1 Introduction

Currently, with the continuous development of diagnostics and imaging fields, fluorescence imaging technology has become an indispensable method for monitoring changes in biochemical indicators and the appearance and development of biomarkers in living systems. Fluorescent probes are an important part of fluorescence imaging. This kind of important tool has received increasing attention from scientists in recent years due to its unique advantages. Fluorescent probes refer to a type of fluorescent molecule that has a characteristic fluorescence response in the ultraviolet–visible-near-infrared (UV-Vis-NIR) region, and their fluorescent properties are sensitive to the nature of the environment. The observation and analysis of the structure of fluorescent probes used in bioimaging and therapy reveal conjugated bonds or conjugated systems in probes with different structures. It is speculated that these structures may be necessary for the probe to emit light. Furthermore, the basic structure of a fluorescent probe is composed of a fluorophore, a linker, and a recognition group unit. Of these, the fluorophore is the most important, as it determines the properties of the fluorescent probe. Therefore, the fluorophore used in bioimaging needs to be low cost, easy to modify and to exhibit good biocompatibility and other characteristics. The receptor unit is used as the binding site of the molecule to be analyzed and achieves biological imaging under the action of electrostatic interactions, p–p interactions, covalent bonds or hydrogen bonds or their combination [1, 2]. Depending on the different substances to be detected by the fluorescent probes, there are some specific reaction mechanisms. For example, the mechanisms fluorescent probes use in detecting biological thiols include Michael addition, aldehyde cyclization, the conjugate addition cyclization of acrylic resin, natural chemical linkage, the cleavage of sulfonamide and sulfonate, the cleavage of disulfide bonds or selenium, aromatic substitution rearrangement, supramolecular interactions, etc. [3, 4].

Among the fluorescent probes used for bioimaging, there are three common photophysical mechanisms that promote fluorescence changes [5]: photoinduced electron transfer (PET), intracellular charge transfer (ICT), and Förster fluorescence resonance energy transfer (FRET) (Fig. 1a). In PET, electrons are transferred from the donor to the excited-state fluorophore with strong fluorescence quenching. After the donor group binds to analyte recognition, the PET action is blocked, thereby turning on fluorescence. Yanfeng Tang's group [6] designed a “turn-on” fluorescent probe CTB for the detection of Cd2+, which uses a tris-(2-aminoethyl)-amine moiety as a recognition unit and an electron-donor unit. The presence of a PET interaction between the fluorophores resulted in fluorescence quenching. After complex formation with Cd2+, the PET effect was blocked and the fluorescence turned on (Fig. 1b). ICT is a change in fluorescence caused by electron transfer within a fluorophore molecule. In this mechanism, the recognition group acts as a part of the electron donor or acceptor. When the recognition group binds to the analyte, the electronic push–pull ability of the recognition group changes, and the electronic structure of the system is redistributed, resulting in a change in spectral properties. Changli Zhang et al. [7] designed the hydrogen polysulfide (H2Sn) ratio sensing probe BDP-PHS, which leads to ICT inhibition under the action of strong electron-withdrawing groups. After interaction with H2Sn, the strong electron-pulling group is removed, and the ICT recovery leads to a change in the emission spectrum, enabling ratiometric sensing of H2Sn (Fig. 1c). FRET occurs when the emission spectrum of the donor fluorophore overlaps the excitation spectrum of the acceptor fluorophore and the distance between the two is 7–10 nm. FRET has an obvious distance effect, and FRET does not occur when the distance between the two fluorophores is too large or too small (Fig. 1d). It is a common mechanism in fluorescent probes with a donor–acceptor p-conjugated system [8].

Fig. 1
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a Three main luminescence mechanisms of fluorescent probes. (i) Photoinduced electron transfer (PET). The transfer direction is usually the electron donor to the excited state fluorophore, which produces a strong quenching effect on fluorescence. Once combined with the guest, PET is inhibited and the fluorophore emits fluorescence. (ii) Intramolecular charge transfer (ICT). Electron donors or electron acceptors are usually part of the recognition group. When the recognition group specifically recognizes and binds to the substance to be tested, the electrical properties of the group changes, which leads to the redistribution of the system’s electronic structure, showing a red shift or blue shift in the spectrum. (iii) Fluorescence resonance energy transfer (FRET). FRET occurs when the emission spectrum of the donor fluorophore and the excitation spectrum of the acceptor fluorophore overlap and are separated by a distance of 7–10 nm. The donor fluorescent molecule can excite the acceptor molecule to emit fluorescence, and at the same time the intensity of the donor fluorescent molecule itself is attenuated. b Sensing mechanism of the PET-based “turn-on” fluorescent probe. c ICT-based ratiometric fluorescent probe sensing mechanism. d Three conditions for FRET to occur: the donor–acceptor fluorophore distance is between 7 and 10 nm; the donor–acceptor fluorophore has a suitable relative position; the donor fluorophore emission spectrum overlaps with the acceptor fluorophore excitation spectrum

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Compared with traditional imaging techniques, such as positron emission tomography (PET), magnetic resonance imaging (MRI), and single-photon emission computed tomography (SPECT) [9], there are many advantages of fluorescent probes, such as real-time imaging, deep visualization, high selectivity and sensitivity, high spatial and temporal resolution, small size, easy modification, little damage to biological samples, and no need for in vivo sampling [3, 10,11,12], and they can accurately diagnose many diseases or detect biological substances through noninvasive, real-time, high-resolution imaging, therefore making fluorescent probes the “main force” in bioimaging methods. Fluorescent probes are also used widely in bioimaging due to their unique advantages; they can be used to detect inorganic ions, DNA, RNA, hydrazine, biological thiols, reactive oxygen species (ROS), ATP, amino acids and other small molecules, enzymes, antigen antibodies, and protein macromolecules [13,14,15,16,17,18,19]. Moreover, they are also used to monitor changes in intracellular pH or changes in mitochondrial membrane potential [20]. In short, detecting the level of disease biomarkers to make an accurate and sensitive diagnosis of diseases makes it possible to treat some difficult-to-detect diseases early.

Since the emergence of the concept of fluorescent probes, chemists and biologists have been working on the transformation of the structure of fluorescent probes. Based on the premise of following the basic principles of biocompatibility and nonimmunogenicity and ensuring low enough cytotoxicity and good cell permeability [21], the design of fluorescent probes is moving towards the goal of achieving “more sensitive, more selective, less damage and better biocompatibility”. Therefore, a variety of new types of single-color probes (on–off, off–on), proportional probes, two-photon (TP) probes, and other new probes have been developed [22, 23]. In addition, the interdisciplinary model also promotes the development of fluorescent probes. In this context, scientists are satisfied not only with the single function of fluorescent probes for diagnosis but also with molecular modification, adding linking groups and other means to connect fluorescent probes with drug molecules to synthesize fluorescent probes with a dual function of diagnosis and treatment [24,25,26]. Based on this idea, the use of fluorescent probes in combination with targeted modification and nanometer-scale modification in elementary complex technologies and other emerging therapeutic fields, such as immunotherapy imaging, has brought the research of fluorescent probes to a new level [27, 28]. In the future, this will become an important direction for fluorescent probe research. Even so, compared with nanoprobes undergoing rapid development in recent years, small-molecule fluorescent probes have better pharmacokinetic properties and biocompatibility and have a clear chemical structure that can be modified easily by structural modification. The photophysical properties of probes have been optimized, making their operation clearer and easier [22].

UV–visible small-molecule fluorescent probes, with an emission wavelength less than 650 nm, are being increasingly studied. In bioimaging, compared with traditional instrument detection methods, UV–visible small-molecule fluorescent probes have many advantages, such as real-time monitoring, strong selectivity, high sensitivity, and no need for sampling. They provide more accurate and advanced methods for cell imaging, disease biomarker detection, and biological substance index detection. According to reports in the literature, there are many classic types of UV–visible fluorophores, such as coumarin, fluorescein, rhodamine, BODIPY, pyrene, and naphthalene. Each fluorophore has its unique advantages. Depending on the substance to be detected, the use of probes with different fluorophores can achieve the effect of maximizing strengths and avoiding weaknesses and can thus make the detection results more accurate.

In this review, we classified UV–visible fluorescent probes based on different widely used fluorophore structures and introduced the development of UV–visible fluorescent probes with various structures. This review focuses on the structural characteristics, design strategies, advantages, representative probes, and the latest developments in the application fields of fluorescent probes over 3–5 years. At the end, we look forward to future development of fluorescent probes from the perspective of bioimaging and diagnostics.

2 UV–Visible Probes Based on Conventional Fluorophores

2.1 Coumarin

Coumarin (2H-1-benzopyran-2-one) and its derivatives are among the most intensively studied fluorophores among UV–visible fluorescent probes (Fig. 2a). It has many advantages, such as good solubility, high quantum yield, good cell permeability, low toxicity, simple structure, easy synthesis, good photostability, and large stokes shift, which make it the first choice for traditional fluorescent probe skeletons [29, 30]. As shown in Fig. 2b, the general structure of fluorescent coumarin probe design is to connect diethylamino or hydroxyl groups at the 7-position to improve the physical properties of the probe (to increase water solubility) or to act as a recognition group, and different functional groups connect at the 3-position for the detection of various substances. When the test substance and the reactive group undergo addition, elimination, complexation and other reactions, the purpose of imaging can be achieved by turning on or off the fluorescence response or changing the emission wavelength [31,32,33,34,35,36,37,38]. Additionally, the electron-donating group at position 6 and position 7 and the electron-withdrawing group at position 3 and position 4 will cause the emission wavelength to be red shifted [39, 40]. Connecting a recognition group to the 8-position is also a design strategy used for some coumarin probe structures [41, 42]. Some probe designs exploit special structures, that is, the coumarin group is combined with other functional receptors to build. For example, Bin Yu et al. [43] developed a probe that uses coumarin-fused coumarin as a fluorophore to detect biological thiols. Cell imaging results showed that the probe YB successfully imaged biothiols, and the in vitro responses to H2S and GSH were time-dependent and concentration-dependent (Fig. 2c); Van-Nghia Nguyen et al. [44] replaced the lactone carbonyl oxygen atom with a sulfur atom to achieve a higher selectivity for detecting ClO− in organisms. Studies have shown that when the probe concentration is 20 μM, clearer cell imaging results can be obtained at λem = 465–495 nm (Fig. 2d); He Meng et al. [38] designed a probe that responds quickly to HOCl by coupling coumarin and rhodamine, and introduced a morpholine structure to achieve accurate targeting of the probe to lysosomes. In addition, they performed colocalization imaging experiments with 1 μM CR-Ly and 0.2 μM commercial lysosome stain Lyso-Tracker Deep Red, and obtained good colocalization results, indicating the specificity of the probe to lysosomes targeting (Fig. 2e). These probes have a great imaging effect on the corresponding detection substance in vitro.

Fig. 2
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c Reproduced with permission [43]. Copyright 2018, Elsevier B.V. d Reproduced with permission [44]. Copyright 2020, Elsevier B.V. e Reproduced with permission [38]. Copyright 2019, Elsevier B.V. f Reproduced with permission [46]. Copyright 2019, The American Chemical Society. g Reproduced with permission [48]. Copyright 2021, The American Chemical Society

a Coumarin fluorescent probe core. b General design strategy of coumarin fluorescence probe. c (i) Proposed mechanism YB for H2S and GSH. (ii) Time-dependent images of HeLa cells treated with YB (60 μM) and images of HeLa cells treated with different concentrations of YB for 60 min. d (i) Proposed hypochlorite sensing mechanism of CZCN-S. (ii) After the probe was treated with different concentrations of imaging ClO− in live HeLa cells, fluorescence images were obtained at λex = 405 nm and λem = 465–495 nm. Bar 30 μm. e (i) Sensing mechanism of CR-Ly for HOCl. (ii) Co-localization experiments with RAW264.7 cells. Cells were co-stained with 1 μM CR-Ly and 0.2 μM Lyso-Tracker Deep Red. f (i) Design and sensing mechanism of the new N2H4 probe. (ii) Fluorescence images of the probe in A549 and zebrafish following addition of N2H4. (Red Channel: λem = 570–625 nm; Green Channel: λem = 500–545 nm). g (i) The sensing mechanism of CMHC. (2) Fluorescence imaging of CMHCH (5 μM, 30 min) in live SiHa cells with differing pH values; Fluorescence imaging of CMHCH (5 μM, 30 min) in live SiHa cells under different stimulations. (Oleic acid is known to be a nutrient-rich substance to stimulate the production of lipid droplets (LDs) in live cells; LDs could be metabolized rapidly in the absence of glucose; Chloroquine (CQ) is sometimes used to disrupt the acidic microenvironment of lysosomes.) (red channel λem = 600–650 nm; green channel:λem = 500–550 nm).

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In practical applications based on the coumarin sensor platform, Duxia Cao et al. [45] comprehensively and critically reviewed many specific examples of coumarin sensors, which are used to detect anions, metal cations, reactive sulfur species, biothiols and selenocysteine, and ROS, pH and intracellular physical and chemical properties. There are also some newer applications of coumarin sensors.

Hydrazine (N2H4) induces hepatotoxicity, neurotoxicity and mutagenicity, causing serious harm to human health. Therefore, the detection of hydrazine in vitro and in vivo has always been the focus of toxicant detection. There are two problems with the fluorescent probes currently used for hydrazine detection: many high-selectivity and high-sensitivity probes that have been designed have the defect of poor water solubility, and their applicability in actual sample analysis is not strong. At present, most probes are open or closed probes, which have strong background interference [46]. To overcome these problems, Jun Li et al. [46] introduced oligoethylene glycol into fluorescent probes to increase hydrophilicity. In addition, the N,N-diethylamine group, conjugated cyano group and pyridine side chain form a strong electronic push–pull system, it can make the emission wavelength of the probe produce a greater degree of red shift, thereby effectively solving the problem of the probe’s own fluorescence background interference. The C3 position is used as the recognition site of hydrazine. After the reaction, the side chain is removed, and the emission wavelength of the probe is blue-shifted, thereby sensing the hydrazine. The in vitro and in vivo imaging effects of the probe were investigated in A549 cells and zebrafish models, respectively. Photographs were taken at 30 min, 60 min and 90 min after the addition of N2H4. It was found that the experimental results of the probe in vitro and in vivo were consistent, that is, with the increase of time, the emission wavelength of the probe shifted blue, and gradually changed from red to green. This is in line with the sensing mechanism of the probe (Fig. 2f). Experiments show that the probe can be used successfully for hydrazine vapor detection and zebrafish imaging.

The pH stability of the organelles is of great significance to the normal physiological activities of the human body. Among organelles, lipid droplets (LDs) and lysosomes are responsible for the important tasks of storage and digestion of intracellular substances, respectively. However, when LDs are damaged, they are transported to the lysosome for degradation and fat phagocytosis occurs [47]. This process is usually related closely to metabolic diseases and chronic inflammation. Fangfang Meng et al. [48] designed a probe CMHCH based on coumarin derivatives. It can make use of the different emission wavelengths of probes caused by the difference of its conjugation length under different pH conditions and can simultaneously distinguish and identify lysosomes and LDs. It can also monitor the dynamic process of fat phagocytosis. The probe molecule uses a coumarin derivative as a fluorophore and has a pH-sensitive intramolecular spirocyclic side chain as a sensing group. Under neutral or alkaline conditions, the molecular absorption and emission wavelengths are blue-shifted due to the breaking of conjugated bonds, while under acidic conditions, the molecules exhibiting red-shift absorption and emission. In addition, the pH-dependent change of the intramolecular spiro ring can provide a basis for the development of new fluorescent probes. Based on this principle, the probe CMHCH has great potential for elucidating LD-lysosome interactions and diagnosing metabolic-related diseases. Fangfang Meng’s group used SiHa cells as the experimental cell line to image under different pH conditions (pH 4.0, 5.0, 6.0, 7.0, 8.0). The results showed that with the increase of pH, the emission wavelength of the probe blue-shifted. This demonstrates the feasibility of the probe to measure pH response in vitro (Fig. 2g). To further evaluate the ability of probe CMHCH to monitor LD and lysosomal changes, three exogenous agonists were applied to induce LD and lysosomal changes. After the addition of oleic acid (OA), obvious green fluorescence enhancement and enlarged LD can be observed, indicating that the probe can observe the production of LD stimulated by OA; under starvation culture conditions without glucose, LD was metabolized rapidly, and the green fluorescence in the imaging image was reduced significantly while the characteristic red fluorescence of lysosomes did not change significantly, indicating the high responsiveness of CMHCH to starvation-stimulated changes in LD; finally, stimulation with chloroquine (CQ) reduced lysosomal acidity and the red fluorescence was significantly attenuated while the green fluorescence signal of LD was unaffected, indicating that CMHCH could respond accurately to CQ-induced lysosomal changes (Fig. 2g).

Coumarin as a fluorescent sensing platform backbone has strong adaptability in bio-optical imaging. The smaller structure gives coumarin-based fluorescent probes a huge advantage in terms of synthesis and cell penetration. In addition, coumarin itself is considered to be a molecule with pharmacological properties, and several drugs containing coumarin already exist. Therefore, coumarin fluorescent probes have great potential in the development of integrated platforms for diagnosis and treatment and drug discovery. This still requires intensive studies on the acute and long-term toxicity of the platform. However, the coumarin sensing platform also has certain limitations in bioimaging applications. First, there is still a lot of room for improvement in terms of probe biocompatibility; second, coumarins tend to aggregate by π–π stacking in solvents with a high proportion of water, resulting in fluorescence quenching. In addition, the shorter excitation wavelength and emission wavelength have the problems of greater light damage to biological samples and difficulty in detection due to poor penetration. Therefore, the coumarin scaffold should be altered to allow excitation and emission in red and NIR. With the development of new materials and new sensing mechanisms, the coumarin framework can be used to prepare new aggregation-induced emission (AIE) or TP sensing platforms, and can also be used in conjunction with nanotechnology to prepare a variety of nano-sensing probes, which is promising for overcoming the shortcomings of the current coumarin framework.

2.2 Fluorescein

Fluorescein is another frequently used fluorophore in ultraviolet–visible fluorescent probes. Because of its high quantum yield, stable fluorescence signal, greater water solubility, and special structure, it is often chosen as one of the luminescence sources of fluorescent probes [49]. Fluorescein has two variable structures, a spiro ring structure and an open ring structure (Fig. 3a). Imaging based on opening or closing the fluorescein spiro ring has become a common principle used to change the luminescence of fluorescein fluorescent probes. Therefore, in the design of “OFF–ON” fluorescein probes, fluorescein or its derivative molecules are usually bonded to the reactive group. After entering the body, the reactive group reacts with the substance to be detected and is removed, and the spiro ring in the fluorescein molecule opens and is excited, resulting in fluorescence [50,51,52,53]. In addition, the connection of open-ring fluorescein with the reactive group to quench the fluorescence of the probe by the principle of PET is also a reasonable design scheme based on the fluorescein “OFF–ON” probe. Fanyong Yan and his colleagues [54] developed two fluorescein probes, FDA and FDH, based on fluorescein aldehyde and nitroaniline derivatives. Among them, FDA is based on ligand-to-metal charge transfer (LMCT) designed “closed” probes for the sensing of metal ion Ce4+. The excitation of LMCT after the complexation of Ce4+ with FDA results in the fluorescence switch-off of the probe. FDH is a ClO−-specific “turn-on” probe designed based on the principle of PET. Due to the presence of PET, the probe fluorescence of FDH is quenched. After interacting with ClO−, the fluorescein lactone ring is opened, the PET process is inhibited, and the fluorescence is turned on. Spectrometry results show that FDA and FDH fluorescence intensities are concentration-dependent on their respective analytes (Fig. 3b). The probe used for the substance to be detected in a certain organelle is also connected to the organelle-specific targeting group in the structure, such as the lysosomal-specific targeting group morpholine, which can improve the specificity and targeting ability of the probe [55, 56]. In addition, Xilang Jin et al. [57] and Chao-Rui Li et al. [58] used the strategy of reacting fluorescein with hydrazine to replace the lactone structure with a lactam structure when designing the probe, and the free amino group was connected with the recognition group to form a probe (Fig. 3c). The probe of this structure fluoresces by complexing with intracellular ions and opening the spiro ring. It is speculated that the purpose of the N–N bond introduced in the structure is to restrict the reactive group from leaving the fluorescein molecule after the reaction. In the past 3 years, there have been fewer designs of probes with fluorescein as the fluorophore, which may be because the fluorescein probes are mainly “OFF–ON” probes; occasionally, “ON–OFF-ON” probes have been developed, but they have certain limitations.

Fig. 3
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b Reproduced with permission [54]. Copyright 2019, Elsevier B.V. c Reproduced with permission [57]. Copyright 2018, Elsevier B.V.

a Fluorescein structure conversion between open loop and closed loop. b (i) Proposed mechanism between FDA and Ce4+, FDH and OCl−. (ii) Fluorescence spectra of Ce4+ added to FDA and OCl− added to FDH. Inset: the relationship between the F0-F fluorescence intensity ratio at 505 nm and the Ce4+ concentration and the relationship between the F–F0 fluorescence intensity ratio at 515 nm and the OCl− concentration. c (i) Conventional fluorescein containing lactone structure and fluorescein with lactam structure. (ii) The Zn2+/ATP-sensing mechanism of probe of Xilang Jin group. (iii) The proposed mechanism for the response of probe of Chao-Rui Li group to Mg2+.

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One of the biggest advantages of fluorescein-based fluorescence sensing platforms is that they provide more active modification sites. For probes used for ion detection, the existence of multiple binding sites can greatly improve the detection sensitivity and reduce the detection limit, and has the characteristics of simplicity, high efficiency and good selectivity. Double quenching mechanism probes can also block the fluorescence of fluorescein by introducing two functional groups such as ICT and FRET mechanisms. This overcomes the defects of high detection limit and excessive background noise caused by incomplete quenching of fluorescence. In addition, the lipophilic modification by blocking the hydroxyl group of fluorescein increases the cell membrane penetration of the probe, which expands the application range and can be used for the sensing of biological small molecules and macromolecules. However, most fluorescein-based fluorescence sensing platforms that have been developed so far rely on sensing under conditions of high proportions of organic solvents, which may result in lethal toxic damage to biological samples. In addition, most probes for sensing by binding to analytes have low detection accuracy and only stay on the detection of small molecular analytes. Fluorescein is one of only a few fluorescent dyes approved by the FDA. Possibly due to unexplored potential biological toxicity, it is used clinically only for ophthalmic diagnostics. Therefore, the development of fluorescein probes with higher biocompatibility and water solubility for bioimaging is the future development direction.

2.3 Rhodamine

Rhodamine is a catechol fluorophore similar in structure to fluorescein and is classified into rhodamine B, rhodamine 6G, and rhodamine 123 (Fig. 4a). Generally, rhodamine B is the most widely used bioimaging probe, followed by rhodamine 6G. Because of their excellent photophysical properties, rhodamines are ideal fluorophores for the development of fluorescent probes. Similar to the luminescence mechanism of fluorescein, rhodamine and its derivatives are interchangeable between nonfluorescent closed-loop spiro and fluorescent open-loop forms (Fig. 4b) [59]. The stability of the spiro ring plays a decisive role in the probe. “OFF–ON” probes have therefore become the main type of UV–visible rhodamine probes. These probes are fluorescently turned off when the spiro ring is formed, and the probe spiro ring is opened by the redox reaction between the analyte and the fluorophore, the fluorescence is restored, and the analyte concentration is indicated by the fluorescence intensity. Figure 4c–f shows the sensing mechanism of several highly specific “Turn on” fluorescent probes and the imaging results of in vitro analysis. The results show that these probes have strong sensing capabilities in both sample analysis and cell imaging [60,61,62,63]. Within contrast to fluorescein, rhodamine changes the hydroxyl groups on the two benzene rings in xanthenes to amino/ethylamino/diethylamino groups and replaces spironolactone with spironolactone. This allows rhodamine to have better water solubility, and the spirolactam structure generally does not separate from the recognition group when the ring opens and the fluorophore emits light so that the probe has a larger emission wavelength. Due to its structural similarity with fluorescein, the design strategy of rhodamine fluorescent probes is roughly the same as that of fluorescein probes. Rhodamine is a common fluorophore used in NIR fluorescent probes in recent years.

Fig. 4
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c Reproduced with permission [60]. Copyright 2019, Elsevier B.V. d Reproduced with permission [61]. Copyright 2018, Elsevier B.V. e Reproduced with permission [62]. Copyright 2019, Elsevier B.V. f Reproduced with permission [63]. Copyright 2020, Elsevier B.V. g Reproduced with permission [64]. Copyright 2021, The American Chemical Society

a Nucleus structure of several rhodamine fluorescent probes. b Rhodamine and its derivatives show interchangeability between non-fluorescent closed-loop spiro and fluorescent open-loop forms. c The response mechanism of fluorescent probe RHHP-PN for ONOO− and absorption responses of probe RHHP-PN (5 μM) in the presence of ONOO− (10 μM) at 25 °C. Inset Image of the probe (20 μM) solution with/without ONOO− (100 μM). d (i) Mechanism of RL1 for sensing HOCl. (ii) Fluorescence images of RAW264.7 cells co-stained with RL1 (1 mM) and Lyso Tracker Deep Red (0.2 mM) in the presence of lipopolysaccharide (LPS) and phorbol myristate acetate (PMA). e Proposed mechanism of 6G-ClO with ClO− and in vivo and in vitro experiments of the probe. f (i) The structure of probe RhP. (ii) The probe's selectivity to Fe3+ and intracellular imaging. g (i) Structure analysis of the probe Rd1, Rd2 and proposed mechanism of Rd1. (ii) Fluorescence imaging of the probe at the cell, tissue, and animal level.

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Endogenous ROS play an important role in various physiological activities. Excessive levels of active oxygen in the body can lead to a series of related diseases. In many cases, the progress of the disease can be judged in time by detecting indicators of active oxygen. Therefore, endogenous ROS are important biomarkers for many diseases. Meng-Yang Li et al. [64] synthesized an immobilized fluorescent probe Rd1. The probe has rhodamine as the skeleton and triphenyl phosphate (TPP) as the mitochondrial targeting group, which is used for imaging of mitochondrial endogenous ClO−. In addition, they introduced methoxymaleamide as a fixed group in the probe and found that by comparing the probe Rd2 without a fixed group, Rd1 is more conducive to long-term stable existence in aqueous solution. Bioimaging experiments show that Rd1 exhibits better retention and better imaging characteristics in cell and tissue mitochondria, and enables clear in vivo imaging of zebrafish models (Fig. 4g).

Rhodamine structure-based xanthenes dyes have been essential fluorophores in biosensing and imaging. Generally, rhodamine fluorescent probes have emission wavelengths in the visible region due to the larger conjugated system. Similar to fluorescein, rhodamine also has more modification sites. At present, many fluorescent probes for biological tracking are modified by rhodamine and its derivatives. In addition, the replaceability of rhodamine central oxygen atom makes rhodamine a highly competitive fluorophore for the development of ultra-long emission wavelength probes and NIR fluorescent probes. However, many current rhodamine probes suffer from low tissue penetration, high light scattering, and low resolution of the resulting images due to their short emission wavelengths. In addition, some modified structures of rhodamine may make the opening of lactone structures difficult, which limits the development of rhodamine-based “off–on” fluorescent probes. In terms of probe stability, rhodamine probes are insufficient for long-term and dynamic monitoring assays at the subcellular level. In terms of synthesis, many complex modified rhodamines have lengthy synthetic steps, which lead to high development costs. In the past decades, researchers have been working on the rational modification design of rhodamine fluorescent probes and developing bioimaging sensing platforms with high stability and high biocompatibility. In the future, imaging tools for emission in the NIR II will be the development direction of rhodamine fluorophores.

2.4 BODIPY

BODIPY (fluoroboron dipyrrole) is a polycyclic heterocyclic aromatic compound with excellent light stability, chemical stability, a high molar extinction coefficient, and insensitivity to pH [65] that is used widely in the design and synthesis of UV–visible fluorescent probes and NIR fluorescent probes (Fig. 5a). With the continuous in-depth study of BODIPY, the low water solubility of the BODIPY fluorophore has been discovered—a major defect that hinders its application in imaging living cells. In this regard, Lian-Xun Gao et al. [66] designed four “turn-on” sensing probes based on different water-soluble R groups for the rapid detection of biothiols. Biothiols react with nitroalkenes via Michael addition, so that the PET and ICT processes are blocked, thereby turning on fluorescence. The difference in water solubility of the four probes with different R groups led to the difference in the reaction rate with biothiols (Fig. 5b). As shown in Fig. 5b(ii), in order to explore the effect of hydrophilicity on the reaction rate, the group measured the reaction kinetic parameters of four sensing platforms with GSH in HEPES buffer. The results showed that the hydrophilicity of the probe was correlated positively with the reaction rate constant, and the order was BDP-QA > BDP-OH > BDP-OEG > BDP. This suggests that the hydrophilicity of fluorescent probes is extremely important in bioimaging applications. Therefore, efforts to design probes with high water solubility and high biocompatibility facilitate rapid and sensitive detection of target analytes. Additionally, the electronegativity of the side chain groups in the BODIPY probe will also greatly affect the selectivity and sensitivity of the probe to biological substances. For example, Nannan Wang et al. [67] explored the response characteristics of the different electronegativities of the side chain R group to cysteine or homocysteine (Cys/Hcy) when designing a probe for detecting biological thiols. They analyzed the time-dependent fluorescence responses of the three probes to Cys and Hcy. It was found that the reaction rate of probe 1 with the electron-withdrawing substituent trifluoromethyl was the fastest, followed by probe 2 with neutral hydrogen atom, and the slowest reaction rate was probe 3 with an electron-giving substituent. Since the reaction of the biothiol with the probe is a nucleophilic reaction, probe 1 with electron withdrawing group is more favorable for nucleophilic attack (Fig. 5c). There is a fixed pattern in the design strategy of BODIPY-based fluorescent probes; that is, many fluorescent probes based on BODIPY connect different types of recognition groups with the BODIPY core in the design, and through the mechanism of intramolecular PET or ICT, the fluorescence of the probe is quenched. After the probe enters the organism, the recognition group reacts with the substance to be tested, which causes the pathway of the two mechanisms to be blocked, and, finally, fluorescence turns on [55, 68,69,70]. Based on this strategy, Fang-Fang Wang et al. [71] summarized the design strategy of BODIPY-based biothiol probes (Fig. 5d). On the one hand, the introduction of 2,4-dinitrobenzenesulfonic acid and nitroalkene double reactive groups greatly improves the sensitivity and reactivity of the probe. On the other hand, it also overcomes the obstacle of incomplete quenching of fluorescence encountered in the design of BODIPY-based probes. When the probe interacts with the biothiol, the dual reactive groups undergo Michael addition and sulfonate cleavage, respectively, and the intramolecular electron system is rearranged, resulting in the shutdown of PET and ICT, and the recovery of probe fluorescence. In addition, the team also explored the spectral properties of probe DB-1 for different detected substances. The results show that DB-1 can clearly detect different biological thiols with high selectivity and sensitivity.

Fig. 5
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b Reproduced with permission [66]. Copyright 2020, Elsevier B.V. c Reproduced with permission [67]. Copyright 2018, Elsevier B.V. d Reproduced with permission [71]. Copyright 2018, Elsevier B.V

a Core of the BODIPY fluorescent probe. b (i)The structures and proposed mechanism of the BODIPY-based fluorescent probes. The R group is modified for water solubility. The nitroolefin unit is designed for sensing biothiols. (ii) The kinetic parameters of probes reacted with glutathione (GSH) in HEPES buffer. The data are the mean of three independent experiments; the better the water solubility of the probe, the faster the reaction rate. c (i) Chemical structure of designed probe for detecting Cys and Hcy in vitro. (ii) The influence of side chain electronegativity on probe reaction rate. The results show that the greater the electronegativity of the side chain group is beneficial to the nucleophilic reaction between the probe and the biothiol, which is manifested as a faster reaction rate. d (i) Design and mechanism of probe DB-1. (ii) The difference between the probe's absorption wavelength and emission wavelength for different detection substances.

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The high hydrophobicity of BODIPY leads to its possible non-specific interactions with cellular components with a higher probability, and a solution to the high hydrophobicity of the BODIPY core is currently available. BODIPY has the ability to modify the boron site, the pyrrole site and the central site, and different sites have their own advantages in different applications. Therefore, the design and development of BODIPY-based multifunctional sensing imaging probes with high water solubility and high biocompatibility has become a hot research direction. Additionally, BODIPY is the scaffold of choice (high cell membrane permeability and stability) for commercial cell tracers due to its electrically neutral core and insensitivity to temperature and environmental changes. BODIPY-based endoplasmic reticulum imaging probes and mitochondrial tracing probes already exist on the market. One of the reasons for the high photostability of BODIPY is the extremely low triplet yield, which minimizes the production of singlet oxygen molecules and greatly avoids photodegradation of the dye. However, in terms of spectral properties, the narrow UV absorption spectrum of BODIPY limits its capture of more photons, which is less desirable in fluorescent dyes. Therefore, researchers have developed various schemes to improve the properties of BODIPY probes. In addition to the common site modification, there are also heteroatom replacement of boron and fluorine atoms, and the incorporation of BODIPY into molecular-level devices such as various nanomaterials. All of these can have a huge impact on the properties of BODIPY dyes, and also broaden the application fields of BODIPY-based probes.

2.5 Pyrene and Naphthalene

Both pyrene and naphthalene are fluorophores with a rigid molecular plane, and there are multiple electrophilic substitution sites in the molecule, which can be relatively easily modified to adjust their fluorescence characteristics (Fig. 6a). As a fluorophore, pyrene has a large absorption coefficient, a long single-peak lifetime, and a high fluorescence quantum yield. After molecular modification, its emission wavelength can reach the visible light region [72]; naphthalene shows attractive light stability and chemical stability, as well as high fluorescence quantum yield and excellent biocompatibility [73]. In the design strategy of such fluorescent probes, pyrene and naphthalene are usually directly connected to carbonyl groups, carbon–carbon double bonds, sulfur atoms, nitrogen atoms, etc., and p–π conjugates or π–π conjugates occur in the molecule, so the emission wavelength of the fluorescent probe can be redshifted to achieve less cell damage and stronger tissue penetration ability. Additionally, in the design of naphthalene probes; its derivative, 1,8-naphthalimide, is also often used as a fluorophore [74,75,76,77] (Fig. 6b).

Fig. 6
figure 6figure 6

c Reproduced with permission [78]. Copyright 2019, The American Chemical Society. d Reproduced with permission [79]. Copyright 2018, The American Chemical Society. e Reproduced with permission [80]. Copyright 2018, The American Chemical Society. f Reproduced with permission [81]. Copyright 2021, The American Chemical Society

a Pyrene, naphthalene and naphthalene derivatives as cores of fluorescent probes. b The structure of several traditional ultraviolet (UV)–visible fluorescent probes based on 1,8-naphthalimide. c (i) Detection mechanism of coumarin-naphthylamine fusion probe for β-galactosidase detection. (ii) Fluorescence spectra of 10 μM CG after the addition of serious concentrations of β-galactosidase (0–3 U ml−1) for 15 min (λex = 420 nm) and Kinetics behaviors of fluorescent probe CG (10 μM) to β-galactosidase (3 U ml−1) at different times. d Fluorescence spectrum of fluorescent probe B6S. Inset Proposed mechanism of fluorescent probe B6S and color change of dimer and monomer. e (i) Structure design of the probe and the product of its reaction with ONOO−. (ii) Fluorescence imaging of the probe in SH-SY5Y cells, primary cortical neuron cells and rat hippocampal tissue. f (i) Design strategy of the N-methyl-d-aspartic acid (NMDA) receptor probe. (ii) two-photon fluorescence microscopy (TPM) images of primary cortical neuron cells and rat hippocampal slices.

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From the point of view of molecular structure, the fluorophore has a rigid three-ring structure and a large-conjugated structure of the nitrogen atom-carbonyl-naphthalene ring system. Therefore, compared with naphthalene, 1,8-naphthalimide has better stability and a longer emission wavelength. Second, the two carbonyl oxygen atoms form hydrogen bonds more easily, which allows the probe to have better water solubility. Because of these advantages, the design of probes for pyrene and naphthalene has also been a research hotspot in UV–visible fluorescent probes in recent years.

The development of proportional fluorescent probes can effectively reduce false positive results that may occur due to environmental impacts. It can correct for environmental interference and enhance the dynamic range of fluorescence measurement. Xiuqi Kong et al. [78] designed a smart ratio fluorescent probe CG based on the dual mechanism of ICT-FRET for the sensitive detection of β-galactosidase (Fig. 6c). The ICT mechanism can control the quantum yield of fluorescent probes by adjusting the electron donating ability of the group; FRET transfers energy from the donor group to the acceptor group, which is another powerful mechanism for constructing proportional probes. Therefore, the dual mechanism of ICT-FRET is beneficial to increase the emission intensity of the probe and realize highly sensitive detection of β-galactosidase. In the probe CG, coumarin and naphthylamine are used as the backbone, and β-d-galactopyranoside is used as the hydrolysis site of β-galactosidase to couple with naphthimide. Due to the effect of ICT-FRET, before and after β-galactosidase hydrolysis, the fluorescence of the probe changes from the blue of the coumarin donor to the yellow of the naphthylamine acceptor, thereby realizing the ratio imaging. Furthermore, as shown in Fig. 6c(ii), when exploring the concentration-dependent response of CG to β-galactosidase, they found that, with the increase in β-galactosidase concentration, the emission of CG at 500 nm decreased gradually, and the emission at 570 nm increased sharply, verifying the probe ICT-FRET synergy mechanism effect. The kinetic behavior of CG on β-galactosidase was explored later, and it was found that the probe CG had the characteristics of high affinity and ultra-fast response to β-galactosidase. This probe can be used for ultra-sensitive imaging of β-galactosidase in biological systems. Yen Leng Pak et al. [79] developed a TP fluorescent probe based on pyrene ratio, B6S, which can be used to monitor endogenous OCl− with a TP microscope, in which the imidazoline-2-thione group is the recognition and monitoring group. The probe exists in the form of a dimer molecule and emits blue-green fluorescence (λem = 482 nm). After reacting with OCl−, it becomes a monomer, and its emission wavelength is blue-shifted and enhanced at λem = 378 nm. It has the ability to detect endogenous OCl− sensitively (Fig. 6d).

1,8-Naphthalimide is also a TP fluorophore with excellent performance. Dayoung Lee et al. [80] used it to design and synthesize a TP fluorescent probe for monitoring ONOO− near the N-methyl-d-aspartic acid (NMDA) receptor in order to solve the problem of the lack of biomarker detection methods in neurodegenerative diseases such as Parkinson’s disease (PD), stroke, Alzheimer’s disease, cardiovascular disease, cancer and other diseases; this lack meaning that these diseases cannot be diagnosed quickly and accurately. In particular, the development of detection tools for ONOO− in the NMDA receptor region can effectively obtain more accurate information and help understand the role of ONOO− in the nervous system. The probe uses the boronic acid moiety as the recognition group of ONOO−, and the NMDA receptor antagonist as the specific targeting group, which has the advantages of high-water solubility, rapid response and high selectivity. In vitro experiments have proved that the probe is used to detect ONOO− near the NMDA receptor in living cells and tissues (Fig. 6e). In addition, glutathione (GSH) is another important indicator for early diagnosis of neurological diseases. Nahyun Kwon et al. [81] also designed a probe targeting NMDA. The probe is designed with a classic structure in which fluorophores, recognition groups and targeting groups are connected. The probe uses 1,8-naphthalimide as the TP fluorophore and the ifenprodil group as the targeting group of the NMDA receptor, and show high selectivity for GSH near the NMDA receptor. It can successfully monitor GSH in neurons and hippocampus. Studies have shown that the probe can be used as an effective detection tool to obtain important GSH information in the brain (Fig. 6f).

As aromatic fluorescent effector molecules with well-defined structural and fluorescent properties, Pyrene and naphthalene are two ancient probe backbones for bioimaging and sensing. Aromatic fluorophore monomers exhibit strong intrinsic fluorescence in the near-UV region, and the stacking of aromatic ring systems endows them with unique excimer fluorescence properties in the visible region. This dual luminescence mode has powerful applications for the development of novel ratiometric fluorescent probes. The dual emission spectral characteristics make the pyrene-based probe extremely sensitive to the detection environment, the intrinsic emission fluorescence can provide relevant information about the detection microenvironment, and the excimer fluorescence reflects the approach of another pyrene in the system. In addition, the high molar extinction coefficient is also a major advantage of aromatic fluorophores. This requires extremely low analyte concentration in practical bioimaging. In terms of structural modification, similar to other fluorescent probe backbones, various photophysical properties of aromatic fluorophores can be improved by fine-tuning the structure. Among them, 1,8-naphthalimide allows the introduction of various functional group modifications at the naphthalene moiety and the N-imide site. Third, highly sensitive and selective detection enables fast and accurate detection of analytes by aromatic probes in various mixed media or in a single medium with the addition of a mixture. Macromolecular labeling of fluorophores is a common strategy for in vivo tracing. However, the use of pyrene to label macromolecules such as proteins may result in the stacking of aromatic ring systems affecting intermolecular interactions, so it is not desirable in certain circumstances. In addition, the fluorescence of pyrene can be quenched by oxygen molecules, requiring strict control of oxygen partial pressure and temperature changes in the preparation and use of fluorescent probes.

2.6 UV–Visible Probes Based on Other Fluorophores

In addition to UV–visible fluorescent probes developed based on conventional fluorophores, some probes with excellent optical properties and biological imaging capabilities have also been developed in recent years. Among them, AIE probes and TP fluorescence probes are new types of probes developed in this century. They use innovative fluorescent materials to overcome, to some extent, some of the disadvantages of traditional UV–visible fluorescent probes, and further to lay the foundation for the development of probes with high clinical applicability.

Generally, conventional organic fluorophores have a tendency to spontaneously aggregate in solution. They usually exhibit strong fluorescence emission in dilute solutions, while in concentrated solutions, fluorescence quenching (ACQ) occurs due to aggregation. Since the beginning of the twenty-first century, people have discovered dyes with special properties: AIE luminescence agents (AIEgens), which emit strong light when the helical molecules aggregate in a concentrated solution or in a solid state [82,83,84,85,86,87]. Using the AIE characteristics of AIEgens, a new type of fluorescent probe was designed, that is, the AIE fluorophore was reasonably modified to make it have greater water solubility and fluorescence quenching properties in solution, and, after interacting with the target compound, it forms an aggregated cross-linked network and emits strong fluorescence. Among them, tetrastyrene (TPE) is a typical fluorophore with AIE characteristics. Jun Li et al. [88] introduced a mitochondrial-specific targeting group triphenylphosphonium (TPP) on the AIE group tetrastyrene and constructed a probe for accurately detecting changes in mitochondrial membrane potential. Compared with previous studies, the TPP is conjugated with AIE through a double bond to make the probe have a longer emission wavelength, which can reduce the background interference in the cell. The co-localization experiments showed that the AIE probe and the commercially available mitochondrial tracker had good overlap in the imaging of mitochondria in Hela cells, which demonstrated the high specificity of the probe to mitochondria and the feasibility of its application (Fig. 7a). In addition, the probe has good anti-interference ability against biological thiols and ROS in vivo.

Fig. 7
figure 7figure 7

a Reproduced with permission [88]. Copyright 2017, The American Chemical Society. b Reproduced with permission [89]. Copyright 2021, The American Chemical Society.

a Chemical structure of the aggregation-induced emission (AIE) probe (top); co-localization imaging of the AIE probe and mitochondrial tracker for mitochondrial staining in Hela cells (bottom). b Schematic diagram of the ionization state of glycopeptide modified AIE probe TGO under acidic and basic conditions; tranmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of TGO assembly morphology at different pH values. c (i) Reaction mechanism of Azulene-based fluorescent probe for ONOO−. (ii) TPM images of RAW 264.7 macrophmages and rat hippocampal slices. d Sensor design of fluorescent probe NUUs and proposed mechanism towards HClO.

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In the direction of biological pH imaging, the design of AIE probes faces the challenge of having both pH sensitivity and lighting characteristics. Fangling Zhang et al. [89] introduced a new glycopeptide-modified AIE probe (TGO) based on solid-phase peptide synthesis. The probe is composed of glycopeptide motif and AIE molecule TPA-1. Because the two have opposite hydrophilicity, the molecule is amphiphilic. It can induce self-assembly and switch between nanosheets and nanomicelles according to different pH values. That is, as the pH increases, the probe switches from nanosheets to nanomicelles, and the pH-induced charge changes in the peptides will greatly affect the microenvironment of the AIEgen, resulting in an increase in fluorescence intensity (Fig. 7b). The development of this glycopeptide-AIE probe provides a new idea for the design of pH-sensitive sensing platforms for bioimaging.

Compared with conventional single-photon imaging, TP imaging has many advantages, such as deeper tissue penetration and tissue three-dimensional (3D) image resolution, extended light-emitting time, lower background interference, and better biocompatibility [90, 91]. TP absorption is the process of absorbing TPs at the same time after being excited by TP light of NIR wavelength and making them transition from the ground state to the excited state with twice the photon energy. The TP fluorescent probe developed by TP absorption overcomes a series of shortcomings such as high photobleaching and high tissue phototoxicity of single-photon fluorescent probe, making it more suitable for biological detection and imaging in the fields of life science and medicine. In addition, the general structure, design ideas and mechanism of this probe are not significantly different from single-photon probes. UV–visible TP fluorescent probes based on other fluorophore structures have also been developed continuously in recent years. Their simpler structure and greater advantages of TP imaging have strong competitiveness in UV–visible probes. TP fluorescence imaging has become an indispensable technology for cell imaging. Lloyd C. Murfin et al. [92] reported a new type of TP fluorophore, Azulene, which has not been reported previously in biological imaging applications. Azulene is a nonalternant bicyclic aromatic hydrocarbon, isomeric with naphthalene, yet with appreciably different properties. The probe AzuFluor483-Bpin, designed based on a new fluorophore, is coupled with a boronic ester acceptor motif and substituted azulene for TP imaging of ROS. It has high selectivity to peroxynitrite and has excellent light stability. In addition, Azulene is sensitive to perturbation of linking substituents, so more new TP fluorescent probes can be developed by linking a large number of known receptor motifs. At present, the TP sensing platform based on Azulene has been used successfully to image ROS in RAW264.7 cells and rat hippocampus tissue (Fig. 7c).

Studies have shown that high concentrations of ROS, especially HClO, are closely related to the pathogenesis of PD. However, current research still cannot determine the brain HClO level during the development of PD. Not only that, the lack of a HClO level detector for brain tissue also hinders the study of the role of HClO in the pathogenesis of PD. To this end, Jiali Chen et al. [93] developed a highly sensitive, super-selective and fast-response fluorescent probe, NUU-1, which can successfully image the basal brain HClO and distinguish PD brain tissue from normal tissues (Fig. 7d). In terms of structural design, the phenothiazine part was used as the recognition site of HClO, and a series of open fluorescent probes were designed by combining with different aromatic amines. The probe uses ICT to mask the probe fluorescence. After being recognized and reacted with HClO, the phenothiazine part of the sulfur atom is oxidized, inhibiting ICT, and causing the fluorescence to turn on. In addition, site 2 on the aromatic amine is also a non-specific recognition site for HClO, which gives the probe the ability to recognize the dual sites of HClO and significantly improves the selectivity of the probe.

All in all, both AIE probes and TP probes represent hot directions for novel probes for bioimaging. At present, under the in-depth research of scientific research groups in various fields represented by the research group of Benzhong Tang, the AIE platform is being combined with various technologies, such as photodynamic therapy/photothermal therapy, nanotechnology, multiphoton imaging technology, multiple molecular coupling, etc., enriching bioimaging AIE sensing platforms/systems and applications at an astonishing rate, and developing probes that can theoretically accomplish any imaging task. In addition, TP and even multi-photon technology has promoted the development of multi-photon microscopy at an astonishing speed, and various imaging parameters are constantly being refreshed towards higher and more precise targets. In the future, novel imaging platforms based on AIE platform and multiphoton imaging will push forward the frontiers of biological imaging.

3 Application Areas of Fluorescent Probes

Currently, an increasing number of UV–visible fluorescent probes with different structures are being designed and developed and applied to all aspects of biological imaging. The detection of various metal ions (Cu2+, Al3+, Fe3+, Mg2+, Hg2+, Zn2+, etc.) in organisms and cells is one of the most popular applications of probes [33, 57,58,59, 63, 70]. Generally, the nitrogen atoms and oxygen atoms in the probe undergo a complex reaction with the ion to be detected to change the electron transfer mechanism in the probe to cause the probe to fluoresce by excitation or quenching, thereby realizing qualitative or quantitative ion detection. Moreover, many small molecules are closely related to the physiological state of the organism or the occurrence of diseases. For example, the sensitive and selective detection of hydrogen sulfide, hydrogen polysulfide, hypochlorous acid, formaldehyde, hydrazine, NO, biothiols, amino acids, etc. is helpful for the diagnosis and treatment of diseases [46, 49, 52, 65, 79, 94,95,96,97,98]. pH probes are also an important type of fluorescent probe that can detect the pH of a specific area in the body through the difference in the fluorescence characteristics of the probes under different pH conditions, and they also have great application prospects in the medical field [99, 100]. The development of protein fluorescent probes is one of the important methods used for the discovery of disease biomarkers. The positioning and imaging of enzymes or specific protein receptors on the cell surface can help doctors correctly determine the development of the disease to quickly treat symptoms [76, 78, 80, 81, 101]. In organelle imaging, the targeted probes developed in recent years target mainly mitochondria and lysosomes, as well as the endoplasmic reticulum. By adding specific targeting groups to the molecule, the two organelles can be targeted, and small-molecule probes will enter then the organelle to image related substances [38, 48, 56, 61, 64, 88, 102]. Moreover, the design of related nanoprobes has also progressed. Narode et al. [103] developed rhodamine 6G gold-covered nanoparticles for the highly selective detection of the free radical-induced oxidation of glutathione to quantitatively detect the concentration of glutathione in cells (Table 1).

4 Conclusion and Outlook

Fluorescent probes play an irreplaceable role in biological imaging. Compared with conventional biological imaging methods, fluorescent probes have many advantages, such as high resolution, less damage to biological samples, large modification space, and real-time tracking imaging [3]. Small-molecule fluorescent probes have penetrated the field of ion detection, disease biomarker tracking, and cell imaging due to their good pharmacokinetic properties and biocompatibility, as well as their diverse structural designs, and their modification strategies and the maturity of the technology. Small-molecule fluorescent probes are also one of the key development directions of fluorescent probes. UV–visible probes have been developed vigorously in the early days of fluorescent probe research due to their simple synthesis, easy availability of fluorophores, good water solubility, and high quantum yield. Today, many UV–visible probes with novel designs and excellent imaging effects are still being developed and applied and occupy the mainstream in the field of fluorescent probes.

However, with the continuous development of the field of fluorescent probes, some shortcomings of UV–visible probes can no longer meet the application requirements in biological imaging and diagnostics. Due to the short wavelength and poor penetration of UV–visible light, the fluorescence background interference when the probe is used for in vivo imaging is strong and the response is poor when used for deep tissue imaging. This reduces the ability to recognize and mark markers and lesions in deep tissues, which greatly limits the clinical application of UV–visible probes. Additionally, the energy of UV–visible light is relatively high, and when the probes used for bioimaging are light-excited, they will cause greater light damage to tissues and organs.

NIR fluorescent (NIRF) probes refer to probes with emission wavelengths of 650–900 nm. Compared with UV–visible fluorescent probes, NIRF probes have many advantages [4]. One of the most obvious is that they cause less damage to tissues and biological samples. Additionally, the long emission wavelength of the NIRF probe makes it have lower tissue absorption and reflection and higher photon emission, which is more conducive to penetrating deep tissue. This allows it to expand the detection range of the fluorescent probe to the diseased tissue so that it can be diagnosed more accurately, and it is more dominant in vivo imaging. Moreover, because the autofluorescence wavelength of tissue cells is closer to the UV–visible wavelength, background fluorescence interference is also an important factor considered by fluorescent probes. NIRF probes have less background interference and a lower signal-to-noise ratio due to the large difference in wavelength from the background fluorescence. Therefore, the development and application of NIRF probes have an indispensable position in the future of bioimaging and diagnostics. In addition to small molecule fluorescent probes, the development of nanotechnology in recent years has opened up new approaches in the field of biological imaging. Nanotechnology has breakthrough advantages in biosensing: high-throughput screening, high detection limit, label-free detection, significantly reduced sample size and strong applicability [104, 105]. A variety of nano probes for biological imaging have been developed, including quantum dots [106], gold nanoparticles [107], dye-doped materials [108], and metal organic frameworks [109]. In particular, carbon quantum dots (CQD) and graphene quantum dots (GQD), as zero-dimensional nanomaterials, have received more and more attention because of their higher photostability, low toxicity and better biocompatibility [110].

Even so, small-molecule UV–visible probes have occupied an unshakable position in biological imaging due to the diversity of fluorophore types, the variability of probe structure and the ease of synthesis. Therefore, the future development of small molecule UV–visible fluorescent probes has the following aspects. First, link a variety of UV–visible fluorophores through chemical bonds to develop new probes with proportional fluorescence or TP fluorescence properties. Second, redshift the fluorescence emission wavelength through structural complication and modification to develop related far-red, NIR and far-infrared fluorescent probes. Third, make use of the unique advantages of nanotechnology to prepare nano-UV–visible fluorescent probes. In addition, based on the development of organic chemistry, more structurally innovative chemical structures with fluorescent properties have been discovered, filling the dye library of UV–visible fluorophores, and making a great contribution to the development of more new probes.

Table 1 Application areas and representative probes of ultraviolet (UV)–visible fluorescent probes
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