Fluorescent Probes for Sensing and Imaging within Specific Cellular

Sep 23, 2016 - SYTO RNASelect is commercially available for nucleolar staining but suffers from low photostability and has undisclosed chemical struct...
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Fluorescent Probes for Sensing and Imaging within Specific Cellular Organelles Hao Zhu, Jiangli Fan, Jianjun Du, and Xiaojun Peng* State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, Dalian 116024, China CONSPECTUS: Fluorescent probes have become powerful tools in biosensing and bioimaging because of their high sensitivity, specificity, fast response, and technical simplicity. In the last decades, researchers have made remarkable progress in developing fluorescent probes that respond to changes in microenvironments (e.g., pH, viscosity, and polarity) or quantities of biomolecules of interest (e.g., ions, reactive oxygen species, and enzymes). All of these analytes are specialized to carry out vital functions and are linked to serious disorders in distinct subcellular organelles. Each of these organelles plays a specific and indispensable role in cellular processes. For example, the nucleus regulates gene expression, mitochondria are responsible for aerobic metabolism, and lysosomes digest macromolecules for cell recycling. A certain organelle requires specific biological species and the appropriate microenvironment to perform its cellular functions, while breakdown of the homeostasis of biomolecules or microenvironmental mutations leads to organelle malfunctions, which further cause disorders or diseases. Fluorescent probes that can be targeted to both specific organelles and biochemicals/microenvironmental factors are capable of reporting localized bioinformation and are potentially useful for gaining insight into the contributions of analytes to both healthy and diseased states. In this Account, we review our recent work on the development of fluorescent probes for sensing and imaging within specific organelles. We present an overview of the design, photophysical properties, and biological applications of the probes, which can localize to mitochondria, lysosomes, the nucleus, the Golgi apparatus, and the endoplasmic reticulum. Although a diversity of organelle-specific fluorescent stains have been commercially available, our efforts place an emphasis on improvements in terms of low cytotoxicity, high photostability, near-infrared (NIR) emission, two-photon excitation, and long fluorescence lifetimes, which are crucial for long-time tracking of biological processes, tissue and body imaging with deep penetration and low autofluorescence, and time-resolved fluorescence imaging. Research on fluorescent probes with both analyte responsiveness and organelle targetability is a new and emerging area that has attracted increasing attention over the past few years. We have extended the diversity by developing organelle-specific responsive probes capable of detecting changes in biomolecular levels (reactive oxygen species, fluoride ion, hydrogen sulfide, zinc cation, thiol-containing amino acids, and cyclooxygenase-2) and the microenvironment (viscosity, polarity, and pH). Future research should give more considerations of the “low-concern” organelles, such as the Golgi apparatus, the endoplasmic reticulum, and ribosomes. In addition, given the tiny sizes of subcellular organelles (20−1000 nm), we anticipate that clearer visulization of the cellular events within specific organelles will rely on superresolution optical microscopy with nanoscopic-scale resolution.

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mediators of oxidative stress,1,2 while irregular ROS production is involved in mitochondrial uncoupling and cardiac problems.3 Consequently, monitoring of biological species and the microenvironment within specific organelles is crucial to provide accurate information to understand their roles in the physiopathology of organelles. Fluorescent probes are powerful molecular tools for both analytical sensing and optical imaging because of their high sensitivity, specificity, fast response, and technical simplicity. They can provide direct visualization and dynamic information concerning the localization and quantity of biomolecules of interest.4 In addition, by taking advantage of the effect of the local environment on the fluorescence properties of fluorophores, highly environmentally sensitive fluorescent probes

INTRODUCTION Organelles are specialized subunits within cells that are usually separately enclosed by their own lipid bilayers. Major eukaryotic organelles include the nucleus, mitochondria, lysosomes, the endoplasmic reticulum, and the Golgi apparatus (Figure 1), with typical sizes from 20 to 1000 nm. Each of these organelles plays a specific and indispensable role in cellular processes. For example, the nucleus regulates gene expression, mitochondria are responsible for aerobic metabolism, and lysosomes digest macromolecules for cell recycling. A certain organelle needs specific biological species and the appropriate microenvironment to function; therefore, disruptions in the homeostasis of these species or microenvironmental mutations contribute to dysfunctions of the organelle, further causing disorders and diseases. For example, mitochondria are a primary source of endogenous cellular reactive oxygen species (ROS), which act as physiological signaling molecules and © 2016 American Chemical Society

Received: June 13, 2016 Published: September 23, 2016 2115

DOI: 10.1021/acs.accounts.6b00292 Acc. Chem. Res. 2016, 49, 2115−2126

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Accounts of Chemical Research

fluorescent probe that localizes in the Golgi apparatus for tumor imaging.

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MITOCHONDRIA-LOCALIZED PROBES Mitochondria, the principal energy-producing compartments in most eukaryotes, play critical roles in a number of vital cellular processes, such as ATP production, central metabolism, and apoptosis.11 One way to selectively target mitochondria to attract the negative potential of the mitochondrial membrane by cations. In particular, delocalized lipophilic cations (DLCs), such as triphenylphosphonium (TPP), are effective at crossing the hydrophobic membranes and preferentially accumulate in mitochondria of living cells.12 TPP has been extensively adopted for the uptake of small-molecule fluorescent dyes into mitochondria.7,13−16 However, toxicity is a particular concern for the use of some DLCs, as they have been shown to compromise mitochondrial function at high concentrations.12 To address this, we introduced a pyridinium-based mitochondria-targeting group with low cytotoxicity. Furthermore, we have developed dual-functional mitochondrial probes that respond to ROS, H2S, F−, viscosity, and polarity. OBEP17 was reported as an effective mitochondrial stain in which positively charged pyridinium, an established mitochondrial tag,18 was tethered to a difluoroboron dipyrromethene (BODIPY) fluorophore at the meso position (Figure 2). The absorption and fluorescence spectra of OBEP were slightly bathochromically shifted (λabs = 520 and λem = 541 nm), presumably because of charge transfer between the pyridinium moiety and the BODIPY core. Importantly, OBEP showed excellent lightfastness (Figure 2B) and extremely low cytotoxicity (Figure 2C), which are critical for long-term tracking of biological events. The swollen mitochondria resulting from different degrees of cell damage by irradiation were readily labeled by OBEP (Figure 2D). Compared with the UV−vis light, near-infrared (NIR) light (650−900 nm) is preferred for biological imaging because of minimal photodamage to biological samples, deep tissue penetration, and minimum interference from background autofluorescence.19 However, NIR dyes are generally vulnerable to light and exhibit rapid photobleaching. By introducing a quaternized pyridine moiety to a π-extended BODIPY fluorophore with phenyl and thienyl substituents at the 3,5-

Figure 1. Major organelles of an animal cell.

have been exploited to report on microenvironmental changes.5 Confocal microscopy with increasing optical resolution has significantly facilitated the bioimaging applications of compartment-specific fluorescent probes in live cells. To date, a diverse array of organelle-staining fluorescent probes are commercially available and can be used in biological investigations of respiration, mitosis, substrate degradation and detoxification, and intracellular transport, among others.6 However, issues of cytotoxicity, photostability, short excitation and emission wavelengths, and fast fluorescence decay limit their applications in long-term fluorescence tracking of cellular events, tissue and body imaging with deep penetration and low autofluorescence, and time-resolved imaging. Over the past few years, there has been an upsurge in interest in responsive organelle-targetable fluorescent probes that fulfill the demand for reporting changes in biomolecular concentration or microenvironment within specific organelles, as exemplified by the mitochondria-targeted H2O2-selective probe,7 protein-labeled zinc sensors that localize to mitochondria or the Golgi apparatus,8 the lysosomal molecular rotor,9 and polarity-sensitive sensors accumulated in the endoplasmic reticulum.10 Still, further development of diversities in types of organelles and analytes is in demand. In this Account, we introduce our efforts to develop organellespecific fluorescence labels and responsive probes, highlighting low-toxicity organelle tags, highly photostable near-infrared dyes, a long-lived fluorescein derivative, the first example of a mitochondrial polarity-sensitive probe, a DNA-specific redemissive dye, and a cyclooxygenase-2 (COX-2)-responsive

Figure 2. (A) Structure of OBEP. (B) Photofadings of OBEP, MitoTracker Red CMXRos, and MitoTracker Green FM in buffer. (C) Cytotoxicity of OBEP and MitoTracker Red CMXRos on HeLa cells after 12 h. (D) Various damaged mitochondrial forms of HeLa cells after incubation with OBEP for 1 h. (b−d) show enlarged images of representative cells indicated by the red squares in (a). Adapted with permission from ref 17. Copyright 2012 Royal Society of Chemistry. 2116

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fluorescence enhancement of PQ due to the monomerization of PQ and the formation of a dye inclusion complex. Complexation with CB[8] conferred ultraphotostability on PQ, which enabled long-term monitoring of mitochondria without obvious photobleaching (Figure 4B). ROS, produced as byproducts from the electron transport chain in mitochondria,1 modulate a variety of disparate physiological and pathological events in biological systems.2 We have developed PTZ-Cy2, a mitochondria-targeted fluorescent probe for highly reactive oxygen species (hROS), including ClO− and ·OH, based on a hybrid cyanine− phenothiazine platform (Figure 5A).24 PTZ-Cy2 exhibited ratiometric absorption (A470nm/A550nm) and “off/on” fluorescence (I470nm and I595nm) responses toward ClO− (Figure 5C) and ·OH, which were ascribed to oxidation of the sulfur atom in the phenothiazine moiety and the concomitant destruction of the π conjugation of the cyanine. PTZ-CY2 enabled the ratiometric visualization of hROS generation in cellular mitochondria (Figure 5E). To design a HClO-specific fluorescent probe, we introduced an oxime group at the 2-position of BODIPY, affording MitoClO (Figure 5B).25 Initially, the fluorescence of MitoClO was quenched because of the CN isomerization. The oxime group could react rapidly with NaClO to generate a carboxy group, which restricted the CN isomerization and revived the fluorescence. The fluorescence enhancement of MitoClO by NaClO could be completed within dozens of seconds. The use of MitoClO enabled the imaging of endogenous HClO, stimulated by phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharides (LPS), in the mitochondria of macrophages (Figure 5D). Fluoride is one of the most important biologically relevant anions. Studies suggest that a high exposure to fluoride ions causes oxidative stress damage to mitochondria and decreases mitochondrial respiratory chain efficiency.26 We reported, FP, the first fluorescent probe for the selective determination of fluoride ions in cellular mitochondria (Figure 6).27 Free FP is nonfluorescent. The addition of fluoride ions triggers the cleavage of the Si−O bond in FP, followed by intramolecular cyclization (Figure 6A), resulting in a profound fluorescence enhancement (Figure 6B). The detection limit was as low as

and 1,7-positions, respectively, we developed a highly photostable NIR mitotracker, M-DPT (Figure 3).20 Both the

Figure 3. (A) Structure of M-DPT. (B) Normalized absorption and fluorescence spectra of M-DPT. (C) Confocal fluorescence images of dual-stained HeLa cells: (a) MitoTracker Green FM; (b) M-DPT; (c) overlap of (a) and (b). Reproduced with permission from ref 20. Copyright 2013 Royal Society of Chemistry.

absorption and emission spectra of M-DPT lay in the NIR region (λabs = 670 nm and λem = 718 nm, Figure 3B). In comparison with Mitotracker Deep Red FM, M-DPT possessed much superior photostability. In live-cell imaging experiments, M-DPT was found to localize in mitochondria (Figure 3C) and was then applied to demonstrate the various forms of mitochondrial morphologies. Cucurbit[n]urils (CB[n], n = 5−8) have large hydrophobic cavities with polar carbonyl groups surrounding the two portals, which enable positively charged dyes to be encapsulated within them.21 In addition, CB[n] have been reported to have no intrinsic cytotoxicity and good uptake by many human cancer cell lines.22 We have established a supramolecular approach for mitochondrial staining by complexing a cationic NIR dye, PQ, with CB[8] (Figure 4).23 Free PQ aggregates in the aqueous solution by cation−π interactions with weak fluorescence at 645 nm. The addition of CB[8] resulted in a 30-fold

Figure 4. (A) Disaggregation of PQ stacks in water upon addition of CB[8]. (B) Confocal fluorescence images of MCF-7 cells after incubation with a PQ/CB[8] complex at different time points. Adapted with permission from ref 23. Copyright 2014 Royal Society of Chemistry. 2117

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Figure 5. Proposed sensing mechanisms of (A) PTZ-Cy2 and (B) MitoClO. (C) Changes in the fluorescence spectra of PTZ-Cy2 upon addition of NaClO. Reproduced with permission from ref 24. Copyright 2012 Royal Society of Chemistry. (D) Confocal fluorescence images of MitoClOstained RAW264.7 cells (a) without and (b) with treatment of LPS and PMA. Reproduced with permission from ref 25. Copyright 2013 Royal Society of Chemistry. (E) Confocal fluorescence images of PMA-stimulated HeLa cells after incubation with PTZ-Cy2: (a) green channel (470 ± 20 nm); (b) red channel (590 ± 20 nm). Reproduced with permission from ref 24. Copyright 2012 Royal Society of Chemistry.

Figure 6. (A) Proposed sensing mechanism of FP. (B) Changes in the fluorescence spectra of FP upon addition of F−. (C) Paper test strips for fluoride detection. (D) Confocal fluorescence images of COS-7 cells incubated with FP (a) before and (b, c) after the addition of tetrabutylammonium fluoride (b) and NaF (c). Reproduced with permission from ref 27. Copyright 2014 Royal Society of Chemistry.

1.67 × 10−7 M. Fluoride-dependent fluorescence could be readily visualized using FP in test strips and live cells (Figure 6C,D). Mitochondrial hydrogen sulfide (H2S) has been shown to exert protective effects in oxidative stress, leading to dysfunction and cell death.28 To monitor H2S with high sensitivity and fast response within mitochondria, we recently developed an NIR ratiometric fluorescent probe, FD-H2S, derived from flavylium (Figure 7).29 Initially, FD-H2S emitted NIR fluorescence with a maximum at 707 nm. Nucleophilic attack of H2S on the benzopyrylium moiety of FD-H2S interrupted the π conjugation, and as a result, the emission at 707 nm decreased along with the emergence of coumarin fluorescence at 487 nm (Figure 7B). The detection limit was

calculated to be as low as 68.2 nM, and the recognition process could be completed within 15 s. Confocal imaging results demonstrated the utility of FD-H2S in rapid detection of exogenous and endogenous mitochondrial H2S in living cells and, for the first time, in mice (Figure 7C). Intracellular viscosity is a critical factor governing the transport of mass and signal as well as the interactions between biomacromolecules.30 In particular, the viscosity of the mitochondrial matrix is closely related to the respiratory state of the mitochondria.16 For the measurement of mitochondrial viscosity, we designed and synthesized two ratiometric fluorescent molecular rotors, Mito-V and Caz-Cy2 (Figure 8). Mito-V was derived from a thiazole-Cy5 dye by the incorporation of an aldehyde group at the meso position of 2118

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Figure 7. (A) Proposed sensing mechanism of FD-H2S. (B) Changes in fluorescence spectra of FD-H2S upon addition of H2S. (C) Imaging of H2S in mice using FD-H2S: (a) mouse without any treatment used as a control; (b−h) fluorescence images collected at different time points after injection of NaHS into a mouse that was prestained with FD-H2S. λex = 630 nm. Emission channel: 700 ± 20 nm. Adapted with permission from ref 29. Copyright 2015 Royal Society of Chemistry.

monitor mitochondrial viscosity during apoptosis via both fluorescence ratiometry and lifetime imaging (Figure 8D). Two-photon fluorescence microscopy uses low-energy NIR laser excitation and allows deep penetration with low photodamage and autofluorescence.32 Caz-Cy2, a carbazolebased cyanine derivative, was developed as a ratiometric twophoton viscosity sensor.33 The fluorescence spectrum of CazCy2 possesses two emission bands centered at 380 and 580 nm. As the solvent viscosity increased, both the blue and red fluorescence were enhanced (Figure 8C). Caz-Cy2 proved to be an excellent two-photon probe, capable of reporting on the viscosity distribution within the mitochondria in live cells as well as tissues at depths of 60−130 μm (Figure 8E). Polarity is another significant environmental parameter that controls the interaction activity of proteins and initiates signal transduction processes.34 On the basis of the donor−π-bridge− acceptor (D−π−A) design philosophy, we developed BOB, an environment-sensitive fluorescent probe for the ratiometric determination of solvent polarity (Figure 9).35 BOB has two emission maxima, at 467 and 642 nm, arising from the coumarin moiety and the π conjugation in BOB, respectively. The fluorescence ratio (I467nm/I645nm) was linearly enhanced with decreasing solvent polarity (Figure 9B). Importantly, BOB

Figure 8. (A) Structures of Mito-V and Caz-Cy2. (B) Changes in the fluorescence lifetime of Mito-V with increasing viscosity. Reproduced with permission from ref 31. Copyright 2013 Elsevier. (C) Changes in the fluorescence spectra of Caz-Cy2 as a function of solvent viscosity. Reproduced with permission from ref 33. Copyright 2013 John Wiley & Sons. (D) Fluorescence lifetime images of Mito-V-stained HeLa cells after stimulation by etoposide at different time points. Adapted with permission from ref 31. Copyright 2013 Elsevier. (E) Twophoton images of a fresh rat hepar slice after incubation with Caz-Cy2 at a depth of 110 μm (λex = 720 nm): (a) blue channel (375−440 nm); (b) red channel (575−640 nm); (c) overlap of (a), (b), and the bright-field image; (d) ratiometric image. Reproduced with permission from ref 33. Copyright 2013 John Wiley & Sons.

the pentamethine chain.31 In nonviscous media, the aldehyde group can freely rotate, providing relaxation of the electronically excited dye by a nonradiative process. Such rotation is restrained in viscous environments, accompanied by increases in the fluorescence intensity (62.6-fold at 658 nm) and lifetime (from 202.0 to 2374.3 ps) (Figure 8B). Mito-V was applied to

Figure 9. (A) Structure of BOB. (B) Linear response of BOB (I467nm/ I645nm) to solvent polarity. (C) Confocal fluorescence images of (a) COS-7, (b) RAW 264.7, (c) HepG2, and (d) MCF-7 cells after incubation with BOB. Adapted with permission from ref 35. Copyright 2015 John Wiley & Sons. 2119

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Figure 10. (A) Proposed sensing mechanism of LysoSH. (B) Structures of NBM and LysoZn-1. (C) Fluorescence spectra of LysoZn-1 upon titration with Zn2+. Adapted with permission from ref 39. Copyright 2013 Royal Society of Chemistry. (D) Changes in the fluorescence intensity of LysoSH as a function of time in the presence of Cys, Hcy, and GSH. Reproduced from ref 41. (E) Confocal fluorescence images of dual-stained HeLa cells: (a) LysoTracker Green; (b) NBM; (c) overlap of (a) and (b). Reproduced with permission from ref 37. Copyright 2013 Royal Society of Chemistry. (F) Reversible detection of endogenous Zn2+ by LysoZn-1 in NSCs: (a) fluorescence ratiometric images (I575−620nm/I655−755nm) of NSCs incubated with LysoZn-1; (b) after treatment of H2O2; (c) after further addition of N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Reproduced with permission from ref 39. Copyright 2013 Royal Society of Chemistry. (G) Dynamic imaging of the nucleus and lysosomes during hepatic cell division using Hoechst (blue) and NBM (red), respectively. Reproduced with permission from ref 37. Copyright 2013 Royal Society of Chemistry. (H) Two-photon images of liver, heart, kidney, lung, and spleen from mice intravenously injected with LysoSH. Reproduced from ref 41.

proved to be the first mitochondrial probe for polarity sensing. Ratiometric fluorescence imaging with BOB showed that the microenvironment within cancer cells is more polar than that in normal cells (Figure 9C), providing a potential approach to distinguish cancer cells from normal cells.

(Figure 10E) in various types of live cells at a very low concentration (25 nM). More importantly, NBM was first used to observe the disappearance and reproduction of lysosomes during cell division (Figure 10G). Lysosomal membrane permeabilization (LMP) has an important function in zinc-induced neuronal death under oxidative stress.38 To investigate lysosomal zinc function in neurons with high spatial and temporal reliability, we developed the ratiometric probe LysoZn-1 (Figure 10B).39 Upon Zn2+ binding, LysoZn-1 exhibited obvious fluorescence enhancement (I578nm) and ratio (I578nm/I680nm) changes (Figure 10C). The fluorescence of LysoZn-1 and LysoZn-1 + Zn2+ did not change significantly at lysosomal pH ranging from 4.5 to 6.0. Live-cell imaging experiments demonstrated that LysoZn-1 could reliably detect labile Zn2+ changes in the presence of exogenous or endogenous (H2O2 stimulation) Zn2+ in neural stem cells (NSCs) (Figure 10F). Amino acids containing a thiol group, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), are closely associated with proteolysis in lysosomes.40 We have designed and synthesized LysoSH, a fluorescent probe that binds lysosomal thiols (Figure 10A).41 Initially, the fluorescence of

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LYSOSOME-LOCALIZED PROBES The lysosome is the main digestive compartment of the cell, where all sorts of macromolecules are delivered for degradation. Lysosomes contain a high proton concentration and more than 50 hydrolases with an optimum pH below 6.36 In this section, we highlight our research on amine-free lysosome targeting; thiol-, zinc-, and pH-responsive lysosomal probes; and the thermally activated delayed fluorescence of a fluorescein derivative that localizes in cell lysosomes. NBM is an NIR lysotracker in which Nile Blue was selected as the fluorophore because of its distinguished photophysical properties and 2-morpholinoethylamine served as the lysosome targeting group (Figure 10B).37 The absorption and emission maxima of NBM in water appeared at 638 and 678 nm, respectively. Co-staining assays demonstrated the utility of NBM as a highly selective and biocompatible lysosome tracker 2120

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heavy atoms, DCF-MPYM (Figure 12),48 which showed longlived luminescence (22.11 μs in deaerated ethanol; Figure

LysoSH was quenched (ΦF = 0.015) as a result of photoinduced electron transfer (PET) between 1,8-naphthalimide and 2,4-dinitrobenzenesulfonyl (DNBS). When exposed to thiols, DNBS underwent cleavage to generate the free piperazine form in concert with a dramatic fluorescence enhancement (Figure 10D). LysoSH was applied to monitor thiol levels in mouse tissues such as liver, heart, kidney, lung, and spleen at depths of 20−140 μm by two-photon microscopy (Figure 10H). Many lysosome-specific probes have side chains of weak base, such as 4-(2-hydroxyethyl)morpholine and N,N-dimethylethylenediamine, which render the probes acidotropic and lead to their accumulation in acidic vesicles upon protonation.6,9,42−45 Unfortunately, these amine-containing lysosome probes can exert an “alkalizing effect” on lysosomes, such that long incubation with these probes can induce an increase in lysosomal pH.6 To address this issue, we first introduced an amine-free lysosome-locating group, methylcarbitol. By appending it to a rhodamine fluorophore, we constructed the fluorescent probe Rlyso to report on changes in lysosomal pH (Figure 10).46 The pH titration curve of Rlyso yielded a pKa value of 5.47, with a sharp incline in the lysosomal pH range (Figure 11B). The specific lysosomal accumulation of Rlyso

Figure 12. (A) Structure of DCF-MPYM. (B) Emission decays of DCF-MPYM under anaerobic (red) and aerobic (black) conditions. (C) Time-resolved luminescence images of MCF-7 cells after incubation with DCF-MPYM and BSA. (D) Confocal fluorescence images of dual-stained MCF-7 cells: (a) LysoSensor Green DND-189; (b) DCF-MPYM; (c) overlap of (a) and (b). Reproduced from ref 48. Copyright 2014 American Chemical Society.

12C). The singlet−triplet energy gap (ΔEST) of 28.36 meV for DCF-MPYM is small enough to allow efficient intersystem crossing (ISC) and reverse ISC, leading to efficient thermally activated delayed fluorescence at room temperature. The long lifetime of DCF-MPYM enabled its use in time-resolved luminescence imaging (Figure 12B). Confocal fluorescence imaging indicated that DCF-MPYM specifically stains lysosomes.

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NUCLEUS-LOCALIZED PROBES The nucleus is the control center of the cell, and its main functions are to maintain the integrity of genes and to control the activities of the cell by regulating gene expression. The molecular basis of genes is deoxyribonucleic acid (DNA), and transcription by ribonucleic acids (RNA) is the first step of gene expression. DAPI and Hoechst 33258 are presently used for DNA-specific staining; however, they require UV excitation, which can lead to cellular damage under lengthy irradiation. SYTO RNASelect is commercially available for nucleolar staining but suffers from low photostability and has undisclosed chemical structures. Our research focused on the development of red/NIR-emitting, highly photostable, and DNA/RNAselective fluorescent probes for nuclear imaging. DEAT-TO-3 is a TO-3 analogue with long-wavelength excitation and emission (λabs(DNA) = 626 nm and λem(DNA) = 649 nm) that was developed for live-cell DNA imaging and quantification (Figure 13).49 DEAB-TO-3 shows very low intrinsic fluorescence in aqueous solution. Upon binding to calf thymus (CT) DNA with a great contribution of the minor groove, the quantum yield of DEAB-TO-3 increased by 97.3fold. DEAB-TO-3 exhibited much less fluorescence enhancement in the presence of Saccharomyces cerevisiae RNA and almost no change in fluorescence in response to bovine serum albumin (BSA) (Figure 13B). The live-cell imaging assay demonstrated that DEAB-TO-3 is membrane-permeable and stains nuclear chromatin with faint cytoplasmic staining (Figure 13D). The DNA specificity of DEAB-TO-3 enabled its

Figure 11. (A) Structure of Rlyso. (B) pH titration curve of Rlyso. (C) Confocal fluorescence images of Rlyso-stained RAW 264.7 macrophages (a) before and (b) after treatment with chloroquine and (c) relative fluorescence intensities of Rlyso extracted from (a) and (b). Adapted with permission from ref 46. Copyright 2012 Royal Society of Chemistry.

was numerically proved using the logarithm of the water/ octanol partition coefficient (log P). The log P value for Rlyso in the spiro form is 4.9, suggesting its membrane permeability and easy entry into lysosomes. In the low-pH lysosomal environment, Rlyso is transformed to the corresponding ringopened species with a log P value of −0.8, which is not very membrane-permeable and becomes trapped within the lysosomes. Rlyso was able to quantitatively determine the chloroquine-induced increase in lysosomal pH (Figure 11C) and monitor changes in the acidity of lysosomes during apoptosis in live cells. Compared with fluorescence imaging utilizing fluorophores with lifetimes on the order of nanoseconds, time-resolved fluorescence microscopy is more advantageous in monitoring target fluorescence by eliminating the short-lived background fluorescence and providing high signal-to-noise ratios.47 We reported a fluorescein derivative without rare-earth metals or 2121

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Figure 13. (A) Structures of DEAB-TO-3, TO-3, and TO-3-CN. (B) Fluorescence spectra of DEAB-TO-3 in the absence and presence of CT DNA, S. cerevisiae RNA, and BSA in buffer. Reproduced with permission from ref 49. Copyright 2011 John Wiley & Sons. (C) Photofadings of TO-3 and TO-3-CN in buffer. Reproduced with permission from ref 50. Copyright 2014 Royal Society of Chemistry. (D) Live-cell staining with DEABTO-3. (E) Cell cycle and apoptosis analysis using DEAB-TO-3. Reproduced with permission from ref 49. Copyright 2011 John Wiley & Sons. (F) Confocal fluorescence images of dual-stained MCF-7 cells: (a) costained with SYTO-9; (b) TO3-CN; (c) overlap of (a) and (b). Reproduced with permission from ref 50. Copyright 2014 Royal Society of Chemistry.

application in cell-cycle analysis by flow cytometry and the quantitation of DNA without RNase digestion (Figure 13E). Low photostability is a big concern for DEAB-TO-3 because of its long and unsubstituted trimethine chain. To solve this problem, TO3-CN (Figure 13) was synthesized by introducing a cyano group on the trimethine chain of the classical redemitting dye TO-3,50 which decreased the electron density of polymethine chains and, as a result, reduced the reactivity of the dyes with singlet oxygen.51 Compared with the control compound TO-3, TO3-CN showed marked hypsochromic shifts in the maximum absorption (543 nm) and emission (611 nm) wavelengths, an increased Stokes shift (67 nm), and much better lightfastness (Figure 13C). Unlike DEAB-TO-3, TO3CN produced undifferentiated fluorescence responses to DNA and RNA in buffer solution but preferred to bind with RNA in cells (colocalized with the commercial RNA dye SYTO-9, as depicted in Figure 13F), probably because of the halfintercalation binding mode with DNA. Hsd contains two benzothiazolium-2-ylvinyl moieties at the 2- and 4-positions of dimethylaminobenzene (Figure 14).52

The absorption peak of Hsd is at 458 nm, while the emission is extended into the NIR region (682 nm). The fluorescence intensity of Hsd is enhanced 24-fold with a red shift of 12 nm and 65-fold with a blue shift of 8 nm in the presence of DNA and RNA, respectively. Counterstaining with Hoechst 33258 and performing DNase and RNase digestion tests demonstrated that Hsd preferentially stains RNA in fixed cells. Although Hsd is not membrane-permeable, it can be drawn into live cells with the assistance of CB[7]. Metal coordination complexes can also serve as candidates for the development of nucleolar probes.53 The heterodinuclear metal complex OsIr was designed for use as a nucleolar stain (Figure 15).54 Upon irradiation with visible light, OsIr exhibits dual-color emission with peaks at 534 and 721 nm, corresponding to the iridium and osmium activating centers, respectively. The interactions of the iridium moiety with proteins or RNA trigger significant luminescence enhancements along with an alteration of the emission of the osmium moiety, providing ratiometric responses by OsIr. The cellular uptake of

Figure 15. (A) Structure of OsIr. (B) Confocal fluorescence images of MCF-7 cells after incubation with OsIr for 2 h: (a) red channel (685− 755 nm); (b) green channel (510−560 nm). Reproduced from ref 54. Copyright 2014 American Chemical Society.

Figure 14. Proposed RNA-staining mechanism of Hsd with the assistance of CB[7]. Reproduced with permission from ref 52. Copyright 2013 Elsevier. 2122

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Figure 16. (A) Structures of ANQ-IMC-6 and Niblue-C6-IMC. (B) Fluorescence and (C) two-photon spectra of ANQ-IMC-6 in the absence or presence of COX-2 in buffer. (D) Confocal fluorescence images of dual-stained HeLa cells: (a) ANQ-IMC-6; (b) BODIPY TR C5-ceramide; (c) overlap of (a) and (b). (E) Two-photon images of ANQ-IMC-6-labeled Golgi apparatus during apoptosis at different time points (a, 0 h; b, 6 h; c, 24 h). Reproduced from ref 60. Copyright 2013 American Chemical Society. (F) Fluorescence images of Niblue-C6-IMC-stained mice (a) without and (b) with pretreatment with celecoxib. Reproduced with permission from ref 61. Copyright 2014 Royal Society of Chemistry.

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ER-LOCALIZED PROBES The endoplasmic reticulum (ER) is primarily responsible for the proper sorting of lipids and proteins in cells. Most of the probes for the ER are either lipids or chemicals that affect protein movement.6 We have prepared two fluorene-derived two-photon fluorescent probes for targeting the ER (TPFLER) and lysosomes (TPFL-Lyso) by introducing a chlorine group and a morpholine group, respectively (Figure 17A).62 Because of their similar molecular structures, TPFL-ER and TPFL-Lyso are suitable for simultaneous imaging of the ER and lysosomes without affecting protein movements (Figure

OsIr examined by confocal microscopy revealed an apparent nucleolar and cytoplasmic staining (Figure 15B).

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GOLGI-LOCALIZED PROBES

The Golgi apparatus is a key structure for the transport and secretion of some important proteins and enzymes. It has been suggested that when the stress-signaling threshold is exceeded, the Golgi apparatus automatically initiates apoptotic processes, resulting in the accumulation of cyclooxygenase-2 (COX-2) in the Golgi apparatus.55 In addition, COX-2 is a biomarker of virtually all cancer cell lines. Recently, increasing research on enzyme-activated fluorescent probes for cancer imaging has been reported;56−59 however, none of them were designed to target specific organelles. We used indomethacin as a COX-2 binding site and designed Golgi-targeted two-photon or NIR “off/on” fluorescent probes for the rapid identification of cancer cells. ANQ-IMC-6 was constructed by linking indomethacin to the acenaphtho[1,2-b]quinoxaline (ANQ) fluorophore with a sixcarbon spacer (Figure 16A).60 In an aqueous buffer solution, free ANQ-IMC-6 is present in the folded state where its fluorescence is quenched through PET between the fluorophore and indomethacin. Upon binding to COX-2, ANQ-IMC6 is forced to adopt the unfolded state, which results in revival of the fluorescence by restraining the PET process (Figure 16B). The push−pull charge-transfer structure of ANQ-IMC-6 provides significant two-photon properties (Φδmax = 118 GM; Figure 16C), permitting thick-tissue imaging (50−550 μm). ANQ-IMC-6 has been successfully used to rapidly differentiate cancer cells from normal cells using flow cytometry and oneand two-photon fluorescence microscopy. Furthermore, the dynamic changes of the Golgi apparatus during cancer cell apoptosis were visualized via ANQ-IMC-6 (Figure 16E). By replacing ANQ with Nile Blue, we developed the first Golgitargeted NIR fluorescent probe (Niblue-C6-IMC) for COX-2 (Figure 16A).61 By taking advantage of the NIR property, Niblue-C6-IMC was further exploited to image tumors in a mouse model (Figure 16F).

Figure 17. (A) Structures of TPEL-ER and TPEL-Lyso. (B) Fluorescence spectra resolved from images of MCF-7 cells costained with TPEL-ER and TPEL-Lyso. (C) Confocal fluorescence images of dual-stained MCF-7 cells: (a) TPEL-ER; (b) ER-Tracker; (c) overlap of (a) and (b). (D) Fluorescence images of (a) Kinesin-Cy2-stained kinesin and (b) MAP2-Cy3.5-stained microtubules after incubation with TPEL-ER and TPEL-Lyso; (c) overlap of (a) and (b). Reproduced with permission from ref 62. Copyright 2013 Royal Society of Chemistry. 2123

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Accounts of Chemical Research 17D). The specific ER localization of TPFL-ER (Figure 17C) is likely due to binding of the chlorine pump in the ER by the chlorine group of TPFL-ER.6

Jianjun Du is an associate professor at the State Key Laboratory of Fine Chemicals at Dalian University of Technology. He received his B.S. and Ph.D. degrees in Applied Chemistry from Dalian University of Technology in 2004 and 2010, respectively. After his postdoctoral fellowship work at Nanyang Technological University (Singapore) in 2010−2013, he started his independent research career at Dalian University of Technology. His research is focused on organic/ inorganic nanoparticles with functional organic molecules.

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CONCLUSION AND PERSPECTIVE This Account summarizes our recent work on the development of targetable fluorescent probes that allow biological imaging and sensing in specific subcellular organelles, including mitochondria, lysosomes, the nucleus, the Golgi apparatus, and the endoplasmic reticulum. We have developed organellestaining probes with upgraded properties by exploiting new organelle tags, encapsulating positively charged dyes in the cavities of cucurbit[n]urils, and adopting NIR, two-photon, or long-lifetime fluorophores. Furthermore, we have presented responsive organelle probes capable of detecting changes in biomolecular levels (ROS, F−, H2S, Zn2+, thiol-containing amino acids, and COX-2) and the microenvironment (viscosity, polarity, and pH). Other researchers have also devoted extensive endeavors to the development of available organelle-specific fluorescent probes during the past few years. However, a majority of the present probes are designed to target mitochondria or lysosomes, and examples of probes for other organelles, such as the Golgi apparatus, the endoplasmic reticulum, and ribosomes, are much fewer. Thus, in the future more consideration should be given to the development of probes for staining these “low-concern” organelles and monitoring their related activities. In addition, a basic feature of organellespecific probes is their selective accumulation in a particular region, which necessitates microscopy imaging with high resolution. Given that the general diameters of organelles are in the range of 20−1000 nm and the resolution of conventional light microscopy cannot reach below 250 nm, we anticipate that further investigations of cellular events within specific organelles using fluorescent probes will benefit from the development of super-resolution optical microscopy with nanoscopic-scale resolution, such as stimulated emission depletion (STED), photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM).

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Xiaojun Peng received his Ph.D. in 1990 at Dalian University of Technology. After completing postdoctoral research at Nankai University (China), he has worked at the Dalian University of Technology since 1992. In 2001 and 2002 he was a visiting scholar at Stockholm University and Northwestern University (USA). Currently he is a professor and the director of the State Key Laboratory of Fine Chemicals at Dalian University of Technology. His research interests cover dyes for fluorescent bioimaging/labeling and digital printing/ recording.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21136002, 21421005, 21422601 and 21576037), and the National Basic Research Program of China (2013CB733702).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Hao Zhu obtained his Bachelor of Science degree from Dalian University of Technology (DUT) in 2006. He received his Ph.D. from the State Key Laboratory of Fine Chemicals at DUT under the supervision of Prof. Xiaojun Peng and Prof. Jiangli Fan in 2016. He is currently a postdoctoral fellow at The University of Tokyo. His research interests focus on small-molecule fluorescent probes for bioimaging and sensing. Jiangli Fan received her Ph.D. from Dalian University of Technology in 2005. In 2010 she attended the University of South Carolina as a visiting scholar. She is currently a professor at the State Key Laboratory of Fine Chemicals at Dalian University of Technology. Her research is focused on fluorescent-dye-based probes and their biological applications. 2124

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