Squaraine Dyes Exhibit Spontaneous Fluorescence Blinking That Enables Live-Cell Nanoscopy.

Hampered by their susceptibility to nucleophilic attack and chemical bleaching, electron-deficient squaraine dyes have long been considered unsuitable for biological imaging. This study unveils a surprising twist: in aqueous environments, bleaching is not irreversible but rather a reversible spontaneous quenching process. Leveraging this new discovery, we introduce a novel deep-red squaraine probe tailored for live-cell super-resolution imaging. This probe enables single-molecule localization microscopy (SMLM) under physiological conditions without harmful additives or intense lasers and exhibits spontaneous blinking orchestrated by biological nucleophiles, such as glutathione or hydroxide anion. With a low duty cycle (∼0.1%) and high-emission rate (∼6 × 104 photons/s under 400 W/cm2), the squaraine probe surpasses the benchmark Cy5 dye by 4-fold and Si-rhodamine by a factor of 1.7 times. Live-cell SMLM with the probe reveals intricate structural details of cell membranes, which demonstrates the high potential of squaraine dyes for next-generation super-resolution imaging.

Design of Stimuli-Responsive Phenothiazine Derivatives with Triplet-Related Dual Emission and High-Contrast Mechanochromism Guided by Polymorph Prediction.

The development of high-contrast stimulus-responsive materials with excited triplet emission is of great significance for anti-counterfeiting, sensor and memory applications, but remains a challenge. Here, we report a strategy for the rational design of stimulus-responsive phenothiazine derivatives with triplet-related dual emissions and high-contrast mechanochromism guided by Polymorph Prediction. The designed phenothiazine derivatives have the characters of simple structures, a facile synthetic procedure, and a good crystalline nature. We found that the crystals of those derivatives with the potential to form both quasi-axial (ax) and quasi-equatorial (eq) conformations could undergo conformation transition and show significant emission difference (Δλem >100 nm) under mechanical force. Meanwhile, all these phenothiazine derivatives exhibit aggregation-induced emission and emit room-temperature phosphorescence or thermally activated delayed fluorescence. The significant luminescent change of these materials under different stimuli gives them promise for applications in encryption and anti-counterfeiting.

Synergistic Lysosomal Activatable Polymeric Nanoprobe Encapsulating pH Sensitive Imidazole Derivative for Tumor Diagnosis.

Developing optical tumor imaging probes with minimal background noise is very important for its early detection of small lesions and accurate diagnosis of cancer. To overcome the bottleneck of low signal to noise ratio and sensitivity, it needs further improvement in fluorescent probe design and understanding of tumor development process. Recent reports reveal that lysosome's acidity in cancer cells can be below 4.5 with high Na+ /H+ exchange activity, which makes it an ideal target intracellular organelle for cancer diagnosis based on the variation of pH. Herein, a boron 2-(2'-pyridyl) imidazole complex derivative (BOPIM-N) is developed, with the ability to show a pH-activatable "OFF-ON" fluorescent switch by inhibiting twisted intramolecular charge transfer upon protonation at pH 3.8-4.5, which is studied for its selective viable cancer cell imaging ability in both in vitro and in vivo experiments. Interestingly, BOPIM-N can specifically emit green fluorescence in lysosomes of cancer cells, indicating its promising cancer cell specific imaging ability. More importantly, nanoformulated BOPIM-N probes can be specifically light-ON in tumor bearing site of nude mice with resolution up to cellular level, indicating its potential application in tumor diagnosis and precision medicine.

A Water-Soluble Fluorescent Probe for SO2 Derivatives in Aqueous Solution and Serum Based on Phenanthroimidazole Dye.

A water-soluble fluorescent SO2 derivatives probe PI-SO 2 based on a phenanthroimidazole dye, and a sensitive SO2 recognition site, aldehyde was constructed. The probe PI-SO 2 exhibits desirable properties such as high sensitivity, high selectivity and good water-solubility. Significantly, we have demonstrated that the probe PI-SO 2 is suitable for rapidly fluorescence detecting of SO2 derivatives in aqueous solution and serum. The application of the novel probe PI-SO 2 proved that it was not only a useful tool for the detection of SO2 derivatives in vitro, but also a potential assay for investigating the effects of SO2 derivatives, and demonstrating its value in practical applicationin of complex biological samples.

One Stone, Three Birds: Design and Synthesis of "All-in-One" Nanoscale Mn-Porphyrin Coordination Polymers for Magnetic Resonance Imaging-Guided Synergistic Photodynamic-Sonodynamic Therapy.

Multifunctional nanomaterials with potential applications in both bioimaging and photodynamic-sonodynamic therapy have great advantages in cancer theranostic, but the design and preparation of "all-in-one" type of multifunctional nanomaterials with single component remains challenging. Herein the "all-in-one" type of Mn-PpIX (Protoporphyrin IX) coordination polymers (MnPPs) was reported as efficient nano-photo/sonosensitizers. The MnPPs had an average size of âˆ¼ 110 nm. Upon light/US (ultrasound) irradiation for 5 min, 61.8 % (light) and 32.4 % (US) of DPBF (1.3-diphenyl isobenzofuran) was found to be oxidized by MnPPs, which showed effective ROS (reactive oxygen species) generation for photodynamic/sonodynamic therapy (PDT/SDT). In addition, MnPPs revealed excellent biosafety and could be engulfed by cells to produce intracellular ROS under light/US excitation for efficient killing tumor cells. When MnPPs was injected into mice, the tumor could be monitored via MRI (magnetic resonance imaging). In addition, tumor growth could be significantly inhibited by the synergistic PDT-SDT. Therefore, the present study not only represents MnPPs as an "all-in-one" type of multifunctional nanomaterials for MRI-guided PDT-SDT therapy, but also provides some insights for designing other PpIX-related molecules with the similar structure for bioapplication.

From biomaterials to biotherapy: cuttlefish ink with protoporphyrin IX nanoconjugates for synergistic sonodynamic-photothermal therapy.

Biologically produced nanomaterials capable of therapeutic purposes have received increasing interest in tumor therapy because of their intrinsic biocompatibility. In this study, we made cuttlefish ink (extracted from cuttlefish) and protoporphyrin IX (PpIX) nanoconjugates (CIPs) where PpIX was an endogenous organic compound. In the case of CIPs, PpIX could be triggered by ultrasound (US) for sonodynamic therapy (SDT), and the cuttlefish ink could be excited by a near-infrared laser for photothermal therapy (PTT). Thereafter, tumor growth was greatly inhibited through synergistic SDT-PTT in comparison to single SDT or PTT. In addition, in vivo administration of CIPs showed no noticeable side effects for mouse blood and chief organs, providing an effective strategy for developing biologically produced biomaterials and using them for biotherapy.

Enhanced cell death imaging using multivalent zinc(II)-bis(dipicolylamine) fluorescent probes.

There is a clinical need for imaging technologies that can accurately detect cell death in a multitude of pathological conditions. Zinc(II)-bis(dipicolylamine) (Zn2BDPA) coordination complexes are known to associate with the anionic phosphatidylserine that is exposed on the surface of dead and dying cells, and fluorescent monovalent Zn2BDPA probes are successful cell death imaging agents. This present study compared the membrane targeting ability of two structurally related deep-red fluorescent probes, bis-Zn2BDPA-SR and tetra-Zn2BDPA-SR, with two and four appended Zn2BDPA units, respectively. Vesicle and cell microscopy studies indicated that a higher number of Zn2BDPA targeting units improved probe selectivity for phosphatidylserine-rich vesicles, and increased probe localization at the plasma membrane of dead and dying cells. The fluorescent probes were also tested in three separate animal models, (1) necrotic prostate tumor rat model, (2) thymus atrophy mouse model, and (3) traumatic brain injury mouse model. In each case, there was more tetra-Zn2BDPA-SR accumulation at the site of cell death than bis-Zn2BDPA-SR. The results indicate that multivalent Zn2BDPA probes are promising molecules for effective imaging of cell death processes in cell culture and in living subjects.

Convenient Synthesis of Multivalent Zinc(II)-Dipicolylamine Complexes for Molecular Recognition.

A pair of novel dipicolylamine ligands bearing isothiocyanate groups were used as conjugation reagents to prepare multivalent molecules with anionic recognition capability. The isothiocyanates were reacted with two classes of dendritic scaffolds bearing primary amines, squaraine rotaxanes and PAMAM dendrimers, and the products were converted into water soluble zinc(II) coordination complexes. The multivalent squaraine rotaxanes exhibit high fluorescence quantum yields in water and are very well suited for biological imaging applications.

Water-soluble, deep-red fluorescent squaraine rotaxanes.

Eight fluorescent squaraine rotaxanes with deep-red absorption/emission wavelengths were prepared and assessed for chemical stability and suitability as water-soluble, fluorescent tracers. The most stable squaraine rotaxanes have four large stopper groups attached to the ends of the encapsulated squaraine, and two members of this structural class have promise as highly fluorescent tracers with rapid renal clearance and very low tissue uptake in living mice.

Utilization of Nonradiative Excited-State Dissipation for Promoted Phototheranostics Based on an AIE-Active Type I ROS Generator.

As for effective multimodal phototheranostic AIEgens, it is important to find strategies for manipulating photophysical dissipation to achieve optimized performance. Herein, a "all-in-one" phototheranostic AIEgen, (E)-3-(2-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)naphtho[1,2-d]thiazol-1-ium-1-yl)propane-1-sulfonate (NS-STPA) was constructed by a rigid coplanar grafting flexible rotor. NS-STPA nanoparticles (NPs) exhibited NIR fluorescent luminescence (FL) with φFL 2.78%. Upon 660 nm irradiation, the high photothermal conversion efficiency (39.01%) and effective reactive oxygen species (ROS) generation (5.28 times to Ce6) indicated the nonradiative decays are valuable in phototherapy. High •OH outputs showed NS-STPA NPs were outstanding type I ROS generators. The twisted D-A structure induced a large spin-orbit coupling (SOC), and insertion of thiophene decreased the S1-T1 energy gap (ΔEST). The nanoaggregate prolonged the triplet-state lifetime (τT). These all facilitate the intersystem crossing (ISC) for NS-STPA NPs. The photoinduced electron transfer resulted in •O2- and then •OH generation. In vivo evaluation indicated the promising application of NS-STPA NPs in FL and photothermal dual imaging-guided synergistic photodynamic and photothermal therapies.

In vivo targeting of cell death using a synthetic fluorescent molecular probe.

A synthetic, near-infrared, fluorescent probe, named PSS-794 was assessed for its ability to detect cell death in two animal models. The molecular probe contains a zinc(II)-dipicolylamine (Zn(2+)-DPA) affinity ligand that selectively targets exposed phosphatidylserine on the surface of dead and dying cells. The first animal model used rats that were treated with dexamethasone to induce thymic atrophy. Ex vivo fluorescence imaging and histological analysis of excised organs showed thymus uptake of PSS-794 was four times higher than a control fluorophore that lacked the Zn(2+)-DPA affinity ligand. In addition, the presence of PSS-794 produced a delayed and higher build up of dead and dying cells in the rat thymus. The second animal model employed focal beam radiation to induce cell death in tumor-bearing rats. Whole-body and ex vivo imaging showed that the amount of PSS-794 in a radiation-treated tumor was almost twice that in a non-treated tumor. The results indicate that PSS-794 may be useful for preclinical optical detection of tumor cell death due to therapy.

In vivo optical imaging of acute cell death using a near-infrared fluorescent zinc-dipicolylamine probe.

Cell death is a fundamental biological process that is present in numerous disease pathologies. Fluorescent probes that detect cell death have been developed for a myriad of research applications ranging from microscopy to in vivo imaging. Here we describe a synthetic near-infrared (NIR) conjugate of zinc(II)-dipicolylamine (Zn²+-DPA) for in vivo imaging of cell death. Chemically induced in vivo models of myopathy were established using an ionphore, ethanol, or ketamine as cytotoxins. The Zn²+-DPA fluorescent probe or corresponding control was subsequently injected, and whole animal fluorescence imaging demonstrated probe uptake at the site of muscle damage, which was confirmed by ex vivo and histological analyses. Further, a comparative study with a NIR fluorescent conjugate Annexin V showed less intense uptake at the site of muscle damage and high accumulation in the bladder. The results indicate that the fluorescent Zn²+-DPA conjugate is an effective probe for in vivo cell death detection and in some cases may be an appropriate alternative to fluorescent Annexin V conjugates.

High-contrast colourimetric probes for fluoride and trace water based on tautomerization of naphthalimide and application in fingerprint imaging.

Three probes for fluoride ion and trace water based on naphthalimide were designed and synthesized. A new sensing mechanism based on naphthalimide tautomerization induced by fluoride ion and water was explored in the aprotic organic solvent. In the fluoride ion sensing process, the probes exhibited a remarkable absorption peak centred at 560 nm in the visible range of 400-700 nm. When trace water presented, the newly formed absorption peak centred at 560 nm gradually disappeared. The sensitive colour variation of the probe also was used in fingerprint imaging. Accordingly, the significant changes in chemical shift of dept135 and 1HNMR spectrum confirmed the structural transformation of the probes with high contrast. Furthermore, this work also presented an optimization strategy for the sensitivity of the probe based on regulatory tautomerization.

Optical imaging of mammary and prostate tumors in living animals using a synthetic near infrared zinc(II)-dipicolylamine probe for anionic cell surfaces.

In vivo optical imaging shows that a fluorescent imaging probe, comprised of a near-infrared fluorophore attached to an affinity group containing two zinc(II)-dipicolylamine (Zn-DPA) units, targets prostate and mammary tumors in two different xenograft animal models. The tumor selectivity is absent with control fluorophores whose structures do not have appended Zn-DPA targeting ligands. Ex vivo biodistribution and histological analyses indicate that the probe is targeting the necrotic regions of the tumors, which is consistent with in vitro microscopy showing selective targeting of the anionic membrane surfaces of dead and dying cells.

Retinal-based polyene fluorescent probe for selectively detection of Cu2+ in physiological saline and serum.

Retinal is a flexible natural chromophore and widely present in organisms. The slender conjugated polyene structure retinal is conducive to entering protein structure. In this work, a novel turn-on fluorescent probe for Cu2+ based on retinal and phenylenediamine was designed and synthesized. The probe achieved recognition of copper ions in human serum complex protein environment. Furthermore, the high sensitivity, selectivity for Cu2+ and the sensing mechanism was also investigated.

Functionalized thiophene-based [7]helicene: chirooptical properties versus electron delocalization.

The functionalized, enantiomerically pure [7]helicene 1 derived from bis(benzodithiophene) functionalized with four heptyl groups is prepared from 1,8-dibromo-4,5-diheptylbenzo[1,2-b:4,3-b']dithiophene building block 2. Such [7]helicene structure, functionalized with bromines at the terminal positions of the helicene inner rim and multiple solubilizing alkyl groups, is an attractive building block for long [n]helicenes and oligo[7]helicenes. Chirooptical properties and the degree of electron delocalization are determined and compared to those of analogous carbon-sulfur [7]helicene and [7]helicenes derived from benzodithiophene to provide a correlation between chirooptical properties and the degree of electron delocalization. [7]Helicene 1 possesses a moderately increased electron delocalization, but its chirooptical properties are similar to those for analogous [7]helicenes with relatively lower electron delocalization, indicating that chirooptical properties are not significantly affected by electron delocalization for this series of [7]helicenes. Molecular structures of racemic [7]helicene 1 and its benzodithiophene building block 2 are confirmed by single-crystal X-ray analysis. Crystals of 2 are chiral and adopt the shape of long, flexible, flat needles that can be readily bent.

Intramolecular cyclization of thiophene-based [7]helicenes to quasi-[8]circulenes.

Intramolecular cyclization in a series of thiophene-based dibromo[7]helicenes (4-6) with different helix structures is investigated by vacuum pyrolysis, tin- and palladium-mediated C-C bond forming reactions. The product with the cyclic structure of the annelated aromatic rings, which resembles [8]circulene devoid of an atom linkage, is referred to as quasi-[8]circulene. Vacuum pyrolysis of 4 gives insoluble, unidentified products, while 5 and 6 yield the corresponding quasi-[8]circulenes under similar conditions. Thermogravimetry (TG) and differential scanning calorimetry (DSC) analyses for 4 indicate complex reaction pathways, while those for 5 and 6 show a single process corresponding to a loss of 1 equiv of Br(2) at about 330 degrees C. Pd-mediated reductive cyclization provides quasi-[8]circulenes for all three [7]helicenes, though only 4 gives a good isolated yield. Tributyltin hydride-mediated radical cyclization of 4-6 provides quasi-[8]circulenes in excellent yields, and it is practically insensitive to the helix structure. Experimental and calculated UV-vis absorption spectra for quasi-[8]circulenes and [8]circulenes are reported. The results suggest that the lack of atom linkage in quasi-[8]circulene does not significantly affect properties and conformation, compared to those for the corresponding [8]circulenes.

Aggregation-induced emission enhancement (AIEE)-active boron-difluoride dyes with reversible mechanochromic fluorescence.

A novel class of solid-emissive boron-difluoride derivatives, using phenanthrenequinone hydrazone as ligands, were designed and efficiently synthesized. These dyes exhibit weak fluorescence in dilute solutions, but much higher fluorescence efficiency in aggregate states with a large stokes shift (over 70 nm) due to the their aggregation-induced emission enhancement (AIEE) characteristics. According to their photophysical properties and X-ray single crystal structure analysis, the AIEE was ascribed to the H(J)-aggregate formation aided by multiple intermolecular interactions to restrict intramolecular motion in the solid state. Moreover, their solid emissions could be reversibly tuned between "on" and "off" by mechanical grinding and recrystallization, due to the stacking model transition between H(J)-aggregation with loose molecular packing and J-aggregation with intense intermolecular interactions.

A novel 2,5-bis(benzo[d]thiazol-2-yl)phenol scaffold-based ratiometric fluorescent probe for sensing cysteine in aqueous solution and serum.

An efficient and novel 2,5-bis(benzo[d]thiazol-2-yl)phenol scaffold-based ratiometric fluorescent probe BTP-Cys for the sensing of cysteine has been developed. The probe BTP-Cys with acrylates moiety, as recognition site, has been successfully constructed on account of the excited state intramolecular proton transfer (ESIPT) mechanism. Upon the treatment with Cys (0-250 µM), this probe BTP-Cys exhibits a dramatic fluorescent intensity ratios enhancement (from 0.03 to 18.3) and a large emission shift (113 nm). The detection limit of this probe is as low as 3.8 × 10-7 M. Importantly, the concentration and time dependent of Cys in bovine serum albumin (BSA) has also been measured, indicating that BTP-Cys could be a biocompatible and rapid probe for Cys in vitro.

Amphiphilic diarylethene as a photoswitchable probe for imaging living cells.

This communication reports a unique example of water-soluble and fluorescent-switchable amphiphilic diarylethene. This compound performs stable vesicle aggregation in water and shows aggregation-dependent emission in its open form. The fluorescence can be effectively switched by alternating between UV and visible light irradiation. This compound thus can stain KB cells for switchable living cell imaging with excellent resistance to fatigue.