Unlocking new frontiers in bioimaging: The power of FLIM-AIE integration

Abstract

The frontiers of bioimaging encompass cutting-edge technologies and methodologies that enhance the visualization, analysis, and interpretation of biological structures and processes with unprecedented precision. In this context, fluorescence lifetime imaging microscopy (FLIM) has emerged as a powerful technique that enables quantitative and concentration-independent imaging by measuring the fluorescence decay time of molecular probes. Unlike intensity-based fluorescence methods, FLIM provides robust data on molecular interactions, environments, and biomarker presence, making it invaluable for bioimaging. However, its widespread adoption remains constrained by technical complexity, the high cost of instrumentation, and the underdevelopment of fluorescence lifetime probes with AIE characteristics. A promising solution to these challenges lies in aggregation-induced emission (AIE) fluorophores, which overcome issues such as aggregation-caused quenching and photobleaching. Despite their potential, AIE fluorophores have been rarely integrated with FLIM, leaving a significant gap in bioimaging research. Integrating FLIM with AIE probes could revolutionize bioimaging, enabling precise, stable, and environment-sensitive lifetime imaging for diagnostics, metabolism, and live-cell monitoring. In this perspective, we examine the potential of FLIM-AIE integration in bioimaging, highlighting its advantages, identifying key barriers, and proposing strategies to promote its use in biomedical research. We highlight the critical role of interdisciplinary collaboration, enhanced training programs, and targeted funding initiatives in accelerating innovation in the field of bioimaging.

Main text

Driving innovation in the visualization, analysis, and interpretation of biological structures and processes remains a major challenge in bioimaging. Advancements in this field have profound implications for medicine, pharmacology, nanomedicine, and molecular biology. In this context, fluorescence lifetime imaging microscopy (FLIM) has emerged as a powerful tool, offering insights into biomolecular interactions and environmental conditions. Unlike conventional fluorescence microscopy, which relies on intensity-based measurements affected by fluorophore concentration, excitation power, and photobleaching, FLIM operates independently of these variables. It measures fluorescence lifetime, the mean time fluorophores remain in the excited state before emitting photons. This parameter is sensitive to environmental factors such as pH, viscosity, and molecular binding, making FLIM a robust technique for quantitative imaging. Due to its unique ability to generate absolute, concentration-independent data, FLIM is widely applied in biomedicine. It offers advantages over intensity-based fluorescence microscopy: (1) it effectively differentiates fluorophore signals from cellular autofluorescence; (2) it is less susceptible to photobleaching, ideal for biological imaging; and (3) it reduces photodamage through pulsed excitation with short breaks while enabling precise biomarker quantification in complex systems.¹

Recent advances in FLIM technology, including faster acquisition systems and improved detectors, are enhancing its potential for high-resolution, high-throughput imaging in both fundamental research and clinical diagnostics. Historically, the high cost and technical skill required for FLIM have limited its adoption to specialized facilities. However, recent advances in detectors and wide-field FLIM systems have significantly cut acquisition times, making high-throughput and real-time imaging increasingly feasible. This progress has prompted greater interest in FLIM as a tool for disease diagnostics, drug development, and physiological research.² By measuring fluorescence lifetimes, researchers can map local variations in key physiological parameters such as pH, viscosity, oxygen levels, and molecular interactions. These lifetime maps provide high-resolution visualization of molecular changes within cells and tissues, facilitating the study of dynamic processes, including protein folding, enzyme activity, intracellular signaling, biomarker quantification, and metabolic imaging. Such advancements support real-time monitoring of cellular dynamics and drug treatment efficacy, offering precise molecular-level insights into therapeutic responses. Other time-resolved techniques, like time-resolved X-ray and Raman microscopy, focus on materials characterization rather than bioimaging. In contrast, FLIM can compete with super-resolution imaging techniques, particularly when combined with stimulated emission depletion (STED) microscopy. The STED-FLIM approach constitutes a powerful bioimaging tool that has enabled the high-resolution visualization of subcellular structures, including the inner mitochondrial membrane and the nuclear envelope-endoplasmic reticulum interface. Phosphorescence lifetime imaging microscopy (PLIM) has recently emerged as a complementary technique to FLIM, enabling the use of phosphorescent probes in time-resolved bioimaging. PLIM is best implemented with piezoelectric scanners, which offer the slower and more precise scanning required for these probes. Consequently, phosphorescent materials can also be integrated as lifetime probes, representing a significant advancement in areas such as the development of biomimetic photosensitizers.^3,4

FLIM technology relies on efficient probes that meet specific requirements to ensure accurate fluorescence lifetime measurements. Ideally, FLIM-based probes should exhibit long and stable fluorescence lifetimes, resistance to fluorescence quenching upon aggregation, low cytotoxicity, and minimal interference with biological functions. However, many conventional fluorophores experience significant fluorescence quenching and reduced emission intensity when aggregated or confined within dense biological environments, limiting their effectiveness in advanced bioimaging applications. To address these limitations, the emergence of aggregation-induced emission (AIE) fluorophores presents a promising solution. Unlike traditional fluorophores, which often suffer from fluorescence quenching upon aggregation, AIE-based probes remain non-emissive or weakly emissive in molecularly dissolved states but exhibit strong fluorescence upon aggregation, generally due to the restriction of intramolecular motions causing non-radiative decays. The term AIE was coined in 2001 by B.Z. Tang and co-workers, who explained it through the restriction of intramolecular rotations (RIR) in fluorophores like hexaphenylsilole (HPS) and tetraphenylethylene (TPE).⁵ Fluorescence enhancement or quenching also depends on excitonic interactions. In Kasha’s exciton model, J-type aggregates are commonly linked to AIE behavior. Hestand and Spano introduced the concept of effective short-range exciton coupling due to wave function overlaps of the interacting molecules, allowing a deeper understanding of the relationship between intermolecular interactions and photophysical properties. The restricted access to a conical intersection (RACI), the blocking of twisted intramolecular charge transfer (TICT), excited-state intramolecular proton transfer (ESIPT), and the restriction of photoisomerization are other molecular mechanisms commonly responsible for the AIE phenomenon. The fluorescence emission of AIE-based probes is enhanced not only with the molecular aggregation but also in highly viscous media due to the restriction of intramolecular motions. Thus, the AIE phenomenon not only prevents quenching but actively enhances signal strength, making these fluorophores particularly suitable for fluorescence imaging in complex biological systems containing regions of high molecular density or viscous cellular environments. The integration of FLIM with AIE fluorophores confers robust photostability, as AIE-based probes maintain high emission and resist photobleaching. When coupled with FLIM’s capacity to detect lifetime perturbations enables highly detailed mapping of molecular and cellular processes. Ongoing research continues to optimize AIE-fluorescence lifetime probes for environmental sensing, protein interactions, and in vivo cellular imaging. By combining AIE’s unique emission properties with FLIM’s quantitative precision, this innovative approach paves the way for exciting new possibilities in bioimaging.

Despite the immense potential of FLIM-AIE in bioimaging, its widespread adoption faces several challenges. First, the high cost of instrumentation and the need for specialized expertise create significant barriers. Second, the lack of standardized protocols complicates reproducibility, such as variations in sample preparation, imaging parameters, and instrumentation settings, making it difficult to validate results. Additionally, insufficient awareness and training contribute to hesitation among researchers. Many of them perceive FLIM as overly complex due to its specialized data analysis and mathematical modeling requirements, leading them to default to traditional intensity-based bioimaging methods, even though fluorescence lifetime data provide superior precision and contrast. Moreover, the limited development of fluorescence lifetime probes with AIE characteristics reduces the availability of suitable probes to fully leverage FLIM-AIE synergy. For effective bioimaging, longer fluorescence lifetimes than cellular autofluorescence are typically needed. Different approaches have been reported that achieve fluorescence lifetime modulation but depend on the deep knowledge of the molecular mechanisms associated with the non-radiative decays (TICT, ESIPT, RIR, etc.).⁶ Overcoming these hurdles through innovation in instrumentation, standardized methodologies, and improved training could unlock the full potential of FLIM-AIE in advanced bioimaging.

Several studies highlight the potential of combining AIE fluorophores with FLIM, showcasing its impact on bioimaging and diagnostic applications. Pacheco-Liñán et al. demonstrated how an anthracene derivative enhanced its fluorescence emission upon aggregation due to the formation of T-shaped dimers in the excited state (AIE-excimer).⁷ When combined with FLIM, these excimers enable high-contrast bioimaging and precise identification of subcellular structures thanks to distinctive lifetime profiles and stable emission signals. Similarly. Chen et al. were among the first to apply FLIM technology for intracellular viscosity mapping, employing a TPE derivative as an AIE probe sensitive to viscosity via the RIR mechanism.⁸ Meso-aryl-BODIPYs have also been explored as fluorescence lifetime viscosity probes by Yu et al.⁹ In high-viscosity environments, their excited state remains planar and emits fluorescence, whereas in low-viscosity conditions, it undergoes the TICT process, becoming non-fluorescent. Beyond viscosity sensing, Ho et al. used a styryl dye as an AIE probe to monitor cellular responses to mechanical stimuli with FLIM.¹⁰ Similarly, Ripoll et al. illustrated how AIE-active anthracene derivatives can be used as nanothermometers in living cells. FLIM enables accurate correlation between fluorescence lifetime and cellular temperature.¹¹ Additionally, Gao et al. developed a nanothermometer based on an AIE fluorophore (HPS) in a butter matrix stabilized with DSPE-PEG-biotin.¹² Fluorescence intensity increased as temperature decreased due to enhanced viscosity restricting molecular motions. The photostability of these excimers ensures reliable long-term temperature monitoring under physiological conditions. Together, these studies underscore the potential of FLIM-AIE synergy. Specifically, this application of AIE probes for intracellular temperature monitoring could become a flagship approach, enabling thermal mapping in metabolically active regions. Calibration using a temperature-controlled stage or live-cell incubator allows precise thermal modulation. Under these conditions, the fluorescence lifetime of the AIE probe is correlated with temperature, providing a reference for intracellular thermometry in metabolic studies. It holds potential for studying processes such as protein denaturation or folding, transcriptionally active genomic regions, oxidative stress, inflammatory responses, thermoregulatory mechanisms, or mitochondrial heat production. These insights could contribute to the design of thermally activated therapies, heat-triggered treatments, or even nanosystems for controlled drug release in response to thermal stimuli. Combining FLIM’s lifetime mapping with AIE’s stable emission enables real-time cellular and molecular analysis, advancing bioimaging applications.

There is a pressing need for studies focusing on more complex systems like 3D models, tissues, or small animals. However, these systems face challenges, including the aggregation of the studied compounds, which can occur due to various factors, such as accumulation within specific organelles, leading to a significant increase in local compound concentration. Furthermore, the inherent complexity of these systems can actively promote molecular aggregation in specific regions, a critical consideration for the accurate interpretation of experimental results. Importantly, interactions between probes and various biomolecules—such as proteins—can significantly contribute to this aggregation phenomenon through binding-induced clustering, profoundly influencing fluorescence behavior and ultimately affecting the reliability and reproducibility of bioimaging data. Barroso et al.¹³ underscores the potential of 3D organoid models as effective platforms for FLIM. Currently, few fluorescent probes are available for studying metabolism in 3D systems. Some of these probes are used to study the glycolysis process and are based on the analysis of extracellular pH, redox processes measured by FLIM, or the autofluorescence of tryptophan, which can also be assessed using FLIM. Another metabolic process that can be studied using these probes is oxidative phosphorylation, which can be assessed through mitochondrial membrane potential analysis, the fluorescence lifetime of NAD(P)H or FAD using FLIM, or intracellular temperature variations. In this context, for example, the use of an exogenous AIE-based temperature probe capable of simultaneously monitoring intracellular temperature could serve as a synergistic tool to validate and enhance the accuracy of the obtained data.

In complex systems, the use of conventional fluorophores is significantly hindered by fluorescence quenching upon aggregation, limiting the ability to accurately study metabolic processes in these environments. AIE fluorophores overcome this by becoming highly emissive upon aggregation, ensuring strong signals in dense or compartmentalized regions. This unique property allows for reliable metabolic analysis in 3D systems, where FLIM-based fluorescence lifetime measurements can be used to investigate glycolysis, oxidative phosphorylation, and other key cellular processes without the limitations of quenching. It is worth noting that certain AIE probes may induce undesired interferences, such as non-specific interactions with intracellular biomolecules. These challenges can be effectively addressed by surface functionalization to minimize off-target binding or by encapsulating the probes within nanocarriers. In this regard, emerging AIE dots hold significant promise, as they not only reduce non-specific interactions and enhance biocompatibility but also improve cellular internalization and enable multifunctionality. These nanoplatforms can be co-loaded with therapeutic agents, nucleic acids, peptides, or proteins, thereby expanding their applicability in bioimaging and targeted drug delivery.¹⁴

By harnessing the aggregation-induced fluorescence enhancement of AIE fluorophores, researchers can achieve high-contrast and stable signals, enabling the precise investigation of dynamic metabolic pathways in physiologically relevant models with improved accuracy and sensitivity. Figure 1 illustrates how the integration of FLIM with AIE systems enhances bioimaging capabilities.

Figure 1.

Enhancing bioimaging with FLIM-AIE integration for real-time cellular analysis and disease detection

Based on all of the above, we forecast that FLIM is now ready to take the next step toward more complex systems, with the potential to revolutionize diagnostic techniques and precision medicine. Recent progress has improved its accessibility by lowering costs and simplifying interpretation. However, a key challenge remains: the identification of suitable probes, particularly for complex environments such as 3D models and tissues. AIE fluorophores offer a promising solution to many of these challenges, providing bright, stable signals even in demanding conditions. Despite this potential, there is still a significant gap in the development of AIE-based probes specifically optimized for FLIM applications. The successful integration of FLIM with AIE fluorophores has the potential to greatly improve bioimaging, boosting diagnostic accuracy and expanding its use in both research and clinical settings. To fully unlock this potential, the scientific community must focus on developing, validating, and refining these probes while fostering collaboration across disciplines. By doing so, FLIM-AIE can become a game-changing tool, offering deeper insights into complex biological systems and shaping the future of non-invasive imaging.

Funding and acknowledgments

This work was supported by the Ministerio de Ciencia e Innovación and Agencia Estatal de Investigación, Spain (RED2022-134287-T), as well as CPP2021-008597, funded by MICIU/AEI/10.13039/501100011033 and the European Union NextGenerationEU/PRTR. Additional funding was provided by JCCM through SBPLY/21/180501/000050 and SBPLY/23/180502/000013 and by the ACEPAIN Foundation. S.B.-G. acknowledges UCLM for the predoctoral grant 2024-PRED-21958.

Declaration of interests

The authors declare that they have no competing interests.

Published Online: August 18, 2025