A tetraphenylethene-substituted pyridinium salt with multiple functionalities : synthesis , stimuli-responsive emission , optical waveguide and speci fi c mitochondrion imaging †

HKUST-Shenzhen Research Institute, No. 9 Nanshan, Shenzhen, 518057, China. E-mail Department of Chemistry, Institute for A Functional Materials and Division of Bi University of Science & Technology (HKU Kong, China CAS Key Laboratory of Photochemistry, Be Science, Institute of Chemistry, Chinese P. R. China Department of Physics, HKUST, Clear Wate Guangdong Innovative Research Team, S State Key Laboratory of Luminescent Mater of Technology, Guangzhou, 51640, China † Electronic supplementary information UV-vis and PL emission spectra, TEM im and uorescent images of living cells. See Cite this: J. Mater. Chem. C, 2013, 1, 4640


Introduction
Development of new materials with advanced functionalities is a crucial step for future technological innovations.Luminescent materials have attracted much interest because of their wide applications in electronics, 1 optics, 2 storage media 3 and biological science. 4Whereas luminescence behaviours of conjugated molecules and polymers are normally investigated in the solution state, they are utilized in the solid state (e.g., as thin lms) for real-world applications, where the luminophores tend to form aggregates.It is known that aggregation of organic luminophores oen results in partial or even complete quenching of their light emission. 5This effect of aggregationcaused quenching (ACQ) has limited the scope of their technological applications.We and other groups, however, observed a phenomenon of aggregation-induced emission (AIE) in some propeller-like molecules such as tetraphenylethene (TPE) and silole that is exact opposite to the ACQ effect. 6Instead of quenching, aggregate formation has made these luminogens to emit efficiently though they are almost non-uorescent in the solution state.Based on theoretical and experimental studies, the restriction of intramolecular rotation (RIR) is considered to be the main cause for the AIE phenomenon. 7Since AIE luminogens are highly emissive in the aggregated state, this unique characteristic differentiates them from conventional luminophores and makes them promising materials for high-technological applications in the practically useful solid state. 8As a result, various AIE dyes have been developed and their utilities in many elds such as organic light-emitting diodes, 9 bioprobes, 10 chemosensors 11 and cell imaging 12 have been explored, thanks to the enthusiastic efforts of the scientists.
Most of AIE dyes prepared so far show blue or green emission. 13For biological scientists, they prefer luminogens with longer-wavelength emissions as these luminescent materials suffer little interferences from optical self-absorption and autouorescence from the background. 14For red-emissive dyes, they are generally constructed from merged planar rings with extended conjugation or possess a strong intramolecular charge transfer (ICT) effect caused by the interaction between the electron-donating and accepting groups. 15However, it is synthetically difficult to prepare luminogens with highly conjugated structures, while materials with the ICT feature can offer only weak emissions, particularly in a polar medium. 16ecently, we succeeded in synthesizing a red-emissive luminogen by melding a benzothiazolium unit with TPE. 17 Although the hybrid molecule exhibits the ICT characteristic, it emits intensely in the aggregated or solid state due to the prevailed AIE effect contributed by the TPE unit.Such a result shows that the synergy interplay between the ICT and AIE attributes has led to efficient solid-state emitters with emissions at the longer wavelengths.On the other hand, many AIE dyes prepared previously are based on their potential utility in a specic single aspect such as organic light-emitting diodes or uorescent sensors.9b,11a Few of them, however, exhibit multi-functionality or purpose. 18Thus, development of efficient luminescent materials in the solid state with potential high-tech applications in various elds through a simple synthetic procedure is of interest from the concept of convenience and efficiency in the modern society.Furthermore, few AIE molecules can stain the organelles, especially mitochondrion, which is an important organelle in living cells, 12a although many of them work as good uorescent visualizers for cytoplasmic imaging.12b,c Based on the strategy discussed above, in this paper, we report the synthesis of a new yellow-emissive AIE dye (TPE-Py, Scheme 1) by attaching a pyridinium unit to TPE through vinyl functionality, and present its multi-functional properties including morpho-and mechano-chromism, optical waveguide and mitochondrion imaging, which are extraordinary, if not unprecedented.

Results and discussion
Synthesis and optical property TPE-Py was synthesized according to the synthetic route shown in Scheme 1. 17 1,4-Dimethylpyridinium iodide (1) and 4-(1,2,3triphenylvinyl)benzaldehyde (2) were prepared by the synthetic procedures as shown in Scheme S1 in the ESI † and heated under reux in ethanol, which gave TPE-Py in a reasonable yield aer counter anion exchange and purication by column chromatography.Detailed information on their synthesis and characterization data can be found in the ESI.† The dilute THF solution (20 mM) of TPE-Py exhibits an absorption maximum at 403 nm (Fig. S1 †).Photoexcitation of the solution induces a weak red emission at 625 nm, giving a Stokes shi as large as 222 nm (Fig. S1 †).The uorescence quantum yield (F F ) estimated using coumarin 153 (F F ¼ 58% in ethanol) is merely 3.5%, suggesting that TPE-Py is a weak emitter in the solution state.When the measurements are carried out in other solvents with increasing polarity, the emission spectrum moves to the longer-wavelength region accompanying with a decrease in the emission intensity (Fig. S2 †), which is demonstrative of an ICT effect caused by the interaction between the electron-donating TPE unit and the electron-accepting pyridinium moiety. 4The emission was weakened progressively when up to 90% water was added to the THF solution but started to swily increase aerwards.At 99% water content, the emission intensity is more than 4-fold higher than that in pure THF solution (Fig. 1).Since TPE-Py is insoluble in water, its molecules must have been aggregated in aqueous mixtures with high water contents.Evidently, TPE-Py is AIE-active.The formation of aggregates has activated the RIR process, thus making the dye more emissive in the aggregated state.However, the gradual addition of water into the THF solution has increased the polarity of the solvent mixture, which has intensied the ICT effect and hence weakened the light emission.The ICT effect seems to be dominated at water fraction #90%.Aerwards, the AIE process prevails, which converts TPE-Py into a strong emitter.It is noteworthy that the aggregates suspended in the aqueous mixture emit bluer color than their isolated species in THF solution because of the reduction in the solvent effect on their photophysical properties.
Interestingly, the emission intensity and color of a freshly prepared 95% aqueous mixture change when standing at room temperature with time.As depicted in Fig. 2A, the emission peak at 600 nm becomes weaker progressively as time elapses.Meanwhile, a new peak with enhanced intensity appears at 512 nm.Aer 30 min, the peak at 600 nm disappears completely and the emission spectrum is dominated by the peak at 512 nm.At the same time, the emission color changes from yellow to green under 365 nm UV irradiation, as suggested by the photographs shown in the inset of Fig. 2B.Similar observation was found in 90% aqueous mixture, though the time to reach Scheme 1 Synthetic route to TPE-Py.equilibrium and the extent of emission enhancement are different (Fig. S3 †).
To decipher the mechanism of such a phenomenon, we analyzed the aggregates suspended in 95% and 90% aqueous mixtures by transmission electron microscopy (TEM) and electron diffraction (ED).The TEM image of a freshly prepared 95% aqueous mixture exhibits spherical nanoparticles with varied sizes (Fig. 3A).Only a diffuse halo was observed in the associated ED pattern (Fig. 3B), suggesting that the aggregates are amorphous in nature.However, entirely different results were obtained aer the mixture was standing at room temperature for 30 min.Flaky aggregates, rather than nanoparticles, were observed in the TEM image (Fig. 3C).The aggregates are crystalline, as revealed by the numerous diffraction spots in the ED pattern.Evidently, crystallization leads to such emission color and intensity changes, which is exactly the same as that observed in our previous publication. 17The aggregates formed in 90% aqueous mixture are amorphous but crystalline when the mixture is le at room temperature for 135 min (Fig. S4 †).Aqueous mixtures with water contents higher than 95% exhibit no such time-dependent phenomenon, probably due to the tighter packing of the formed aggregates, which hampers the reorientation and repacking of the TPE-Py molecules in a more ordered fashion.Crystallization generally red-shis the emission spectrum and weakens the light emission.The unusual phenomenon observed here may be due to the more conformational twisting of the TPE-Py molecules in order to t into the crystal lattice.The stronger interaction between the TPE-Py molecules in the crystal state may, on the other hand, stiffen the molecular conformation and hinder intra-and inter-molecular motions, thus making the crystalline aggregates stronger emitters. 19

Mechanochromic luminescence
Mechanochromic luminophores are smart materials and show a sensitive response to external stimuli.They have received considerable attention in recent years for their potential applications in the optical information storage media and memory system. 20Some AIE luminogens with good mechanochromic properties have been prepared in view of their facile transformation between the crystalline and amorphous states in response to external stimuli, the emission contrast, however, is low in most cases. 21Since the amorphous and crystalline aggregates of TPE-Py exhibit different emission behaviours, it encourages us to investigate its mechanochromic property.Pale-yellow crystals of TPE-Py were readily obtained by slow evaporation of its dichloromethane (DCM)-hexane mixture at room temperature.Unfortunately, the sizes of the crystals are too small to be analysed by crystal X-ray diffraction.UV irradiation of the TPE-Py crystals gives a strong green emission at 515 nm with a F F value of 31.8% (Fig. 4A).Aer gentle grinding the crystals using a metal spatula, yellow powders are formed.The powders emit at 600 nm, giving a large emission contrast of $85 nm.At the same time, the F F value drops to 20.4%.Such a transformation is reversible aided by fuming with acetone vapor for 10 min or heating at 150 C for 10 min.The conversion between the green-and yellow-emissive solids as well as their   corresponding emission spectra can be repeated many times without fatigue because these external stimuli are nondestructive in nature (Fig. 5B).
To demonstrate their useful practical application, TPE-Py crystals were spread on a lter paper as a thin lm (Fig. 4B).Under 365 nm UV irradiation, the TPE-Py lm exhibits strong green emission.Letters of "AIE" are written on the lm using a metal spatula, which appear yellow and thus can be readily discernible from the background due to their high contrast.Fuming the lm with acetone vapour for 10 min erases the letters and reinstalls the original green background, allowing new letters of "TPE" to be written on the lm using the same method.These results show that TPE-Py is potential to be used as a recyclable optical storage medium.
To prove that the same mechanism is responsible for the mechanochromism of TPE-Py, we analyzed TPE-Py in different aggregated states by powder X-ray diffraction (XRD).As shown in Fig. 6, the X-ray diffractogram of TPE-Py crystals shows many sharp diffraction peaks, which are indicative of their wellordered structure.Aer grinding, almost all the diffraction peaks disappear, suggesting that the ground sample possesses low crystallinity or is even amorphous.When such a sample was thermal-treated or fumed by solvent vapor, sharp diffraction peaks emerge again due to the recrystallization of the TPE-Py molecules, though the spectral pattern is different from that of the untreated one.
Analysis by differential scanning colorimetry (DSC) also supports the above claim (Fig. 6B).The heating scan of the ground sample detects an exothermic peak at 139 C. Since TPE-Py degrades at temperatures higher than 200 C as revealed by the TGA analysis, the transition at 139 C thus should not be due to the thermal decomposition of the dye molecule but is associated with its crystallization process.No signals are detected in untreated, thermal-annealed and fumed samples as they are in the crystalline state.Due to the propeller-like TPE unit, non-covalent interactions may only exist between the TPE-Py molecules, which can be readily destroyed in the presence of mechanical perturbation and recovered through thermal and solvent-fuming processes.

Optical waveguide
Development of organic crystals in the eld of photoelectric material is a hot research topic for their well-ordered arrangement and high stability and mobility. 22As crystalline AIE molecules show high solid-state emission, they are thus promising photoelectric materials.Indeed, a few micro/nanocrystalline AIE dyes have been fabricated and utilized as optical waveguide and amplied spontaneous light-emitting materials. 23When observed under the uorescent microscope, the crystalline microrods of TPE-Py formed by self-organization in the DCM-hexane mixture exhibit bright green emission (Fig. 7A).Careful investigation shows that the two ends emit more intensely, suggesting that TPE-Py exhibits optical waveguide behaviour.22b,23b,24 To prove this, a distance-dependent uorescent image of a single microrod was measured on a near-eld scanning optical microscope.As shown in Fig. 7B, the chosen microrod on the glass coverslip is excited using a uniform focused laser (351 nm) at six different local positions  along its length.Interestingly, except for the excited sites, green emission was also observed at both ends (since the microrod is long, only ends labelled with numbers 1-5 are shown).This phenomenon may originate from the absorption of the excitation light by TPE-Py molecules and propagation of the light emission to the rod end.Clearly, the appearance of outcoupling light demonstrates the strong waveguide property.Thus, according to previous reports, TPE-Py can be classied as active waveguide materials as the light is generated from the light emission process.22b The corresponding emission spectra taken at varied excitation positions and xed emitted ends are shown in Fig. 7C.By lengthening the distance between the excited site and the emitted end, the emission at the rod end becomes weaker due to the optical loss during the propagation process but causes no change in the spectral pattern.The optical loss coefficient (a) is an important parameter to determine the property of waveguide materials. 25To determine the a value, the emission intensity at the xed end (I end ) and the excited site (I body ) are recorded.By tting the curve in Fig. 7D using a single exponential tting, an equation in the form of I end /I body ¼ Aexp Àax was obtained, 25b where x is the distance between the excited site and the emitted end and A is the ratio of the light that has escaped from the excitation spot and that of light propagated along the rod.Herein, the a value of TPE-Py is determined to be 0.032 dB mm À1 , which is quite low compared with the previous reported values.23a,25a,26 The low optical loss coefficient of TPE-Py may be related to its intrinsic property of large Stokes shi, which helps decrease re-absorption.Moreover, the smooth surface and well-ordered molecular arrangement of the crystalline microrods, as suggested by powder X-ray diffraction, may also contribute to such good optical waveguide behaviour, which is rarely reported for TPE derivatives.

Mitochondrion staining
We also explored the utilization of TPE-Py as a uorescent visualizer for intracellular imaging. 12,27The nanoaggregates of TPE-Py were prepared in the minimum essential mediumdimethyl sulfoxide mixture and the HeLa cells were imaged using a standard cell-staining protocol.The living cells were incubated with TPE-Py (5 mM) for 15 min at 37 C and then washed three times with phosphate buffered saline solution.As shown in Fig. S5, † the living cells grow healthy and display their normal morphology, demonstrating that TPE-Py is biocompatible.Amusingly, when investigated under a uorescent microscope, distinct yellow emission likely originated from the mitochondria was observed.
To conrm the above speculation, we co-incubated the living cells with TPE-Py and MitoTracker Red CMXRos (100 nM), a widely utilized mitochondrion-targeted dye.We took uorescent images of one cell stained by TPE-Py and MitoTracker Red, which show yellow and red emissions, respectively (Fig. 8A and  B).Merging both photos generates an orange image, from which the position, shape and amount of mitochondria stained by TPE-Py are found to be the same as those by MitoTracker Red.Clearly, TPE-Py can specically stain the mitochondria of living cells.
To work as a good intracellular visualizer, it should possess high resistance to photobleaching.With such regard, the photostability of TPE-Py as well as MitoTracker Red was examined under the same experimental conditions.Aer continuous excitation for 180 s, the emission from the living cells stained with MitoTracker Red is largely quenched (Fig. 9A).The measurement of the change in the emission intensity with time shows that the magnitude at 180 s is merely $20% of the initial value owing to photobleaching (Fig. 9B).Amazingly, aer continuous excitation at identical power for the same time, strong light was still observed in living cells incubated with TPE-Py as the emission intensity drops only 30% under such circumstances.Evidently, TPE-Py possesses a stronger photostability or higher resistance to photobleaching than Mito-Tracker Red.
It is well-known that a mitochondrion shows a large negative potential on the matrix side of the membrane.Thus, mitochondrion-targeted uorescent dyes are generally lipophilic and cationic in character. 4,12b,28 TPE-Py possesses both such characteristics and thus makes it work as a good staining agent for specic targeting of mitochondrion in a living cell.

Conclusions
In summary, a heteroatom-containing luminogen with multifunctionality was synthesized in a reasonable yield by melding a pyridinium unit with TPE.Whereas TPE-Py is weakly emissive in solution, it becomes a strong emitter when aggregated, demonstrating a phenomenon of aggregation-induced emission.Crystallization generally weakens and red-shis the light emission.The crystalline aggregates of TPE-Py, however, show stronger and bluer light than their amorphous aggregates.Its  solid-state emission can be reversibly switched between green and yellow color with a contrast by grinding and fuming or heating processes due to the morphological change from the thermodynamically stable crystalline phase to the metastable amorphous state.Crystalline microrods of TPE-Py show an excellent optical waveguide property with low optical loss.Thanks to its cationic and lipophilic nature, TPE-Py works as a uorescent visualizer for specic staining of mitochondria in living cells with high photostability.It is anticipated that the ready synthesis of TPE-Py coupled with its multi-potential applications will trigger new research enthusiasm and effort for the creation of new AIE materials with improved or new properties for more high-technological applications.

Fig. 1
Fig. 1 (A) Emission spectra of TPE-Py in THF-water mixtures with different water fractions (f w ).Solution concentration: 20 mM; excitation wavelength: 403 nm.(B) Plot of the relative emission intensity (I/I 0 ) versus the composition of the aqueous mixture of TPE-Py.I 0 ¼ emission intensity in pure THF solution.Inset in (B): photographs of TPE-Py in THF-water mixtures with f w values of 0, 90 and 99% taken under 365 nm UV irradiation.

Fig. 2
Fig. 2 Change in the emission spectrum of TPE-Py in 95% aqueous mixture with standing time at room temperature.Solution concentration: 20 mM; excitation wavelength: 403 nm.(B) Plot of wavelength and emission intensity versus the standing time from 0 to 30 min.Inset in (B): photographs of TPE-Py in 95% aqueous mixture taken at 0 and 30 min under 365 nm UV illumination.

Fig. 3 (
Fig. 3 (A and C) TEM images and (B and D) ED patterns of (A and B) amorphous and (C and D) crystalline aggregates of TPE-Py formed in 95% aqueous mixture (A and C) before and (B and D) after standing at room temperature for 30 min.

Fig. 4 (
Fig. 4 (A) Switching the solid-state emission of TPE-Py by the grinding-fuming/ heating process.(B) Fluorescent images of TPE-Py thin films on filter papers (a) without and (b) with letters of "AIE" being written by using a metal spatula.The photograph in (c) was obtained by fuming the film in (b) with acetone vapour for 10 min, while that in (d) was obtained by writing the letters of "TPE" on the TPE-Py film in (c) using a metal spatula.All the photos were taken under 365 nm UV irradiation.

Fig. 5
Fig. 5 (A) Change in the emission spectrum of TPE-Py crystals by the grindingfuming/heating process.(B) Repeated switching of the solid-state fluorescence of TPE-Py by repeated grinding and fuming/heating cycles.

Fig. 7 (
Fig. 7 (A) Fluorescent image of crystalline microrods of TPE-Py taken under UV irradiation on a fluorescent microscope.(B) Fluorescent micrographs obtained by exciting an identical TPE-Py microrod at different positions, where the up arrow indicated the excited site and the down arrow showed the emitted end.Scale bar: 20 mm.(C) Corresponding emission spectra of the emitted ends labelled with numbers 1-5 in (B).(D) Plot of emission intensity at the end versus the distance between the excited site and the emitted end.

Fig. 9
Fig. 9 (A) Fluorescent images of HeLa cells stained with TPE-Py and MitoTracker Red taken under continuous UV excitation at 405 nm and 560 nm, respectively, for 0 and 180 s.Scale bar: 20 mm.(B) Luminescence decay curves of TPE-Py and MitoTracker Red.