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Luminescent probes for luminescence lifetime sensing and imaging in live cells: a narrative review

Nie, Kaixuan; Peng, Xiao; Yan, Wei; Song, Jun; Qu, Junle

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doi: 10.1097/JBR.0000000000000081
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Luminescence lifetime is an intrinsic property of luminescent materials and varies with the microenvironment. Moreover, the luminescence lifetime of a probe is independent of its concentration and other factors that influence the luminescence intensity (eg, the measurement method, excitation intensity, and wavelength). Recently, luminescence lifetime measurement has been applied for monitoring of dynamic interactions between luminescence-labeled molecules and micro-scale changes in biological environments.[1] Fluorescence lifetime imaging microscopy (FLIM) has been successfully applied in bioimaging, including identification of reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in live cells,[2] study of dynamic intracellular processes,[3] and measurement of protein–protein interactions.[4] Compared with conventional intensity-based steady-state fluorescence microscopy, FLIM is a good technique to observe the same fluorescence-associated phenomenon while also providing supplementary information; thus, FLIM yields more reliable results.[5]

FLIM is an ideal tool for studying systems that exhibit auto-luminescence, such as NADH-NADPH transformations.[2] However, most cellular microenvironments and in vivo biological processes lack detectable luminescence signals, necessitating the introduction of specific optical probes. These probes can act as not only specific markers for biological molecules or cell organelles, but also as indicators for intrinsically non-luminescent species or parameters, including ions, viscosity, and temperature. The former type of probe possesses a chemical unit that targets molecules or cell organelles; these probes usually require a constant luminescence lifetime from which the background fluorescence can be subtracted.[6] The latter type of probe has a photoluminescence lifetime that changes in the presence of analytes and under certain environmental conditions.[7] Compared with luminescence intensity-based probes, luminescence lifetime-based probes exhibit signals that are independent of the probe concentration or excitation method. The main advantage of luminescence lifetime-based probes is that, unlike luminescence intensity-based probes, ratiometric detection relative to an internal or external reference is not needed.

In 2010, Achilefu and Berezin presented a detailed introduction of fluorescence lifetime-based measurement and its biological applications.[1] Our group presented a lasting and effective discussion of FLIM and its biological applications.[8–12] In this review, we introduce the design approaches for luminescence lifetime-based probes and summarize representative examples of biological applications such as sensing of intracellular pH, cation/anion concentrations, oxygen levels, biomolecule contents, viscosity, and temperature, as well as live-cell FLIM. Studies based on direct FLIM analysis of endogenous molecules are not included in this review. With the rapid development of luminescence lifetime-based probes, more and more biological applications using FLIM techniques are being developed, and we review these recent developments here.

Database search strategy

The articles described in this narrative review were based on the design and application of fluorescence lifetime-based probes for sensing and imaging. An electronic search of the Web of Science database for literature describing FLIM-based probes from 2008 to 2020 was performed using the following conditions: FLIM (fluorescence lifetime imaging) and probe type or type of analytes to be tested. The results were further screened by title and abstract to only include synthetic probes, such as organic small molecules/polymers, metal−organic frameworks, and quantum dots. Non-FLIM applications and review articles were excluded. In addition, an electronic search of the Web of Science for cell imaging only was also completed. This included publications from 2014 to 2020, with the following search criteria: biological FLIM imaging, inducing organelle, cancer cells, tissue, and animals. Articles reporting FLIM imaging with endogenous molecules or traditional dyes were excluded.

Basic concepts in the design of luminescence lifetime-based probes

The luminescence lifetime is the time required for the energy of a population of excited fluorophores to decrease to 1/e of the maximum intensity via energy loss through luminescence (fluorescence or phosphorescence) and other nonradiative processes.[1] In general, the photoluminescence lifetime is mainly influenced by the internal molecular structure and conformation of the probe, and the external environment (eg, the presence of an energy acceptor for a nonradiative process), thus establishing the foundation of lifetime-based sensing. The design of probes for FLIM analysis is diverse because variations in emission wavelength and intensity can be ignored. Luminescence lifetime-based probes can be divided into two types: those based on chemical reactions and those based on photophysical changes without a fixed chemical structure. The typical strategy used in the design of these probes is shown in Figure 1. Though this strategy is also commonly used to design fluorescence intensity-based probes with changeable fluorescence spectra, the difference is obvious. Because the indistinguishable change in fluorescence spectra of FLIM probes before and after the addition of the analyte is dispensable for FLIM analysis.

Figure 1
Figure 1:
Typical models of luminescence lifetime-based probes based on (A) chemical reactions and (B) photophysical processes. MLCT = metal-to-ligand charge transfer, PET = photo-induced electron transfer, TADF = thermally activated delayed fluorescence.

Chemical reactions, such as protonation/deprotonation, oxidation, and metal–ligand complexation, between a probe and an analyte in a living cell are the most common factor affecting the photoluminescence lifetime of a probe. Such probes always contain a fluorescent group and a selective receptor for the analyte (eg, an ion or organic biomolecule). These probes undergo evident intramolecular photophysical processes, such as photo-induced electron transfer (PET), metal-to-ligand charge transfer (MLCT), and thermally activated delayed fluorescence (TADF), which affects their luminescence lifetime (Fig. 1A). For example, in Förster resonance energy transfer (FRET)-based probes, the lifetime of the energy donor declines as a result of nonradiative processes between donor and acceptor. The lifetime of the acceptor, which is also a receptor for the analyte, is enhanced after connection with the donor.[13] When the analyte is added, the FRET process is hindered as the receptor is consumed. Thus, the luminescence lifetime of the donor is recovered, and changes in lifetime can be measured for quantitative analysis of the analyte.

The second type of probe undergoes changes not in its molecular structure, but in its molecular conformation or intramolecular interactions. For example, when the excitation energy of a probe is lost internally via molecular vibrations and rotations,[14] or externally via energy transfer outside of the molecule,[15] its lifetime decreases. The concept of molecular rotation is the most efficient strategy in designing these probes (Fig. 1B). Components that can rotate (so-called molecular rotors) serve as sources of nonradiative relaxation, thus imparting probes with short photoluminescence lifetimes.[11] In viscous environments, the rotations are slowed, so both the luminescence intensity and lifetime of the probe are strongly enhanced. The enhancement of intramolecular interactions is another useful concept in the design of this type of luminescence lifetime-based probe. A typical probe can form an excimer in response to changes in the external environment. Excimers of some rigid aromatic compounds (eg, pyrene) formed when an excited molecule interacts with the same molecule in the ground state possess longer lifetimes than monomers.[16] In addition, organic molecules that exhibit FRET, aggregation-induced quenching, aggregation-induced emission, or other photophysical processes can be used in the design of such luminescence lifetime-based probes.

Sensing in live cells

pH sensing

Intracellular pH is a crucial parameter that is closely associated with cellular behaviors and pathological conditions. Accordingly, visualization of changes in intracellular pH by FLIM technology has attracted the interest of scientists.[17–20] Borisov et al[21] developed a phenolic-based small molecule for H+/OH sensing based on the regulation of an intramolecular PET process. PET is in the “ON” state in basic solutions, in which no fluorescence signal is emitted, but in the “OFF” state in acidic solutions, in which the fluorescence signal is recovered. PET-based probes also exhibit pH-dependent changes in lifetime,[22] which makes them suitable for studying two-dimensional pH distributions using FLIM techniques. Fluorescent proteins[23,24] and quantum dots[25,26] are good lifetime-based probes for pH. Probes that emit near-infrared (NIR) fluorescence and have long decay times can be used to generate signals that can be readily distinguished from intrinsic cell autofluorescence (<10 ns). Ruedas-Rama et al[25] developed the first quantum dot-based probes for pH sensing in cells using FLIM. The average photoluminescence lifetime of the dots varied from 8.7 ns (pH < 5) to 15.4 ns (pH > 8) with protonation and deprotonation of the carboxylic acid caps on the surface of dots (Fig. 2). Based on this strategy, Chen produced novel pH-responsive doped quantum dots with NIR emissions and long lifetimes (∼1 μs) by combining CuCl2, CdCl2, Zn(OAc)2, and L-glutathione.[26] These quantum dots showed excellent pH sensitivity and exhibited a linear response in the pH range from 5.5 to 7.0, which corresponds to changes in the fluorescence lifetime up to 600 ns. Recently, Ning et al[27] reported a wide-range pH-sensitive molecular NIR probe based on Yb3+ porphyrinate for FLIM. Results showed that this lanthanide complex is useful as a FLIM probe for interrogating deep tissue.

Figure 2
Figure 2:
Working mechanisms, FLIM images, and recovered lifetime histograms of quantum dots in the cytoplasm of CHO-k1 cells after incubation with nigericin in extracellular buffers with different pH values.[25] FLIM=fluorescence lifetime imaging microscopy. Reprinted with permission from reference [25]. Copyright (2013) American Chemical Society.

Cation and anion sensing

Sensing and imaging of intracellular concentrations of cations (eg, Ca2+, Mg2+, Zn2+, Na+, and K+) and anions (eg, Cl, ClO, and HCO3) are very important in medical and pharmaceutical research. Among these ions, Ca2+ is challenging to detect in biological systems using traditional intensity-based imaging, but FLIM technology provides a promising alternative.[28,29] One commercial Ca2+ indicator, Oregon Green BAPTA-1 (OGB-1) dye, has been widely used in Ca2+-distribution studies in cells.[30] This probe is insensitive to factors such as viscosity, temperature, and pH, which commonly affect other probes. Moreover, the fluorescence lifetime of this dye increases from 0.73 to 4 ns upon Ca2+ binding. A recent FLIM study showed that OGB-1 is sensitive to Ca2+ concentrations within the 10 to 500 nM range in rat brain slices.[31] In addition, our team designed Ca2+-selective nanospheres composed of Pluronic F127, (4-carboxybutyl) triphenylphosphonium bromide, and chromoionophore III for FLIM imaging of intracellular Ca2+ in both mitochondria and lysosomes.[32]

FLIM probes for other ions are also gradually being developed.[33–35] Iotti's group synthesized a diaza-18-crown-6 ethers-based probe for Mg2+ imaging in cells using FLIM.[36] When Mg2+ binds to the diaza-18-crown-6 ethers group, the lifetime of the probe decreases. FLIM maps showed that the amplitude term of the bi-exponential equation describing the fluorescence lifetime decay of this probe is related to the intracellular Mg2+ concentration. Tian et al[37] reported a novel probe, P–Zn, with a two-photon absorption cross-section (δ) of 516 ± 77GM that enables Zn2+ sensing in cells, which is meaningful for studying brain neurotransmission. Coordination of the probe with Zn2+ in cells increases the probe's lifetime, and this change can be detected by two-photon FLIM analysis (Fig. 3). Fluorescence lifetime-based probes for K+[38] and ClO[39] have also been reported. These studies demonstrate that the measurement of fluorescence lifetime can overcome the various challenges involved in fluorescence intensity-based imaging, such as instability of the light source and photobleaching effects.

Figure 3
Figure 3:
Working mechanisms and fluorescence lifetime imaging microscopy (FLIM) images of P–Zn probe in cells. (A) Structure of the P–Zn probe and corresponding metal complex.[37] (B) Fluorescence lifetime image of SHSY-5Y cells incubated with 5 μM P–Zn probe for 90 minutes and Zn2+ for 30 minutes at different concentrations (in μM): (a) 0, (b) 5, (c) 10, and (d) 25; (e–h) histograms of fluorescence lifetime in whole SHSY-5Y cells shown in a–d. (C) Representative lifetime decay curves and (D) fluorescence lifetimes from cytoplasm areas deemed region of interest (ROI) 1, ROI 2, ROI 3, and ROI 4 (delineated in a–d, respectively). Reprinted with permission from reference [37]. Copyright (2017) American Chemical Society.

Oxygen sensing

Oxygen (O2) is an essential molecule for cell growth and many vital physiological processes, including the production of adenosine triphosphate (ATP) and reactive oxygen species in mitochondria.[40] O2 effectively quenches TADF[41] and phosphorescence.[42] Xu et al[43] reported a phosphorescence/fluorescence dual-emissive nanoscale metal-organic framework (NMOF) nanoparticle, R-UiO, as an intracellular O2 sensor for live cells. The O2-sensitive ligand in this probe is a Pt-porphyrin complex with a long-lived triplet state at ambient temperature, allowing for sensing based on variations in both luminescence intensity and lifetime. In combination with Rhodamine-B, which is insensitive to O2 and, thus, functions as a reference probe, this probe can be used to detect O2 using a ratiometric method (Fig. 4). Yu et al[44] developed a similar nanosensor with iridium(III) complexes as active part and gold nanoclusters as an internal reference probe for O2. Papkovsky et al[45] created a similar ratiometric FLIM O2 nanosensor, which is comprised of a Pt-based complex (TFPP), fluorophore [poly(9,9-dioctylfluorene)] acting as a FRET donor, and two-photon antennae. This nanosensor can be used for ratiometric phosphorescence intensity- and lifetime-based sensing of O2 under one-photon or two-photon excitation. Reportedly, the iridium(III) complex is also useful for detection of reactive oxygen species (ROS), which is of great interest to researchers.[46] Although many studies have reported fluorescent intensity-dependent probes for ROS sensing,[47–49] FLIM probes for ROS are still lacking.

Figure 4
Figure 4:
Preparation and performance of the O2 probe, M-UiO NMOF.[43] (A) Synthesis of the mixed-ligand M-UiO NMOF and its post-synthesis modification from the R-UiO NMOF. (B) Emission spectra [excitation wavelength (λ ex): 514 nm] and (D) phosphorescent decays (λ ex = 405 nm) of R-UiO in Hank's balanced salt solution (HBSS) under various O2 partial pressures. Plots of R I 0 /R I (C) and τ 0 (E) as functions of the O2 partial pressure. NMOF = nanoscale metal-organic framework, RITC = Rhodamine-B isothiocyanate. Reprinted with permission from reference [43]. Copyright (2016) American Chemical Society.

Endogenous molecule sensing

Proteins, nucleic acids, and small organic molecules are attractive to researchers in many fields, including analytical chemistry and drug chemistry. Probes that interrogate changes in intermolecular interactions (such as PET, FRET, and MLCT) in the presence of analytes are ideal for FLIM analysis.[50–54] Yang's group developed a triarylboron-based Cu2+ complex with strong PET properties for H2S sensing using FLIM.[55] In addition, a non-fluorescent Zn2+-cyanine complex developed by Tian can be used for phosphorylated tau (p-tau) protein sensing and mapping of Zn2+ distribution at the single-cell level with FLIM imaging (Fig. 5).[56] Sun et al[57] reported fluorescence-quenched nanoflares with FRET properties with the ability to detect specific intracellular mRNA in cancer cells, which they used to conduct fluorescence lifetime-based imaging in living cells. Zhang's team achieved photoluminescence lifetime-based imaging of synthesized proteins in living cells using an iridium–alkyne complex with MLCT properties.[58] Later on, using the same concept, they synthesized an iridium–ligand complex as a photoluminescence lifetime-based probe for carboxylesterases in cells.[59] Furthermore, a variety of pure organic molecules with chemical bonds reactive to analytes are used in this field.[60,61]

Figure 5
Figure 5:
(A) Illustration of the selective and accurate detection of p-tau protein using a τ-p-tau probe.[56] (B) Image of a single neuron captured by time-correlated single photon counting FLIM with excitation at 600 nm. Neurons were prepared by incubation with 50 nM OA for 2, 4, and 6 hours, followed by loading with 5 μM τ-p-tau probe for imaging. Scale bar = 25 μm. (C) Lifetime decay curves and (D) average fluorescence lifetime in the selected areas (ROI 1−4 as denoted in a). Aβ = beta-amyloid peptide, ATP = adenosine triphosphate, OA = okadaic acid, p-tau = phosphorylated tau protein, ROI = region of interest. Reprinted with permission from reference [56]. Copyright (2019) American Chemical Society.

Physiological parameter sensing

Intracellular physiological parameters, including the local refractive index (RI), viscosity, temperature, membrane tension, and oxidative stress are also important for vital cellular activities. RIs in cells are chiefly influenced by local concentrations of macromolecules, mainly proteins and ribonucleoproteins. Our team demonstrated the measurement of intracellular RIs based on the fluorescence lifetime of histone H2B-GFP protein. FLIM images showed an apparent reduction in the fluorescence lifetime of H2B-GFP with increasing intracellular RI.[12] Colloidal quantum dots have also been demonstrated as useful lifetime-based probes for local RI measurements as the RI significantly impacts the fluorescence dynamics of these quantum-dot nano-emitters.[62]

Molecules with rotors are ideal viscosity sensors for fluorescence intensity- and lifetime-based sensors, as viscosity can impact the rate of rotations.[63] Kuimova et al[64] proposed the first dipyrromethene boron difluoride (BODIPY) rotor for FLIM analysis of viscosity in living cells. Confocal fluorescence images and FLIM images captured with BODIPY probes show cell viscosity with both spatial resolution and precision. This kind of electrically neutral BODIPY molecules can be easily endocytosed by cells and is only beneficial for the detection of viscosity inside cells. Later, this group reported a new BODIPY-based molecular rotor with double-positive charge located on its hydrocarbon tail. This tail prevents endocytosis of cells and retains the rotor function for FLIM mapping of viscosity in plasma membranes.[65] Based on these foundations, Guo et al[66] synthesized a new BODIPY-based molecule for ultrasensitive FLIM detection of intracellular viscosity (1.8–6.0cP) via a bond-energy transfer process. Xiao et al[67] and Yu et al[68] introduced organelle-targeting groups into the molecular skeleton of a BODIPY probe and reported their effectiveness in monitoring mitochondrial and lysosomal viscosity in combination with FLIM (Fig. 6). Recently, our team designed a new BODIPY-based probe for the quantification of local viscosity changes in the endoplasmic reticulum during intracellular autophagy.[69] With the development of FLIM technology and new probes, analysis of organelle membrane viscosity is becoming easier.[70–74]

Figure 6
Figure 6:
Working mechanisms and fluorescence lifetime imaging microscopy (FLIM) images of BODIPY probe in cells. (A) Working principle of the lysosomal viscosity probe Lyso-B and cell imaging.[68] Confocal (B, D) and FLIM (C, E) images of HeLa cells stained with 5 μM Lyso-B with (B, C) and without (D, E) coincubation with dexamethasone. (F) Lifetime histograms of images C and E; (G) Fluorescence lifetime-based spectral of images C and E. FLIM images were taken using fluorescence detection at 590 ± 15 nm after pulsed excitation at 561 nm. PET = photo-induced electron transfer. Reprinted with permission from reference [68]. Copyright (2018) American Chemical Society.

In contrast to organic probes, Hao et al[75] and Anh et al[76] developed novel iridium(III) and terbium(III) complexes with rotatable groups and long-lifetime emission as viscosity probes for FLIM. The lifetimes of some metal complexes and molecular rotors are also sensitive to temperature. Zhang et al[77] synthesized a novel polymer composed of iridium(III) complexes as temperature-sensitive units and europium(III) complexes as reference units. Sensitive and reversible temperature-induced changes have been used to conduct self-calibrating ratiometric imaging and lifetime-based imaging in HeLa cells and zebrafish.

Membrane tension is a significant factor regulating cell processes such as motility, endocytosis, and cell division, but it is hard to measure in real-time. Thus, the visualization of membrane tension in live cells using FLIM probes is meaningful and challenging. Matile's group is one of the few groups working on this topic.[78] They designed a planarizable push–pull fluorescent probe, called FliptR, for the detection of cell membrane tension.[79] The fluorescence properties of this probe change in response to lipid-packing changes, which makes it an ideal membrane-tension probe. More recently, the team modified this probe to allow for selective labeling of intracellular organelles, such as lysosomes, mitochondria, and the endoplasmic reticulum, enabling sensitive and specific detection of changes in the membrane tensions of specific organelles.[80]

An imbalance of cellular redox status, commonly called oxidative stress, is closely related to the development of neurodegenerative diseases. FLIM is a powerful tool for imaging of the oxidative stress state.[81] In addition to the endogenous molecule (eg, NADH and NADPH), synthetic small molecules sensitive to endogenous ROS are used as FLIM probes. For example, two novel iridium(III)-nitroxide conjugates have been synthesized by Jiang for imaging of mitochondrial oxidative stress.[82] Cyclic nitroxides with bulky substituents (NO) in the probe can be translated to diamagnetic non-radical species (NOR, R = H, CH3, etc.) in response to cellular oxidative stress. These conjugates display enhanced phosphorescence intensity and elongated phosphorescence lifetimes in A549 cells following pre-treatment of cells with ROS inducers, offering a visual image of cellular oxidative stress states.

Cell imaging

In addition to sensing, optical probes can also be used to label cells or specific organelles. However, contemporary FLIM is fundamentally limited by the nanosecond-scale lifetimes of available probes. Interference arises mainly from the autofluorescence of endogenous biomolecules, which exhibit visible-light emission like most exogenous labels and have lifetimes of several nanoseconds. Hence, fluorophores with long-lifetime fluorescence emission are highly desirable to reduce the interference of background fluorescence from molecules with lifetimes shorter than 10 ns.[83] To the best of our knowledge, there are few studies on single FLIM imaging. Instead, FLIM imaging-involved multi-mode imaging is one of the main streams of biological research.

Fluorophores with thermally activated delayed fluorescence (TADF) display long fluorescence lifetimes, which is useful for eliminating background signals associated with FLIM images. Li et al[84] developed smart organic dots with a TADF molecule, CPy, for both time-resolved fluorescence imaging and FLIM in vivo. These dots also show aggregation-induced emission (AIE) characteristics, which make them brighter than common hydrophobic dyes in FLIM images. Yang's team is also interested in developing TADF molecules for luminescence lifetime-based imaging of living cells.[85] PXZT, a novel TADF fluorophore with terpyridine as the acceptor and phenoxazine as the donor, was rationally synthesized and used in FLIM and time-gated imaging of HeLa cells. In addition to TADF dyes, transition-metal complexes with long-lifetime phosphorescence would also be suitable probes for FLIM with living cells.[86]

Probes exhibiting fluorescence in the NIR spectral region are attractive because they enable deep bioimaging in vivo. Recently, aggregation-induced emission dots with NIR emission have been used in FLIM imaging. Yu et al[87] prepared novel AIE-NIR dots with an emission peak at 975 nm and large tail extending beyond 1000 nm. TB1, the fluorophore in the dots, comprises an electron donor (tetraphenylethylene or triphenylamine) and electron acceptor (benzothiadiazole derivative). The strong intramolecular electron push-pull effect reduces the molecular band gap, thus yielding a fluorescence emission wavelength in the infrared region. These dots were used as near-infrared-II (NIR-II) fluorescent probes for FLIM imaging of cerebral vessels in mice, which is the first example of in vivo NIR-II FLIM imaging.


FLIM, a technique that provides spatially resolved images based on the distribution of fluorophore lifetime, has been applied to a series of biological applications in live cells, such as identifying analytes (eg, NADH and NADPH) as well as dynamic intracellular processes (eg, FRET-based protein interactions). Optical probes (including dyes, metal complexes, fluorescent nanoparticles, and fluorescent proteins) for luminescence lifetime-based analysis have garnered significant attention with the rapid development of FLIM techniques. In this review, we focused on the main design concepts for luminescence lifetime-based probes for biological applications, including sensing of intracellular pH, cation/anion concentrations, oxygen levels, organic biomolecule contents, and physiological parameters, as well as live-cell imaging. Biological imaging with endogenous organic molecules, one advantage of FLIM technology, is not included in this mini review. However, probes used only for live-cell imaging are briefly described in this review. Notably, luminescent molecules with typical photophysical effects (eg, PET, aggregation-induced emission, TADF, FRET, and MLCT) are desirable for designing analyte-reactive probes for FLIM. In addition, probes exhibiting longer luminescent lifetimes or absorption/emission at longer wavelengths are in high demand to minimize the interference of background fluorescence for in vivo FLIM.

In theory, the probe for FLIM analysis is not affected by the intensity and range of fluorescence wavelength of the probe. In addition, to get a FLIM image, the lifetime signal from the probe in a cell must be strong enough. Thus, the probes for FLIM should have good water solubility, fluorescent quantum efficiency, and NIR emission. Despite the variety of FLIM probes available, there is a lack of probes sensitive to membrane tension, so measuring forces inside cells remains particularly challenging. In the future, probes with rotor structures are expected to play an important role in observing vital processes involving biological membranes.



Author contributions

XP and WY contributed to the writing of the review, and provided the writing guidance and manuscript revision. KN contributed to the writing of the first draft. JS and JQ provided valuable suggestions to manuscript revisions. All authors reviewed and approved the final manuscript.

Financial support

This work was supported by the National Key R&D Program of China (No. 2018YFC0910602), the National Natural Science Foundation of China (Nos. 31771584, 61975127, 61775145, 61525503, 61620106016, and 61835009), National Science Foundation for Postdoctoral Scientists of China (No. 2019M663032), China Postdoctoral Science Foundation (No. 2019M663032), Project of Department of Education of Guangdong Province of China (No. 2016KCXTD007), Shenzhen Basic Research Project (No. JCYJ20170818100153423), and Science Foundation of Shenzhen University of China (No. 2017000193).

Conflicts of interest

The authors declare that they have no conflicts of interest.


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biological application; fluorescence lifetime imaging microscopy; luminescence lifetime; luminescence probe; sensing and imaging

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