Fluorophore Species Research Articles

Simultaneous spectral differentiation of multiple fluorophores in super-resolution imaging using a glass phase plate.

By engineering the point-spread function (PSF) of single molecules, different fluorophore species can be imaged simultaneously and distinguished by their unique PSF patterns. Here, we insert a silicon-dioxide phase plate at the Fourier plane of the detection path of a wide-field fluorescence microscope to produce distinguishable PSFs (X-PSFs) at different wavelengths. We demonstrate that the resulting PSFs can be localized spatially and spectrally using a maximum-likelihood estimation algorithm and can be utilized for hyper-spectral super-resolution microscopy of biological samples. We produced superresolution images of fixed U2OS cells using X-PSFs for dSTORM imaging with simultaneous illumination of up to three fluorophore species. The species were distinguished only by the PSF pattern. We achieved ∼21-nm lateral localization precision (FWHM) and ∼17-nm axial precision (FWHM) with an average of 1,800 - 3,500 photons per PSF and a background as high as 130 - 400 photons per pixel. The modified PSF distinguished fluorescent probes with ∼80 nm separation between spectral peaks.

Spontaneous formation of carbon dots helps to distinguish molecular fluorophores species

Carbon dots (CDs) display photophysical features that appear similar, if not identical, despite the variety of synthesis protocols used to obtain them. The unique design of CDs toward the desired properties requires an exhaustive understanding of the structure itself. This work offers a fundamental insight into the structure and formation of the carbon dots that allows distinguishing the existence of molecular fluorophore species. Remarkably, density functional theory calculations of the elucidated structures support the spontaneous formation of a compact network of molecular species. In addition, as a proof-of-principle, our work comprehensively studies the photoluminescence properties (i.e., concentration, pH and solvent dependencies), which pave the way for developing CD-based logic gate fluorescent sensors.

Two-photon MINFLUX with doubled localization precision

Achieving localization with molecular precision has been of great interest for extending fluorescence microscopy to nanoscopy. MINFLUX pioneers this transition through point spread function (PSF) engineering, yet its performance is primarily limited by the signal-to-background ratio. Here we demonstrate theoretically that two-photon MINFLUX (2p-MINFLUX) could double its localization precision through PSF engineering by nonlinear effect. Cramér-Rao Bound (CRB) is studied as the maximum localization precision, and CRB of two-photon MINFLUX is halved compared to single-photon MINFLUX (1p-MINFLUX) in all three dimensions. Meanwhile, in order to achieve same localization precision with 1p-MINFLUX, 2p-MINFLUX requires only 1/4 of fluorescence photons. Exploiting simultaneous two-photon excitation of multiple fluorophore species, 2p-MINFLUX may have the potential for registration-free nanoscopy and multicolor tracking.

Lifetime-based analysis of binary fluorophores mixtures in the low photon count limit

SummarySingle biomolecule sensing often requires the quantification of multiple fluorescent species. Here, we theoretically and experimentally use time-resolved fluorescence via Time Correlated Single Photon Counting (TCSPC) to accurately quantify fluorescent species with similar chromatic signatures. A modified maximum likelihood estimator is introduced to include two fluorophore species, with compensation of the instrument response function. We apply this algorithm to simulated data of a simplified two-fluorescent species model, as well as to experimental data of fluorophores' mixtures and to a model protein, doubly labeled with different fluorophores' ratio. We show that 100 to 200 photons per fluorophore, in a 10-ms timescale, are sufficient to provide an accurate estimation of the dyes' ratio on the model protein. Our results provide estimation for the desired photon integration time toward implementation of TCSPC in systems with fast occurring events, such as translocation of biomolecules through nanopores or single-molecule burst analyses experiments.

Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection.

Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about intermolecular interactions but is usually limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion, and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus.

Giga-voxel multidimensional fluorescence imaging combining single-pixel detection and data fusion.

Time-resolved fluorescence imaging is a key tool in biomedical applications, as it allows to non-invasively obtain functional and structural information. However, the big amount of collected data introduces challenges in both acquisition speed and processing needs. Here, we introduce a novel technique that allows to acquire a giga-voxel 4D hypercube in a fast manner while measuring only 0.03% of the dataset. The system combines two single-pixel cameras and a conventional 2D array detector working in parallel. Data fusion techniques are introduced to combine the individual 2D and 3D projections acquired by each sensor in the final high-resolution 4D hypercube, which can be used to identify different fluorophore species by their spectral and temporal signatures.

Single-Molecule, Non-Chromatic Discrimination in Binary Fluorophores Mixtures

SummarySingle biomolecule sensing often involves quantification of multiple fluorescent species, coupled for example to different DNAs or proteins resides. While chromatic separation of the fluorescent species is well-established and broadly used it involves several limitations, particularly when the total photon budget is limited. Here we theoretically and experimentally explore the use of time-resolved fluorescence via Time Correlated Single Photon Counting (TCSPC) to accurately quantify multiple fluorescent species. A modified maximum likelihood estimator (MLE) is introduced to include two fluorophore species, with the compensation of the instrument response. We apply this algorithm to simulated data of a simplified two fluorescent species model, as well as to experimental data of free fluorophores mixtures and doubly labelled proteins in order to estimate the errors in the two species ratios as a function of photon budget available. Moreover, focusing on the case of labeled proteins in which the ratios of the two species per molecule is a discrete variable subject to the chemical labeling numbers of the two dyes, we obtain that between 1,000 to 2,000 photons can already provide an accurate estimation of the dyes’ ratio in labelled Ovalbumin proteins. These values correspond to roughly 100 to 200 photons per fluorophore suggesting a realistic path for dyes ratio determination from a single passage of a protein in the detection volume in roughly 10 ms timescale. Our results highlight the way in which temporal limitations of the TCSPC technique could hinder its implementation in systems with fast occurring events such as translocation of biomolecules through nanopores, or fast diffusion in single-molecule burst analyses experiments and provide estimation for the desired photon integration time.

Fluorescent Silica Hybrid Film-Forming Materials Based on Salicylaldazine

Fluorescent film-forming materials were obtained by embedding salicylaldazine (SAA) in silica hybrids generated by sol–gel processes from different silane precursors in acid catalysis. Tuned local environments for the fluorophore were generated in the hosting network by modifying silica sols with organic groups through the co-condensation of tetraethylortosilicate (TEOS) and different alkoxysilanes hydrolysis products. The photophysical properties of the luminescent hybrid films were studied in direct relationship with structural, textural, and surface properties and based on interactions between SAA species and the silica hosting network. Film-forming materials were studied in order to determine differences in absorption and fluorescence emission due to the environments around the fluorophore. The variations recorded in the fluorescence emission spectra of the hybrid films were related to interactions established between the fluorophore species and their sterically hindered surroundings of the host hybrid silica, where free molecular motions are restricted. The influence of the type and amount of network modifier and of the fluorophore loading on the transparency of the films and fluorescence intensity was also investigated. The study carried out led to the elucidation of the necessary conditions for obtaining luminescent film-forming materials with high luminescence intensity and transparency useful for the design of new light concentrators.

Spectral cross-cumulants for multicolor super-resolved SOFI imaging

Super-resolution optical fluctuation imaging provides a resolution beyond the diffraction limit by analysing stochastic fluorescence fluctuations with higher-order statistics. Using nth order spatio-temporal cross-cumulants the spatial resolution and the sampling can be increased up to n-fold in all spatial dimensions. In this study, we extend the cumulant analysis into the spectral domain and propose a multicolor super-resolution scheme. The simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing increases the spectral sampling. The number of discriminable fluorophore species is thus not limited to the number of physical detection channels. Using two color channels, we demonstrate spectral unmixing of three fluorophore species in simulations and experiments in fixed and live cells. Based on an eigenvalue/vector analysis, we propose a scheme for an optimized spectral filter choice. Overall, our methodology provides a route for easy-to-implement multicolor sub-diffraction imaging using standard microscopes while conserving the spatial super-resolution property.

The solvent effect on a styryl‐bodipy derivative functioning as an AND molecular logic gate

AbstractWe study via DFT and TDDFT calculations the photophysical processes of a styryl‐bodipy derivative, (1), of its monometallic complexes 1‐M 2+ (M = Ca, Zn, and Hg), and its trimetallic complex (2) unprotonated, protonated and complexed with water molecules in water solvent and in acidic conditions. The main targets of this study are to gain information regarding published reports on fluorophore species mentioning that fluorescent switching results from trace water, to study how 1 behaves in water solvent which is a common used solvent for molecular logic gates (MLG), and how it behaves in acidic conditions. We conclude that in water solvent, as in acetonitrile solvent (which was found before both theoretically and experimentally) there will be a quenching of emission spectra in 1 and 1‐M 2+ and a retaining of emission in 2. However, contrary to acetonitrile solvent, in water, a weak peak will be observed for 1 and 1‐M 2+, due to a small ratio of reversible protonation, showing that in acetonitrile 1 acts as a better MLG candidate than in water solvent. On the other hand, in acidic conditions all five species will emit and as a result, 1 will not be an AND MLG, showing that the selection of the solvent conditions is crucial for a species to act as an MLG candidate. Finally, we conclude that the retaining of emission is accomplished by the simultaneous tetrahedral geometry of all three aniline N atoms.

Sequential super-resolution imaging using DNA strand displacement.

Sequential labeling and imaging in fluorescence microscopy allows the imaging of multiple structures in the same cell using a single fluorophore species. In super-resolution applications, the optimal dye suited to the method can be chosen, the optical setup can be simpler and there are no chromatic aberrations between images of different structures. We describe a method based on DNA strand displacement that can be used to quickly and easily perform the labeling and removal of the fluorophores during each sequence. Site-specific tags are conjugated with unique and orthogonal single stranded DNA. Labeling for a particular structure is achieved by hybridization of antibody-bound DNA with a complimentary dye-labeled strand. After imaging, the dye is removed using toehold-mediated strand displacement, in which an invader strand competes off the dye-labeled strand than can be subsequently washed away. Labeling and removal of each DNA-species requires only a few minutes. We demonstrate the concept using sequential dSTORM super-resolution for multiplex imaging of subcellular structures.

Electronic tuning of self-healing fluorophores for live-cell and single-molecule imaging.

Bright, long-lasting organic fluorophores enable a broad range of imaging applications. "Self-healing" fluorophores, in which intra-molecularly linked protective agents quench photo-induced reactive species, exhibit both enhanced photostability and biological compatibility. However, the self-healing strategy has yet to achieve its predicted potential, particularly in the presence of ambient oxygen where live-cell imaging studies must often be performed. To identify key bottlenecks in this technology that can be used to guide further engineering developments, we synthesized a series of Cy5 derivatives linked to the protective agent cyclooctatetraene (COT) and examined the photophysical mechanisms curtailing their performance. The data obtained reveal that the photostability of self-healing fluorophores is limited by reactivity of the COT protective agent. The addition of electron withdrawing substituents to COT reduced its susceptibility to reactions with molecular oxygen and the fluorophore to which it is attached and increased its capacity to participate in triplet energy transfer. Exploiting these insights, we designed and synthesized a suite of modified COT-fluorophores spanning the visible spectrum that exhibited markedly increased intra-molecular photostabilization. Under ambient oxygen conditions, the photostability of Cy3 and Cy5 fluorophore derivatives increased by 3- and 9-fold in vitro and by 2- and 6-fold in living cells, respectively. We further show that this approach can improve a silicon rhodamine fluorophore. These findings offer a clear strategy for achieving the full potential of the self-healing strategy and its application to the gamut of fluorophore species commonly used for biomedical imaging.

Approaches to Characterize and Quantify Oligomerization of GPCRs.

Fluorescence resonance energy transfer (FRET) is an approach widely used to detect protein-protein interactions in live cells. This approach is based on the sensitization of an "acceptor" molecule by the energy transfer from a "donor" when there is an overlap between the emission spectrum of the "donor" and the excitation spectrum of the "acceptor" and close proximity between the two fluorophore species (in the region of 8 nm). Various methods exist to quantify FRET signals: here, we describe the application of homogeneous time-resolved FRET (htrFRET) combined with Tag-lite™ technology and its application to determine not only protein-protein interactions but also the capability of GPCR mutant variants to form homomers compared to the wild type GPCR within the plasma membrane of transfected cells.

Multicolor Super-Resolution Imaging using a Laser Line Scanning Hyperspectral Microscope

Single-molecule super-resolution imaging techniques have proven to be successful in observing cellular structures with a resolution below the diffraction limit. However, these experiments often restrict observations to a region of shallow z-depth near the cover slip due to TIRF imaging or else suffer from high backgrounds due to wide field excitation. Confocal microscopy, which offers good background rejection, is prohibitively slow and cannot be used effectively for single-molecule localization based super-resolution. Here we introduce a new method that uses a fast, laser line scanning system that can be used to image a z-section anywhere within a cell and is not restricted to regions near the cover slip. The line scanning system allows a trade-off between acquisition speed and background rejection. Emission light is passed through a prism spectrometer, and a complete spectrum is collected for each spatial pixel. We exploit this hyperspectral emission path to make simultaneous measurements of multiple fluorophore species in the same sample.

Dual-View Imaging System Using a Wide-Range Dichroic Mirror for Simultaneous Four-Color Single-Molecule Detection

A dual-view imaging system for simultaneous four-color single-molecule (SM) detection was developed. As for the detection procedure, four species of SM fluorophores, namely, Alexa 488, 555, 647, and 680, are immobilized on different slides and excited by evanescent-wave illumination. Fluorescence emitted from an SM fluorophore is split by a wide-range dichroic mirror (WR DM) in a dual-view optics and imaged as two SM fluorescence spots (SM spots) on an electron-multiplying charge-coupled device (EM-CCD) at 100 Hz. The transmittance of the WR DM changes gradually over the wavelength range of 500 to 700 nm so that the signal ratios of the two SM spots for the four fluorophore species differ. A method for classifying SM fluorophores into four species in accordance with their signal ratios was developed. It was used to classify 597 SM fluorophores at an accuracy of above 98% for all the species. This accuracy is comparable to that of a conventional four-color SM detection system. To classify four species, the conventional system disperses SM fluorescence with a prism and provides an elongated SM spot that uses more pixels of an EM-CCD chip than that of the developed system. The developed system can thus detect 1.5-fold more SM spots with the same-size EM-CCD chip, so it can achieve 1.5-fold higher throughput. Moreover, the developed system is based on a simple and practical approach, namely, replacing an ordinary dichroic mirror in a commercially available dual-view optics with a WR DM. This replacement transforms a dual-view imaging system for two-color detection into a system for four-color detection. The developed system is suitable for detection systems of next-generation DNA sequencers and DNA microarray-chip analyzers.

Simultaneous four-color imaging of single molecule fluorophores using dichroic mirrors and four charge-coupled devices

We developed a total-internal-reflection (TIR) fluorescence microscopy using three dichroic mirrors and four charge-coupled devices (CCDs) to detect simultaneously four colors of single-molecule (SM) fluorophores. Four spectrally distinct species of fluorophores (Alexa 488, Cy3, Cy5, or Cy5.5) were each immobilized on a different fused silica slide. A species of fluorophores on the slide was irradiated simultaneously, by two excitation beams from an Ar ion laser (488 and 514.5 nm) and a diode laser (642 nm) through TIR on the slide surface. Fluorescence emitted from the fluorophores was spectrally resolved into four components by the dichroic mirrors, and four images were generated from them simultaneously and continuously, with the four CCDs at a rate of 10 Hz. A series of images was thus obtained with each CCD. Fluorescence spots for a species were observed mainly in the series of images recorded by its respective-color CCD. In the first image in the series, we picked out the spots as continuous pixel regions that had the values greater than a threshold. Then we selected only those spots that exhibited single-step photobleaching and regarded them as SM fluorescence spots. Pixel values of SM fluorescence spots widely differed. Some SM fluorophores had pixel values smaller than the threshold, and were left unpicked. Assuming the pixel values of SM fluorescence spots differed with a Gaussian profile, we estimated the ratios of unpicked fluorophores to be less than 20% for all the species. Because of the spectral overlaps between species, we also observed cross-talk spots into CCDs other than the respective-color CCDs. These cross-talk SM fluorescence spots can be mistaken for correct species. We thus introduced the classification method and classified SM fluorescence spots into correct species in accordance with two kinds of four-dimensional signal vectors. The error rates of fluorophore classification were estimated to be less than 3.2% for all the species. Our system is suitable for the biological studies that desire to simultaneously monitor the four colors of SM fluorophores.

Synthesis of cubic ordered mesoporous YPO4:Ln3+ and their photoluminescence properties

Cubic ordered mesoporous yttrium phosphate (YPO4) and lanthanide-doped yttrium phosphates (YPO4:Ln3+, Ln3+ = Eu3+, Tb3+ and Ce3+) have been synthesized successfully through the nanocasting route by employing the Y(NO3)3:Ln(NO3)3/H3PO4/HNO3 system as a guest unit and KIT-6 silica as a hard template host, and their textural and structure properties were characterized by X-ray diffraction, transmission electronic microscope and nitrogen adsorption at low temperature. The results show that the cubic ordered mesoporous YPO4 and YPO4:Ln3+ materials exhibit high surface area, large pore volume and uniform pore size distribution. Photoluminescence (PL) measurements exhibit their optical properties such as red, green and blue emission, and their PL intensities can be altered by the dopant Ln3+ concentration, without the need for doping or grafting external fluorophore species.

Dual-Mode Fluorophore-Doped Nickel Nitrilotriacetic Acid-Modified Silica Nanoparticles Combine Histidine-Tagged Protein Purification with Site-Specific Fluorophore Labeling

We present the first example of a fluorophore-doped nickel chelate surface-modified silica nanoparticle that functions in a dual mode, combining histidine-tagged protein purification with site-specific fluorophore labeling. Tetramethylrhodamine (TMR)-doped silica nanoparticles, estimated to contain 700-900 TMRs per ca. 23 nm particle, were surface modified with nitrilotriacetic acid (NTA), producing TMR-SiO2-NTA-Ni2+. Silica-embedded TMR retains very high quantum yield, is resistant to quenching by buffer components, and is modestly quenched and only to a certain depth (ca. 2 nm) by surface-attached Ni2+. When exposed to a bacterial lysate containing estrogen receptor alpha ligand binding domain (ERalpha) as a minor component, these beads showed very high specificity binding, enabling protein purification in one step. The capacity and specificity of these beads for binding a his-tagged protein were characterized by electrophoresis, radiometric counting, and MALDI-TOF MS. ERalpha, bound to TMR-SiO2-NTA-Ni++ beads in a site-specific manner, exhibited good activity for ligand binding and for ligand-induced binding to coactivators in solution FRET experiments and protein microarray fluorometric and FRET assays. This dual-mode type TMR-SiO2-NTA-Ni2+ system represents a powerful combination of one-step histidine-tagged protein purification and site-specific labeling with multiple fluorophore species.

Time-resolved fluorescence spectroscopy of fulvic acid and fulvic acid complexed with Eu 3+ – a comparative study

In this work a systematic characterization of the fluorescence kinetics of fulvic acid by means of a single-photon time correlation technique is reported. The data were extracted from both single decay curves and time-resolved emission spectra and analyzed by the exponential series method. The results strongly suggest the existence of a tri-modal decay time distribution with peak decay time values centred around 0.7, 3 and 10 ns. The relative amplitudes of the decay time distributions were shown to depend on the emission wavelength, indicating a three-species mixture with slightly different emission maxima and widths. As sustained by time-resolved emission spectra the same species of fluorophores are responsible for the fluorescence kinetics behaviour of fulvic acid at all excitation wavelengths used in the experiments. For the first time these techniques and methods were applied in investigating the effects of the Eu 3+ complexation upon fluorescence kinetics of the fulvic acid. Single decay analysis indicates that the peak decay time values are not affected by Eu 3+ addition at all emission and excitation wavelengths used in the experiments. On the contrary, the dependency of relative amplitudes on emission wavelength was greatly reduced in comparison with the uncomplexed fulvic acid. This effect is further evidenced by analysing the barycenter transients of time-resolved emission spectra. Finally, it is also indicated that within the temporal resolution of the experimental set up used the singlet states play no role as donor states in intramolecular energy transfer from the organic fulvic acid ligands to the Eu 3+ energy levels.

A fluorescence quenching study on protoporphyrin IX in a model membrane system

The interaction of protoporphyrin IX (3,7,12,15-tetramethyl-8,13-divinyl -2,18-porphyrine-dipropionic acid) (PPIX) with unilamellar dimyristoyl-L-α-phosphatidylcholine (DMPC) phospholipid vesicles has been studied by means of steady-state fluorescence quenching spectroscopy. The method of fluorescence-quenching-resolved spectroscopy has been applied in order to resolve the complex emission spectrum of a membrane-bound PPIX into two component spectra, attributed to distinct fluorophore species with different accessibilities to the iodide quencher. It is shown that PPIX associated with liposomes exists in two different microenvironments. One part of the fluorophore is embedded inside the lipid bilayer and is inaccessible to iodide. Its fluorescence spectrum exhibits the maximum characteristic of protoporphyrin found in the apolar medium. The other fraction of PPIX is located near the membrane surface, close to the polar phospholipid heads. Its emission is blue-shifted, resembling that of PPIX in a polar environment. It is quenched by iodide, although it reveals significant shielding from the quencher as compared to a buffer PPIX solution. Fluorescence quenching using l-oxyl-4-oxo-2,2,6,6-tetramethyl-piperidine (TEMPONE) does not discriminate between the two protoporphyrin species. However, the accessibility of protoporphyrin IX to this quencher is much lower in a liposome system than in water.