Spiropyran-based photochromic polymer nanoparticles with optically switchable luminescence

Polymer nanoparticles of 40-400 nm diameter with spiropyran-merocyanine dyes incorporated into their hydrophobic cavities have been prepared; in contrast to their virtually nonfluorescent character in most environments, the merocyanine forms of the encapsulated dyes are highly fluorescent. Spiro-mero photoisomerization is reversible, allowing the fluorescence to be switched "on" and "off" by alternating UV and visible light. Immobilizing the dye inside hydrophobic pockets of nanoparticles also improves its photostability, rendering it more resistant than the same dyes in solution to fatigue effects arising from photochemical switching. The photophysical characteristics of the encapsulated fluorophores differ dramatically from those of the same species in solution, making nanoparticle-protected hydrophobic fluorophores attractive materials for potential applications such as optical data storage and switching and biological fluorescent labeling. To evaluate the potential for biological tagging, these optically addressable nanoparticles have been delivered into living cells and imaged with a liquid nitrogen-cooled CCD.     

Electrochemical Modulation of the Fluorescence of Cyanine Dye Cy5

Organic fluorescent dyes are widely used in single molecule localization microscopy, where their performances are determined by the photophysical properties. Herein, we utilized a sensitive method to modulate the fluorescence of organic dyes by external potentials using a combination of electrochemical cell and super‐resolution fluorescent microscopy. Cy5 (cyanine dye) was chosen as a model molecule considering its wide application and commercial availability. We applied different potentials on the Au electrode to change the Coulombic charge microenvironment of Cy5. When the electrode potential was adjusted negatively, Cy5 displayed a better photostability. This method is proved effective in adjusting the fluorescence of organic dyes.     

Photophysics of New Water‐Soluble Terrylenediimide Derivatives and Applications in Biology

The photophysical properties of three new water‐soluble terrylenediimide (WS‐TDI) derivatives are investigated and their utilization in biological experiments is demonstrated. Each of these dyes can be excited in the far red region of the visible spectrum, making them good candidates for in‐vivo studies. Single‐molecule techniques characterize their photophysics that is, the number of emitted photons, blinking characteristics and survival times until photobleaching takes place. All three dyes exhibit bright fluorescence, as well as an extremely high resistance against photodegradation compared to other well‐known fluorophores. Due to their different characteristics the three new WS‐TDI derivatives are suitable for specialized biological applications. WS‐TDI dodecyl forms non‐fluorescent aggregates in water which can be disrupted in a hydrophobic environment leading to a monomeric fluorescent form. Due to its high lipophilicity WS‐TDI dodecyl anchors efficiently in lipid bilayers with its alkyl chain and hence can be ideally used to image membranes and membrane‐containing compartments in living cells. In contrast, the positively charged WS‐TDI pyridoxy is a new type of chromophore in the WS‐TDI family. It is fully solubilized in water forming fluorescent monomers and is successfully used to label the envelope of herpes simplex viruses. Finally, it is shown that a WS‐TDI derivative functionalized with N‐hydroxysuccinimide ester moiety (WS‐TDI/NHS ester) provides a versatile reactive dye molecule for the specific labelling of amino groups in biomolecules such as DNA.     

Reversible Live-Cell Labeling with Retro-engineered HaloTags Enables Long-Term High- and Super-Resolution Imaging

Self-labeling enzymes (SLE) such as the HaloTag have emerged as powerful tools in high and super-resolution fluorescence microscopy. Newly developed fluorogenic SLE substrates enable imaging in the presence of excess dye. To exploit this feature for reversible labeling, we engineered two variants of HaloTag7 with restored dehalogenase activity. Kinetic studies in vitro showed different turnover kinetics for reHaloTagS (≈0.006 s⁻¹) and reHaloTagF (≈0.055 s⁻¹). Imaging by confocal and stimulated emission depletion microscopy yielded 3-5-time enhanced photostability of reHaloTag labeling. Prominently, single molecule imaging with reHaloTags enabled controlled and stable labeling density over extended time periods. By combination with structured illumination, simultaneous visualization of single molecule diffusion and organellar dynamics was achieved. These applications highlight the potential of reHaloTag labeling for pushing the limits of advanced fluorescence microscopy techniques.     

Photoactivatable Carbo- and Silicon-Rhodamines and Their Application in MINFLUX Nanoscopy

New photoactivatable fluorescent dyes (rhodamine, carbo- and silicon-rhodamines [SiR]) with emission ranging from green to far red have been prepared, and their photophysical properties studied. The photocleavable 2-nitrobenzyloxycarbonyl unit with an alpha-carboxyl group as a branching point and additional functionality was attached to a polycyclic and lipophilic fluorescent dye. The photoactivatable probes having the HaloTagTM amine (O2) ligand bound with a dye core were obtained and applied for live-cell staining in stable cell lines incorporating Vimentin (VIM) or Nuclear Pore Complex Protein NUP96 fused with the HaloTag. The probes were applied in 2D (VIM, NUP96) and 3D (VIM) MINFLUX nanoscopy, as well as in superresolution fluorescence microscopy with single fluorophore activation (VIM, live-cell labeling). Images of VIM and NUPs labeled with different dyes were acquired and their apparent dimensions and shapes assessed on a lower single-digit nanometer scale. Applicability and performance of the photoactivatable dye derivatives were evaluated in terms of photoactivation rate, labeling and detection efficiency, number of detected photons per molecule and other parameters related to MINFLUX nanoscopy.     

新型水溶性不对称五甲川菁染料的合成、光稳定性及蛋白质的荧光标记

合成并表征了系列水溶性五甲川菁染料, 研究了其在不同溶剂中的光谱性能. 结果表明, 染料在水中的最大吸收和荧光光谱在647~665 nm波长范围内, 荧光量子产率达到0.1左右. 考察了N位取代基对染料水溶液光稳定性的影响, 结果表明, 在N原子上引入带有苯环结构和大体积的磺酸基, 可以提高染料的光稳定性. 高效液相色谱(HPLC)分析结果表明, 染料4a的N-羟基琥珀酰亚胺(NHS)活性酯标记牛血清白蛋白(BSA)的检测限为1.2×10^-8 mol/L, 与紫外检测相比, 检测灵敏度提高了近2个数量级.     

Geminate Recombination as a Photoprotection Mechanism for Fluorescent Dyes

Despite common presumption due to fast photodestruction pathways through higher excited states, we show that further improvement of photostability is still achievable with diffusion‐limited photoprotection formulas. Single‐molecule fluorescence spectroscopy reveals that thiolate ions effectively quench triplet states of dyes by photoinduced electron transfer. Interestingly, this reaction rarely yields a radical anion of the dye, but direct return to the ground state is promoted by an almost instantaneous back electron transfer (geminate recombination). This type of mechanism is not detected for commonly used reductants such as ascorbic acid and trolox. The mechanism avoids the formation of radical cations and improves the photostability of single fluorophores. We find that a combination of β‐mercaptoethanol and classical reducing and oxidizing systems yields the best results for several dyes including Atto532 and Alexa568.     

Fluorogenic and Cell-Permeable Rhodamine Dyes for High-Contrast Live-Cell Protein Labeling in Bioimaging and Biosensing

The advancement of fluorescence microscopy techniques has opened up new opportunities for visualizing proteins and unraveling their functions in living biological systems. Small-molecule organic dyes, which possess exceptional photophysical properties, small size, and high photostability, serve as powerful fluorescent reporters in protein imaging. However, achieving high-contrast live-cell labeling of target proteins with conventional organic dyes remains a considerable challenge in bioimaging and biosensing due to their inadequate cell permeability and high background signal. Over the past decade, a novel generation of fluorogenic and cell-permeable dyes has been developed, which have substantially improved live-cell protein labeling by fine-tuning the reversible equilibrium between a cell-permeable, nonfluorescent spirocyclic state (unbound) and a fluorescent zwitterion (protein-bound) of rhodamines. In this review, we present the mechanism and design strategies of these fluorogenic and cell-permeable rhodamines, as well as their applications in bioimaging and biosensing.     

Red‐Emitting Rhodamine Dyes for Fluorescence Microscopy and Nanoscopy

Fluorescent markers emitting in the red are extremely valuable in biological microscopy since they minimize cellular autofluorescence and increase flexibility in multicolor experiments. Novel rhodamine dyes excitable with 630 nm laser light and emitting at around 660 nm have been developed. The new rhodamines are very photostable and have high fluorescence quantum yields of up to 80 %, long excited state lifetimes of 3.4 ns, and comparatively low intersystem‐crossing rates. They perform very well both in conventional and in subdiffraction‐resolution microscopy such as STED (stimulated emission depletion) and GSDIM (ground‐state depletion with individual molecular return), as well as in single‐molecule‐based experiments such as fluorescence correlation spectroscopy (FCS). Syntheses of lipophilic and hydrophilic derivatives starting from the same chromophore‐containing scaffold are described. Introduction of two sulfo groups provides high solubility in water and a considerable rise in fluorescence quantum yield. The attachment of amino or thiol reactive groups allows the dyes to be used as fluorescent markers in biology. Dyes deuterated at certain positions have narrow and symmetrical molecular mass distribution patterns, and are proposed as new tags in MS or LC‐MS for identification and quantification of various substance classes (e.g., amines and thiols) in complex mixtures. High‐resolution GSDIM images and live‐cell STED‐FCS experiments on labeled microtubules and lipids prove the versatility of the novel probes for modern fluorescence microscopy and nanoscopy.     

Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores

To obtain high-resolution information on position or conformation of a molecule and at the same time apply forces to it, one can combine optical trapping with single-molecule fluorescence microscopy. The technical challenge in such an experiment is to discriminate a minute fluorescence signal from the much larger background signals caused by the trap and the fluorescence excitation laser light. We show here that this is feasible even when the fluorophore is directly attached to the trapped particle, by using optimized optical filters. We found, however, that the photostability of the fluorophores we tested suffered from the presence of the additional laser light used for trapping. We found that bleaching rates increased linearly with both the intensity of the trapping laser and the intensity of the fluorescence excitation light. Photobleaching rates were unaffected by the presence or absence of oxygen, but were significantly diminished in the presence of antioxidants. Our results indicate that the enhanced photobleaching is caused by the absorption of a visible photon followed by the excited-state absorption of a near-infrared photon. The higher excited singlet states generated in this way readily form nonfluorescent dye cations. We found that different dyes suffer to a different extent from the excited-state absorption, with Cy3 being worst and tetramethylrhodamine least affected.     

On the photostability of some benzanthrone dye derivatives and their copolymers with methylmethacrylate

The photostability of five benzanthrone dye derivatives containing unsaturated allylic group(s) was investigated and the structure-photostability relationship was found. Their copolymers with methylmethacrylate had an intense colour, and were stable to solvents and fluorescence, due to the chemical bonding of the dye in the polymer chain. Photostability of the dyes thus included in the polymer molecule increased and their influence on the photostability of the polymers was determined.     

Molecular assemblies of perylene bisimide dyes in water

Perylene bisimides are among the most valuable functional dyes and have numerous potential applications. As a result of their chemical robustness, photostability, and outstanding optical and electronic properties, these dyes have been applied as pigments, fluorescence sensors, and n‐semiconductors in organic electronics and photovoltaics. Moreover, the extended quadrupolar π system of this class of dyes has facilitated the construction of numerous supramolecular architectures with fascinating photophysical properties. However, the supramolecular approach to the formation of perylene bisimide aggregates has been restricted mostly to organic media. Pleasingly, considerable progress has been made in the last few years in developing water‐soluble perylene bisimides and their application in aqueous media. This Review provides an up‐to‐date overview on the self‐assembly of perylene bisimides through π–π interactions in aqueous media. Synthetic strategies for the preparation of water‐soluble perylene bisimides and the influence of water on the π–π stacking of perylene bisimides as well as the resulting applications are discussed.     

Characterization of a single molecule DNA switch in free solution

We have studied a donor-acceptor fluorophore-labeled DNA switch where the acceptor is Alexa-647, a carbocyanine dye, in solution at the single molecule level to elucidate the fluorescence switching mechanism. The acceptor, which is in an initial high fluorescence trans state, undergoes a photoisomerization reaction resulting in two additional states during its sub-millisecond transit across the probe volume. These two states are assigned to a nonfluorescent triplet trans state that strongly quenches the donor emission and a singlet cis state that blocks the fluorescence resonance energy transfer (FRET) pathway and gives rise to donor-only fluorescence. The formation of these states is faster than the transit time, so that all three states are approximately equally populated under our experimental conditions. The acceptor dye can stick to the DNA in all these states, with the rate of unsticking determining the rate of isomerization into the other states. Measurement of the rate of change of the FRET signal therefore provides information about the fluorophore-DNA intramolecular dynamics. These results explain the large zero peak in the proximity ratio, often seen in single molecule FRET experiments, and suggest that photoinduced effects may be important in single molecule FRET experiments using carbocyanine dyes. They also suggest that for fast photoinduced switching the interactions of the acceptor dye with the DNA and other surfaces should be prevented.     

A Highly Photostable Near‐Infrared Labeling Agent Based on a Phospha‐rhodamine for Long‐Term and Deep Imaging

Various fluorescence microscopy techniques require bright NIR‐emitting fluorophores with high chemical and photostability. Now, the significant performance improvement of phosphorus‐substituted rhodamine dyes (PORs) upon substitution at the 9‐position with a 2,6‐dimethoxyphenyl group is reported. The thus obtained dye PREX 710 was used to stain mitochondria in living cells, which allowed long‐term and three‐color imaging in the vis‐NIR range. Moreover, the high fluorescence longevity of PREX 710 allows tracking a dye‐labeled biomolecule by single‐molecule microscopy under physiological conditions. Deep imaging of blood vessels in mice brain has also been achieved using the bright NIR‐emitting PREX 710‐dextran conjugate.     

Squaraine-derived rotaxanes: highly stable, fluorescent near-IR dyes

Squaraines are fluorescent, near-IR dyes with promising photophysical properties for biomedical applications. A limitation with these dyes is their inherent reactivity with nucleophiles, which leads to loss of the chromophore. Another drawback is their tendency to form nonfluorescent aggregates in water. Both problems can be greatly attenuated by encapsulating the dye inside an amide-containing macrocycle. In other words, the squaraine becomes the thread component in a Leigh-type rotaxane, a permanently interlocked molecule. Two new rotaxanes are described: an analogue with four tri(ethyleneoxy) chains on the squaraine to enhance water solubility, and a rotaxane that has an encapsulating macrocycle with transposed carbonyl groups. An X-ray crystal structure of the latter rotaxane shows that the macrocycle provides only partial protection of the electrophilic cyclobutene core of the squaraine thread. The stabilities of each compound in various solvents, including serum, were compared with a commercially available cyanine dye. The squaraine rotaxane architecture is remarkably resistant to chemical and photochemical degradation, and likely to be very useful as a versatile fluorescent scaffold for constructing various types of highly stable, near-IR imaging probes.     

Polymeric fluorescent dyes for labeling of proteins and nucleic acids

In order to increase the sensitivity of fluorescence labeling in biochemical reactions and diagnostic procedures a labeling technique with polymeric fluorescence dyes was established and tested for its applicability. The fluorescence dye is based on the fluorophor coumarine and was covalently linked to the model proteins strepavidine and IgG. The dye was synthesized by radical polymerization of three different types of functional monomers to ensure water solubility, covalent coupling to proteins, and fluorescence. The molecular weight range was between 20 and 200 kDa. Fractions of narrow molecular weight distribution were prepared by gel filtration on Superdex 200. The relationship between size and charge of the different fractions was analyzed by gel electrophoresis. Covalent conjugation to proteins was carried out by formation of a peptide bond between a carboxylic group of the functional monomers and an amino group of the protein mediated by 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC). A novel type of gel electrophoresis was developed in order to analyze and optimize the conjugation reaction; the results were in agreement with those from analytical ultracentrifugation with fluorescence detection. Hydrodynamic studies of the uncoupled dye and the protein-dye conjugates exhibited a drastic decrease of Stokes radius of the dye due to the coupling to the protein. Under optimum conditions the fluorescence intensity of a protein-polymeric dye conjugate was enhanced 40-fold compared to a monomeric dye. Biotin binding to the protein streptavidin was not affected significantly by the conjugation with the polymeric dye. At present, the applicability of the polymeric dye in biochemical and diagnostic reactions seems to be limited due to strong but unspecific hydrophobic interactions which might be overcome by using fluoresceine as monomeric dye.     

近红外聚乙烯醇荧光高分子材料的制备及性能

以一种方酸菁染料、水溶性石墨烯和聚乙烯醇为原料,设计合成了在近红外区具有强荧光(660~665 nm)特性及良好的光热稳定性的方酸菁/聚乙烯醇二元和方酸菁/石墨烯/聚乙烯醇三元高分子材料;与在水中相比,方酸菁染料在聚乙烯醇中的最大紫外-可见吸收和荧光波长红移,荧光强度和光稳定性大幅提高;石墨烯的存在增强了材料的光稳定性。     

Intriguing H-aggregate and H-dimer formation of coumarin-481 dye in aqueous solution as evidenced from photophysical studies

Photophysical properties of coumarin-481 (C481) dye in aqueous solution show intriguing presence of multiple emitting species. Concentration and wavelength dependent fluorescence decays and time-resolved emission spectra and area-normalized emission spectra suggest the coexistence of dye monomers, dimers, and higher aggregates (mostly trimers) in the solution. Because of the efficient intramolecular charge transfer (ICT) state to twisted intramolecular charge transfer (TICT) state conversion, the dye monomers show very short fluorescence lifetime of ~0.2 ns. Fluorescence lifetimes of dimers (~4.1 ns) and higher aggregates (~1.4 ns) are relatively longer due to steric constrain toward ICT to TICT conversion. Observed results indicate that the emission spectra of the aggregates are substantially blue-shifted compared to monomers, suggesting H-aggregation of the dye in the solution. Temperature-dependent fluorescence decays in water and time-resolved fluorescence results in water-acetonitrile solvent mixtures are also in support of the dye aggregation in the solution. Though dynamic light scattering studies could not recognize the dye aggregates in the solution due to their small size and low concentration, fluorescence up-conversion measurements show a relatively higher decay tail in water than in water-acetonitrile solvent mixture, in agreement with higher dye aggregation in aqueous solution. Time-resolved fluorescence results with structurally related non-TICT dyes, especially those of coumarin-153 dye, are also in accordance with the aggregation behavior of these dyes in aqueous solution. To the best of our knowledge, this is the first report on the aggregation of coumarin dyes in aqueous solution. Present results are important because coumarin dyes are widely used as fluorescent probes in various microheterogeneous systems where water is always a solvent component, and the dye aggregation in these systems, if overlooked, can easily lead to a misinterpretation of the observed results.     

Characterization of a novel water-soluble 3,4,9,10-perylenetetracarboxylic diimide in solution and in self-assembled zirconium phosphonate thin films

The properties of N,N'-bis(2-phosphonoethyl)-3,4,9,10-perylenetetracarboxylic diimide (PPDI), a water-soluble perylene dye, have been studied in solution and in thin films. Absorption spectra showed that PPDI exists in the monomeric form in water/ethanol (1:1) and water/dimethyl sulfoxide (DMSO) (3:7) mixtures, but forms dimers in water and higher aggregates in ethanol. The PPDI monomer is highly fluorescent, in contrast to the dimers and aggregates, which are nonfluorescent. The monomer/dimer equilibrium was conveniently followed in a water/ethanol (7:3) mixture by varying the dye concentration. An equilibrium constant of K = 1.25 x 10(5) M(-1) was estimated for the dimerization process in this solvent mixture. The addition of cetyl trimethylammonium bromide (CTAB), a cationic surfactant, to aqueous solutions of PPDI resulted in the dissociation of the dimers, showing that the dye was incorporated into the micellar phase. Self-assembled thin films of PPDI were grown on both silica gel particles and flat surfaces, using zirconium phosphonate chemistry. The growth of multilayered films on flat surfaces was monitored by ellipsometry (silicon substrates) and UV/Vis spectroscopy (quartz slides), and was linear with the number of deposition cycles. No fluorescence was detected from the PPDI films, and the absorption spectra of the films were quite similar to the spectrum of the compound in ethanol, indicating that the dye molecules were stacked in the films. Mixed monolayers containing PPDI and N,N'-bis(2-phosphonoethyl)-1,4,5,8-naphthalenediimide (PNDI) on quartz were also prepared. Monolayers obtained by codeposition from solutions containing both PPDI and PNDI were richer in PPDI, even when the solution contained a large excess of the naphthalene derivative, showing that pi-stacking of PPDI is an important driving force in the formation of the films.     

Single molecule fluorescence correlation spectroscopy of single apoptotic cells using a red-fluorescent caspase probe

The detection of single molecules in single cells has enabled biochemical analyses to be conducted with high sensitivity and high temporal resolution. In this work, detection of apoptosis was studied by single molecule fluorescence correlation spectroscopy (FCS) in single living cells. Caspase activity was assayed using a new red fluorogenic probe that avoids the spectral overlap of green fluorescent probes and cell autofluorescence. This new probe, 2SBPO-Casp, was synthesized by coupling a water-soluble Nile Blue derivative (2SBPO) to an aspartic acid residue. Upon apoptosis induction and caspase activation, free 2SBPO dye is shown to accumulate inside the cell after probe cleavage. In previous work in our lab, single molecule fluorescence in single apoptotic cells was detected 45 min after induction using a rhodamine 110-based probe. However, significant statistical analysis was needed to exclude false positives. The use of 2SBPO-Casp overcomes the autofluorescence problem and offers a steady fluorescence signal. In our single molecule FCS measurements, Ramos cells were determined apoptotic on the basis of their correlation coefficient value (R(2)). Cells that contain an R(2) ≥ 0.65 were identified as highly correlated and therefore determined to be apoptotic. Single apoptotic cells identified in this manner were found as early as 30 min after induction and the number of apoptotic cells reached a peak value at the 3rd hour, which is consistent with other techniques. Using single molecule techniques and a new apoptosis probe, the temporal dynamics were elucidated with better sensitivity and resolution than in previous studies.