•A two-photon fluorescent probe for sensing H2S was developed.•The probe shows a large turn on signal (120-fold enhancement).•The probe is suitable for fluorescence imaging of H2S in living cells and tissues.•The probe was capable of detecting H2S up to 170 μm depth in live tissues.A two-photon fluorescence turn-on H2S probe GCTPOC–H2S based on a two-photon platform with a large cross-section, GCTPOC, and a sensitive H2S recognition site, dinitrophenyl ether was constructed. The probe GCTPOC–H2S exhibits desirable properties such as high sensitivity, high selectivity, functioning well at physiological pH and low cytotoxicity. In particular, the probe shows a 120-fold enhancement in the presence of Na2S (500 μM), which is larger than the reported two-photon fluorescent H2S probes. The large fluorescence enhancement of the two-photon probe GCTPOC–H2S renders it attractive for imaging H2S in living tissues with deep tissue penetration. Significantly, we have demonstrated that the probe GCTPOC–H2S is suitable for fluorescence imaging of H2S in living tissues with deep penetration by using two-photon microscopy. The further application of the two-photon probe for the investigation of biological functions and pathological roles of H2S in living systems is under progress.

The long wavelength (far-red to NIR) analyte-responsive fluorescent probes are advantageous for in vivo bioimaging because of minimum photo-damage to biological samples, deep tissue penetration, and minimum interference from background auto-fluorescence by biomolecules in the living systems. Thus, great interest in the development of new long wavelength analyte-responsive fluorescent probes has emerged in recent years. This review highlights the advances in the development of far-red to NIR fluorescent probes since 2000, and the probes are classified according to their organic dye platforms into various categories, including cyanines, rhodamine analogues, BODIPYs, squaraines, and other types (240 references).

Fluorescence imaging has emerged as a powerful tool for monitoring biomolecules within the context of living systems with high spatial and temporal resolution. Researchers have constructed a large number of synthetic intensity-based fluorescent probes for bio-imaging. However, intensity-based fluorescent probes have some limitations: variations in probe concentration, probe environment, and excitation intensity may influence the fluorescence intensity measurements. In principle, the use of ratiometric fluorescent probes can alleviate this shortcoming. Förster resonance energy transfer (FRET) is one of the most widely used sensing mechanisms for ratiometric fluorescent probes. However, the development of synthetic FRET probes with favorable photophysical properties that are also suitable for biological imaging applications remains challenging.In this Account, we review the rational design and biological applications of synthetic FRET probes, focusing primarily on studies from our laboratory. To construct useful FRET probes, it is a pre-requisite to develop a FRET platform with favorable photophysical properties. The design criteria of a FRET platform include (1) well-resolved absorption spectra of the donor and acceptor, (2) well-separated emission spectra of the donor and acceptor, (3) donors and acceptors with comparable brightness, (4) rigid linkers, and (5) near-perfect efficiency in energy transfer.With an efficient FRET platform in hand, it is then necessary to modulate the donor–acceptor distance or spectral overlap integral in an analyte-dependent fashion for development of FRET probes. Herein, we emphasize our most recent progress on the development of FRET probes by spectral overlap integral, in particular by changing the molar absorption coefficient of the donor dyes such as rhodamine dyes, which undergo unique changes in the absorption profiles during the ring-opening and -closing processes. Although partial success has been obtained in design of first-generation rhodamine-based FRET probes via modulation of acceptor molar absorption coefficient, further improvements in terms of versatility, sensitivity, and synthetic accessibility are required. To address these issues with the first-generation rhodamine-based FRET probes, we have proposed a strategy for the design of second-generation probes. As a demonstration, we have developed FRET imaging probes for diverse targets including Cu2+, NO, HOCl, cysteine, and H2O2. This discussion of the methods for successfully designing synthetic FRET probes underscores the rational basis for further development of new FRET probes as a molecular toolbox for probing and manipulating a wide variety of biomolecules in living systems.

A new family of boron difluoride-rigidified dyes, phenanthro[9,10-d]imidazole-quinoline boron difluoride (PQBD), with solid-state fluorescence has been designed and synthesized. The novel series of PQBD are advantageous over the typical boron difluoride-rigidified dyes such as BODIPYs in terms of large Stokes shift and red fluorescence in the solid state.

A new NIR fluorescent probe, NIR-Pd, for palladium species was designed and synthesized, based on a HD NIR fluorophore and deprotection of aryl propargyl ethers by palladium. The probe NIR-Pd displayed either a large NIR fluorescence turn-on or ratiometric response to palladium with high sensitivity and selectivity. Additionally, the novel NIR probe can monitor palladium species in live HeLa cells by NIR fluorescence imaging.

•A new coumarin–quinoline-based ratiometric fluorescent pH probe is described.•One major feature of the probe is that two emission peaks separate by >100 nm.•The probe is useful for monitoring pH variations in living cells.It is highly desirable to develop ratiometric pH probes with a well-resolved emission spectrum for biological imaging applications. In this work the design, synthesis, photophysical properties and cellular imaging studies of a new linked coumarin–quinoline probe is described. The major features of the novel probe include two well-resolved (>100 nm) emission peaks, strong fluorescence under acidic conditions, a pKa of 4.69, low cytotoxicity and good cell membrane permeability; features which render the probe useful for monitoring pH variations from neutral to acidic conditions in living cells.

A new NIR fluorescent probe for monitoring hydrazine in blood samples and live cells was designed and synthesized.

Near-infrared (NIR) fluorescent sensors have emerged as promising molecular tools for imaging biomolecules in living systems. However, NIR fluorescent sensors are very challenging to be developed. Herein, we describe the discovery of a new class of NIR fluorescent dyes represented by 1a/1c/1e, which are superior to the traditional 7-hydroxycoumarin and fluorescein with both absorption and emission in the NIR region while retaining an optically tunable hydroxyl group. Quantum chemical calculations with the B3LYP exchange functional employing 6-31G(d) basis sets provide insights into the optical property distinctions between 1a/1c/1e and their alkoxy derivatives. The unique optical properties of the new type of fluorescent dyes can be exploited as a useful strategy for development of NIR fluorescent sensors. Employing this strategy, two different types of NIR fluorescent sensors, NIR-H2O2 and NIR-thiol, for H2O2 and thiols, respectively, were constructed. These novel sensors respond to H2O2 or thiols with a large turn-on NIR fluorescence signal upon excitation in the NIR region. Furthermore, NIR-H2O2 and NIR-thiol are capable of imaging endogenously produced H2O2 and thiols, respectively, not only in living cells but also in living mice, demonstrating the value of the new NIR fluorescent sensor design strategy. The new type of NIR dyes presented herein may open up new opportunities for the development of NIR fluorescent sensors based on the hydroxyl functionalized reactive sites for biological imaging applications in living animals.

We have constructed a novel NIR fluorescent turn-on hydrogen sulfide probe suitable for fluorescent imaging in living cells based on thiolysis of dinitrophenyl ether.

Coumarin–rhodamine-based compounds 1a,b were rationally designed and synthesized as novel FRET ratiometric fluorescent chemodosimeters. Ratiometric chemodosimeters 1a,b exhibit several favorable features, including a large variation in the emission ratio, well-resolved emission peaks, high sensitivity, high selectivity, low cytotoxicity, and good cell membrane permeability. Importantly, these excellent attributes enable us to demonstrate ratiometric imaging of Cu2+ in living cells by using these novel ratiometric fluorescent chemodosimeters.

We have constructed a novel NIR fluorescent turn-on Cu+ probe suitable for imaging endogenous Cu+ ions in living cells based on a tricarbocyanine scaffold and a high affinity Cu+ receptor.

We have constructed a new fluorescence turn-on chemosensor for hydrogen sulfide based on a phenanthroimidazole scaffold, and the novel sensor is suitable for imaging hydrogen sulfide in living cells.

Hydrogen peroxide (H2O2) acts as a signaling molecule in a wide variety of signaling transduction processes and an oxidative stress marker in aging and disease. However, excessive H2O2 production is implicated with various diseases. Nitric oxide (NO) serves as a secondary messenger inducing vascular smooth muscle relaxation. However, mis-regulation of NO production is associated with various disorders. To disentangle the complicated inter-relationship between H2O2 and NO in the signal transduction and oxidative pathways, fluorescent reporters that are able to display distinct signals to H2O2, NO, and H2O2/NO are highly valuable. Herein, we present the rational design, synthesis, spectral properties, and living cell imaging studies of FP-H2O2-NO, the first single-fluorescent molecule, that can respond to H2O2, NO, and H2O2/NO with three different sets of fluorescence signals. FP-H2O2-NO senses H2O2, NO, and H2O2/NO with a fluorescence signal pattern of blue–black–black, black–black–red, and black–red–red, respectively. Significantly, we have further demonstrated that FP-H2O2-NO, a single fluorescent probe, is capable of simultaneously monitoring endogenously produced NO and H2O2 in living macrophage cells in multicolor imaging. We envision that FP-H2O2-NO will be a unique molecular tool to investigate the interplaying roles of H2O2 and NO in the complex interaction networks of the signal transduction and oxidative pathways. In addition, this work establishes a robust strategy for monitoring the multiple ROS and RNS species (H2O2, NO, and H2O2/NO) using a single fluorescent probe, and the modularity of the strategy may allow it to be extended for other types of biomolecules.

Fluorescence imaging is one of the most powerful techniques for monitoring biomolecules in living systems. Fluorescent sensors with absorption and emission in the near-infrared (NIR) region are favorable for biological imaging applications in living animals, as NIR light leads to minimum photodamage, deep tissue penetration, and minimum background autofluorescence interference. Herein, we have introduced a new strategy to design NIR functional dyes with the carboxylic-acid-controlled fluorescence on–off switching mechanism by the spirocyclization. Based on the design strategy, we have developed a series of Changsha (CS1–6) NIR fluorophores, a unique new class of NIR functional fluorescent dyes, bearing excellent photophysical properties including large absorption extinction coefficients, high fluorescence quantum yields, high brightness, good photostability, and sufficient chemical stability. Significantly, the new CS1–6 NIR dyes are superior to the traditional rhodamine dyes with both absorption and emission in the NIR region while retaining the rhodamine-like fluorescence ON-OFF switching mechanism. In addition, we have performed quantum chemical calculations with the B3LYP exchange functional employing 6-31G* basis sets to shed light on the structure-optical properties of the new CS1–6 NIR dyes. Furthermore, using CS2 as a platform, we further constructed the novel NIR fluorescent TURN-ON sensor 7, which is capable of imaging endogenously produced HClO in the living animals, demonstrating the value of our new CS NIR functional fluorescent dyes. We expect that the design strategy may be extended for development of a wide variety of NIR functional dyes with a suitable fluorescence-controlled mechanism for many useful applications in biological studies.

We have judiciously constructed a novel ICT-based ratiometric OCl−probe capable of ratiometric imaging in the live cells based on the new OCl−-promoted de-diaminomaleonitrile reaction.

A new NIR fluorescent probe for monitoring hydrazine in blood samples and live cells was designed and synthesized.

We have constructed a novel NIR fluorescent turn-on hydrogen sulfide probe suitable for fluorescent imaging in living cells based on thiolysis of dinitrophenyl ether.

We have constructed a new fluorescence turn-on chemosensor for hydrogen sulfide based on a phenanthroimidazole scaffold, and the novel sensor is suitable for imaging hydrogen sulfide in living cells.

A new NIR fluorescent probe, NIR-Pd, for palladium species was designed and synthesized, based on a HD NIR fluorophore and deprotection of aryl propargyl ethers by palladium. The probe NIR-Pd displayed either a large NIR fluorescence turn-on or ratiometric response to palladium with high sensitivity and selectivity. Additionally, the novel NIR probe can monitor palladium species in live HeLa cells by NIR fluorescence imaging.

We have judiciously constructed a novel ICT-based ratiometric OCl−probe capable of ratiometric imaging in the live cells based on the new OCl−-promoted de-diaminomaleonitrile reaction.

We have constructed a novel NIR fluorescent turn-on Cu+ probe suitable for imaging endogenous Cu+ ions in living cells based on a tricarbocyanine scaffold and a high affinity Cu+ receptor.

The long wavelength (far-red to NIR) analyte-responsive fluorescent probes are advantageous for in vivo bioimaging because of minimum photo-damage to biological samples, deep tissue penetration, and minimum interference from background auto-fluorescence by biomolecules in the living systems. Thus, great interest in the development of new long wavelength analyte-responsive fluorescent probes has emerged in recent years. This review highlights the advances in the development of far-red to NIR fluorescent probes since 2000, and the probes are classified according to their organic dye platforms into various categories, including cyanines, rhodamine analogues, BODIPYs, squaraines, and other types (240 references).