Coumarins are deployed in numerous bioimaging and biosensing applications. Among various coumarin derivatives, 6-aminocoumarins attract increasing attention for their red-shifted emissions, mega Stokes shifts, and significant solvatochromism. These spectral characteristics together with weak emission intensities have historically been ascribed to the formation of the twisted intramolecular charge transfer (TICT) state in 6-aminocoumarins. In this work, we demonstrate that it is actually substantial intramolecular charge transfer (ICT) that is responsible for these fluorescent properties. Based on this new understanding, we reanalyzed the sensing mechanism of a 6-aminocouarmin based fluorescent probe and obtained close agreement with experimental data. Our results lead to a deeper understanding of the photophysics of 6-aminocoumarins and will inspire the rational development of novel fluorescent probes.

Fluorescence thermometry based on organic dyes affords high spatial and temporal resolution with a simple system design and low cost, for measuring temperatures in microenvironments. Many fluorescent thermometers consist of two types of fluorophores with distinct temperature responses, and the ratios of their fluorescence intensities afford accurate temperature information. Yet, the reliability of these ratiometric thermometers is vulnerable to photobleaching induced system variations. In this paper, we have proposed and demonstrated a new strategy, to achieve ratiometric temperature measurements from 15 °C to 75 °C, based on ground-state conformational isomers of a single type of dye. These ground-state conformers emit bright fluorescence, in contrast to excited-state conformational changes that generally quench emissions. Moreover, thermal equilibrium of these conformers and their distinct spectra lead to ratiometric temperature readings that are not affected by photobleaching. We expect that our design strategy has significant implications for developing fluorescence thermometry with outstanding reliability.

Protein labeling by using a protein tag and tag-specific fluorescent probes is increasingly becoming a useful technique for the real-time imaging of proteins in living cells. SNAP-tag as one of the most prominent fusion tags has been widely used and already commercially available. Recently, various fluorogenic probes for SNAP-tag based protein labeling were reported. Owing to turn-on fluorescence response, fluorogenic probes for SNAP-tag minimize the fluorescence background caused by unreacted or nonspecifically bound probes and allow for direct imaging in living cells without wash-out steps. Thus, real-time analysis of protein localization, dynamics and interactions has been made possible by SNAP-tag fluorogenic probes. In this review, we describe the design strategies of fluorogenic probes for SNAP-tag and their applications in cellular protein labeling.In this mini-review, we described the design strategies of SNAP-tag fluorogenic probes with turn-on fluorescence responses, which minimized the fluorescence background and allowed for direct imaging in living cells without wash-out steps. These probes can apply in real-time analysis of protein localization, dynamics, and protein–protein interactions in living cells. Furthermore, the excellent fluorescent properties made it possible to apply some of the probes in super-resolution fluorescence imaging.Download high-res image (73KB)Download full-size image

•The probe displayed a fluorescence turn-on response to label SNAP-tag.•The probe exhibited the fast record labeling rate among known fluorogenic probes.•The probe enables no-wash protein imaging in live cells.SNAP-tag is one of most popular genetically encoded protein tags that can be labeled with fluorescent molecules for visualizing a protein of interest in live cells. Fluorogenic probes keep dark until they label protein tags, significantly improving the signal-to-noise ratio to image proteins without wash-out step. However, most of reported fluorogenic probes for SNAP-tag suffered from the low or mild labeling rate comparing with non-fluorogenic ones. In this paper, we reported a 4-amino-naphthliamide derived fluorogenic probe for SNAP-tag, which exhibited the fast record labeling rate among fluorogenic probes. Finally, we applied this probe to image proteins in mitochondria and nucleus in live cells without wash-out steps.The fluorogenic probe for SNAP-tag displayed a fast labeling rate.Download high-res image (175KB)Download full-size image

A 1,8-naphthalimide-derived fluorogenic probe was reported to label SNAP-tag fusion proteins in living cells. The probe can rapidly label a SNAP-tag and exhibit a fluorescence increase of 36-fold due to the additive effects of environment sensitivity of fluorophores and inhibition of photo-induced electron transfer from O6-benzylguanine to the fluorophore. The labeling of intracellular proteins has been successfully achieved without a wash-out procedure.

Replacing conventional dialkylamino substituents with a three-membered aziridine ring in naphthalimide leads to significantly enhanced brightness and photostability by effectively suppressing twisted intramolecular charge transfer formation. This replacement is generalizable in other chemical families of fluorophores, such as coumarin, phthalimide, and nitrobenzoxadiazole dyes. In highly polar fluorophores, we show that aziridinyl dyes even outperform their azetidinyl analogues in aqueous solution. We also proposed one simple mechanism that can explain the vulnerability of quantum yield to hydrogen bond interactions in protonic solvents in various fluorophore families. Such knowledge is a critical step toward developing high-performance fluorophores for advanced fluorescence imaging.

A fluorescent sensor for halogenated solvents termed AMN is reported. AMN shows strong fluorescence in most halogenated solvents (QE > 0.1) but weak fluorescence (QE<0.01) in most non-halogenated solvents. In chlorinated solvents, the fluorescence intensity decreased with the reduction of chlorine content. On the contrary, in brominated solvents the fluorescence intensity increased with the reduction of bromine content. It is worth mentioning that AMN displayed fluorescence emission centered at 520 nm in CCl4 with a quantum yield of 0.607, at 556 nm in CHCl3 with a quantum yield of 0.318, at 584 nm in CH2Cl2 with a quantum yield of 0.128, whereas in CHBr3 was centered at 441 nm with a quantum yield of 0.012. AMN was shown to have the ability to differentiate CCl4, CHCl3, CH2Cl2 and CHBr3 halogenated solvents.

•Cd2+ can trigger amide tautomerization in the amide-DPA receptor.•The probe is extremely sensitive to Cd2+ with 65-fold emission enhancement.•The probe has an excellent selectivity for Cd2+.•The probe can recognize Cd2+ across a wide pH range from 4.5 to 11.5.•The probe can image Cd2+ in living cells.An NBD-derived fluorescent sensor termed CdTS was reported to sense Cd2+ with very high binding selectivity and significant fluorescence turn-on signal selectivity (65 fold enhancement). The amide/di-2-picolylamine receptor binds Cd2+ in an imidic acid tautomeric form, but binds most of other metal ions in an amide tautomeric form. The transformable ability makes CdTS have the specific selectivity for Cd2+. Additionally, CdTS can fluoresently and colorimetricly recognize Cd2+ across a wide pH range from 4.5 to 11.5. Finally, we applied CdTS to detect Cd2+ in living cells.CdTS can fluoresently recognize Cd2+ across a wide pH range.

Nitric oxide has played an important role in many physiological and pathological processes as a kind of important gas signal molecules. In this work, a new fluorescent probe LysoNO-Naph for detecting NO in lysosomes based on 1,8-naphthalimide was reported. LysoNO-Naph has sub-groups of o-phenylenediamine as a NO reaction site and 4-(2-aminoethyl)-morpholine as a lysosome-targetable group. This probe exhibited good selectivity and high sensitivity (4.57 μmol/L) toward NO in a wide pH range from 4 to 12. Furthermore, LysoNO-Naph can be used for imaging NO in lysosomes in living cells.A 1,8-naphthalimide-derived fluorescent probe for imaging lysosomal NO was reported.Download full-size image

Metal ions play an important role in various biological processes, their abnormal homeostasis in cells is related to many diseases, such as neurodegenerative disease, cancer and diabetes. Fluorescent imaging offers a unique route to detect metal ions in cells via a contactless and damage-free way with high spatial and temporal fidelity. Consequently, it represents a promising method to advance the understanding of physiological and pathological functions of metal ions in cell biology. In this highlight article, we will discuss recent advances in fluorescent imaging of metal ions by small-molecule sensors for understanding the role of metals in related diseases. We will also discuss challenges and opportunities for the design of small-molecule sensors for fluorescent detection of cellular metal ions as a potential method for disease diagnosis.

The emission intensities of coumarin 545 solution exhibit a low temperature dependence, with a record-low temperature coefficient of only ∼0.025% per °C. This monomer-aggregate coupled fluorescence system can be used for ratiometric temperature measurements with high spatial and temporal resolutions; three different working modes have been demonstrated.

Hydrogen sulfide (H2S) is an important endogenous signalling molecule in cellular physiology and pathology. Accordingly, sensitive, selective and reliable methods for H2S detection are in high demand. Fluorescence-based methods thus have received great attentions in recent years. Herein we describe the design, synthesis and application of a probe for H2S detection based on the reduction of azide to amine. This probe is highly sensitive and selective toward H2S over biothiols and other biologically relevant species. Finally, the probe was successfully applied for H2S fluorescent imaging in living cells.

The emission intensities of a fluorescent monomer–aggregate coupled system, based on 7-(dimethylamino)-coumarin-3-carbaldehyde, exhibit ultra-low temperature dependence with a low temperature coefficient of only 0.05% per °C, by judicious selection of the excitation wavelength. This finding has significant implications to temperature-sensitive fluorescent applications.

The fluorescence intensity of N,N-dimethyl-4-((2-methylquinolin-6-yl)ethynyl)aniline exhibits an unusual intensification with increasing temperature, by activating more vibrational bands and leading to stronger TICT emissions upon heating in dimethyl sulfoxide. Based on the different temperature dependence at various wavelengths, as shown in the TICT fluorescence spectrum, this dye can be employed to ratiometrically detect temperature.

Hydrogen sulfide (H2S) is an endothelial gasotransmitter which has been extensively studied recently in various physiological processes. H2S can induce lysosomal membrane destabilization leading to an autophagic event of precipitation apoptosis coupled with calpain activation, thus ensuring cellular demise. In this study, we developed a lysosome-targetable fluorescent probe for the recognition of H2S with considerable fluorescence enhancement. Through introducing a lysosome-targetable group 4-(2-aminoethyl)-morpholine into the H2S probe N-imide termus of 4-azide-1,8-naphthalimide, the new compound Lyso-AFP can recognize H2S in lysosomes. This probe emerges as a more biocompatible analysis tool with low poison by-product than reported H2S fluorescent probes.

Three imidazolium derivatives 3–5 were designed and synthesized, in which naphthaimidazolium group acted as both fluorophore and anion receptor. Compound 3 exhibited high selectivity for F− in CH3CN solution over all the other anions and acted as a ratiometric fluorescent probe for F− with an enhanced blue-shift in emission. However, the fluorescence of compound 4 and 5 displayed a quenched blue-shift in emission with fluoride ion and could be quenched by some other tested anions, where the degree of quenching depended on the characteristic of the anions. More importantly, only compound 3 could detect F− in DMSO–water (95:5, v/v) aqueous solution ratiometrically. Based on the analysis of the results of 1H-NMR and 19F-NMR, it was deduced that compound 3 bound with F− mainly by the force of hydrogen bonding, while compound 4 and 5 coordinated with F− through electrostatic interaction.

In this work, a fluorescein-derived fluorescent probe for H2S based on the thiolysis of dinitrophenyl ether is reported. This probe exhibits turn-on fluorescence imaging of H2S in living cells and bulk solutions with excellent selectivity. The reaction mechanism was explained by means of absorption, fluorescence and HPLC–MS.A fluorescein-derived fluorescent probe for H2S based on the thiolysis of dinitrophenyl ether is reported.

In this work, a 1,8-naphthalimide-derived fluorescent probe for H2S based on the thiolysis of dinitrophenyl ether is reported. This probe exhibits turn-on fluorescence detection of H2S in bovine serum and lysosome-targetable fluorescent imaging of H2S with excellent selectivity.

•A two-photon fluorescent probe for hydrogen sulfide was synthesized.•The probe is extremely sensitive to H2S with 37-fold emission enhancement.•The probe has an excellent selectivity for H2S.•The probe can detect H2S in bovine serum.•The probe can image H2S in living cells.Fluorescent probes for hydrogen sulfide have received considerable attention because of the biological significance of H2S recognized recently. Two-photo microscopy offers advantages of increased penetration depth, localized excitation, and prolonged observation time. However, two-photon fluorescent probes for H2S are still rare. In this work, we introduced a dinitrophenyl ether group into the 4-position of 1,8-naphthalimide, which acts as the H2S reactive site, to efficiently yield compound NI–NHS as a two-photo fluorescent probe for H2S. The probe NI–NHS has a high selectivity for H2S over competitive anions and sulfide-containing analytes. This probe exhibits turn-on fluorescence detection of H2S in bovine serum and two-photon fluorescent imaging of H2S in living cells.

•A near-infrared fluorescent probe for hydrogen sulfide was synthesized.•The addition of H2S induced an appearance of emission centered at 655 nm.•The probe is extremely sensitive to H2S with 354-fold emission enhancement.•The probe has an excellent selectivity for H2S.•The probe can sense H2S in living cells.Fluorescent probes for hydrogen sulfide have received considerable attention because of the biological significance of H2S recognized recently. However, near-infrared fluorescent probes for H2S are still rare. In this work, a new near-infrared fluorescent probe for H2S was developed based on the reduction reaction of azide with H2S to amine based on the fluorophore of dicyanomethylene-4H-chromene because of its long excitation and emission wavelength. The probe has a high selectivity for H2S over competitive anions and sulfide-containing analytes. Finally, the probe was applied to sense H2S in living cells.

A fluorescent chemosensor N-n-butyl-3,4-diamino-1,8-naphthalimide (DAN) for detecting NO was developed on the basis of 1,8-naphthalimide with a similar structure of o-phenylenediamine as a NO reaction site. Due to the interaction between the photoinduced electron transfer (PET) and the intramolecular charge transfer (ICT), the chemosensor exhibits a remarkable enhancement in the emission intensity that is ca. 160-fold increase and a blue shift in the emission wavelength after the addition of NO. Meanwhile, it displays a colorimetric response accompanied with a color change from yellow to colorless. The chemosensor DAN shows a high selectivity for NO in the presence of various reactive nitrogen species (RNS) and reactive oxygen species (ROS). Furthermore, DAN can be used for bioimaging of NO in living cells.Graphical abstractHighlights► A fluorescent chemosensor for detecting nitric oxide (NO) was synthesized. ► The chemosensor was extremely sensitive to NO with about 160-fold fluorescence enhancement. ► It can rapidly and selectively detect NO in a colorometric way. ► The molecule can penetrate cell membrane easily and make fast fluorescence bioimaging.

A new ratiometric fluorescent probe for fluoride ions was developed which complexed fluoride by a tridentate receptor of boronic acid and imidazolium. In the current study, a tridentate receptor 1 with one ortho boronic acid and two imidazolium groups was designed. The boron center can co-operate with imidazolium to bind F−. The formation of B–F complex stabilizes the interaction between fluoride and imidazolium which induces a ratiometric fluorescence response. With the addition of F−, a strongly increased fluorescent emission centered at 370 nm appears at the expense of the fluorescent emission centered at 445 nm.

Hydrogen sulfide has emerged as an important biological messenger and much attention has been paid to the design of fluorescent probes for H2S to meet the requirement of accurate measurement of H2S. In this work, a new red emission fluorescent probe for H2S was developed based on the reduction reaction of azide with H2S to amine with the fluorophore of dicyanomethylenedihydrofuran because of its long excitation and emission wavelength. The probe has a high selectivity for H2S over competitive anions and sulfide-containing analytes. Finally, the probe was applied to sense H2S in living cells.

Understanding the molecular origins of the optoelectronic properties of fluorophores provides rational guidelines for chemists to synthesize better-performing dyes. Factors affecting the UV–vis absorption spectral shift, molar extinction coefficients, and Stokes shift of fluorophores are herein examined at the molecular level, via both (time-dependent) density functional theory-based calculations and the empirical harmonic-oscillator-stabilization-energy (HOSE) and bond-length-alternation (BLA) models. The importance of these factors is discussed using six coumarin dyes as exemplars. In particular, a special focus is devoted to the Stokes shift, a critical parameter in fluorophores. It is demonstrated that incorporating a “rotational” substituent in a fluorophore molecule with tailored steric hindrance effects and resonance effects leads to a substantial increase in the Stokes shift, not only in coumarins but also in other chemical dye families: boron-dipyrromethenes (BODIPYs), cyanines, and stilbenes. Structure–property relationships concerning the rotational substituent are discussed in detail with examples of several dye families. These findings lead to the proposal of molecular design criteria that enable one to tune the Stokes shift. Such criteria provide a foundation for the molecular engineering of fluorophores with improved optoelectronic properties.

We report a coumarin-derived fluorescent sensor for Zn2+ termed CTS. CTS shows excellent binding selectivity for Zn2+ over competing metal ions due to the transformable ability of CTS, that is the displacement of other metal ions by Zn2+, which induces transformation of chelation from an amide to an imidic acid tautomeric form.

We report a coumarin-derived fluorescent sensor for Zn2+ termed CTS. CTS shows excellent binding selectivity for Zn2+ over competing metal ions due to the transformable ability of CTS, that is the displacement of other metal ions by Zn2+, which induces transformation of chelation from an amide to an imidic acid tautomeric form.

A 1,8-naphthalimide-derived fluorogenic probe was reported to label SNAP-tag fusion proteins in living cells. The probe can rapidly label a SNAP-tag and exhibit a fluorescence increase of 36-fold due to the additive effects of environment sensitivity of fluorophores and inhibition of photo-induced electron transfer from O6-benzylguanine to the fluorophore. The labeling of intracellular proteins has been successfully achieved without a wash-out procedure.

Metal ions play an important role in various biological processes, their abnormal homeostasis in cells is related to many diseases, such as neurodegenerative disease, cancer and diabetes. Fluorescent imaging offers a unique route to detect metal ions in cells via a contactless and damage-free way with high spatial and temporal fidelity. Consequently, it represents a promising method to advance the understanding of physiological and pathological functions of metal ions in cell biology. In this highlight article, we will discuss recent advances in fluorescent imaging of metal ions by small-molecule sensors for understanding the role of metals in related diseases. We will also discuss challenges and opportunities for the design of small-molecule sensors for fluorescent detection of cellular metal ions as a potential method for disease diagnosis.

The fluorescence intensity of N,N-dimethyl-4-((2-methylquinolin-6-yl)ethynyl)aniline exhibits an unusual intensification with increasing temperature, by activating more vibrational bands and leading to stronger TICT emissions upon heating in dimethyl sulfoxide. Based on the different temperature dependence at various wavelengths, as shown in the TICT fluorescence spectrum, this dye can be employed to ratiometrically detect temperature.

The emission intensities of a fluorescent monomer–aggregate coupled system, based on 7-(dimethylamino)-coumarin-3-carbaldehyde, exhibit ultra-low temperature dependence with a low temperature coefficient of only 0.05% per °C, by judicious selection of the excitation wavelength. This finding has significant implications to temperature-sensitive fluorescent applications.

A fluorescent sensor for halogenated solvents termed AMN is reported. AMN shows strong fluorescence in most halogenated solvents (QE > 0.1) but weak fluorescence (QE<0.01) in most non-halogenated solvents. In chlorinated solvents, the fluorescence intensity decreased with the reduction of chlorine content. On the contrary, in brominated solvents the fluorescence intensity increased with the reduction of bromine content. It is worth mentioning that AMN displayed fluorescence emission centered at 520 nm in CCl4 with a quantum yield of 0.607, at 556 nm in CHCl3 with a quantum yield of 0.318, at 584 nm in CH2Cl2 with a quantum yield of 0.128, whereas in CHBr3 was centered at 441 nm with a quantum yield of 0.012. AMN was shown to have the ability to differentiate CCl4, CHCl3, CH2Cl2 and CHBr3 halogenated solvents.