity of novel functional groups inserted within fluorophore scaffolds. As a proof‐of‐principle, fluorophores containing a borinate ( RF₆₂₀ ) or silanediol ( SiOH2R ) functionality at the bridging position of the xanthene ring system are developed as endogenous H₂O₂ sensors. Both these fluorophores display far‐red to near‐infrared excitation and emission prior to reaction. Upon oxidation by H₂O₂ both sensors are chemically converted to tetramethylrhodamine, producing significant (≥66 nm) blue‐shifts in excitation and emission maxima. This work provides a new concept for the development of ratiometric probes.     

A fluorescence anisotropy method for measuring protein concentration in complex cell culture media

The rapid, quantitative analysis of the complex cell culture media used in biopharmaceutical manufacturing is of critical importance. Requirements for cell culture media composition profiling, or changes in specific analyte concentrations (e.g. amino acids in the media or product protein in the bioprocess broth) often necessitate the use of complicated analytical methods and extensive sample handling. Rapid spectroscopic methods like multi-dimensional fluorescence (MDF) spectroscopy have been successfully applied for the routine determination of compositional changes in cell culture media and bioprocess broths. Quantifying macromolecules in cell culture media is a specific challenge as there is a need to implement measurements rapidly on the prepared media. However, the use of standard fluorescence spectroscopy is complicated by the emission overlap from many media components. Here, we demonstrate how combining anisotropy measurements with standard total synchronous fluorescence spectroscopy (TSFS) provides a rapid, accurate quantitation method for cell culture media. Anisotropy provides emission resolution between large and small fluorophores while TSFS provides a robust measurement space. Model cell culture media was prepared using yeastolate (2.5 mg mL^–1) spiked with bovine serum albumin (0 to 5 mg mL^–1). Using this method, protein emission is clearly discriminated from background yeastolate emission, allowing for accurate bovine serum albumin (BSA) quantification over a 0.1 to 4.0 mg mL^–1range with a limit of detection (LOD) of 13.8 μg mL^–1.     

Parallel factor analysis of spider fluorophores

Fluorophores from the hemolymph of yellow sac spiders (Cheiracanthium mildei) have been characterized using excitation emission matrix (EEM) fluorescence spectroscopy. This approach provides characterization of fluorophores present in the organism without having to isolate pure samples. Minimal variation occurs between individual samples and each EEM has two distinct peaks, suggesting two fluorophores may be present in the hemolymph. Parallel factor analysis reveals that three fluorophores (with excitation and emission maxima at 270/319, 330/389, and 350/465 nm) best explains the sample to sample variation. By comparing the spectra of the three individual components to fluorophores found in scorpions it is shown that these spiders possess different fluorophores than scorpions. Furthermore, the fluorescence observed is not consistent with beta-carboline or 4-methyl-7-hydroxycoumarin, two compounds previously described in scorpions.     

Suppression of secondary PL emission by indirect photoexcitation

A blend of a newly synthesized polyfluorene(PDHBF) and polyvinylcarbazole(PVK) exhibits a photoluminescence(PL) emission spectrum of PDHBF without an increase in the PL intensity on photoexcitation at 340 nm, the UV-visible absorption maximum of PVK, despite of a substantial spectrum overlap. However, the indirect photoexcitation of the blend suppresses the secondary emission of the PL with the maximum at 520 nm. The chromophores generating the secondary emission are formed when the chromophores are photoexcited above the critical energy level of an excited state. The chromophores formed by the energy transfer have energy lower than the critical energy and fail to form the excimers. A low temperature PL study of the blend in a cryogenic chamber proves that the energy transfer in the system takes place mainly between the excimers of PVK generated by the partially eclipsed dimeric states of two carbazole units and the fluorophores of PDHBF.     

Modulated anisotropy fluorescence for quantitative determination of carbaryl and benomyl

Two individual components in mixtures have been resolved by frequency domain fluorescence technique by measuring the observable quantities which characterize the anisotropy decay; differential anisotropy phase and modulated anisotropy ratio (MAR), which in turn are related to the rotational correlation time. The method presented here is capable of directly resolving binaries mixtures of fluorophores on the basis of differences in their rotational diffusion rates. Our results demonstrate that modulation anisotropy ratio measurements can be used for quantitative determination of small analytes, carbaryl and benomyl, having identical or nearly identical fluorescence spectra. This methodology can be applied with good results when the fluorophores have a suitable MAR difference.     

Enhanced fluorescence from o-phthalaldehyde and fluorescamine fluorophores using Triton and β-cyclodextrin

Mexiletine— and lysine hydrochloride—o-phthalaldehyde and mexiletine hydrochloride—, cysteine—, cysteamine—, homocysteine— and lysine hydrochloride—fluorescamine derivatives were subjected to Triton and β-cyclodextrin enhancement treatments. Of several classical fluorescence-enhancing reagents tested (Triton, β-cyclodextrin, sodium dodecyl sulphate, Brij), Triton provided the best results, followed by β-cyclodextrin. Increases in fluorescence emission by a factor of up to about 10 (mexiletine—fluorescamine—Triton X-100) were observed, with a generally negligible influence of the enhancing reagents on the excitation and emission maxima. Fluorescence enhancement by the addition of a suitable reagent solution to the final analyte solution may, in specific instances, enhance the detectability of native or chemically induced fluorophores.     

Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores

Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in super-resolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push-pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.     

Installation/modulation of the emission response via click reaction

We have demonstrated the installation of a fluorescence property into a nonfluorescent precursor and modulation of an emission response of a pyrene fluorophore via click reaction. The synthesized fluorophores show different solvatochromicity and/or intramolecular charge transfer (ICT) feature as is revealed from the UV-visible, fluorescence photophysical properties of these fluorophores, and DFT/TDDFT calculation. We observed that some of the synthesized fluorophores showed purely ICT character while emission from some of them arose from the LE state. A structureless and solvent polarity-sensitive dual emission behavior was observed for one of the triazolylpyrene fluorophores that contains an electron-donating -NMe(2) substituent (fluorophore, 7a). Conversely, triazolylpyrene with an electron-withdrawing -CN group (fluorophore, 7b) showed a solvent polarity-independent vibronic emission. The effect of ICT on the photophysical properties of these fluorophores was studied by fluorescence emission spectra and DFT/TDDFT calculations. Fluorescence lifetimes were also measured in different solvents. All of our findings revealed the delicate interplay of structure and emission properties and thus having broader general utility. As the CT to LE intensity ratio can be employed as a sensing index, the dual emissive fluorophore can be utilized in designing the molecular recognition system too. We envisage that our investigation is of importance for the development of new fluorophores with predetermined photophysical properties that may find a wide range of applications in chemistry, biology, and material sciences.     

Characterization and Diagnosis of Cancer by Native Fluorescence Spectroscopy of Human Urine

Urine is one of the diagnostically important bio fluids, as it has different metabolites in it, where many of them are native fluorophores. Native fluorescence characteristics of human urine samples were studied using excitation–emission matrices (EEMs) over a range of excitation and emission wavelengths, and emission spectra at 405 nm excitation, to discriminate patients with cancer from the normal subjects. The fluorescence spectra of urine samples of cancer patients exhibit considerable spectral differences in both EEMs and emission spectra with respect to normal subjects. Different ratios were calculated using the fluorescence intensity values of the emission spectra and they were used as input variables for a multiple linear discriminant analysis across different groups. The discriminant analysis classifies 94.7% of the original grouped cases and 94.1% of the cross‐validated grouped cases correctly. Based on the fluorescence emission characteristics of urine and statistical analysis, it may be concluded that the fluorophores nicotinamide adenine dinucleotide (NADH) and flavins may be considered as metabolomic markers of cancer.     

聚集诱导发光机理研究

与传统荧光生色团聚集后导致荧光猝灭相反,有一类化合物在单分子状态下荧光微弱甚至观察不到荧光,而在聚集状态下荧光显著增强,这就是聚集诱导发光(AIE)现象。AIE现象独特的优越性使得众多研究组开发出越来越多的新AIE体系,其机理也被广泛而深入地研究。本文总结了目前为止已经提出的AIE机理,包括分子内旋转受限、分子内共平面、抑制光物理过程或光化学反应、非紧密堆积、形成J-聚集体以及形成特殊激基缔合物等;着重评述了目前研究最为全面、适用范围最广的分子内旋转受限机理。同时介绍了一些基于这些机理设计的新AIE体系。     

Biodegradable Polypeptide-based Vesicles with Intrinsic Blue Fluorescence for Antibacterial Visualization

Fluorescence imaging has been an indispensable tool to provide dynamic information about the localization and quantity of organisms.Meanwhile,due to the intrinsic hollow structure and modularized biofunctionalities,polymer vesicles have been widely applied in biomedical field.However,most polymer vesicles are embedded with organic fluorophores for fluorescence imaging,which have certain drawbacks such as leakage and possible cytotoxicity.Here,we present a biodegradable polypeptide-based vesicle with intrinsic blue fluorescence without introducing any fluorophore for real-time visualization of antibacterial process.Through modular design to integrate multiple functional fragments,poly(ε-caprolactone)-block-poly(tryptophan)-block-poly(lysine-stat-phenylalanine)[PCL25-b-PTrP2-b-P(Lys13-stat-Phe4)]was synthesized,where PCL chains form the hydrophobic membrane,P(Lys-stat-Phe) and PTrp provide intrinsic fluorescence and broad-spectrum antibacterial activity.It is noteworthy that the fluorescence emission was shifted from invisible ultraviolet range of amino acids to visible range (emission maximum at 436 nm),which makes it possible to visualize the antibacterial process.In addition,through utilizing the intrinsic fluorescence of vesicles,confocal fluorescent imaging of vesicles with bacteria validated the specific adhesion of vesicle towards bacteria,and the bacterial death through membrane disruption.Overall,we provided a novel approach to developing biodegradable fluorescent polypeptide-based vesicles for real-time visualization of antibacterial process.