AIE Luminogens for Visualizing Cell Structures and Functions - ACS

Chapter 8

AIE Luminogens for Visualizing Cell Structures and Functions Downloaded by MIAMI UNIV on October 17, 2016 | http://pubs.acs.org Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ch008

Sean Lim,1 Ben Zhong Tang,2 and Yuning Hong*,1 1School

of Chemistry, The University of Melbourne, Parkville, Victoria 3010, Australia 2Department of Chemistry, Hong Kong University of Science and Technnology, Clear Water Bay, Kowloon, Hong Kong *E-mail: [email protected]

Organic fluorogens with aggregation-induced emission (AIE) characteristics have demonstrated their potential to be ideal candidates for live cell imaging. Opposite to conventional organic dyes, the AIE luminogens are nonluminescent when molecularly dissolved but highly emissive upon aggregation. As small molecules, the AIE luminogens normally enter cells through diffusion, accumulate in the target location, and generate light emission. Inherently, they possess large Stokes shift with appreciable brightness and they are resistant to photobleaching, owing to the formation of aggregates inside the cells. The utilization of AIE dyes for visualizing the structures and dynamics of subcellular organelles such as mitochondria, lysosomes, and lipid droplets and for monitoring cell functions such as intracellular pH and viscosity will be discussed in this chapter.

Introduction Cells are the smallest units of life yet highly organized. To decode the sophisticated cellular processes and their association with diseases, it is essential to analyze changes of the microenvironment and dynamics of the intracellular compartments on site and in time (1). Among many analytical tools, fluorescence imaging is a non-invasive method that allows direct visualization with superior sensitivity and unraveling spatiotemporal resolution, which is well suited for © 2016 American Chemical Society Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Downloaded by MIAMI UNIV on October 17, 2016 | http://pubs.acs.org Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ch008

studying events at the cellular and molecular levels (2–4). However, even with the recent advancement in instrumentation, many cellular processes still remain invisible, and this calls for further exploration of versatile fluorescent imaging agents (5). In general, fluorescent protein, synthetic dyes, and fluorescent nanoparticles are the three prominent types of fluorescent probes for imaging (6–8). Genetically encoded fluorescent proteins enjoy the high specificity to the target of interest. Despite of the unwanted oligomerization problem, most of the fluorescent proteins are photolabile as compared to synthetic dyes and nanoparticles (9). Fluorescent nanoparticles such as quantum dots demonstrate excellent photostability and high luminescence quantum efficiency (10). However, they usually enter the cells via endocytosis processes, which limit their distributions and sensing regions to the endocytic compartments instead of the entire cytoplasm, not to mention that some of them are even cytotoxic. Organic fluorophores are the most widely used agents for cell imaging because of their simple operation and rich variety (7). They are small in size and less likely to perturb the functions of target molecules. Through robust organic synthesis, the chemical and photophysical properties of the organic fluorophores could be tailored for a specific application (11). Although numerous structures with different functionalities have been made, most of them are built on a limited selection of the conventional chromophores, such as fluorescein, coumarin, cyanine and boron-dipyrromethene (BODIPY) (12). These derivatives inherit the shortcomings of the parental fluorophores and suffer from small Stokes shift, concentration-quenching and poor photostability problems. Therefore, it is desirable if we can expand the pool of the core chromophores and develop new fluorescent probes that can inherently overcome these problems. In our search of the alternatives, luminogens with aggregation-induced emission (AIE) characteristics have attracted our attention. Opposite to conventional dyes, the AIE luminogens are non-emissive when molecularly dissolved, but become highly fluorescent in the aggregate state owing to the restriction of their intramolecular motions (13–16). The AIE dyes collect the merits of both small organic dyes and fluorescent nanoparticles. They can enter the cells through diffusion as small molecules and no complicated and time-consuming transfection process is required. Inside the cells, the dye molecules may accumulate in the target location to form aggregates and generate light emission. As aggregates, they are more resistant to photo-bleaching than conventional dyes which have to be used in very dilute solutions (15). These compelling attributes of the AIE luminogens may provide a new platform for the development of a new generation of fluorescent probes for cell imaging. In this chapter, a series of AIE luminogens and their applications in specific organelle imaging and intracellular environment sensing will be discussed.

Fluorescence Imaging of Cell Structures Mitochondria Mitochondria are the membrane-bound organelle widely existing in most eukaryotic cells, with various pivotal functions under both physiological 200 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


status and pathological conditions (17). Prominently, mitochondria, known as “powerplants” for the cell, supply the most of cellular energy currency - adenosine triphosphate (ATP). The production of ATP involves a series of electron-transport systems to the oxidative phosphorylation reaction. Accompanying with this process, reactive oxygen species (ROS) are generated; the accumulation of ROS in mitochondria will raise the risk of cell damage and cell death (18). The programmed cell death, as called apoptosis, is initiated by the permeabilization of the mitochondrial outer membrane and the release of the pro-apoptotic proteins to the cytosol (19). Owing to the central role in the life and death of the cell, the characteristics of mitochondria have become a important indicator for cell functions.

Morphology Tracking Mitochondria are dynamic organelles that often change their shapes and distribution (20). Under normal physiological conditions, mitochondria form physically interconnected networks in order to deliver nutrition, transfer metabolites and redistribute calcium. However, in pathological (e.g. hyperglycemia, apoptosis, etc.) or diseased conditions (Alzheimer’s ischemic and hemorrhagic stroke, etc.), the mitochondria can undergo fragmentation to form short, round mitochondria (19). Tracking the dynamics of mitochondria morphology can thus provide direct evidence for studying the physiological changes and pathological mechanism of mitochondria disordered diseases (21). To follow the change of mitochondria morphology over time, fluorescent probes with high specificity and photostability are highly desirable. However, most of the conventional dyes suffer from poor photostability and hence imaging over an extended period of time is not possible. The AIE fluorogens, which often consist of multiple phenyl rings, are hydrophobic and tend to form nanoaggregates when dispersed in aqueous buffer solution or cell culture media (22). These nanoaggregates possess better photostability than single fluorescent molecules because even the outermost layer of the nanoaggregates are damaged by excitation light, it forms a protective layer to avoid the contact of the inner part to oxygen species and hamper further photobleaching. The photostability of the AIE active dyes is improved, which allow the tracking of mitochondria dynamics for a long period of time. Triphenylphosphonium (TPP) moiety was found to be a mitochondriatargeting group. Linking TPP with tetraphenylethene (TPE), a typical AIE luminogen, generated a mitochondria-targeting AIE fluorogen (1, Figure 1) (23). Cytotoicity of 1 was evaluated using an MTT assay, which showed only little effect of 1 on cell viability. The selectivity of 1 to mitochondria was examined by the co-staining experiment with the commercial MitoTracker Red FM (MT) (Figure 1A-C). The results suggested that the perfect colocalization of the signals from 1 and MT with the Pearson’s correlation coefficient (Rr) of 0.96. The photostability was investigated using confocal fluorescent imaging to produce continuous scans of HeLa cells stained with 1 or MT. The loss of emission intensity (%) from the cells was measured over the time frame. The signal loss 201 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


from 1 was less than 20% during 50 scans, while for MT, about 75% of the signals lost during only 6 scans (Figure 1D). The results demonstrated the superior photostability of the AIE fluorogen.

Figure 1. Fluorescent images of HeLa cells stained with (A) 1 and (B) MitoTracker Red FM (MT). (C) Merged image. (D) Signal loss (%) of fluorescent emission of 1 (solid circle) and MT (open circle)with increase number of scans. Inset: fluorescent imagings of 1-stained cells with increase number of scans. Reproduced with permission from ref. (23). Copyright (2013) American Chemical Society. The approach demonstrated above allows the use of high fluorophore concentration to compensate the photobleaching effect encountered by most conventional probes (23). In order to enrich the palette of the AIE mitochondria dyes, a yellow-emissive AIE dye, 2 was developed (24). With the positive pyridinium moiety, 2 exhibits excellent specificity to mitochondria, comparable to that of MT. Similar as 1, 2 exhibited excellent photostability and biocompatibility. The morphological dynamics of mitochondria was monitored by using 2 under 202 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


physiological and pathological conditions. To model the changes in mitochondrial morphology in cells of diabetes patients, 2 was used to staine HeLa cells, which then were exposed to high concentration of D-glucose. Under such hyperglycemia condition, short, vesicular structure of mitochondria were found, which were completely different from the elongated, tubular structure in the untreated cells (Figure 2). This mitochondrial fragmentation was found to be reversible, as after treatment of the cells the mitochondria gradually returned to its elongated state.

Figure 2. Hyperglycemic-induced mitochondria fragmentation of HeLa cells. The cells was stained with 2 and then incubated with 40 mM d-glucose solution for (A,B) 0, (C,D) 15, (E,F) 60 and (G,H) 120 min, respectively.

Membrane Potential Measurement The potential across the mitochondrial membrane is measured by the parameter ΔΨm, reflecting the proton gradient that is maintained to drive respiration and ATP synthesis in mitochondria (25). Changes in this potential is therefore indicative of mitochondrial health and is closely related to cellular function. Existing dyes that are selective to mitochondria exhibit aggregation-caused quenching (ACQ) properties, hence only a dilute solution of the dye can be used without causing quenching. This in turn leads to photobleaching by the excitatory light source and reduced emission. Furthermore, using such dyes to measure ΔΨm means that their concentrations in the mitochondria is constantly changing based 203 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


on the proton gradient – if there is an increased concentration of dye within the cell, emission readings could go either way, leading to inaccurate measurements of ΔΨm (26).

Figure 3. (A) Changes in fluorescence intensity of 3-stained HeLa cells upon treated with oligomycin and then CCCP. Inset: snapshots of the cell images taken upon the addition of the stimulants. (B) Fluorescent intensity of the unstained HeLa cells (blank), oligomycin treated and CCCP treated 3-stained HeLa cells analysed by flow cytometry. (C) Confocal images of mouse sperm cells stained with 3 showing the mitochondria and (D) Hoechst 33342 indicating nuclei. (E) The merged picture of panel C and D and the bright field image. Scale bar: 20 μm. Reproduced with permission from ref. (27). Copyright (2015) Royal Society of Chemistry. The abovementioned AIE-active mitochondria dye, 1 and 2, are basically insensitive to the change in ΔΨm. In our recent finding, the indolium salt of TPE derivative, 3, was reported to be mitochondria specific and sensitive to ΔΨm (27). 3 has long excitation (534 nm) and emission wavelength (585 nm) owing to the 204 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


intramolecular charge transfer between the donor and acceptor groups. Thanks to its AIE property, 3 also possesses superior photostability and when used in cell imaging. The viability of 3 for real-time ΔΨm tracking in HeLa cells was investigated, manipulating ΔΨm prior to the addition of the dye by adding oligomycin or carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to increase and decrease ΔΨm, respectively. As shown in Figure 3A, 3 was able to accumulate in the mitochondria, showing an increase in its fluorescence intensity when oligomycin was added, then a decrease when CCCP was added. The advantage of using the AIE-active 3 is that the fluorescence values can be used to establish a direct correlation between intensity and ΔΨm. The method of measuring ΔΨm change is also applicable by using flow cytometry, showing similar results to those generated by confocal microscopy – highlighting the potential of 3 for high throughput analysis of mitochondrial cells (Figure 3B). Furthermore, ΔΨm is a critical parameter determining the viability and fertilization potential of a sperm, the germ cell of males (28). To demonstrate the utility of 3 in this aspect, 3 was used to stain mouse sperm cells, which contained a large concentration of mitochondria (Figure 3C-E). By utilizing its specificity and sensitivity to ΔΨm, identification of energetic and non-energetic sperm cells was possible by quantifying their fluorescence intensities. Further uses of 3 as a probe will include drug screening for tumors with increased ΔΨm, and apoptotic cells with decreased ΔΨm.

Dual Functional Mitochondrial Probe The use of fluorophores to target and illuminate cellular structures has garnered the interest of researchers in recent times. Related to this is the growing field of photodynamic therapy (PDT) - the use of photosensitive species that produce ROS and induces apoptosis in cells (29). Existing photosensitizers are mostly porphyrin and phenylthiazinium derivatives that suffer from long irradiation time and limited specificity, owing to the permeability of the nucleus being tightly controlled (30). In light of this, targeting the mitochondria as a way to induce apoptosis is a favorable solution owing to the essential role of mitochondria in apoptosis. Isoquinolinium functionalized TPE derivative (4), with the positive charge, can target and visualize mitochondria in live and fixed cells with high selectivity comparable to that of MT (31). Intriguingly, this compound can also serve as a photosensitizer to promote the ROS generation in the mitochondrial region upon photoirradition, which induces cell apoptosis, as exemplified by the fragmention of mitochondria in cells (Figure 4). The photosensitizing property of 4 was quantified by using H2DCF-DA, a commercially available ROS probe which fluoresces at 530 nm in the presence of ROS. When irradiated by 365 nm UV light, emission from H2DCF-DA in a solution containing 4 intensified over time, suggesting the ROS generation by 4.

205 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 4. Fluorescence images of HeLa cells stained with 4 for 10 min and exposed to UV irradiation for 10 s. Reproduced with permission from ref. (31). Copyright (2014) Royal Society of Chemistry.

Cytotoxicity of 4 was evaluated using a lactate dehydrogenase (LDH) assay in HeLa cells, which measures the amount of LDH released from the damaged membrane of dead cells. Results showed that 4 in cells did not alter their viability under the concentration up to 1 μM. However, upon exposure to UV radiation, even 500 nM of 4 can cause significant cytotoxicity with only about 50% of cell viability. The results were further confirmed by using a cell impermeable dye, propidium iodide, as it only stains cells with a damaged membrane. Red emission recorded from cells exposed to UV irradiation indicated that ROS generation had indeed killed these cells, while cells not exposed to UV irradiation could not be stained in this manner. The dual functions of 4, as a mitochondria imaging agent and photosensitizer for PDT, offer the possibility of image-guided therapy by generating ROS to induce cell apoptosis and tracking the mitochondrial dynamics at the same time to monitor the efficiency of the PDT.

Lysosomes Studying lysosomal activities provides important information relating to autophagy – the disassembly of unwanted and unnecessary components that accompany cell destruction – which in turn shows a relationship to ageing, longevity, as well as cancers and neurodegenerative diseases (32). The autophagosomes responsible for the delivery of such components fuses with lysosomes, which are then degraded by hydrolases within them. Hence a lysosome probe will be useful for the study of autophagy activities under pathological conditions.


Visualising Autophagy Processes


A lysosome-targeting AIE dye (5) has been developed based on the fluorophore with excited-state intramolecular proton transfer (ESIPT) property and a morpholino moiety for lysosome targeting (Figure 5) (33). Because of its ESIPT characteristics, 5 exhibits two different wavelengths of yellow emission: keto emission produces a longer wavelength while enol emission produces a shorter one. This is caused by the intramolecular hydrogen bond formed in 5 in its keto form, which is absent in its enol form.

Figure 5. (A−E) Fluorescence images of 5-stained HeLa cells before and after rapamycin treatment (50 µg/mL) for different periods of time. (F) Enlarged region of interest of panel E. Scale bar: (A−E) 30 μm, (F) 10 μm. λex = 400−440 nm. Reproduced with permission from ref. (33). Copyright (2015) Wiley. MTT assay was used to assess the cytotoxicity of 5, which revealed that after 24 hour incubation with 5 the HeLa cells still showed 80% viability that implies low interference of 5 on cell growth. Then the lysosome targeting ability was investigated by staining HeLa cells with both 5 and the commercial LysoTracker Red DND-99 (LTR). The results showed that both signals overlap very well (Rr = 0.90) meaning 5 was indeed lysosome selective. It was found that 5 exhibited higher contrast and brighter emission in comparison with LTR; by introducing both dyes to cells it was found that they compete for the same site but 5 possesses higher contrast, providing evidence for its superior affinity to lysosomes. Furthermore, similar as the other AIE dyes, 5 possesses better photostability than LTR. 207 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

5 was then used to visualize autophagy under physiological conditions by employing the use of mammalian target of rapamycin (mTOR), an autophagy suppressant. Rapamycin is a lipophilic antibiotic that binds to mTOR and is used to induce autophagy in prion diseases, by activating autophagy and prion protein degradation (34). 5 was used to visualize HeLa cells treated with rapamycin; during the autophagy process, lysosome count increased and fused with the autophagosome to form an autolysosome which can be clearly visualized by the emission of 5 (Figure 5).


Lipid Droplets Lipids are stored in cells in the form of lipid droplets (LD), which also regulate lipid metabolism and transfer, protein degradation and signal transduction (35). Failure to regulate these processes are linked to a number of diseases, and abnormal LD amount is used as a biomarker in diseases such as infection by the hepatitis C virus. Hence monitoring LD volume and concentration is important in biomedical research to pinpoint diseases – mainly liver related diseases at the early stage (36).

Figure 6. HeLa cells stained with (A-C) 6 and (D-F) Nile red. Images taken under bright field (A,D) and photoexcitation (B,E) by fluorescence microscopy. Panels C,F show merged images. Reproduced with permission from ref. (38). Copyright (2014) Royal Society of Chemistry. 208 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Current tomography scanning techniques that detect LD levels and liver diseases are relatively insensitive and inaccurate, only able to pick up on diseases in their later stages (37). Fluorescent dyes that can selectively illuminate LDs could improve the sensitivity in the diagnosis process. TPE derivative decorated with the electron donating and accepting groups (6) showing polarity dependent flurorescence were synthesized and used for the staining of LDs (38). The emission of 6 is found to be blue-shifted with the increase of hydrophobicity of the solvents. Due to the lipophilicity of the dye, 6 can quickly target LDs in cells (10-15 minutes after incubation), which can be seen as spherical objects in the cytosol under fluorescence microscopy (Figure 6A-C). A parallel experiment was conducted using Nile red dye, which is commonly used to stain LDs. However, compared to 6, the Nile red dye was not selective to LDs; it also stained other organelles such as mitochondria, as seen in Figure 6D-E. As lipid content is an important parameter to determine the value of algaes as a biofuel, 6 can also be used for the screening of algaes (38). 6 was introduced into the green algae Nano by diffusion, after which the diameter of the emission region can be used to quantify the size of LDs within the cytoplasm. In this way, the viability of algae as an environmentally friendly fuel can be assessed.

Fluorescence Imaging of Cellular Environment Intracellular pH The pH of a cell is an important indicator of its overall health, as it regulates cellular functions such as apoptosis, proliferation and protein signaling (39). Monitoring the intracellular pH is possible through the use of a number of methods such as microelectrodes, NMR spectroscopy and optical microscopy. Fluorescent dyes are powerful pH probes due to their high sensitivity and resolution, yet currently available ones have narrow spans that do not cover the entire pH range in physiological environment (40). Alternative approach by doping multiple pH-sensitive dyes into nanoparticle matrixes has been reported to achieve the full-range pH sensing (41). However, the uneven distribution of nanoparticles in endocytic compartments restricts their sensing area over the entire cytoplasm. To circumvent these problems, an AIE active, pH responsive fluorogen based on TPE-cyanine adduct (7) was synthesized, which is cell permeable and has a broad pH-sensing range (Figure 7) (42). 7 can react with either OH- or H+ to produce red or blue emissions of varying intensity (Figure 7A). The aqueous solution of 7 under neutral condition was weakly fluorescent in red region owing to its amphiphilicity. In acidic conditions, the sulfonate group of 7 was protonated (7A), resulting in poor solubility in water and thus higher fluorescence intensity because of the AIE property. Under alkaline conditions, the OH- can act as a nucleophile to break down the conjugation of 7 (7B), thus changing the emission color from red to blue. The presence of 1,2-dioleoyl-glycero-3-phosphocholine (DOPC), the most abundant phospholipid in cellular membrane, facilitates the nucleophilic reaction and promotes the transition of red-to-blue emission at around pH 6.5, much lower than the one in the absence of DOPC (~pH 10) (Figure 7B). The ratio of the blue-to-red emission 209 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


can thus be correlated to the pH change in the physiological conditions from pH 4.5 to 8. In cell imaging, fluorescence signals of 7 were collected in two channels – the blue channel by excited at 405 nm and the red channel by excited at 488 nm. The intracellular pH can thus be mapped by using the ratio of the fluorescence intensity from the two channels, which revealed the most acidic compartment-lysosomein pseudo red color and the most alkaline compartment-mitochondria in pseudo blue color (Figure 7C). This ratio to pH relationship was further investigated using cells incubated in HEPES buffer, which kept the cell at a constant pH 7; analysis showed less than 15% of the cell was acidic. Introducing acetic acid to the living cells increased this percentage to 20%, while Dulbecco’s Modified Eagle Medium (DMEM, pH 8.5) caused the acidic regions to shrink in size. Flow cytometry can also be used to track intracellular pH by using 7 for a high-throughput pH sensing, which could be of use in a variety of assays.

Figure 7. (A) Fluorescent response of 7 to pH change. Structures of 7 and corresponding 7A and 7B after reacting with H+ and OH- respectively. (B) Plot of the ratio of the blue-to-red emission (I489/I615) of 7 in buffer solutions with different pH in the presence of DOPC. Inset: the corresponding fluorescence photographs. (C) Ratiometric fluorescence image of HeLa cells stained with 7, shown in pseudo colors from red to blue indicating low to high blue-to-red emission ratios. Scale bar 20 µm. Reproduced with permission from ref. (42). Copyright (2013) American Chemical Society. 210 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Intracellular Viscosity Intracellular viscosity is an important indicator of cell function; it affects processes such as transportation and signal transduction, and abnormalities in viscosity is associated with a range of diseases (43). Current methods in place to measure intracellular viscosity include fluorescence recovery after photobleaching (FRAP), which employs the use of a high powered laser to bleach an area of a cell, and by measuring the recovery rate the viscosity can then be deduced (44). However, FRAP can only focus on a small area of interest in the cell and it is not an efficient method to study the intracellular viscosity in the entire cytoplasmic region. Using an AIE active fluorescent probe, it is possible to correlate its fluorescence intensity and lifetime to cell viscosity, due to its mechanism of fluorescence by restriction of intramolecular motions being affected by its surroundings (45). 7 was chosen as a potential probe, which when exposed to basic conditions is transformed into 7B and exhibiting blue emission (Figure 7). The blue emission from 7B was found to be weak in pure ethylene glycol solvent, but increasing the viscosity by increasing the glycerol fractions caused enhanced emission, due to reducing the intramolecular motions of the dye and hence inhibiting the non-radiative pathways of decay. Consistent with the change in fluorescence intensity, the fluorescence lifetime was also prolonged with the increase of viscosity. To mimic the membrane environment in cells, artificial lipid vesicles were fabricated and 7B was used to quantify the fluidity within them. DOPC, 1,2diheptanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC) and 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) with cholesterol – membrane lipid components with different degrees of saturation – were used to represent membranes with different fluidity (Figure 8A). As DOPC has unsaturated bonds, it packs less tightly than DOPC with cholesterol, and the saturated tails of DHPC imply it can pack closer to each other. DSPC with the long saturated fatty acid chains pack the closest and leads to the highest viscosity among these models. Similar as the results in solution, 7B exhibited longer lifetime with the increase of membrane rigidity, or in other word, the decrease of membrane fluidity (Figure 8B). Following this, 7 was introduced into the cytoplasm of live HeLa cells from comparing fluorescent images with Nile red – a lipid droplet (LD) selective stain – was found to accumulate around these LDs as well as other organelles due to the lipophilicity of 7. In order to map the fluorescence lifetime of the stained regions in the cell, two photon excitation was used so as to minimize interference from intrinsic autofluorescence (Figure 8C). From the results gathered, a variety of viscosity was shown to exist within the cytoplasm, with the fluorescence lifetime much shorter in LDs than around structures such as tubular mitochondria. Different from most of the membrane-bound organelles with lipid bilayer membranes, LDs are surrounded by a lipid monolayer, which is less rigid. The hydrophobic AIE dyes can dissolve in the lipid pool stored inside LDs where the dye molecules can enjoy free intramolecular motions, leading to shorter fluorescence lifetime. 211 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 8. (A) The proposed packing modes of membranes with different lipid compositions. (B) Fluorescece lifetime of 7 stained lipid vesicles. (C) FLIM images of HeLa cells stained with 7 by using two-photon excitation. Reproduced with permission from ref. (45). Copyright (2015) Wiley.

Conclusion and Perspective In this chapter, we have introduced a family of imaging agents with aggregation-induced emission (AIE) properties and their applications in visualizing the cell structures and monitoring the change in cellular environment. Conventional fluorescent dyes suffer from concentration-quenching effect, and thus only very dilute concentrations of these imaging agents can be used. The small number of these dye molecules can be easily photobleached under the harsh excitation light source, resulting in poor photostability which is not suitable for long-term tracking of the cell events. As alternatives, the AIE dyes exhibit superior photostability, which allows the observation of the dynamic processes in live cells over a long period of time. These dyes show excellent biocompatibility, posing little effect on the cell viability. Through structural modification, specific targeting for organelle imaging and morphological tracking has been realized. Most of our early work on sensing and imaging were based on the change of single-wavelength fluorescence intensity, which might not be suitable for quantitative analysis. The uneven dye distribution and other technical artifacts may also affect the intensity. To overcome these problems, we later on developed AIE luminogens for ratiometric imaging of intracellular pH and lifetime imaging of intracellular viscosity, from which quantative analysis became possible. Dual functional AIE dyes that can monitor and manipulate cell activities have also been developed. A couple of AIE dyes that can induce cell apoptosis through 212 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


photoexcitation have been found, which could be alternative to the current photodynamic therapy systems. In addition to the few examples shown in this chapter, the small molecular AIE luminogens have also been found for nucleic acids in cells (46), long-term cell tracing (22, 47), evaluation of bacteria viability assay (48), enzyme activity detection in cells (49), etc. On the other hand, AIE dyes functionalized with short peptides for specific targeting of proteins of interest and AIE nanoparticles for in vivo imaging have been reported by Liu et al (50, 51) and will be covered in the other chapters of this book. Because of the multiple advantages of the AIE dyes, new imaging agents based on AIE materials should be developed. For zooming into the cells, with the advancement in imaging techniques such as super-resolution microscopy, these new dyes would be useful to visualize biological structures and dynamic intracellular events beyond the diffraction barriers. For zooming out of the cells, the AIE luminogens could be further modified for tissue and in vivo imaging through multi-photon excitation. In perspective, multifunctional AIE materials synthesized by combining fluorescence with other modalities (e.g., magnetic resonance imaging) or functionalities (e.g., photodynamic therapy) will be explored as new generation of theranostic reagents.

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AIE Luminogens for Visualizing Cell Structures and Functions - ACS

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