Fluorescent Solvatochromic Probes for Long-Term Imaging of Lipid Order in Living Cells.

High-resolution spatio-temporal monitoring of the cell membrane lipid order provides visual insights into the complex and sophisticated systems that control cellular physiological functions. Solvatochromic fluorescent probes are highly promising noninvasive visualization tools for identifying the ordering of the microenvironment of plasma membrane microdomains. However, conventional probes, although capable of structural analysis, lack the necessary long-term photostability required for live imaging at the cellular level. Here, an ultra-high-light-resistant solvatochromic fluorescence probe, 2-N,N-diethylamino-7-(4-methoxycarbonylphenyl)-9,9-dimethylfluorene (FπCM) is reported, which enables live lipid order imaging of cell division. This probe and its derivatives exhibit sufficient fluorescence wavelengths, brightness, polarity responsiveness, low phototoxicity, and remarkable photostability under physiological conditions compared to conventional solvatochromic probes. Therefore, these probes have the potential to overcome the limitations of fluorescence microscopy, particularly those associated with photobleaching. FπCM probes can serve as valuable tools for elucidating mechanisms of cellular processes at the bio-membrane level.

Aggregation-Induced Emission (AIE), Life and Health.

Light has profoundly impacted modern medicine and healthcare, with numerous luminescent agents and imaging techniques currently being used to assess health and treat diseases. As an emerging concept in luminescence, aggregation-induced emission (AIE) has shown great potential in biological applications due to its advantages in terms of brightness, biocompatibility, photostability, and positive correlation with concentration. This review provides a comprehensive summary of AIE luminogens applied in imaging of biological structure and dynamic physiological processes, disease diagnosis and treatment, and detection and monitoring of specific analytes, followed by representative works. Discussions on critical issues and perspectives on future directions are also included. This review aims to stimulate the interest of researchers from different fields, including chemistry, biology, materials science, medicine, etc., thus promoting the development of AIE in the fields of life and health.

Push-Pull Bridged Distyrylbenzene with Highly Bright Solid-State Red-Orange Aggregation-Induced Emission.

Multiple emission colors in solid-state organic fluorophores with the same main skeleton are essential for improving the performance of light-emitting devices/materials. Specifically, emission in the red/near-infrared region is of great importance in the biological field. Previously, we developed di-bridged-distyrylbenzene DBDB[7] with high-brightness solid-state blue and aggregation-induced emissions (AIE) by introducing the bridging structures of a seven-membered ring into the vinylene groups of distyrylbenzene (DSB). Herein, we synthesize MNDBDMeODB[7] (1), which features substituted methoxy and malononitrile groups as donor and acceptor groups, respectively, in DBDB[7]. In solvents more polar than THF, MNDSDMeOB (3), which has the same main skeleton as 1 but without bridges, shows no emission in the solid state, whereas 1 exhibits highly bright red-orange emission in the solid state owing to the suppression of intermolecular electronic interactions by the bridges and the AIE property. We also synthesize MNDSD(EHO)B (2) in which the methoxy groups of 3 are replaced by ethylhexyloxy groups, thus disrupting the crystallinity of the molecule. 2 exhibits positive fluorescence solvatochromism and has a high fluorescence quantum yield in the solid state as a red-emitting DSB derivative. The solid-state emission properties of 1 and 2 will improve the applicability of DSBs and functionalities of light-emitting devices/materials.

Flexible Alkylene Bridges as a Tool To Engineer Crystal Distyrylbenzene Structures Enabling Highly Fluorescent Monomeric Emission.

To design ultrabright fluorescent solid dyes, a crystal engineering strategy that enables monomeric emission by blocking intermolecular electronic interactions is required. We introduced propylene moieties to distyrylbenzene (DSB) as bridges between the phenyl rings either side of its C=C bonds. The bridged DSB derivatives formed compact crystals that emit colors similar to those of the same molecules in dilute solution, with high quantum yields. The introduction of flexible seven-membered rings to the DSB core produced moderate distortion and steric hindrance in the DSB π-plane. However, owing to this strategy, it was possible to control the molecular arrangement with almost no decrease in the crystal density, and intermolecular electronic interactions were suppressed. The bridged DSB crystal structure differs from other DSB derivative structures; thus, bridging affords access to novel crystalline systems. This design strategy has important implications in many fields and is more effective than the conventional photofunctional molecular crystal design strategies.

Rhodium-Catalyzed Intermolecular Cycloaromatization Route to Cycloparaphenylenes that Exhibit Aggregation-Induced Emission.

A high-yielding new route to substituted cycloparaphenylenes has been developed via reductive aromatization of a diyne bearing two cyclohexadiene units giving a cyclophenylene-ethynylene (CPE) followed by the cationic rhodium(I)/dppe complex-catalyzed intermolecular [2+2+2] cycloaddition (cycloaromatization) of the CPE with monoynes. The thus-obtained products, substituted [8]cycloparaphenylene-triphenylenes ([8]CPPTs), exhibited definite aggregation-induced emission (AIE). This molecule is noteworthy as a novel AIE-active cycloarylene that does not have well-known AIE luminogens, such as tetraphenylethene and 1,2,4,5-tetraphenylbenzene skeletons. The single-crystal X-ray diffraction analyses of the AIE-active [8]CPPTs revealed their highly ordered packing structures in which the rotation of the triphenylene moieties is restricted.

Principles of Aggregation-Induced Emission: Design of Deactivation Pathways for Advanced AIEgens and Applications.

Twenty years ago, the concept of aggregation-induced emission (AIE) was proposed, and this unique luminescent property has attracted scientific interest ever since. However, AIE denominates only the phenomenon, while the details of its underlying guiding principles remain to be elucidated. This minireview discusses the basic principles of AIE based on our previous mechanistic study of the photophysical behavior of 9,10-bis(N,N-dialkylamino)anthracene (BDAA) and the corresponding mechanistic analysis by quantum chemical calculations. BDAA comprises an anthracene core and small electron donors, which allows the quantum chemical aspects of AIE to be discussed. The key factor for AIE is the control over the non-radiative decay (deactivation) pathway, which can be visualized by considering the conical intersection (CI) on a potential energy surface. Controlling the conical intersection (CI) on the potential energy surface enables the separate formation of fluorescent (CI:high) and non-fluorescent (CI:low) molecules [control of conical intersection accessibility (CCIA)]. The novelty and originality of AIE in the field of photochemistry lies in the creation of functionality by design and in the active control over deactivation pathways. Moreover, we provide a new design strategy for AIE luminogens (AIEgens) and discuss selected examples.

Bridged Stilbenes: AIEgens Designed via a Simple Strategy to Control the Non-radiative Decay Pathway.

To broaden the application of aggregation-induced emission (AIE) luminogens (AIEgens), the design of novel small-molecular dyes that exhibit high fluorescence quantum yield (Φfl ) in the solid state is required. Considering that the mechanism of AIE can be rationalized based on steric avoidance of non-radiative decay pathways, a series of bridged stilbenes was designed, and their non-radiative decay pathways were investigated theoretically. Bridged stilbenes with short alkyl chains exhibited a strong fluorescence emission in solution and in the solid state, while bridged stilbenes with long alkyl chains exhibited AIE. Based on this theoretical prediction, we developed the bridged stilbenes BPST[7] and DPB[7], which demonstrate excellent AIE behavior.

Synthesis of fluorescent polycarbonates with highly twisted N,N-bis(dialkylamino)anthracene AIE luminogens in the main chain.

A synthetic route to embed aggregation-induced-emission-(AIE)-active luminophores in polycarbonates (PCs) in various ratios is reported. The AIE-active monomer is based on the structure of 9,10-bis(piperidyl)anthracene. The obtained PCs display good film-forming properties, similar to those observed in poly(bisphenol A carbonate) (Ba-PC). The fluorescence quantum yield (Φ) of the PC with 5 mol% AIE-active monomer was 0.04 in solution and 0.53 in solid state. Moreover, this PC is also miscible with commercially available Ba-PC at any blending ratio. A combined analysis by scanning electron microscopy and differential scanning calorimetry did not indicate any clear phase separation. These results thus suggest that even engineering plastics like polycarbonates can be functionalized with AIE luminogens without adverse effects on their physical properties.

The K-Region in Pyrenes as a Key Position to Activate Aggregation-Induced Emission: Effects of Introducing Highly Twisted N,N-Dimethylamines.

A new design strategy to activate aggregation-induced emission (AIE) in pyrene chromophores is reported. In a previous report, we demonstrated that highly twisted N,N-dialkylamines of anthracene and naphthalene induce drastic AIE when these donors are introduced at appropriate positions to stabilize the S1/S0 minimum energy conical intersection (MECI). In the present study, this design strategy was applied to pyrene: the introduction of N,N-dimethylamine substituents at the 4,5-positions of pyrene, the so-called K-region, are likely to stabilize MECIs. To examine this hypothesis, four novel pyrene derivatives, which contain highly twisted N,N-dimethylamino groups at the 4- (4-Py), 4,5- (4,5-Py), 1- (1-Py), or 1,6-positions (1,6-Py) were tested. The nonradiative transitions of 4,5-Py are highly efficient (knr = 57.1 × 107 s-1), so that its fluorescence quantum yield in acetonitrile decreases to Φfl = 0.04. The solid-state fluorescence of 4,5-Py is efficient (Φfl = 0.49). In contrast, 1,6-Py features strong fluorescence (Φfl = 0.48) with a slow nonradiative transition (knr = 11.0 × 107 s-1) that is subject to severe quenching (Φfl = 0.03) in the solid state. These results underline that the chemistry of the pyrene K-region is intriguing, both from a photophysical perspective and with respect to materials science.

Highly Twisted N,N-Dialkylamines as a Design Strategy to Tune Simple Aromatic Hydrocarbons as Steric Environment-Sensitive Fluorophores.

The steric-environment sensitivity of fluorescence of 9,10-bis(N,N-dialkylamino)anthracenes (BDAAs) was studied experimentally and theoretically. A new design strategy to tune simple aromatic hydrocarbons as efficient aggregation-induced emission (AIE) luminogens and molecular rotors is proposed. For a variety of BDAAs, prominent Stokes shifts and efficient solid-state fluorescence were observed. Calculations on BDAA-methyl suggested that in the ground state (S0) conformations, the pyramidal amine groups are highly twisted, so that their lone-pair orbitals cannot conjugate with the anthracene π orbitals. Fluorescence takes place from the S1 minima, in which one or both amine groups are planarized. The stability of the S1 excited state minima as well as destabilization of the S0 state is the origin of large Stokes shift. Experimental measurement of the nonadiabatic transition rate suggests that para disubstitution by dialkylamino (or strongly electron-donating) groups is a key for fast internal conversion. Minimum energy conical intersection (MECI) between S1 and S0 states was found to have a Dewar-benzene like structure. Although this can be reached efficiently in liquid phase for fast internal conversion, a large amplitude motion is required to reach this MECI, which is prohibited in the solid state and caused efficient AIE. This strategy is used to find experimentally that naphthalene analogues are also efficient AIE luminogens. The flexibility of alkyl chains on amino groups is also found to be important for allowed charge-transfer transition. Thus, three points [(1) highly twisted N,N-dialkylamines, (2) substitution at the para positions, (3) with flexible alkyl groups] were proposed for activation of small aromatic hydrocarbons.

Bright and photostable push-pull pyrene dye visualizes lipid order variation between plasma and intracellular membranes.

Imaging lipid organization in cell membranes requires advanced fluorescent probes. Here, we show that a recently synthesized push-pull pyrene (PA), similarly to popular probe Laurdan, changes the emission maximum as a function of lipid order, but outperforms it by spectroscopic properties. In addition to red-shifted absorption compatible with common 405 nm diode laser, PA shows higher brightness and much higher photostability than Laurdan in apolar membrane environments. Moreover, PA is compatible with two-photon excitation at wavelengths >800 nm, which was successfully used for ratiometric imaging of coexisting liquid ordered and disordered phases in giant unilamellar vesicles. Fluorescence confocal microscopy in Hela cells revealed that PA efficiently stains the plasma membrane and the intracellular membranes at >20-fold lower concentrations, as compared to Laurdan. Finally, ratiometric imaging using PA reveals variation of lipid order within different cellular compartments: plasma membranes are close to liquid ordered phase of model membranes composed of sphingomyelin and cholesterol, while intracellular membranes are much less ordered, matching well membranes composed of unsaturated phospholipids without cholesterol. These differences in the lipid order were confirmed by fluorescence lifetime imaging (FLIM) at the blue edge of PA emission band. PA probe constitutes thus a new powerful tool for biomembrane research.

1-, 3-, 6-, and 8-Tetrasubstituted Asymmetric Pyrene Derivatives with Electron Donors and Acceptors: High Photostability and Regioisomer-Specific Photophysical Properties.

The systematic synthesis of five 1-, 3-, 6-, and 8-tetrasubstituted asymmetric pyrenes with electron donor and acceptor moieties is presented, together with an examination of their photophysical properties. Pyrene derivative PA1, containing one formyl and three piperidyl groups, showed bright solvatochromic fluorescence from green (λem = 557 nm, ΦFL = 0.94 in hexane) to red (λem = 648 nm, ΦFL = 0.50 in methanol), suggesting potential applications for PA1 as an environmentally responsive probe. Although the synthesis of simple 1- and 3-disubstituted pyrene derivatives is considered difficult, PA13, with two formyl groups at the 1- and 3-positions and two piperidyl groups at the 6- and 8-positions, could be synthesized successfully. PA13 exhibited less pronounced solvatochromism, but displayed a narrow fluorescent band with high ΦFL in all solvents (ΦFL > 0.75). Moreover, its absorption band displayed an exceptional bathochromic shift compared to the other derivatives (e.g., λabs = 480 and 522 nm in ethanol for PA1 and PA13, respectively), suggesting that such modifications of pyrene may be quite important for the modulation of its energy gap. Additionally, all compounds exhibited exceptionally high photostability, which highlights the advantage of these new dyes and provides new insights on the design of photostable fluorophores.

Directional control of π-conjugation enabled by distortion of the donor plane in diarylaminoanthracenes: a photophysical study.

We designed and synthesized a series of diarylaminoanthracenes in which the planarity of the diarylamine moiety is controlled by methylene- or ethylene- bridges. The X-ray crystallographic structures confirm that the methylene- and ethylene bridges gradually decrease the disorder of the diarylamine planes. To quantitatively analyze the photophysical properties and underlying electronic structures of these compounds, we carried out UV-vis and fluorescence spectroscopy, fluorescence quantum yield, and fluorescence lifetime measurements. The results reveal that enhanced planarity of the diarylamine moiety optically forbids the charge-transfer transition between the diarylamine and anthracene moieties. Although it is generally accepted that a planar π-conjugated system favors electron delocalization, our results indicate that distortion of the donor plane induces interchromophoric conjugation rather than conjugation within the local structure. Density functional theory calculations further illustrate that the frontier orbitals of diarylamine and anthracene interpenetrate as the donor plane is distorted. Additionally, natural bonding orbital analyses reveal that distortion of the donor plane changes the directionality of the π-conjugation of the nitrogen n-orbital from intrachromophoric to interchromophoric.

A novel pyrene-based two-photon active fluorescent dye efficiently excited and emitting in the 'tissue optical window (650-1100 nm)'.

The development of two-photon (TP) active fluorophores remains an important issue. Dyes that can be excited and fluoresce efficiently in the 'tissue optical window' (650-1100 nm) are especially in demand to maximize the underlying performance of two-photon fluorescence microscopy (TPFM) as an advanced optical technique. Ideally, such dyes would be compatible with the 1050 nm femtosecond fibre laser, which has recently been developed as an inexpensive excitation source to make the TPFM technique universal. In this work, we designed and synthesized a novel pyrene-based acceptor-π-acceptor (A-π-A) dye, PY, which exhibited outstanding properties such as bright fluorescence (λem = 650 nm and ΦFL = 0.80) and a large two-photon absorption cross-section (1100 GM (1 GM = 10-50 cm4 per photon per molecule) at 950 nm and 380 GM at 1050 nm) in the tissue optical window. In living mitochondria, PY provided more sensitive microscopic images than current dyes and showed great potential to be a building block of TP active fluorescent probes for the 1050 nm fibre laser. We believe that the exceptional properties of PY will be extended to other fluorescent probes through further chemical modification.

Disassembly-driven fluorescence turn-on of polymerized micelles by reductive stimuli in living cells.

Stimuli-response nanoparticles have emerged as powerful tools for imaging and therapeutic applications. Ideally, they should be assembled from biodegradable materials featuring small size and cooperative response to biological stimuli that trigger particle disassembly and release of an active molecule that could be readily monitored in situ. A concept is developed that consists of organic nanoparticles, assembled from fluorescent amphiphiles and polymerized with a redox-cleavable cross-linker. We obtained 20 nm nanoparticles bearing self-quenched Nile Red dye residues, which can disassemble in living cells into highly fluorescent molecular units owing to an external or internal reductive stimulus. The obtained results pave the way to new stimuli-responsive nanomaterials for applications in background-free imaging as well as in drug delivery, as the concept can be further extended to other active molecules including drugs and to cross-linkers cleavable by other biological stimuli.

Design of weak-donor alkyl-functionalized push-pull pyrene dyes exhibiting enhanced fluorescence quantum yields and unique on/off switching properties.

We designed, synthesized, and evaluated environmentally responsive solvatochromic fluorescent dyes by incorporating weak push-pull moieties. The quantum yields of the push (alkyl)-pull (formyl) pyrene dyes were dramatically enhanced by the introduction of alkyl groups into formylpyrene (1-formylpyrene: Φ(F) =0.10; 3,6,8-tri-n-butyl-1-formylpyrene: Φ(F) =0.90; in MeOH). The new dyes exhibited unique sensitivity to solvent polarity and hydrogen-bond donor ability, and specific fluorescence turn-on/off properties (e.g., 3,6,8-tri-n-butyl-1-formylpyrene: Φ(F) =0.004, 0.80, 0.37, and 0.90 in hexane, chloroform, DMSO, and MeOH, respectively). Here, the alkyl groups act as weak donors to suppress intersystem crossing by destabilizing the HOMOs of 1-formylpyrene while maintaining weak intramolecular charge-transfer properties. By using alkyl groups as weak donors, environmentally responsive, and in particular, pH-responsive fluorescent materials may be developed in the future.

Solvatochromic pyrene analogues of Prodan exhibiting extremely high fluorescence quantum yields in apolar and polar solvents.

True colors: Novel pyrene analogues of Prodan exhibit outstanding photophysical properties with remarkably high fluorescence quantum yield (QY) in solvents ranging from apolar hexane to polar methanol (see figure). This is accompanied by strong solvatochromism and large Stokes shifts. These properties have not been previously achieved in enormous solvatochromic dyes, but are quite useful for emitting materials and imaging tools.

A three-dimensional assembly of pyrene dye on a tetraphenylethane scaffold enhances fluorescence quantum yield.

A three-dimensional pyrene assembly on a tetraphenylethane skeleton enhanced the fluorescence quantum yield compared to the yield obtained when using monomeric species, without changing the shape of the emission spectrum. The unique spacing of the pyrene units may prevent intramolecular fluorescence quenching or non-radiative decay.

Fluorescence enhancement of pyrene chromophores induced by alkyl groups through σ-π conjugation: systematic synthesis of primary, secondary, and tertiary alkylated pyrenes at the 1, 3, 6, and 8 positions and their photophysical properties.

We have systematically synthesized 1-, 3-, 6-, and 8-alkyl-substituted pyrene derivatives using the latest synthesis methods and investigated the effects of alkyl substitution on the photophysical properties of the pyrene chromophore. Like the trimethylsilyl group, which is known to enhance the fluorescence properties of some chromophores through σ*-π* conjugation, alkyl groups (primary, secondary, and tertiary) enhanced the fluorescence quantum yield of the pyrene chromophore through σ-π conjugation in most cases. While these enhancements in the fluorescence quantum yield were beyond expectations, the results were supported by absolute measurements. These results also indicate that ubiquitous alkyl groups can be used to tune the photophysical properties of the pyrene chromophore, as well as to improve the solubility or prevent aggregation. In other words, they can be used to develop new photofunctional materials.