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.

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.

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.

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.

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.

Simultaneous Two-Color Visualization of Lipid Droplets and Endoplasmic Reticulum and Their Interplay by Single Fluorescent Probes in Lambda Mode.

In living systems, subcellular organelles mutually cooperate and closely contact to form organelle interaction networks. Thus, the simultaneous and discriminative visualization of different organelles is extremely valuable for elucidating their distribution and interplay. However, such meaningful investigations remain a great challenge due to the lack of advanced single fluorescent probes (SF-probes) capable of simultaneous and two-color imaging of two targets. Herein, for the first time, we present two excited-state intramolecular proton transfer (ESIPT) based SF-probes (PPC and EPC) for simultaneous two-color fluorescence imaging of lipid droplets (LDs) and the endoplasmic reticulum (ER) under single-wavelength excitation. Due to the strong electron-donating ability of the side substituents, the fluorescence spectra and colors of these ESIPT probes are highly sensitive to the nuance of water contents between LDs and ER, leading to orange and green fluorescence in LDs and ER, respectively, in the Lambda imaging mode. Using the probe PPC or EPC, the morphology, size, and distribution of LDs and ER have been investigated in live cells and tissues. With the aid of in situ and real-time fluorescence imaging in Lambda mode, we observed the generation of newborn LDs near the ER regions and their close apposition and shared identical fluorescence colors, probably providing a valuable proof for the mainstream hypothesis that LDs originate from the ER. The remarkable imaging performances render these SF-probes as powerful tools to decipher LD-ER related biological processes.

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.

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.

Intramolecular Spirocyclization Enables Design of a Single Fluorescent Probe for Monitoring the Interplay between Mitochondria and Lipid Droplets.

The interplay between mitochondria and lipid droplets (LDs) plays a central role in regulating the ß-oxidation and storage of fatty acids (FA) and is also engaged in responding to external stimuli such as nutrient deficiency. However, a single fluorescent probe enabling the discriminative and simultaneous visualization of the two organelles has not been reported yet, which brings limitation for the in-depth study on their interplay. In this work, utilizing the intramolecular spirocyclization reaction of rhodamine dyes that can dramatically change the optical and soluble properties, we have designed a new single fluorescent probe for labeling LDs and mitochondria in clearly separated dual-emission channels. The newly designed "biform" probe, MT-LD, presented in a ring-opened form in mitochondria to give a strong red emission, while it underwent the intramolecular spirocyclization reaction to target LDs showing an intense blue fluorescence. In this manner, MT-LD can label LDs and mitochondria in blue and red fluorescence, respectively. With this robust probe, the increase of mitochondria-LD contact and peridroplet mitochondria (PDM) amount during oleic acid treatment and starvation-induced autophagy has been successfully revealed. The interaction between the two organelles was also visualized in different tissues, which revealed an obviously higher level of mitochondria-LD contact and PDM amount in brown adipose tissue and lung tissue. This work provides a promising molecular tool to investigate the interplay between mitochondria and LDs and promotes studies on FA metabolism and autophagy.

Dual-Emissive Probe for Reversible Visualization of ΔΨm Revealing Voltage Heterogeneity in a Single Mitochondrion.

Mitochondrial membrane potential (ΔΨm) is a fundamentally important parameter in eukaryotic cells playing central roles in various vital biological processes. Precise visualization of ΔΨm depends on the robust ratiometric fluorescent probes. In this work, a new dual-emissive fluorescent probe has been fabricated for ratiometric visualization of ΔΨm. The unique probe can form near-infrared emissive aggregates (∼670 nm) in mitochondria with high ΔΨm, which turned to green-emitting monomers (530 nm) with loss of ΔΨm. The reversible changes of ΔΨm can be clearly observed, and the ultralarge emission shift (∼140 nm) is greatly favorable for the clear observation of voltage distribution under a super-resolution microscope. With the robust probe, the heterogenous voltage distribution in a single mitochondrion has been revealed for the first time, which can facilitate the in-depth understanding of fine structures in mitochondria. The cell damages induced by various reagents were successfully visualized using the innovative probe, demonstrating its pronounced potential for biological research.

Ratiometric Fluorescence Imaging for the Distribution of Nucleic Acid Content in Living Cells and Human Tissue Sections.

The misregulation of nucleic acids behavior leads to cell dysfunction and induces serious diseases. A ratiometric fluorescence probe is a powerful tool to study the dynamic behavior and function relationships of nucleic acids. However, currently, no such effective probe has been reported for in situ, real-time tracking of nucleic acids in living cells and tissue sections. Herein, the unique probe named QPP-AS was rationally designed for ratiometric fluorescence response to nucleic acids through skillful regulation of the intramolecular charge-transfer capabilities of the electron acceptor and donor. Encouraged by the advantages of the selective nucleic acid response, ideal biocompatibility, and high signal-to-noise ratio, QPP-AS has been applied for in situ, real-time ratiometric fluorescence imaging of nucleic acids in living cells for the first time. Furthermore, we have demonstrated that QPP-AS is capable of visualizing the dynamic behavior of nucleic acids during different cellular processes (e.g., cell division and apoptosis) by ratiometric fluorescence imaging. More significantly, QPP-AS has been successfully used for ratiometric fluorescence imaging of nucleic acids in human tissue sections, which provides not only the cell contour, nuclear morphology, and nuclear-plasma ratio but also the nucleic acid content information and may greatly improve accuracy in clinicopathological diagnosis.

Fluorescent Probes for the Visualization of Cell Viability.

Monitoring cell viability is a crucial task essential for the fundamental studies in apoptosis, necrosis, and drug discovery. Cell apoptosis and necrosis are significant to maintain the cell population, and their abnormality can lead to severe diseases including cancer. During cell death, significant changes occur in the intracellular contents and physical properties, such as decrease of esterase activity, depolarization of the mitochondrial membrane potential (ΔΨm), increase of caspase content, dissipation of membrane asymmetry, and loss of membrane integrity. To detect cell viability, the fluorescent probes have been developed by taking advantage of these biological parameters and using various fluorescence mechanisms. These fluorescent probes can serve as powerful tools to facilitate the research in biology and pathology. In this Account, the representative examples of the fluorescent probes for cell viability during the past decades have been summarized and classified into five types based on the biological changes. The basic principle, design strategy, fluorescence mechanisms, and molecular construction of these fluorescent probes have been discussed. Furthermore, the intrinsic characteristics and merits of these probes have been illustrated. Particularly, this Account describes our recent works for the design and synthesis of the fluorescent probes to detect cell viability in the dual-color and reversible modes. The dual-color and reversible fluorescent probes are highlighted owing to their unique benefits in accurate and dynamic detection of cell viability. In general, the dual-color fluorescent probes were constructed based on the loss of esterase activity during cell death. Excited-state intramolecular proton transfer (ESIPT) and intramolecular charge transfer (ICT) process were exploited for the probe design. The construction of such dual-color probes were realized by the acetate of the phenyl group on fluorophores. Esterases in healthy cells hydrolyze the acetate and bring a spectral shift to the probes. Moreover, reversible fluorescent probes for cell viability were designed based on the depolarization of ΔΨm, with relocalization properties dependent on ΔΨm. The probes target mitochondria in healthy cells with high ΔΨm, while they are relocalized into the nucleus in unhealthy cells with depolarized ΔΨm. As ΔΨm is reversibly changed according to the cell viability, these probes reversibly detect cell viability. The reversible and simultaneously dual-color fluorescent probes were developed based on the relocalization mode and aggregation-induced emission shift. The probes target mitochondria to form aggregates with deep-red emission, while they migrate into the nucleus to present in monomers with green fluorescence. In this manner, the probes enable dual-color and reversible detection of cell viability. Fluorescent probes for cell viability based on sensing the membrane integrity, caspase activity, and membrane symmetry are also presented. High-polarity and large-size fluorescent probes impermeable to the intact lipid bilayer selectively target apoptotic cells with a destructive plasma membrane. Fluorescent probes sensing caspases in a turn-on manner exclusively light up apoptotic cells with caspase expression. Membrane-impermeable probes with high affinity to phosphatidylserine (PS) specifically stain the plasma membrane of dead cells, since PS flip-flops to the outer leaflet of the membrane during cell death. In summary, this Account illustrates the basic principles, design strategies, characteristics, and advantages of the fluorescent probes for cell viability, and it highlights the dual-color and reversible probes, which can promote the development of fluorescent probes, apoptosis studies, drug discovery, and other relative areas.

Unique pH-Sensitive RNA Binder for Ratiometric Visualization of Cell Apoptosis.

Fluorescent probes for monitoring cell apoptosis are powerful tools in biological and pathological research. However, ratiometric probes for in situ and real-time visualization of apoptosis with high accuracy are still deficient, which limits the studies relative to cell apoptosis. In this work, a pH-sensitive and positively charged RNA binder was designed and synthesized for the first time for the ratiometric visualization of cell apoptosis. In healthy cells, the probe targets mitochondria with basic matrixes and high membrane potential and displays intense emission in the blue and red channels. During apoptosis, the probe is released from mitochondria, binds to basophilic RNA, and shows emission in only the red channel. Consequently, cell apoptosis caused by drug treatment could be efficiently and clearly monitored in a ratiometric manner. The probe is expected to facilitate the study of cell apoptosis and relative areas.

Förster Resonance Energy Transfer-Based Fluorescent Probe for the Selective Imaging of Hydroxylamine in Living Cells.

Hydroxylamine (HA) is an important product of cell metabolism and plays a significant role in many biological processes, and therefore, real-time imaging of HA is of great importance for the in-depth study of its physiological and pathological functions. However, a HA-specific fluorescent probe is currently lacking primarily because the highly selective HA-responsive site is undeveloped. To address this critical issue, we present a HA-specific FRET-based fluorescent probe (RhChr) for the selective detection of HA in living systems. Inspired by aza-Michael addition, the unsaturated system appended with an iminium ion was employed as the new HA-specific response site. In response to HA, RhChr provided a ratiometric signal output with excellent selectivity toward HA over biothiols and ammonia. We have demonstrated that RhChr could be applied for the ratiometric imaging of endogenous HA in living cells and the evaluation of xanthine oxidase (XOD) activity in living organs.

A two-photon excited red-emissive probe for imaging mitochondria with high fidelity and its application in monitoring mitochondrial depolarization via FRET.

Mitochondria play central roles in plenty of biological processes, such as apoptosis, signaling, and cell differentiation. Mitochondrial depolarization is a significant sign of deterioration of intracellular status. Although many fluorescent probes for visualizing mitochondria have been delivered, two-photon red-emitting mitochondrial probes capable of detecting mitochondrial depolarization remain rare. In this work, by linking a pyrene moiety to a quinoline salt, we have constructed two-photon red-emitting mitochondrial probes, MVQ and HVQ. Between them, HVQ with a longer side chain exhibits higher hydrophobic properties, and can image mitochondria with high-fidelity due to the aggregation caused quenching effect. In cooperation with MTDR, a commercialized mitochondrial probe, HVQ, could be used to image the depolarization of mitochondria induced by a protonophore and hydrogen peroxide. We believe that HVQ can serve as a powerful tool for the investigation of mitochondria and mitochondrial membrane potential in fundamental biological research.

Discriminating Live and Dead Cells in Dual-Color Mode with a Two-Photon Fluorescent Probe Based on ESIPT Mechanism.

Discrimination of live and dead cells is an important task in biological, pathological, medical, and pharmaceutical studies. In this work, we have developed a novel fluorescent probe DACA that can discriminate live and dead cells in a dual-color mode for the first time. DACA can stain dead cells with blue fluorescence peaked at 440 nm, while it can also label live cells with orange emission peaked at 570 nm. Compared with one-color fluorescent probes, such a dual-color probe can efficiently avoid false positive results from cellular autofluorescence and misleading signals brought by inhomogeneous staining, and thus can supply more accurate information in biological applications. By means of DACA, the health status of tumor cells pretreated by H2O2 and ultraviolet radiation has been successfully detected and imaged. Moreover, DACA and the hydrolyzed product exhibit excellent two-photon properties. Live and dead cells, as well as the zebrafishes, have been discriminated with dual emission colors under one- and two-photon microscope. These results demonstrate that DACA is a powerful tool for dual-color distinguishing live and dead cells in vitro and in vivo.