A polarity-responsive lysosomes-nucleus translocation probe for the dual-emissive visualization of cell apoptosis.

Visualization of cell apoptosis is a critical task playing central roles in the fundamental studies in biology, pathology, and biomedicine. Dual-emissive fluorescent probes are desired molecular tools for study on apoptosis, which however were rarely reported. Herein, utilizing the polarity differences between lysosomes and nucleus, a translocation type of fluorescent probe (NA-S) was developed for the dual-color visualization of cell apoptosis. NA-S was designed to be polarity sensitive, bearing alkalescence group, and with DNA affinity. In living cells, NA-S targeted the lysosomes to give blue fluorescence, which translocated into the nucleus during cell apoptosis to give green emission. Thereby, the cell apoptosis could be visualized with NA-S in dual-emissive manner. With the unique probe, the cell apoptosis induced by oxidative stress, UV irradiation, rotenone, colchicine, and paclitaxel have been successfully visualized.

Unique fluorescent probe for the recognition of late apoptosis via translocation from plasma membrane to nucleus.

Cell apoptosis is a crucial physiological process playing central roles in key biological and pathological activities. However, the current fluorescent probes for the detection of late apoptosis were "off-on" probes, which were facilely interfered by false positive signals caused by inhomogeneous staining and other factors. Herein, a unique fluorescent probe (NPn) discriminating late apoptosis from early apoptosis and heathy status with two different sets of fluorescent signals have been prepared, to overcome the possible false positive signals. NPn was designed impermeable to biomembranes and simultaneously with high affinity to DNA/RNA, which localized on the plasma membranes of living and early apoptotic cells, while relocated to the nucleus in late apoptotic cells. The hydrophilic amine unit and small ion radius were responsive for its membrane impermeability, which was confirmed with two control molecules without amine group. Using the probe, we have successfully evaluated the cell apoptosis induced by ultraviolet irradiation, rotenone, colchicine, and paclitaxel, demonstrating its potential application in biological researches.

Single Fluorescent Probe for Multiple Tasks: Illuminating Lipid Droplets and Lysosomes in Dual Channels and Distinguishing Autophagy and Apoptosis.

Lipid droplets (LDs) and lysosomes play key roles in autophagy and cell apoptosis, and the discriminative visualization of the two organelles and simultaneously of autophagy and apoptosis is very helpful to understand their internal relationships. However, fluorescent probes that can concurrently achieve these tasks are not available currently. Herein, we delicately fabricate a robust probe CAQ2 for multiple tasks: illumination of LDs and lysosomes in dual emission colors as well as discriminative visualization of cell apoptosis and autophagy. The probe exhibited both lipophilic and basic properties and displayed different emission colors in neutral and protonated forms; thus, LDs and lysosomes emitted blue and red fluorescence colors, respectively. Because of the lysosomal acidification during autophagy, CAQ2 detected autophagy with evidently enhanced red emission. Because of the lysosomal alkalization during apoptosis, CAQ2 imaged apoptosis with a drastically decreased red fluorescence intensity. With the robust probe, the autophagy under starvation and lipidless conditions was visualized, and the apoptosis induced by H2O2, ultraviolet (UV) irradiation, and rotenone treatment was successfully observed. The efficient detoxification of Na2S against rotenone treatment was successfully revealed.

Cancer-cell-specific Self-Reporting Photosensitizer for Precise Identification and Ablation of Cancer Cells.

Cancer-cell-specific fluorescent photosensitizers (PSs) are highly desired molecular tools for cancer ablation with minimal damage to normal cells. However, such PSs that can achieve cancer specification and ablation and a self-reporting manner concurrently are rarely reported and still an extremely challenging task. Herein, we have proposed a feasible strategy and conceived a series of fluorescent PSs based on simple chemical structures for identifying and killing cancer cells as well as monitoring the photodynamic therapy (PDT) process by visualizing the change of subcellular localization. All of the constructed cationic molecules could stain mitochondria in cancer cells, identify cancer cells specifically, and monitor cancer cell viability. Among these, IVP-Br has the strongest ability to produce ROS, which serves as a potent PS for specific recognition and killing of cancer cells. IVP-Br could translocate from mitochondria to the nucleolus during PDT, self-reporting the entire therapeutic process. Mechanism study confirms that IVP-Br with light irradiation causes cancer cell ablation via inducing cell cycle arrest, cell apoptosis, and autophagy. The efficient ablation of tumor through PDT induced by IVP-Br has been confirmed in the 3D tumor spheroid chip. Particularly, IVP-Br could discriminate cancer cells from white blood cells (WBCs), exhibiting great potential to identify circulating tumor cells (CTCs).

Construction of a Dual-Emissive Probe for Discriminative Visualization of Lysosomal and Mitochondrial Dysfunction.

Discriminatively visualizing mitochondrial and lysosomal dysfunction is crucial for an in-depth understanding of cell apoptosis regulation and relative biology. However, fluorescent probes for the separate visualization of lysosomal and mitochondria damages have not been reported yet. Herein, we have constructed a fluorescent probe [2-(4-hydroxystyryl)-1,3,3-trimethyl-3H-indol-1-ium iodide (HBSI)] for labeling mitochondria and lysosomes in dual emission colors and discriminatively imaging mitochondrial and lysosomal damage in two different sets of fluorescent signals. In living cells, HBSI targeted both lysosomes and mitochondria to give green and red emission, respectively. During mitochondrial damages, HBSI immigrated into lysosomes, and the red emission decreased. During lysosomal damage, HBSI immigrated into mitochondria, and the green emission decreased. With the robust probe, the different damaging sequences of mitochondria and lysosomes under different amounts of H2O2 and chloral hydrate have been revealed. The sequential damage of lysosomes and mitochondria during cell apoptosis induced by rotenone, paclitaxel, and colchicine has been discovered. Furthermore, the regulation of mitochondria, lysosome, and their interplay during autophagy was also observed with the probe.

Development of a high polarity-sensitive fluorescent probe for visualizing the lipid droplets and endoplasmic reticulum with dual colors in living cells.

Lipid droplets (LDs) are unique organelles that control the lipid metabolism in cells. It has been identified that the generations of LDs derive from endoplasmic reticulum (ER) and they have closely related with amount of cellular activities for maintaining homeostasis. To further explore the detail interactions between LDs and ER, we have developed a novel polarity-sensitive fluorescent probe LP with distinct D-π-A-π-D framework and applied it to imaging LDs and ER with dual colors at the same time. Probe LP showed well red-shifted emissions with the increase fraction of water in the 1,4- dioxane due to ICT process. In biological imaging, probe LP could visualize LDs and ER with green and red fluorescence separately. Besides, the dynamic behaviors of LDs and ER were achieved using LP during the oleic acids and starvation stimulations. Therefore, probe LP is a valuable molecular tool for investigating the relationships of LDs and ER in various cellular activities.

Autophagy-Activated Self-reporting Photosensitizer Promoting Cell Mortality in Cancer Starvation Therapy.

Cancer starvation therapy have received continuous attention as an efficient method to fight against wide-spectrum cancer. However, during cancer starvation therapy, the protective autophagy promotes cancer cells survival, compromising the therapeutic effect. Herein, a novel strategy by combination of autophagy-activated fluorescent photosensitizers (PSs) and cancer starvation therapy to realize the controllable and efficient ablation of tumor is conceived. Two dual-emissive self-reporting aggregation-induced emission luminogens (AIEgens), TPAQ and TPAP, with autophagy-activated reactive oxygen species (ROS) generation are prepared to fight against the protective autophagy in cancer starvation therapy. When protective autophagy occurs, a portion of TPAQ and TPAP will translocate from lipid droplets to acidic lysosomes with significant redshift in fluorescence emission and enhanced ROS generation ability. The accumulation of ROS induced by TPAQ-H and TPAP-H causes lysosomal membrane permeabilization (LMP), which further results in cell apoptosis and promotes cell death. In addition, TPAQ and TPAP can enable the real-time self-reporting to cell autophagy and cell death process by observing the change of red-emissive fluorescence signals. Particularly, the efficient ablation of tumor via the combination of cancer starvation therapy and photodynamic therapy (PDT) induced by TPAQ has been successfully confirmed in 3D tumor spheroid chip, suggesting the validation of this strategy.

Ratiometric and Discriminative Visualization of Autophagy and Apoptosis with a Single Fluorescent Probe Based on the Aggregation/Monomer Principle.

Autophagy and apoptosis play a central role in maintaining homeostasis in mammals. Therefore, discriminative visualization of the two cellular processes is an important and challenging task. However, fluorescent probes enabling ratiometric visualization of both autophagy and apoptosis with different sets of fluorescence signals have not been developed yet. In this work, we constructed a versatile single fluorescent probe (NKLR) based on the aggregation/monomer principle for the ratiometric and discriminative visualization of autophagy and apoptosis. NKLR can simultaneously perform two-color imaging of RNA (deep red channel) and lysosomes (yellow channel) in aggregation and monomer states, respectively. During autophagy, NKLR migrated from cytoplasmic RNA and nuclear RNA to lysosomes, showing enhanced yellow emission and sharply decreased deep red fluorescence. Moreover, this migration process was reversible upon the recovery of autophagy. Comparatively, during apoptosis, NKLR immigrated from lysosomes to RNA, and the yellow emission decreased and even disappeared, while the fluorescence of the deep red channel slightly increased. Overall, autophagy and apoptosis could be discriminatively visualized via the fluorescence intensity ratios of the two channels. Meanwhile, the cells in three different states (healthy, autophagic, apoptotic) could be distinguished by three point-to-point fluorescence images via the localization and emission color of NKLR. Therefore, the probe NKLR can serve as a desirable molecular tool to reveal the in-depth relation between autophagy and apoptosis and facilitate the study on the two cellular processes.

Chameleon-Like Fluorescent Probe for Monitoring Interplays between Three Organelles and Reporting Cell Damage Processes through Dramatic Color Change.

The in-depth study of the interplay and cooperation between multiple organelles is an important biological task. Single fluorescent probes for separate visualization of multiple organelles is a desirable molecular tool, but the construction of such a probe is extremely difficult owing to the lack of valid strategies. In this work, utilizing the reversible cyclization reaction and intermolecular π stacking mechanism, a robust fluorescent probe is constructed to discriminatively illuminate lipid droplets (LDs), mitochondria, and lysosomes with blue, green, and red emission colors, respectively. Using the probe, the interplays and cooperation between LDs, mitochondria, and lysosomes are successfully studied, and the critical roles of lysosomes and LDs during mitochondrial fission are successfully revealed. Furthermore, this unique probe reveals the sequential damage of mitochondria and lysosomes during apoptosis through the successive fading of green and red emission. Thereby, the probe enables the discrimination of health state, early apoptosis, and late apoptosis of cells with three different sets of fluorescent signals. Overall, the robust probe is a desirable molecular tool to reveal the interactions between the three organelles, and investigate cell apoptosis and relative areas.

Single Fluorescent Probe for Zero-Crosstalk Discrimination of Lipid Droplets and the Endoplasmic Reticulum Based on Reversible Cyclization Reaction.

The interactions between different organelles are ubiquitous and crucial for life activities. Thus, development of a single fluorescent probe enabling the simultaneous two-color visualization of two organelles is of great significance for the study of organelle interplay. Herein, using the reversible ring-opening/closing reactions of rhodamine dyes, we have fabricated a robust fluorescent probe to distinguish lipid droplets (LDs) and the endoplasmic reticulum (ER) in dual-emission channels with negligible crosstalk. The probe 6'-(diethylamino)-4'-((7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-1',2',3',4'-tetrahydro-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one, which was sensitive to the changes in the water content in the organism, displayed strong green fluorescence in the hydrophobic LDs from its ring-closed form, while it existed in a ring-opened form in the ER to illuminate a strong near-infrared emission. Importantly, the spectral difference was up to 320 nm, and thus the crosstalk between two channels was negligible. With the unique probe, the lipid accumulation in cells treated with different concentrations of oleic acid, cholesterol, and stearic acid has been successfully observed. The changes of LDs and the ER in living cells stimulated by temperature changes and hypoxia stimulation have also been revealed. Meanwhile, the different sizes and distribution of LDs and the ER in various tissues were also studied using the robust probe. This work provides a new approach to the design of dual-emissive probes and contributes to a significant molecular tool to promote the study of organelle interactions.

Permeability-Controlled Probe for Directly Visualizing the Opening of Mitochondrial Permeability Transition Pore in Native Status.

The opening of mitochondrial permeability transition pore (mPTP) plays a fundamental role in cell apoptosis regulation, ischemia-reperfusion injury, and neurodegenerative disorders. However, the molecular tools for detecting mPTP open in cellular native status have not been reported yet. Herein, we de novo designed a robust fluorescent probe mPTP-F to monitor mPTP opening in cellular native status for the first time. The membrane-permeable probe could accumulate into mitochondria and convert to a product poorly permeable to biomembranes, which was trapped in mitochondria to form near-infrared (NIR)-emissive aggregates. After mPTP opening, the product was released from mitochondria through the pore to form green-emissive monomers. Significantly, with mPTP-F, we discovered that formaldehyde, a signaling molecule, could induce mPTP opening. Therefore, the new probe could serve as a desirable molecular tool for the study of ischemia-reperfusion injury, cell apoptosis, and relative areas.

Revealing the Phase Separation in ER Membranes of Living Cells and Tissues by In Situ NIR Ratiometric Imaging.

Biomembranes in the endoplasmic reticulum (ER) play indispensable roles in various bioactivities, and therefore, visualizing the phase separation in ER membranes is crucial for the studies on the fundamental biology of the ER. However, near-infrared (NIR) ratiometric imaging of the phase behaviors of the ER in living cells with different statuses and in diverse tissues has not been investigated. Herein, we developed a polarity-responsive NIR fluorescent probe (DCA) for the visualization of the phase behavior in ER membranes. The probe displayed a large Stokes shift and was highly sensitive to polarity. By direct and native fluorescence imaging at room temperature, the ERo and ERd biomembranes in the ER could be clearly distinguished by dual NIR emission colors. Oxidative damage by H2O2 and homocystein (Hcy)-induced ER stress can efficiently induce the formation of large-scale ERo domains in ER membranes. Moreover, we have also revealed that different tissues exhibited diverse phase behaviors in the ER membranes. The ER membranes in cardiac and skeletal muscle tissues showed no evident phase separation, while large-scale ERo domains existed in the ER of liver tissues and formed at the ER membranes adjacent to lipid droplets (LDs) in white adipose tissues. We expect that the probe could serve as a powerful molecular tool to promote fundamental research studies on ER membranes and relative biomedical areas.

Hot-Band Absorption of a Cationic RNA Probe Enables Visualization of ΔΨm via the Controllable Anti-Stokes Shift Emission.

Mitochondrial membrane potential (ΔΨm) is an important biophysical parameter playing central roles in cell apoptosis, mitochondrial dysfunction, and other biological and pathological processes. Herein, we have rationally designed and fabricated a unique fluorescent probe for convenient ΔΨm visualization based on hot-band absorption and controllable anti-Stokes shift emission. The robust probe was excitable via hot-band absorption and emitted anti-Stokes upconversion emission and Stokes downconversion fluorescence simultaneously. The anti-Stokes emission could be efficiently inhibited upon the binding to RNA. The cationic probe targeted mitochondria in living cells with high ΔΨm and displayed both anti-Stokes green emission and ordinary red fluorescence. After the decrease of ΔΨm, the probe immigrated out of mitochondria to RNA and nucleolus, which showed only red emission owing to the inhibition of anti-Stokes fluorescence. In this manner, the ΔΨm could be visualized in dual-color mode. The probe enabled clearly monitoring the reversible changes in ΔΨm and was successfully employed to visualize oxidative damage of living cells. The decrease of ΔΨm in living tissues was also successfully observed with the newly designed probe.

The diversity of the coordination bond generated a POSS-based fluorescent probe for the reversible detection of Cu(II), Fe(III) and amino acids.

In recent years, it has been found that Cu2+, Fe3+, and amino acids play an irreplaceable and subtle role in organisms and have attracted the considerable attention of many researchers. Therefore, it is vital to design visual indicators to reveal the relationships between metal ions and amino acids. However, there have been few reports on this vigorous subject. Fortunately, based on the different coordination effects between metal ions and boron groups, we have designed an accessible fluorescent probe (PSI-A). Borane was introduced as an ion-sensitive group to form a novel POSS-based fluorescent probe, which achieves fascinating performance, in situ dynamic multiple detection, excellent photostability, and enervative biological toxicity. PSI-A exhibited predominant selectivity and sensitivity to Cu2+/amino acids and Fe3+/amino acids sequence reactions in HepG2 cells and zebrafish. The fluorescence of PSI-A was quenched by Cu2+, which can be recovered by adding Asp, Ser, Arg, Ace or Trp. Additionally, the fluorescence of PSI-A quenched by Fe3+ can be restored after adding Asp. PSI-A is available to monitor Cu2+/amino acids and Fe3+/amino acids sequence reactions and can be repeated for at least three consecutive cycles without a fatigued performance. Therefore, this multifunctional fluorescent probe may have prospective application potentials in the biological field.

Two-Color Visualization of Cholesterol Fluctuation in Plasma Membranes by Spatial Distribution-Controllable Single Fluorescent Probes.

Visualizing cholesterol (CL) fluctuation in plasma membranes is a crucially important yet challenging task in cell biology. Here, we proposed a new imaging strategy based on permeability changes of plasma membranes triggered by different CL contents to result in controllable spatial distribution of single fluorescent probes (SF-probes) in subcellular organelles. Three spatial distribution-controllable SF-probes (PMM-Me, PMM-Et, and PMM-Bu) for imaging CL fluctuation in plasma membranes were rationally developed. These SF-probes target plasma membranes and mitochondria at normal CL levels, while they display solely staining in plasma membranes and mitochondria at increased and decreased CL levels, respectively. These polarity-sensitive probes also show distinct emission colors with fluorescence peaks of 575 and 620 nm in plasma membranes and mitochondria, respectively. Thus, the CL fluctuation in plasma membranes can be clearly visualized by means of the spatially distributed and two-color emissive SF-probes.

Monitoring Mitophagy via the FRET Mechanism: Visualizing Mitochondria, Lysosomes, and Autolysosomes in Three Different Sets of Fluorescence Signals.

Mitophagy is a vital biological process playing central roles in the regulation of metabolic activity and quality control of mitochondria. The presented dual-color fluorescent probes to directly monitor mitophagy were based on the optical response to pH change during mitophagy, but pH fluctuation may lead to interference. To overcome this, herein, two fluorescent probes (G-Mito, R-Lyso) were rationally designed to visualize mitophagy directly in a dual-color manner, relying on the Förster resonance energy transfer (FRET) process for the first time. Green emissive G-Mito targeted and anchored the mitochondria via reaction with protein thiols. Red-emissive R-Lyso exclusively targeted lysosomes. Live cells loaded with the two probes demonstrated strong fluorescence in only the green channel with excitation at 405 nm. After mitophagy, G-Mito in mitochondria was delivered into the lysosomes, and red fluorescence evidently increased due to the FRET process. With the probes, mitochondria, lysosomes, and autolysosomes could be discriminatively visualized in three different sets of signals. Mitophagy induced by starvation and in normal physiological status were successfully observed. The probes revealed that a certain amount of H2O2 could induce mitophagy. We expect that the two probes can serve as molecular tools for validation of mitophagy and promote the development of related areas.

Fabrication of a fluorescent probe for reversibly monitoring mitochondrial membrane potential in living cells.

Mitochondria are important organelles in cells, which play an important role in metabolism and many other vital biological events. Mitochondrial membrane potential (MMP) is a significant biological parameter participating in various procedures. However, fluorescent probes for monitoring MMP are rarely reported, which greatly limited the related studies. Herein, we present the rational design, synthesis, and living cell imaging studies of a fluorescent probe REP for monitoring MMP changes based on organic cationic fluorophores. In live cells with high MMP levels, REP can exclusively light up mitochondria with intense fluorescence. Upon the loss of MMP, the emission of intracellular REP evidently decreased. The reversible changes in MMP have been successfully monitored by REP, and the oxidative damages to live cells have been detected with the probe. The probe is expected to serve as a desired tool in studying MMP and related areas.

A red-emissive and positively charged RNA ligand enables visualization of mitochondrial depolarization and cell damage.

In this work, a red-emissive RNA ligand bearing two positive charges were developed for the visualization of mitochondrial depolarization, via the subcellular localization of the ligand molecules. The ligand with quinolinium moiety and strong electronic donor displays red fluorescence peaked at 630 nm. Meanwhile, the probe is concentrated in mitochondria of live cells due to the high mitochondrial membrane potential, and re-localizes into nucleolus upon mitochondrial depolarization owing to the affinity to RNA. In this manner, the decrease of mitochondrial membrane potential could be real-timely and in-situ monitored with the red-emissive probe. Particularly, two cations were decorated on the probe, which enables the fast response to mitochondrial depolarization with elevated sensitivity. Cell damage induced by H2O2 was also successfully observed with the probe. We expect that the probe can promote researches on mitochondrial membrane potential, cell apoptosis, and relative areas.

Endoplasmic reticulum-specific fluorescent probe for the two-photon imaging of endogenous superoxide anion (O2•-) in live cells and zebrafishes.

ER stress has close relation with various metabolic diseases including obesity and insulin resistance, and could result in the abnormal production of ROS including O2-. Real-time and in situ detection of endogenous O2- in ER is vitally important for revealing the physiological roles of O2- during ER stress. Herein, we present an ER-specific two-photon probe (ER-Rs) for the detection of endogenous O2- in living cells and zebrafishes. The probe ER-Rs employed triflate as the response site for O2-, and used p-methylsulfonamide as the ER-specific moiety. In response to O2-, the triflate group of the probe ER-Rs was transformed to hydroxyl and the turn-on fluorescence was produced. The probe ER-Rs displayed highly sensitive and selective response to O2-, and could be employed as an ER-specific two-photon probe for the visualization of endogenous O2- in live cells, tissues and zebrafishes.