Time-gated Luminescence Imaging Research Articles

Novel Endoplasmic Reticulum-Targeted Luminescent Probe for Visualization of Carbon Monoxide in Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a major hepatic dysfunction commonly caused by hepatotoxic drug overdose, resulting in a considerable number of fatalities worldwide. Recent studies have highlighted the regulatory and hepatoprotective effects of carbon monoxide (CO) during the liver injury process. However, precisely tracking the dynamic changes in the composition of CO in DILI is still a great challenge. In this work, leveraging the innovative "quencher-insertion" strategy, a unique endoplasmic reticulum (ER)-targetable lanthanide complex-based luminescence probe, ER-ANBTTA-Eu3+/Tb3+, has been developed for the selective and accurate monitoring of CO fluxes in live cells and laboratory animals. The new probe is composed of three covalently linked functional moieties: the terpyridine polyacid-Eu3+/Tb3+-mixed chelates as the long-lived luminophore, a p-toluenesulfonamide moiety as the ER-anchoring motif, and an allyloxy-nitrobenzyl ether moiety as the CO-specific recognition unit. Upon reaction with CO in the presence of Pd2+ ions, the Tsuji-Trost reaction leads to the cleavage of the allyloxy-nitrobenzyl group from the Eu3+/Tb3+-mixed chelates, which results in the restoration of Tb3+ emission at 538 nm and the attenuation of Eu3+ emission at 688 nm, leading to a dramatic increase of the I538/I688 ratio. In addition to the exceptional response sensitivity and selectivity toward CO, ER-ANBTTA-Eu3+/Tb3+ also exhibits the outstanding ER-locating capability, which allows the probe to be used for imaging of CO in the ER of live cells. Using this probe, combined with the time-gated luminescence imaging mode, the exogenous and endogenous CO in ER of live cells were monitored without the interference of background autofluorescence. Moreover, the upregulation of hepatic CO in DILI mice was successfully visualized. The results suggested the potential of ER-ANBTTA-Eu3+/Tb3+ for deeply exploring the functions of CO in DILI pathogenesis.

Responsive β-Diketonate-europium(III) Complex-Based Probe for Time-Gated Luminescence Detection and Imaging of Hydrogen Sulfide In Vitro and In Vivo.

As a crucial biological gasotransmitter, hydrogen sulfide (H2S) plays important roles in many pathological and physiological processes. Highly selective and sensitive detection of H2S is significant for the precise diagnosis and evaluation of diverse diseases. Nevertheless, challenges remain in view of the interference of autofluorescence in organisms and the stronger reactivity of H2S itself. Herein, we report the design and synthesis of a novel H2S-responsive β-diketonate-europium(III) complex-based probe, [Eu(DNB-Npketo)3(terpy)], for background-free time-gated luminescence (TGL) detection and imaging of H2S in autofluorescence-rich biological samples. The probe, consisting of a 2,4-dinitrobenzenesulfonyl (DNB) group coupled to a β-diketonate-europium(III) complex, shows almost no luminescence owing to the existence of intramolecular photoinduced electron transfer. The cleavage of the DNB group by a H2S-triggered reaction results in the recovery of the long-lived luminescence of the Eu3+ complex, allowing the detection of H2S in complicated biological samples to be performed in TGL mode. The probe showed a fast response, high specificity, and high sensitivity toward H2S, which enabled it to be successfully used for the quantitative TGL detection of H2S in tissue homogenates of mouse organs. Additionally, the low cytotoxicity of the probe allowed it to be further used for the TGL imaging of H2S in living cells and mice under different stimuli. All of the results suggested the potential of the probe for the investigation and diagnosis of H2S-related diseases.

Multiplexed MicroRNA biomarker detection by bridging lifetime filtering imaging and dynamic chemical labeling

MicroRNAs (miRs) have emerged as promising biomarkers for early disease diagnosis and personalized treatment monitoring. However, their clinical utility has been hampered by technical limitations. Dynamic chemical labeling (DCL) based on capturing abasic PNA probes and reactive nucleobases, known as SMART bases, is a PCR-free approach that has proven very useful for the direct interrogation of circulating miRs. In this work, we expand the palette of tools available for the detection of DCL miR by synthesizing a new SMART nucleobase called SMART-C-Eu. This nucleobase contains a stable lanthanide cryptate. Using this SMART-C-Eu base and time-gated (TG) luminescence imaging, we successfully detect and quantify miR-122–5p in human serum samples. miR-122–5p is a well-known biomarker for drug-induced liver injury. Through a bead-counting analysis approach, statistical robustness is improved and miR-122–5p concentrations are detected in the nanomolar range. Furthermore, we extend this approach to multiplexed detection of three different miRs (miR-371a-3p, miR-451a-5p, and miR-122–5p) using spectral and temporal filtering. Importantly, we designed a user-independent multiplexed analysis using machine learning algorithms for automatic bead classification. Although the sensitivity of this technique must be further improved to detect miRs at lower concentrations, the method represents a significant advancement in miR analysis by combining ML segmentation using lifetime and intensity images. In addition, the technique offers multiplexing capabilities and the potential for automation, paving the way for more accurate and robust clinical applications in the future.

A bimodal time-gated luminescence-magnetic resonance imaging nanoprobe based on a europium(III) complex anchored on BSA-coated MnO2 nanosheets for highly selective detection of H2O2.

A novel nanocomposite, [Eu(BTD)3(DPBT)]-BSA@MnO2, is reported to serve as an effective nanoprobe for bimodal time-gated luminescence (TGL) and magnetic resonance (MR) imaging of H2O2in vitro and in vivo. The nanoprobe was fabricated by immobilizing visible-light-excitable Eu3+ complexes in bovine serum albumin (BSA)-coated lamellar MnO2 nanosheets. The TGL of the Eu3+ complex was effectively quenched by the MnO2 nanosheets. Upon exposure to H2O2, the MnO2 nanosheets underwent reduction to Mn2+, which simultaneously triggered rapid, selective and sensitive "turn-on" responses toward H2O2 in both TGL and MR detection modes. The presence of a protective "corona" formed by BSA enables the nanoprobe to withstand high concentrations of glutathione (GSH), a strong reducing agent of MnO2 nanosheets. This capability allows the nanoprobe to be utilized for detecting H2O2 in living biosamples. The combined utilization of TGL and MR detection modes enables the nanoprobe to image H2O2 across a wide range of resolutions, from the subcellular level to the whole body, without any depth limitations. The results obtained from these modes can be cross-validated, enhancing the accuracy of the detection. The capability of the nanoprobe was validated by TGL imaging of endogenous and exogenous H2O2 in live HeLa cells, as well as bimodal TGL-MR imaging of H2O2 in tumor-bearing mice. The research achievements suggest that the integration of luminescent lanthanide complexes with protein-coated MnO2 nanosheets offers a promising bimodal TGL-MR sensing platform for H2O2in vitro and in vivo.

Mitochondria-Targetable Ratiometric Time-Gated Luminescence Probe Activated by Selenocysteine for the Visual Monitoring of Liver Injuries.

Liver injury can result from various risk factors including diabetes, virus, alcohol, drugs, and other toxins, which is mainly responsible for global mortality and morbidity. Selenocysteine (Sec), as the main undertaker of selenium function in the life system, features prominently in a series of hepatic injuries and has close association with the pathological progression of liver injuries. Here, we report a mitochondria-targetable lanthanide complex-based probe, Mito-NPTTA-Tb3+/Eu3+, that can be used for accurately determining Sec in live cells and laboratory animals via the ratiometric time-gated luminescence (TGL) technique. This probe is composed of 2,2':6',2″-terpyridine-Tb3+/Eu3+ mixed complexes as the luminophore, 2,4-dinitrophenyl (DNP) as the responsive moiety and a lipophilic triphenylphosphonium cation (PPh3+) as the mitochondria-targeting moiety. Upon reaction with Sec, accompanied by the cleavage of DNP from the probe molecule, the I540/I690 ratio of the probe increased by 55 times, which enabled Sec to be detected with the ratiometric TGL method. After being incubated with living cells, the probe molecules were selectively accumulated in mitochondria to allow the mitochondrial Sec to be successfully imaged under the ratiometric TGL mode. Importantly, using this probe coupled with the ratiometric TGL imaging technique, the fluctuations of liver Sec in various liver injuries of model mice induced by diabetes, drug, toxin, and alcohol were precisely monitored, revealing that Sec plays an important antioxidant role during the oxidative stress process in liver injury, and the Sec levels have a close interrelationship with the degree of liver injury. All the results suggest that the new probe Mito-NPTTA-Tb3+/Eu3+ could be a potential tool for the accurate diagnosis of liver injury.

A tumor-targetable probe based on europium(III)/gadolinium(III) complex-conjugated transferrin for dual-modal time-gated luminescence and magnetic resonance imaging of cancerous cells in vitro and in vivo.

The synergy of magnetic resonance imaging (MRI) and time-gated luminescence imaging (TGLI) provides a robust platform with extensive spatial resolution (from submicrometer to hundred-micron) and unlimited penetration depth for visual detection of lesion tissues and target biomolecules. In this work, highly stable lanthanide (Eu3+ and Gd3+) complexes with a terpyridine polyacid ligand, CNSTTA-Ln3+, were chosen as signal reporters for TGLI (Ln3+ = Eu3+) and MRI (Ln3+ = Gd3+), respectively. After conjugating CNSTTA-Ln3+ with a tumor-targetable glycoprotein, transferrin (Tf), the obtained bioconjugate, showed low cytotoxicity and high stability and exhibited strong long-lived luminescence (Tf-CNSTTA-Eu3+, ϕ = 10.8%, τ = 1.27 ms), high magnetic resonance relaxivity (Tf-CNSTTA-Gd3+, r1 = 8.70 mM-1 s-1, r2 = 10.90 mM-1 s-1), and high binding affinity toward Tf receptor-overexpressed cancerous cells. On the basis of these features, a tumor-targetable probe was constructed by simply mixing Tf-CNSTTA-Eu3+ and Tf-CNSTTA-Gd3+, and successfully used for the bimodal TGLI and MRI of tumor cells in tumor-bearing mice. The bimodal imaging simultaneously provided the anatomical and molecular information of the tumor, which enabled the accuracy for tumor diagnosis to be mutually verified, and revealed the potential of Tf-CNSTTA-Gd3+/Eu3+ for the monitoring of cancer cells in vivo.

An activatable nanoprobe based on nanocomposites of visible-light-excitable europium(III) complex-anchored MnO2 nanosheets for bimodal time-gated luminescence and magnetic resonance imaging of tumor cells.

Bimodal imaging probes that combine magnetic resonance imaging (MRI) and photoluminescence imaging are quite appealing since they can supply both anatomical and molecular information to effectively ameliorate the accuracy of detection. In this study, an activatable nanoprobe, [Eu(BTD)3(DPBT)]@MnO2, for bimodal time-gated luminescence imaging (TGLI) and MRI has been constructed by anchoring visible-light-excitable Eu3+ complexes on lamellar MnO2 nanosheets. Due to the luminescence quenching effect and non-magnetic resonance (MR) activity of MnO2 nanosheets, the developed nanoprobe presents quite weak TGL and MR signals. After exposure to H2O2 or GSH, accompanied by the transformation from MnO2 to Mn2+, the nanoprobe exhibits rapid, sensitive, and selective "turn-on" responses towards GSH and H2O2 in TGL and MR detection modes. Furthermore, the nanoprobe displays high stability, low cytotoxicity, good biocompatibility and water dispersion. Given the high contents of GSH and H2O2 in cancer cells, the nanoprobe was used for the identification of cancer cells by TGLI of intracellular GSH and H2O2, as well as for the tracing of tumor cells in tumor-bearing mice by tumor-targeting in vivo MRI and TGLI of tumor tissues. The research outcomes proved the potential of [Eu(BTD)3(DPBT)]@MnO2 as a useful nanoprobe for the tracing and accurate detection of cancer cells in vitro and in vivo via bimodal TGLI and MRI.

Ruthenium(II) complex-based long-lived two-photon luminescence probe for dynamic monitoring of glutathione S-transferases in mouse models of drug-induced liver injury

We report a novel ruthenium(II) complex-based long-lived and two-photon luminescence probe, Ru-NO2, for dynamic monitoring of GSTs fluxes in the process of drug-induced liver injury (DILI). Ru-NO2 shows weak luminescence due to the strong photo-induced electron transfer (PET) process from the Ru(II) center to the electron-withdrawing 3,4-dinitrobenzene (DNB) group. As a result of the specific nucleophilic aromatic substitution triggered by GSTs in the presence of reduced glutathione (GSH), the 4-nitro group of Ru-NO2 is substituted by GSH to form strongly emissive Ru-SG. Ru-NO2 shows high selectivity and sensitivity for responding to GSTs. Notably, one- and two-photon luminescence imaging experiments were conducted to visualize GSTs in living cells and mouse tissues. In addition, Ru-NO2 was applied for background-free time-gated luminescence (TGL) detection and luminescence imaging analysis of GSTs in DILI mouse sera and liver tissues, which showed that GSTs level in sera significantly increased accompanied by the decrease in liver in the DILI process, revealing the possible potential of the probe as an effective tool for clinical diagnosis and treatment monitoring of DILI.

A multifunctional nanoprobe based on europium(iii) complex–Fe3O4 nanoparticles for bimodal time-gated luminescence/magnetic resonance imaging of cancer cells in vitro and in vivo

A multifunctional nanoprobe for tumor-targeting time-gated luminescence and magnetic resonance imaging in vitro and in vivo.

A miniaturized apparatus based on a smartphone for microsecond-resolved luminescence lifetime imaging

Time-resolved luminescence imaging is usually operated on Lab instruments equipped with high-speed excitation sources and detectors, which are always bulky and expensive. In order to miniaturize the instrument and reduce the cost, a FPGA (field programmable gate array) -based digital circuit was designed to control a mechanical chopper and a UV-LED for time-gated luminescence imaging. By utilizing a smartphone as a camera, the luminescence lifetime imaging of mouse kidney sections dyed by two europium complexes was successfully accomplished, demonstrating a temporal resolution of tens of microseconds. It is the first time that microsecond-resolved luminescence lifetime imaging was achieved with a smartphone. We believe this miniaturized apparatus can provide many opportunities for time-resolved luminescence detection and imaging for eliminating the background signals from scattering and short-lived autofluorescence.

Bioconjugates of versatile β-diketonate-lanthanide complexes as probes for time-gated luminescence and magnetic resonance imaging of cancer cells in vitro and in vivo.

Magnetic resonance imaging (MRI) and optical imaging (OI) are attractive for constructing bimodal probes due to their complementary imaging characteristics. The combination of these two techniques could be a useful tool to simultaneously obtain both anatomical and molecular information as well as to significantly improve the accuracy of detection. In this study, we found that β-diketonate-lanthanide complexes, BHHBCB-Ln3+, could covalently bind to proteins to exhibit long-lived and intense luminescence (Ln3+ = Eu3+, τ = 0.52 ms, Φ = 0.40) and remarkably high relaxivity (Ln3+ = Gd3+, r1 = 35.67 mM-1 s-1, r2 = 43.25 mM-1 s-1) with excellent water solubility, stability and biocompatibility. Hence, we conjugated BHHBCB-Ln3+ with a tumor-targetable biomacromolecule, transferrin (Tf), to construct the probes, Tf-BHHBCB-Ln3+, for time-gated luminescence (TGL, Ln3+ = Eu3+) and MR (Ln3+ = Gd3+) imaging of cancerous cells in vitro and in vivo. As expected, the as-prepared probes showed high specificity to bind with the transferrin receptor-overexpressed cancerous cells, to enable the probe molecules to be accumulated in these cells. Using Tf-BHHBCB-Ln3+ as probes, the cultured cancerous cells and the tumors in tumor-bearing mice have been clearly visualized by background-free TGL and in vivo MR imaging. The research outcomes suggested the potential of β-diketonate-lanthanide complexes for use in constructing bimodal TGL/MR imaging bioprobes.

Lanthanide-based resonance energy transfer biosensors for live-cell applications.

Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enable sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living mammalian cells. LRET biosensors are polypeptides that consist of an alpha-helical linker sequence sandwiched between a lanthanide complex-binding domain and a fluorescent protein (FP) with two interacting domains residing at each terminus. Interaction between the terminal affinity domains brings the lanthanide complex and FP in close proximity such that lanthanide-to-FP, LRET-sensitized emission is increased. A recent proof-of-concept study examined model biosensors that incorporated the affinity partners FKBP12 and the rapamycin-binding domain of m-Tor (FRB) as well as p53 (1-92) and HDM2 (1-128). The sensors contained an Escherichia coli dihydrofolate reductase (eDHFR) domain that binds with high selectivity and affinity to Tb(III) complexes coupled to the ligand trimethoprim (TMP). When cell lines that stably expressed the sensors were treated with TMP-Tb(III), TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. Much larger signal changes (>2500%) and Z'-factors of 0.5 or more were observed when cells were grown in 96-well or 384-well plates and analyzed using a TGL plate reader. In this chapter, we elaborate on the design and performance of LRET biosensors and provide detailed protocols to guide their use for live-cell microscopic imaging studies and high-throughput library screening.

Time-gated luminescence imaging of positively charged poly-l-lysine-coated highly microporous silicon nanoparticles in living Hydra polyp.

The development of non-toxic fluorescent agents alternative to heavy metal-based semiconductor quantum dots represents a relevant topic in biomedical research and in particular in the bioimaging field. Herein, highly luminescent Si─H terminal microporous silicon nanoparticles with μs-lived photoemission are chemically modified with a two step process and successfully used as label-free probes for in vivo time-gated luminescence imaging. In this context, Hydra vulgaris is used as model organism for in vivo study and validity assessment. The application of time gating allows to pursue an effective sorting of the signals, getting rid of the most common sources of noise that are fast-decay tissue autofluorescence and excitation scattering within the tissue. Indeed, an enhancement by a factor ~ 20 in the image signal-to-noise ratio can be estimated.

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

SummaryLanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

Fluorescence Lifetime-Tunable Water-Resistant Perovskite Quantum Dots for Multidimensional Encryption

Using temporal dimension in optical multiplexing is a promising method to increase the security of data encryption. However, adjusting the fluorescence lifetime of light-emitting material often results in inevitable changes in their fluorescence spectra, which is unfavorable for confidential information protection. Here, we report the preparation of various perovskite quantum dot/polymer nanospheres (PQD/polymer) with tunable and long fluorescence lifetimes but identical fluorescence spectra, which are ideal multidimensional data encryption materials. This new data encryption strategy utilizes the water sensitivity of perovskite and achieves spatial dimension encryption of information using different water stabilities between uncoated perovskite quantum dots and PQD/polymer. The fluorescence lifetime of PQD/polymer is used as the coding element to achieve temporal dimension data encryption, and the data are decrypted by fluorescence lifetime imaging microscopy and time-gated luminescence imaging techniques. This study shows the potential of PQD/polymer as a new class of materials for advanced data encryption.

Development of a lysosome-targetable visible-light-excited europium(III) complex-based luminescent probe to image hypochlorous acid in living cells

Lanthanide complex-based luminescent probes have shown great utilities in detecting various analytes by time-gated luminescence imaging technology. However, few lanthanide probes are suitable for the monitoring of bioactive species in organelles. Furthermore, the common UV excitation windows of most lanthanide probes limit their application in living biological samples. In this work, a novel visible-light-excited europium(III) complex-based luminescent probe, [Eu(Lyso-CDHH)3(DPBT)], was designed and synthesized for the detection of exogenous and endogenous hypochlorous acid (HOCl) in lysosomes. The probe exhibits a wide excitation window from ultraviolet to visible region with a maximum excitation wavelength at 400 nm and a high luminescence quantum yield (41.7%). Upon reaction with HOCl, the luminescence of the probe can be quenched within 5 s and displays strong resistance to the interferences from other reactive species. The designed probe also shows high sensitivity towards HOCl with a low detection limit at micromolar concentration level. In addition, the good cell-membrane permeability and the lysosome-targetability of the probe enabled its successful application in the time-gated luminescence imaging of exogenous and endogenous HOCl in lysosomes of RAW 264.7 cells. This work will shed lights on the design of visible-light-excited and organelle-targetable lanthanide molecular probes for broader time-gated luminescence application.

Separation of Natural Laboratory-Grown Diamond Using Time-Gated Luminescence Imaging

A technique that expands on the surface luminescence imaging used in the DiamondView instrument has been developed at De Beers Group Technology, Maidenhead, UK. This provides an additional level of imaging information by way of separating prompt and delayed surface luminescence. The technique has the added benefit of quickly and easily distinguishing colorless or near-colorless natural diamond from laboratory-grown diamond. It can be applied when the identification of natural diamond is required in the study of single stones, multiples in batches, set jewelry, or in a fully automated process. The prompt and delayed luminescence characteristics of natural diamond are compared with a range of chemical vapor deposition (CVD) and high-pressure, high-temperature (HPHT) synthetic diamonds. Of significant interest are some of the less common CVD synthetic samples that have been observed in recent years. This article will summarize the luminescence observed in different diamond types, discuss its spectral characteristics, and serve as a useful reference when interpreting such luminescence images.

Time-gated luminescence imaging for background free in vivo tracking of single circulating tumor cells.

Fluorescence imaging is severely limited by the background and autofluorescence of tissues for in vivo detection of circulating tumor cells (CTCs). Time-gated luminescence (TGL) imaging, in combination with luminescent probes that possess hundreds of microsecond emission lifetimes, can be used to effectively suppress this background, which has predominantly nanosecond lifetimes. This Letter demonstrates the feasibility of TGL imaging using luminescent probes for the in vivo real time imaging and tracking of single CTCs circulating freely in the blood vessels with higher accuracy given by substantially higher signal-to-noise ratio. The luminescent probe used in this Letter was a commercial Eu3+ chelate (EuC) nanosphere with a super-long lifetime of near 800 µs, which enabled TGL imaging to achieve background-free detection with ∼5 times higher SNR versus steady state. Phantom and in vivo mouse studies indicated that EuC labeled tumor cells moving in medium or bloodstream at the speed of 1-2 mm/s could be captured in real time.

Auto-Phase-Locked Time-Resolved Luminescence Detection: Principles, Applications, and Prospects.

Time-resolved luminescence measurement is a useful technique which can eliminate the background signals from scattering and short-lived autofluorescence. However, the relative instruments always require pulsed excitation sources and high-speed detectors. Moreover, the excitation and detecting shutter should be precisely synchronized by electronic phase matching circuitry, leading to expensiveness and high-complexity. To make time-resolved luminescence instruments simple and cheap, the automatic synchronization method was developed by using a mechanical chopper acted as both of the pulse generator and detection shutter. Therefore, the excitation and detection can be synchronized and locked automatically as the optical paths fixed. In this paper, we first introduced the time-resolved luminescence measurements and review the progress and current state of this field. Then, we discussed low-cost time-resolved techniques, especially chopper-based time-resolved luminescence detections. After that, we focused on auto-phase-locked method and some of its meaningful applications, such as time-gated luminescence imaging, spectrometer, and luminescence lifetime detection. Finally, we concluded with a brief outlook for auto-phase-locked time-resolved luminescence detection systems.

Responsive Metal Complex Probes for Time-Gated Luminescence Biosensing and Imaging.

The development of reliable bioanalytical probes for selective and sensitive detection of particular analytes in biological systems is essential for better understanding the roles of the analytes in their native contexts. In the last two decades, luminescent metal complexes have greatly contributed to the development of such probes for biosensing and imaging due to their unique spectral and temporal properties, controllable cell membrane permeability, and cytotoxicity. Conjugating an analyte-activatable moiety to the metal complex luminophores allows the production of responsive metal complex probes for this analyte detection. Owing to their long-lifetime emissions, the responsive metal complex probes are accessible to the technique of time-gated luminescence (TGL) detection and imaging. With a delay time after pulsed excitation, the TGL technique allows for collection of only long-lived luminescence from responsive metal complex probes, while filtering out short-lived background autofluorescence, providing a background-free approach for the detection and imaging of the analyte at subcellular and/or molecular levels. Responsive metal complex probes, therefore, have emerged as complementary sensing and imaging tools of organic dye-based fluorescent probes for the in situ detection of analytes in complicated biological environments.In this Account, we describe the advances in the development of metal complex probes and their applications for TGL bioassays with particular focus on our efforts made in this field. We first introduce the photophysical/-chemical properties of luminescent metal complexes, including lanthanide (europium and terbium) and transition metal (ruthenium and iridium) complexes. The luminescence lifetimes (τ) of lanthanide and transition metal complexes are at micro/millisecond (μs/ms) and hundreds/thousands nanosecond (ns) levels, respectively. The emission lifetimes are significantly longer than the autofluorescence lifetime (τ < 10 ns) of biological samples. Such long-lived luminescence of these metal complexes enables our research on demonstrating responsive probes for background-free TGL detection of some reactive biomolecules, such as reactive oxygen/nitrogen species (ROS/RNS) and biothiols.We conclude this Account by outlining the future directions to further develop new generation responsive TGL probes for promoting their practical applications. The responsive TGL probes are expected to be translated for biomedical and/or (pre)clinical investigations of biomolecules in situ. Reversibility, lower toxicity, ability of excitation at longer wavelength, and potential to be translated are key criteria for the development of next-generation probes. We also anticipate that further development of responsive TGL probes will contribute to the bioassay in more challenging biological systems, such as plants that have significant higher background autofluorescence than animals.