Time-resolved Luminescence Imaging Research Articles

Ultralong luminescence lifetime imaging of edible plant tissue for humidity sensing in food packaging by a smartphone

The safety of luminescence sensors and probes used in food packaging should be seriously considered, while most luminescence sensors were artificially synthesized with unclear toxicity, and cannot be directly used as indicators that were in contact with food. To overcome this problem, a humidity indicator based on an edible plant tissue was developed without any chemical processing. We found that garlic bulbs could emit significant persistent luminescence after drying at room temperature. The luminescence lifetime decreases from hundreds of milliseconds to tens of milliseconds as humidity increases. The long-lived luminescence could easily be detected through smartphones without any sophisticated instruments. The edible garlic is expected to be used as a humidity indicator in food packaging without worrying about food safety. Furthermore, the interference of scattered light and short-lived fluorescence from foods and packages can be eliminated in time-resolved luminescence imaging, greatly increasing the signal-to-noise ratio.

Cell-penetrating peptides noncovalently modified red phosphorescent nanoparticles for high-efficiency imaging.

The application of long-lived phosphorescence probes in time-resolved luminescence imaging is limited by their low quantum yield in aqueous solutions. However, sensitization of thermally activated delayed fluorescence (TADF) materials can compensate for this limitation while addressing the issue of insufficient proportion of their own long lifetime. In this study, we utilized the characteristics of phosphorescence and TADF materials simultaneously by doping the receptor iridium complex PMD-Ir into the donor TADF polymer PCzDP-20 through donor-receptor doping method, and successfully prepared highly efficient red phosphorescent nanoparticles. The quantum yield of the nanoparticles obtained by this method reaches up to 30%, and the luminescence lifetime can reach several thousand nanoseconds. Additionally, due to the low concentration doping of PMD-Ir, the risk of transition metal toxicity is greatly reduced. Furthermore, we used non-covalent modification with amphiphilic cell-penetrating peptides (CPPs) to increase the cell membrane permeability of the nanoparticles. The CPPs modified nanoparticles achieve in vivo confocal imaging of zebrafish and intracellular time-resolved imaging by its significantly improved bioimaging capabilities. The functional nanoparticles designing method fully utilizes the characteristics of PMD-Ir, PCzDP-20, and CPPs, solving the problems of low quantum yield and poor membrane permeability of Ir-complex nanoparticles. This will greatly promote the development of time-resolved luminescence imaging.

Fused-Ring Pyrrole-Based Near-Infrared Emissive Organic RTP Material for Persistent Afterglow Bioimaging.

Organic near-infrared room temperature phosphorescence (RTP) materials offer remarkable advantages in bioimaging due to their characteristic time scales and background noise elimination. However, developing near-infrared RTP materials for deep tissue imaging still faces challenges since the small band gap may increase the non-radiative decay, resulting in weak emission and short phosphorescence lifetime. In this study, fused-ring pyrrole-based structures were employed as the guest molecules for the construction of long wavelength emissive RTP materials. Compared to the decrease of the singlet energy level, the triplet energy level showed a more effectively decrease with the increase of the conjugation of the substituent groups. Moreover, the sufficient conjugation of fused ring structures in the guest molecule suppresses the non-radiative decay of triplet excitons. Therefore, a near-infrared RTP material (764 nm) was achieved for deep penetration bioimaging. Tumor cell membrane is used to coat RTP nanoparticles (NPs) to avoid decreasing the RTP performance compared to traditional coating by amphiphilic surfactants. RTP NPs with tumor-targeting properties show favorable phosphorescent properties, superior stability, and excellent biocompatibility. These NPs are applied for time-resolved luminescence imaging to eliminate background interference with excellent tissue penetration. This study provides a practical solution to prepare long-wavelength and long-lifetime organic RTP materials and their applications in bioimaging.

Fluorescent/Phosphorescent Dual-Emissive Probe for Simultaneous and Independent Sensing of Intracellular Hypochlorite and pH via Time-Resolved Luminescence Imaging.

In this work, we present a dual-emissive polymeric probe capable of simultaneous and independent sensing of intracellular hypochlorite and pH. The probe integrates an anthracene-carboxamide-based fluorescent compound with a phosphorescent iridium(III) complex. The fluorescence and phosphorescence spectra are significantly overlapped at 500 to 650 nm, but their lifetimes differ substantially at 14.8 and 1113.6 ns, respectively, thereby allowing for monitoring in different time intervals through time-resolved luminescence analysis. The fluorescence is quenched upon oxidation by hypochlorite, whereas an acidic environment favors protonation of pyridyl groups on the iridium(III) complex, which causes phosphorescence quenching. During hypochlorite sensing, a linear plot of the fluorescence/phosphorescence ratio over hypochlorite concentration was obtained. During pH sensing, the pK a value was calculated to be about 4.6. The utilization of this probe for imaging cellular endogenous hypochlorite and intracellular pH values was demonstrated. The short-lived and long-lived signals were successfully extracted via time-resolved luminescence imaging microscopy, thereby allowing independent, simultaneous, and quantitative analysis of fluorescence and phosphorescence signals and their response toward hypochlorite and pH values. This study offers potential implications for the development of biosensing and bioimaging applications and paves the way for the simultaneous multiplexed detection of physiological and pathological activities within living cells.

Luminescence lifetime tuning of non-conjugated organic clusters through external heavy-atom effect for smartphone-based time-resolved imaging

Clusteroluminescence from non-conjugated organic molecules has attracted extensive attention, and it is extraordinarily important to develop effective methods for tuning the clusteroluminescence lifetime with balanced emission intensity in optical multiplexing for time-resolved luminescence imaging. Herein, chitosan, a renewable polymer, was found to exhibit persistent clusteroluminescence after dehydration. In addition, external heavy-atom effect was successfully utilized for enhancing phosphorescence intensity and regulating its phosphorescence lifetime. By adjusting the doping concentration of bromide salts in chitosan, the phosphorescence lifetime could be gradually tailored from 49 to 647 ms. The heavy atoms could promote phosphorescence transition and avoid the reduction of short-lived luminescence component, which facilitated the lifetime-multiplexed information encryption. This strategy for tuning phosphorescence lifetime could be extended to other phosphorescence materials. Moreover, smartphone-based luminescence imaging of shrimp chitin was realized by enhancing its persistent luminescence, which indicated that our method could be used for label-free luminescence imaging of chitin-containing organisms.

Research and Design of Aggregation-Induced Phosphorescent Materials for Time-Resolved Two-Photon Excited Luminescence Imaging.

Pure organic two-photon excited room temperature phosphorescent (RTP) materials have attracted great attention for time-resolved imaging due to their long emission lifetime and high resolution. The materials with an aromatic carbonyl group exhibit aggregation-induced emission (AIE) and RTP characteristics simultaneously. Here, we deeply explored the nature of aggregation-induced phosphorescence (AIP), especially the relationship between molecular configuration and optical properties. It was found that aggregation effect can suppress geometrical vibrations and regulate energy difference between S1 and T1. The aromatic carbonyl group plays significant roles in changing electronic configuration, resulting in large Stokes shift and spin-orbit coupling. It also leads to small transition dipole moment, decreasing two-photon absorption cross section and radiative decay rate. To improve two-photon absorption properties, we further designed a π-conjugated compound with large two-photon absorption cross section in the biological window (36.40 GM/656 nm) and AIP characteristics, which is a potential material in the application of time-resolved two-photon excited imaging.

Lanthanide-Based Nanoprobes for Time-Resolved Luminescence Imaging on Various Ions and Molecules

Lanthanide-doped upconversion nanoparticles (Ln-UCNPs) have been extensively explored in the biological field. In particular, Ln-UCNPs with near-infrared (NIR) fluorescence have tremendous potential for biological imaging because of their outstanding photo-and chemo-stability, extended photoluminescence lifetimes, low long-term toxicities and narrow photoluminescence bandwidths as well as minimal background interferences. Using predesigned energy transfer routes makes it possible to get upconversion luminescence from lanthanides' 4f-4f optical transitions. This article clarifies the key working principles and superiorities of Ln-UCNPs for bioimaging. A crucial overview of recent advances in biological detection adopting lanthanide-based luminescence resonance energy transfer (LRET) mechanisms is presented while emphasizing the importance of modifying Ln-UCNPs to obtain a more efficient energy transfer mechanism.

Thermally activated delayed fluorescence (TADF) emitters: sensing and boosting spin-flipping by aggregation.

Metal-free organic emitters with thermally activated delayed fluorescence (TADF) characteristics are emerging due to the potential applications in optoelectronic devices, time-resolved luminescence imaging, and solid-phase sensing. Herein, we synthesized two (4-bromobenzoyl)pyridine (BPy)-based donor–acceptor (D–A) compounds with varying donor size and strength: the emitter BPy-pTC with tert-butylcarbazole (TC) as the donor and BPy-p3C with bulky tricarbazole (3C) as the donor unit. Both BPy-pTC and BPy-p3C exhibited prominent emission with TADF properties in solution and in the solid phase. The stronger excited-state charge transfer was obtained for BPy-p3C due to the bulkier donor, leading to a more twisted D–A geometry than that of BPy-pTC. Hence, BPy-p3C exhibited aggregation-induced enhanced emission (AIEE) in a THF/water mixture. Interestingly, the singlet–triplet energy gap (ΔEST) was reduced for both compounds in the aggregated state as compared to toluene solution. Consequently, a faster reverse intersystem crossing rate (kRISC) was obtained in the aggregated state, facilitating photon upconversion, leading to enhanced delayed fluorescence. Further, the lone-pair electrons of the pyridinyl nitrogen atom were found to be sensitive to acidic protons. Hence, the exposure to acid and base vapors using trifluoroacetic acid (TFA) and triethylamine (TEA) led to solid-phase fluorescence switching with fatigue resistance. The current study demonstrates the role of the donor strength and size in tuning ΔEST in the aggregated state as well as the relevance for fluorescence-based acid–base sensing.

Oxygen Quenching-Resistant Nanoaggregates with Aggregation-Induced Delayed Fluorescence for Time-Resolved Mapping of Intracellular Microviscosity.

Microviscosity is a fundamental parameter in the biophysics of life science and governs numerous cellular processes. Thus, the development of real-time quantitative monitoring of microviscosity inside cells is important. The traditional probes for detecting microviscosity via time-resolved luminescence imaging (TRLI) are generally disturbed by autofluorescence or surrounding oxygen in cells. Herein, we developed loose packing nanoaggregates with aggregation-induced delayed fluorescence (FKP-POA and FKP-PTA) and free from the effect of oxygen and autofluorescence for viscosity mapping via TRLI. The feasibility of FKP-PTA nanoparticles (NPs) for microviscosity mapping through TRLI was demonstrated by monitoring the variation of microviscosity inside HepG2 cancer cells, which demonstrated a value change from 14.9 cP to 216.9 cP during the apoptosis. This indicates that FKP-PTA NP can be used as a probe for cellular microviscosity mapping to help people to understand the physiologically dynamic microenvironment. The present results are expected to promote the advancement of diagnostic and therapeutic methods to cope with related diseases.

Photoswitchable Thermally Activated Delayed Fluorescence Nanoparticles for “Double‐Check” Confocal and Time‐Resolved Luminescence Bioimaging

AbstractThermally activated delayed fluorescence (TADF) materials have attracted increasing attention due to their great potential in time‐resolved luminescence imaging (TRLI) in bioimaging. However, the triplet state (T1) of delay fluorescence is quenched readily by oxygen and the autofluorescence of biological systems remains inevitable interference in luminescence imaging, which often reduces signal‐to‐noise ratio during biodetection. To address this issue, the authors design a two‐component photoswitchable TADF polymeric nanoparticle (PDFPNs) by covalently incorporating a well‐designed TADF molecule (PDC‐DA) and a photochromic spiropyran derivative (SPMA). The as‐prepared PDFPNs with a unique luminescent “double‐check” strategy exhibit dual‐color fluorescence and variable fluorescence lifetime upon fluorescence resonance energy transfer (FRET) process through external lights irradiation. The obtained PDFPNs display the advantages of negligible oxygen‐sensitivity, high FRET efficiency, rapid photoresponsiveness, outstanding photoreversibility, and prominent long‐term fluorescence stability. The established PDFPNs show promising application in the high‐contrast dual‐color confocal and time‐resolved luminescence bioimaging in oxygenic living cells.

Aggregation-induced delayed fluorescence for time-resolved luminescence sensing of carboxylesterase in living cells

Thermally activated delayed fluorescence (TADF) materials with long-lived fluorescent emission, have been considered to be promising candidates for time-resolved luminescence imaging (TRLI) and sensing (TRLS). Though the development of the TADF luminophores for TRLI has received considerable attention, the oxygen-caused delayed fluorescence quenching has greatly stagnated the progress of TADF-based TRLS. In this work, we firstly proposed the strategy of in situ generated aggregation-induced delayed fluorescence (AIDF) for TRLS. A new luminophore FAc-Py with multiply twisted geometry and AIDF behavior was designed to offer the long-lived emission for meeting the time-resolved requirement. The carboxylester-capped FAc-Py, namely FAc-Py-Ester, which is non-emissive but shows good liposolubility, was then rationally synthesized for the delayed fluorescence turn-on sensing of carboxylesterase. Carboxylesterase easily promoted the in situ release of hydrophobic FAc-Py from FAc-Py-Ester, accompanying with the activation of air-insensitive AIDF emission and finally paving a way for the functional TRLS of carboxylesterase in living cells.

Enhancing Persistent Luminescence of Cellulose by Dehydration for Label-Free Time-Resolved Imaging

Organic persistent luminescence is usually emitted from synthesized materials containing π units (aromatic rings, double bonds, etc.). It is challenging but desirable to develop organic materials from renewable resources with tunable persistent luminescence to reduce environmental impact. Herein, we proposed an effective method to enhance the luminescence of cellulose, the most abundant natural renewable material free of π units. It was found that removing the crystal water in cellulose would increase the compactness of molecular packing and stabilize the hydroxyl clusters of polysaccharide chains, consequently leading to significant enhancement of phosphorescence with lifetime up to subseconds. This dehydration induced luminescence enhancement was found in not only purified cellulose but also cellulose-contained substances such as papers, cottons, and many other plant tissues. This indicated easier access to cellulose-based luminescence materials without chemical purification. In addition, label-free time-resolved luminescence imaging of plant tissues was achieved on a smartphone-based apparatus, since endogenous cellulose is distributed extensively in plants. Compared with synthetical luminophores, cellulose is a renewable and biodegradable source abundant in nature, not only readily available but also environmentally friendly. We believe this method may be used to develop other sustainable organic luminescence materials with a lack of π units for various optical applications.

Image Processing and Luminescent Probes for Bioimaging Techniques with High Spatial Resolution and High Sensitivity

The balance of microenvironmental factors (including temperature, pH, ROS species, etc.) plays a crucial role in maintaining normal living organisms’ normal physiological activities and physiological functions. Therefore, armed with the unique superiorities of high spatial resolution, non-invasion, high sensitivity, real-time monitoring, and simple operation, luminescent imaging technology has been widely used in real-time and accurate monitoring of microenvironmental factors in these organisms to prevent, diagnose and treat related diseases in time. However, due to its optical imaging characteristics, it is also faced with such interference factors as relatively shallow imaging penetration depth, background fluorescence (biological autofluorescence) interference in a complex environment, uncertain probe concentration, and unstable laser power in the imaging process, which are not related to the analyte. As for the problems in imaging, such as the uncertainty of probe concentration and the fluctuation of instrument laser power, the ratio detection, and imaging technology with self-calibration function can effectively avoid these problems. As for background fluorescence interference in imaging, probes with long-life emission can be used in imaging. The long-life luminescence of probes from background fluorescence can be recognized by time-resolved luminescence imaging technology to reduce its impact. This paper briefly introduces and summarizes the relative research of ratio detection and imaging technology and time-resolved luminescence imaging technology.

Rational design of pyrrole derivatives with aggregation-induced phosphorescence characteristics for time-resolved and two-photon luminescence imaging

Pure organic room-temperature phosphorescent (RTP) materials have been suggested to be promising bioimaging materials due to their good biocompatibility and long emission lifetime. Herein, we report a class of RTP materials. These materials are developed through the simple introduction of an aromatic carbonyl to a tetraphenylpyrrole molecule and also exhibit aggregation-induced emission (AIE) properties. These molecules show non-emission in solution and purely phosphorescent emission in the aggregated state, which are desirable properties for biological imaging. Highly crystalline nanoparticles can be easily fabricated with a long emission lifetime (20 μs), which eliminate background fluorescence interference from cells and tissues. The prepared nanoparticles demonstrate two-photon absorption characteristics and can be excited by near infrared (NIR) light, making them promising materials for deep-tissue optical imaging. This integrated aggregation-induced phosphorescence (AIP) strategy diversifies the existing pool of bioimaging agents to inspire the development of bioprobes in the future.

In vivo high-contrast visualization of upconversion nanoparticle labeled virus using time-resolved approach

Virus labeling is an ideal strategy to explore the interaction between virus and host cells. However, existed probes are limited in in vivo studies due to the poor imaging effects caused by tissue-depth and autofluorescence. Lanthanide-doped upconversion nanoparticles (UCNP) can be effectively excited in deeper bio-tissues, and its long fluorescence life-time is suitable for in vivo imaging using time-resolved luminescence imaging technology. In this study, lanthanide-doped UCNP was used to label viral envelope and nucleic acid, which could be used for in vitro and in vivo virus tracking. The in vitro results demonstrated that the UCNP-labeled Influenza A virus (UCNP-IAV) could be used for the study of virus internalization kinetics, and proved that the movement of UCNP labeled IAV was lysosome- and microtube-dependent. For in vivo study, pre-labeling and in situ labeling strategies were implemented to monitor the IAV infection. The results showed that the UCNP probe could provide high-quality images using time-gated strategy to effectively monitor the viral infection in vivo. Further, simultaneous tracking of IAV and adenovirus type-5 (Ad5) labeled by UCNP with different life-time was realized using time-resolved luminescence imaging technology. Hence, this paper provided a novel labeling method for virus tracking using UCNP, which was optimal to study the mechanism of virus infection in vitro and in vivo.

Editorial: Modern Tools for Time-Resolved Luminescence Biosensing and Imaging

Editorial: Modern Tools for Time-Resolved Luminescence Biosensing and Imaging

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.

Luminescence lifetime imaging of ultra-long room temperature phosphorescence on a smartphone.

Luminescence lifetime imaging plays an important role in distinguishing the luminescence decay rates in time-resolved luminescence imaging. However, traditional imaging instruments used for detecting lifetimes within milliseconds would be time-consuming when imaging ultra-long luminescence lifetimes over subseconds. Herein, we present an accessible and simple optical system for detecting lifetimes of persistent luminescence. A smartphone integrated with a UV LED, a dichroic mirror, and a lens was used for recording the persistent luminescence. With only a few seconds of data acquisition, a luminescence lifetime image could be processed from the video by exponential fitting of the gray level of each pixel to the delay time. Since this approach only requires single excitation, no synchronous control is needed, greatly simplifying the apparatus and saving the cost. The apparatus was successfully used for ultra-long luminescence lifetime imaging of mouse tissue dyed with a persistent luminescence molecule. This miniaturized apparatus exhibits huge potentiality in time-resolved luminescence imaging for luminescence study and biological detection.

B- and N-Embedded π-Conjugation Units Tuning Intermolecular Interactions and Optical Properties of Platinum(II) Complexes.

A new series of neutral and cationic platinum(II) complexes containing a B- or N-embedded π-conjugation unit has been prepared. Notably, significantly different intermolecular interactions (Pt-Pt, π-π, head to tail, and head to head) and interesting optical properties exist in these complexes, which can be attributed to the difference in spatial structures and π-electron properties between B- and N-embedded π-conjugation units. Unexpectedly, under a hypoxic atmosphere, N-embedded neutral complex PtNacac can display a distinct dual-emission with both fluorescence and phosphorescence, whereas only a single fluorescence emission was observed in the air, which is different from the B-embedded neutral complex PtBacac with only a single phosphorescence emission at any atmosphere, as well confirmed by lifetime measurement and oxygen sensing experiments. DFT calculations reveal that unusual ligand-to-metal charge transfer (LMCT) excited state character and low spin orbit coupling (SOC) elements can be found in N-embedded complexes due to the strong electron-donating ability of the N-embedded unit. Based on this, as a novel ratiometric oxygen probe with a simple structure, PtNacac can be successfully used to examine intracellular oxygen levels by monitoring both fluorescence and phosphorescence signals via ratiometric photoluminescence imaging and time-resolved luminescence imaging (TRLI) technology. This work provides a completely new idea for designing fluorescence/phosphorescence dual-emissive complexes.

Phosphine Oxides Manipulate Aggregation‐Induced Delayed Fluorescence for Time‐Resolved Bioimaging

Delayed fluorescence (DF) materials have shown great potential in time‐resolved luminescence imaging (TRLI), due to the relatively elongated emission lifetimes. However, the sensitivity of DF to environmental quenching leads to a challenge to realize TRLI directly using pure thermally‐activated DF (TADF) molecules. Herein, two TADF molecules, namely CzPOTCF and tBCzPOTCF, are designed and synthesized, featuring a diphenylphosphine oxide (DPPO) group substituted at the ortho‐position of the carbazole group. Compared with DPPO‐free CzTCF, the steric hindrance of DPPO groups enlarges dihedral angles between TCF and the carbazole groups in CzPOTCF and tBCzPOTCF; meanwhile, PO provides other channels for forming intermolecular hydrogen bonds. The optimized intermolecular interactions facilitate the dimer formation of CzPOTCF and tBCzPOTCF, which simultaneously enhance aggregation‐induced DF and mitigate disordered packing‐induced quenching. As a consequence, CzPOTCF and tBCzPOTCF show the enhanced aggregation‐induced DF with largely increased lifetimes. Without any additional assistance, the long‐lived fluorescence of CzPOTCF and tBCzPOTCF is successfully observed for cell TRLI, with 1–1.7 μs elongated average lifetimes of 6.69 and 7.41 μs, respectively.