Illuminating liver fibrosis: recent progress in the design and applications of highly sensitive fluorescent probes

Abstract

Liver fibrosis involves excessive, disorganized extracellular matrix deposition in the liver, critically driving progression from chronic liver disease to cirrhosis and determining patient prognosis. Although histological examination of liver tissue biopsies continues to serve as the most reliable diagnostic approach, the development of precise detection methods remains crucial for enabling timely therapeutic interventions and improving patient management. Recent advances in fluorescent probes have transformed the detection of liver fibrosis, enabling real-time, non-invasive visualization of biomarkers and microenvironmental changes. Based on its design strategy, targeted objects and functional characteristics, this review systematically classifies state-of-the-art fluorescent probes into five categories: probes directly targeting liver fibrosis markers, enzyme-activated probes, microenvironment-responsive probes, intracellular targeting probes, and multifunctional theranostic probes. Among them, near-infrared II (NIR-II, 1000–1700 nm) imaging and genetically encoded probes boost molecular precision and resolution, yet clinical application faces challenges from limited tissue penetration and poor biocompatibility. Consequently, future research in this field will concentrate on the development of NIR II probes, the discovery of biomarkers in biofluids, and the design of new therapeutic interventions. By elucidating design principles and applications, this review aims to bridge the gap between molecular innovation and clinical practice, ultimately advancing precision medicine for liver fibrosis.

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Wider impact

The development and application of fluorescent probes for monitoring liver fibrosis hold significant potential to transform the diagnosis and management of chronic liver diseases. By enabling non-invasive, real-time, and high-resolution detection of fibrotic progression, these probes could reduce the reliance on invasive biopsies, improving patient comfort and accessibility to early diagnosis. This technology may also facilitate personalized treatment strategies by allowing dynamic assessment of therapeutic responses, ultimately improving clinical outcomes. Beyond hepatology, advances in molecular imaging could inspire similar approaches for other fibrotic diseases (e.g., pulmonary or renal fibrosis), fostering interdisciplinary innovation. Widespread adoption could lower healthcare costs through early intervention and reduce the global burden of end-stage liver complications, aligning with global health priorities like the WHO's goal to combat non-communicable diseases.

1. Introduction

Liver fibrosis represents a progressive pathological condition marked by abnormal accumulation of extracellular matrix (ECM) proteins, serving as a key transitional stage in various chronic liver disorders, including viral hepatitis, alcohol-associated liver disease, and non-alcoholic steatohepatitis (NASH).¹ Globally, chronic liver diseases impact over 1.5 billion individuals, and without early therapeutic measures, fibrotic progression can lead to irreversible cirrhosis or hepatocellular carcinoma.^2–4

Specifically, the pathophysiological core of hepatic fibrosis lies in the abnormal repair response induced by chronic liver injury, involving dynamic interactions among multiple cellular and molecular mechanisms. Chronic liver injury (e.g., viral hepatitis, alcohol, or metabolic factors) first triggers an inflammatory response, prompting Kupffer cells to release pro-inflammatory cytokines (e.g., TNF-α and IL-6) and chemokines that recruit neutrophils and monocytes, establishing a chronic inflammatory microenvironment.⁵ During this process, the central role of hepatic stellate cells (HSCs) in fibrosis is evidenced by the observation that their activation is pivotal in driving ECM overproduction through complex signaling pathways, such as transforming growth factor-βeta/Sma- and Mad-related proteins and platelet-derived growth factor BB/platelet-derived growth factor receptor βeta (TGF-β/Smad and PDGF-BB/PDGFR-β).^6–11 Under injury signaling, quiescent hepatic stellate cells (HSCs) transition into a myofibroblast-like phenotype, massively secreting extracellular matrix (ECM) components (e.g., type I collagen and fibronectin) while maintaining activation through autocrine loops.¹² Concurrently, ECM metabolic imbalance manifests as suppressed matrix metalloproteinase (MMP) activity and upregulated tissue inhibitors of metalloproteinases (TIMPs), leading to insufficient collagen degradation and abnormal deposition, ultimately forming fibrous septa.¹³ Additionally, oxidative stress and endoplasmic reticulum stress exacerbate HSC activation via the reactive oxygen species-nuclear dactor kappa-B (ROS-NF-κB) pathway, while vascular remodeling (e.g., sinusoidal capillarization) further promotes hypoxia and fibrotic progression.¹⁴ Despite advances in understanding the mechanisms of fibrosis, early diagnosis and accurate staging remain formidable challenges due to the silent nature of early fibrosis and the limitations of conventional diagnostic tools.

The clinical diagnosis and monitoring of liver fibrosis continue to evolve with technological advancements, yet current methods face multiple limitations, driving the exploration of novel imaging techniques. Liver biopsy, as the traditional gold standard, is difficult to repeat due to invasive risks (e.g., hemorrhage incidence ∼0.4%, mortality 0.11%¹⁵) and sampling errors¹⁶ (assessing only ∼1/50 000 of the liver parenchyma), particularly limiting its use in pediatric patients or those with coagulation disorders. Transient elastography (e.g., FibroScan®), while non-invasive and convenient, suffers from insufficient sensitivity for early-stage fibrosis (F1–F2) and is significantly influenced by obesity, hepatic steatosis, and operator experience.¹⁷ Serum biomarkers (e.g., FIB-4 and APRI) can indicate fibrosis risk but lack spatial resolution to localize lesions and are prone to interference from age, platelet count, and extrahepatic diseases.¹⁸ These limitations underscore the pressing need for non-invasive real-time diagnostic techniques that can capture dynamic molecular and microenvironmental changes in fibrotic livers.² In clinical settings, the application of fluorescent probes is progressively transitioning from laboratory research to practical diagnostic and therapeutic tools, demonstrating core value across multiple domains including early disease detection, intraoperative navigation, therapeutic monitoring, and pathological analysis.

In this context, near-infrared (NIR) imaging has emerged as a research hotspot due to its unique ability to visualize molecular and functional activities. Compared to ultrasound elastography, NIR imaging employs targeted probes (e.g., MMP-13-activated fluorescent probes) to specifically label collagen synthesis or degradation, providing dynamic metabolic insights beyond static stiffness measurements. While NIR imaging is inferior to magnetic resonance elastography (MRE) in penetration depth (<2 cm) and whole-organ imaging, its micrometer-level resolution enables precise localization of early fibrotic lesions and differentiation between reversible fibrosis and mature scars (e.g., using LOXL2-targeted probes to mark cross-linked collagen). Additionally, NIR's radiation-free nature and low cost make it promising for long-term follow-up or intraoperative navigation (e.g., guiding precision liver biopsy). However, clinical translation of NIR imaging remains constrained by unvalidated probe targeting efficiency, signal attenuation in deep tissues, and the lack of standardized quantification methods.

Future breakthroughs should focus on multimodal technology integration, such as combining NIR with ultrasound elastography to complement molecular specificity with anatomical resolution, or developing NIR-II window probes (1000–1700 nm) to enhance penetration depth. Concurrently, advancing probes targeting key fibrotic markers (e.g., integrin αvβ3 and PDGFRβ) into clinical trials will accelerate the transition of NIR from the lab to the bedside, ultimately enabling precise, dynamic, and personalized management of liver fibrosis (Table 1).

Table 1 Comparison of medical imaging technologies: technical characteristics and clinical applicability

Technology	Spatial resolution	Molecular specificity	Penetration depth	Dynamic monitoring	Clinical applicability
Ultrasound elastography	∼1–5 mm	None	∼5–8 cm	No	High (widely used)
Magnetic resonance elastography (MRE)	∼0.5–1 mm	Low	Whole-body	No	Medium (requires MRI equipment)
CT/MRI	∼0.5–1 mm	Low	Whole-body	No	High
Near-infrared (NIR) imaging	∼10–100 μm	High	<2 cm	Yes	Low (preclinical stage)

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This review systematically classifies advances in fluorescent probes for liver fibrosis over the past five years, highlighting their design principles, biomedical applications and translational potential. The probes are classified according to detection targets. Following this, we scrutinize key gaps: biocompatibility optimization and challenges in clinical validation. To conclude, we propose future research directions advocating for collaboration across disciplines to bridge the gap between laboratory findings and clinical practice faster.

2. Liver fibrosis fluorescence targeting different detection targets

Liver fibrosis is driven by dynamic changes in molecular and cellular components, so detecting dynamic changes in these components can be a key target for probe design.¹ Depending on the detection goal, this review will describe five classes of fluorescent probes: probes directly targeting fibrosis biomarkers, enzyme-activated probes responding to fibrosis signals, microenvironmental-responsive probes, intracellular targeting probes, and multifunctional theranostic probes. This section will systematically review the design principles and applications of these probes (Table 2).

Table 2 Liver fibrosis fluorescence targeting different detection targets

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2.1. Direct targeting of fibrosis biomarkers

Liver fibrosis is characterized by two critical pathological processes: the activation of hepatic stellate cells (HSCs) and the dynamic reorganization of extracellular matrix (ECM) components.¹⁹ The direct targeting of fibrosis biomarkers, including collagen isoforms, cell fibronectin (cFN) and hematopoietic stem cell surface markers, can facilitate the accurate location of fibrotic lesions. These probes typically rely on ligand–receptor interaction or antibody–antigen binding, exhibiting high specificity. However, it is crucial to optimize the affinity and biodistribution for optimal performance.

2.1.1. Collagen-specific probes (type I/III/IV and denatured collagen). Non-alcoholic steatohepatitis (NASH) is characterized by progressive hepatic fibrosis, which can advance to liver failure and fatal outcomes.^20,21 Andrew L. Wang et al.²² developed and characterized an autonomously assembling protein nanomembrane, designated as type I collagen-binding thermo-responsive assembling protein (Col1-TRAP), which exhibits nanomolar affinity for type I collagen in vitro. The Col1-TRAP nanomembrane demonstrates a high degree of type I collagen-binding avidity in vitro, utilizing multivalent interactions to achieve this while maintaining biocompatibility. For in vitro visualization, Col1-TRAP is labelled with a near-infrared fluorescent dye (NIR-Col1-TRAP). The labelling of protein micelles with NIR fluorescent dyes does not result in any adverse effects on their structural or functional properties. Surface plasmon resonance (SPR) analysis revealed that Col1-TRAP and NIR-Col1-TRAP bound to type I collagen with ∼3.8-fold greater affinity compared to TRAP alone. Furthermore, in NASH murine models, NIR-Col4-TRAP displayed markedly reduced plasma retention time and enhanced liver accumulation relative to healthy controls at 1 h post-injection. Therefore, NIR-Col1-TRAP displays promise as a NASH imaging probe with in vivo targeting capabilities in mice following administration. Furthermore, protein-based imaging probes possess distinctive advantages over other probes, the most significant of which include both environmental degradability and the feasibility of large-scale biological synthesis. The clearance of NIR-Col1-TRAP from the serum was observed to occur at a more rapid rate in these mice, with a notable accumulation in fibrotic livers when compared to the control mice (Fig. 1).

Fig. 1 Targeted imaging of type I collagen in liver fibrosis using the Col1-TRAP fluorescent probe. (A) Schematic of the Col1-TRAP fluorescent probe. (B) Representative imaging of mouse liver. The figures were adapted from Wang, A.L., et al., 2024²² with permission from Elsevier, copyright 2024.

The accumulation of type I collagen, occurring from the early (S1) to the late (S4) stages of fibrosis, drives the formation of fibrous septa, thereby exacerbating the fibrotic state.^23,24 Concurrently, the rise in type IV collagen results in the disruption of liver structure throughout the fibrotic stage. The presence of denatured collagen (a marker of collagen degradation and turnover) provides further evidence of the continued progression of fibrosis. Linge Nian et al.²⁵ engineered peptide-based SF-I, SF-IV, and SF-D probes, which are tailored for targeted imaging of type I, type IV and degenerate collagen. These probes are capable of high spatial resolution fingerprinting of collagen in liver fibrosis. The probes comprise silver nanoparticles (Ag NPs), Raman reporter genes, and FAM-labelled collagen-targeting peptides. They exhibit distinct Raman peaks at 2227, 2154, and 2102 cm, enabling multiplexed imaging without cross-talk. The results of fluorescence-guided SERS imaging demonstrate the capacity of the method to identify, localize and quantify collagen, thereby offering new insights into the role of collagen in the development of liver fibrosis. The incorporation of Asp residues is intended to maintain the negative charge state of the CTP, which induces electrostatic repulsion and thus ensures significant dispersion and stability of the SERS probe.

Pre-type III collagen N-terminal peptide (P-III-NP), a biomarker linked to fibrotic progression in the liver and heart, can be detected using a novel single-step fluorescent immunosensor developed by Yi et al.²⁶ Their approach utilized a quenchbody (Q-body) system, where an anti-P-III-NP single-chain variable fragment (scFv) was engineered with a Cys-tag for site-specific fluorophore conjugation. To generate the scFv, total mRNA was isolated from hybridoma cells, followed by cDNA synthesis, PCR amplification of the variable region, and subsequent sequencing. Recombinant expression in E. coli Shuffle T7 yielded 9.8 mg L⁻¹ of the purified scFv. Fluorescence labeling was achieved via maleimide–thiol coupling, with different spacer arm lengths tested to optimize signal response. Among the tested configurations, the TAMRA-C6-conjugated Q-body exhibited antigen-dependent fluorescence quenching, enabling sensitive detection of P-III-NP—with a limit of detection (LOD) of 0.46 ng mL⁻¹ in buffer and 1.0 ng mL⁻¹ in 2% human serum. This one-step immunoassay eliminates the need for washing steps, offering rapid and efficient quantification of the procollagen type III N-terminal peptide, a key indicator of fibrotic disease.

Peptide-based probes SF-I, SF-IV, and SF-D are engineered for specific targeting of type I collagen, type IV collagen, and denatured collagen, respectively. Utilizing distinct Raman signatures, these probes enable high spatial resolution multiplexed imaging of collagen subtypes without signal crosstalk. Crucially, fluorescence-guided SERS imaging with these probes allows identification, localization, and quantification of collagen alterations in fibrotic livers, revealing characteristic changes compared to healthy tissue architecture.

2.1.2. Cellular fibronectin (cFN)-targeted aptamer probes. Fibronectin (FN) is a key component of the ECM and exhibits high expression during ECM deposition in tissues (organs) following the resolution of the inflammatory response.^23,27 The deposition rate of FN can serve as a valuable indicator for assessing the progression of fibrosis. At present, commercially available assays are unable to distinguish between plasma fibronectin (pFN) and cellular fibronectin (cFN), which restricts the in vivo detection of non-pFN and the precise staging of tissue fibrosis. It is therefore of great scientific importance and clinical value to develop ligand molecules targeting cFN for the monitoring of the onset and development of early fibrosis in vivo.

Recent advances in molecular imaging have led to the development of novel fluorescence probes utilizing a nucleic acid aptamer (ZY-1) specific to intracellular fibronectin (cFN), a critical ECM component whose expression markedly increases during hepatic fibrogenesis.²⁸In vitro studies using human hepatic stellate cells (HSCs) confirmed ZY-1's high binding specificity for cFN. Building upon this finding, investigators engineered multiple ZY-1-conjugated fluorescent markers and evaluated their dynamic imaging capabilities in murine models with carbon tetrachloride-induced hepatic fibrosis at various progression stages (Fig. 2). As shown in Fig. 2A, upregulation of cellular fibronectin (cFN) enables specific binding of the DNA aptamer ZY-1 to activated LX-2 hepatic stellate cells in vitro. Through real-time imaging and area-under-the-curve (AUC) analyses, ZY-1 fluorescent probes achieve early detection and staging of liver fibrosis by distinguishing CCl₄-induced murine models from healthy controls. Notably, these ZY-1-derived probes achieved unprecedented discrimination between initial fibrotic changes (stage 6 of Ishak 3) and severe fibrosis (stage 6 of Ishak 5), representing a significant breakthrough in fibrosis staging technology. This foundational research establishes ZY-1-based imaging agents as potential clinical tools for timely detection and progression monitoring of liver fibrosis, offering new possibilities for diagnostic approaches in hepatic and other fibrotic disorders.²⁹

Fig. 2 Fluorescent probes conjugated to the ZY-1 aptamer allow specific visualization of intracellular fibronectin (cFN) for assessment of liver fibrosis progression. (A) Real-time imaging and AUC analysis of ZY-1-based fluorescent probes in normal mice and CCl₄-induced liver fibrosis mice allow early detection and staging of liver fibrosis. (B) and (C) In vivo molecular imaging of liver fibrosis with fluorescent probes and ex vivo imaging of the corresponding major organs. The figures were adapted from Ge, M., et al. 2024²⁹ with permission from Elsevier, copyright 2024.

2.1.3. Apolipoprotein L2 (APOL2)-directed molecular probes. A recent study has demonstrated that researchers have screened a library of natural diterpenoids derived from the Euphorbiaceous family and identified 12-deoxyphorbol 13-palmitate (DP) as an effective agent against liver fibrosis. The photoaffinity labelling method was employed to identify apolipoprotein L2 (APOL2) as a direct target of DP (Fig. 3), which is predominantly located within the endoplasmic reticulum (ER). The results of mechanistic studies demonstrated that APOL2 is induced in response to the activation of hepatic stellate cells and binds to sarcoplasmic/ER calcium ATPase 2 (SERCA2). This interaction leads to the activation of ER stress and the subsequent activation of the PERK-HES1 axis, which plays a crucial role in the promotion of liver fibrosis. Inhibition of APOL2 by DP or APOL2 ablation can prevent this signaling and attenuate fibrosis. This study not only established APOL2 as a new potential therapeutic target for fibrotic liver disease but also highlighted the potential of DP as a promising lead compound for the treatment of liver fibrosis.³⁰

Fig. 3 Targeted imaging of APOL2 in liver fibrosis using DP fluorescent probes. (A) Schematic design of DP fluorescent probes. (B) and (C) Immunofluorescence images showing co-localization of APOL2 (green) and α-SMA (red) in hepatic tissues from both murine and human samples. The figures were adapted from Gan, L., et al. 2025³⁰ with permission from Springer Nature, copyright 2025.

2.1.4. Hepatic stellate cell (HSC) surface marker targeted probes. Excessive activation of hepatic stellate cells (HSCs) plays a pivotal role in promoting extracellular matrix accumulation during fibrotic liver progression, and the development of molecular probes targeting platelet-derived growth factor receptor beta (PDGFR-β), a surface marker of HSCs, is crucial for understanding the disease mechanism and precise intervention.^31,32 Overactivation of the PDGF-BB/PDGFR-β pathway promotes proliferation, migration and fibrotic phenotypic transformation of HSCs, but the off-target effects of conventional tyrosine kinase inhibitors are often caused by target generalization.^33,34 The recently discovered natural cyclic peptide destruxin A5 provides an innovative strategy for targeting PDGFR-β: it blocks ligand–receptor binding and downstream signaling activation by selectively binding to the PDGF–BB interaction interface in the extracellular structural domain of PDGFR-β (instead of the ATP-binding pocket). Using Biacore T200 surface plasmon resonance, heat transfer assay and micro thermophoresis, Destruxin A5 demonstrated nanomolar affinity for PDGFR-β, significantly inhibited HSC activation and collagen synthesis in a liver fibrosis model, and was shown to be effective in reversing the fibrotic process in animal studies.

Researchers have explored the potential of destruxin A5 as a fluorescent probe for HSCs, due to its unique targeting properties.³⁵ Coupling destruxin A5 with near-infrared fluorescent moieties allows for the construction of molecular probes capable of real-time tracing of activated HSCs. These probes have three advantages: (1) high specificity: based on the interfacial blocking mechanism, the probes can accurately identify activated HSCs with high PDGFR-β expression, avoiding the non-specific labelling of hepatic parenchymal cells; (2) dynamic monitoring: combining with the in vivo imaging technology, the spatial distribution of HSCs and the changes in activation status can be monitored and can be visualized in real time in fibrotic livers; (3) evaluation of the therapeutic efficacy: through the probes, the signal intensity and the degree of fibrosis can be quantified. Quantitative correlation between signal intensity and the fibrosis degree, and the therapeutic response of antifibrotic drugs (e.g., destruxin A5 derivatives) can be objectively assessed. In addition, the natural cyclic peptide backbone of destruxin A5 gives the probe good biocompatibility, which provides the basis for its clinical translation.

Despite the encouraging indications, further development of the optimization of destruxin A5 probes is required to address the challenges associated with pharmacokinetic characterization and multimodal imaging integration. The identification of destruxin A5 represents a significant advancement, offering a highly selective instrument for the study of PDGF-BB/PDGFR-β signaling. Furthermore, it provides a novel avenue for translating molecular mechanisms into clinical applications, facilitating targeted diagnosis and treatment of liver fibrosis.

2.2. Fluorescent probes based on enzyme activity

The progression of liver fibrosis is closely related to the abnormal regulation of specific enzyme activities. These enzymes play a key role in extracellular matrix remodeling, oxidative stress and inflammatory signaling pathways. Fluorescent probes based on enzyme activity can detect enzyme activity changes in the pathological microenvironment in real time and in situ by designing enzyme-responsive molecular switches, providing dynamic molecular information for disease staging and treatment evaluation. This section systematically reviews the fluorescent probes developed in recent years for liver fibrosis-related enzymes, focusing on their design principles and application breakthroughs in preclinical models. By combining quantitative imaging data with histopathological verification, these probes not only reveal the role of temporal and spatial heterogeneity of enzyme activity in fibrosis, but also lay a molecular foundation for the development of enzyme-targeted therapy (Table 3).

Table 3 Fluorescent probes based on enzyme activity

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2.2.1. γ-Glutamyl transpeptidase (GGT)-responsive probes. Elevated levels of γ-glutamyl transpeptidase (GGT) have been consistently associated with hepatic pathological conditions, including chronic liver damage, fibrotic progression, and hepatocarcinogenesis.²¹ This established relationship positions GGT as a potential biomarker for tracking fibrotic development in hepatic tissues.³⁶ Nevertheless, traditional methods for GGT quantification present several drawbacks, such as technical complexity, substantial operational costs, and lack of capacity for real-time biological monitoring.³⁷ In response to these challenges, Wang and colleagues³⁶ developed an innovative fluorescent detection system employing the Rho-GGT probe in conjunction with glutamic acid 5-hydrazide to enable precise measurement of GGT enzymatic activity. This represents the inaugural instance of a substrate being introduced with the objective of reducing spatial site resistance. Spectroscopic experiments demonstrated that Rho-GGT exhibited an increased red-emitting fluorescence at 632 nm in response to GGT. Furthermore, the researchers discovered that Rho-GGT demonstrated greater selectivity for GGT in comparison to other species, exhibiting an exceptional capacity for mitochondrial targeting with minimal cytotoxicity.

The Rho-GGT probe demonstrated effective detection of endogenous GGT activity in both LX-2 cell cultures and zebrafish models. Importantly, this imaging agent successfully monitored dynamic GGT changes in murine models of both acute hepatic damage and fibrotic liver disease. When applied to pharmacologically treated fibrotic mice, Rho-GGT provided quantitative assessment of GGT activity that correlated with established clinical parameters for fibrosis staging and therapeutic monitoring. Notably, the probe's imaging performance in hepatic tissue shows significant translational potential, as it can be integrated with conventional serum biomarkers and diagnostic modalities. Histopathological analysis confirmed consistent fluorescence patterns and morphological features between liver specimens from experimental mice and human patients with fibrosis (Fig. 4). This successful validation suggests that Rho-GGT enables accurate spatial mapping of GGT activity in hepatic injury, offering promising applications for both preclinical research and clinical diagnostics.

Fig. 4 Fluorescent probe Rho-GGT for the detection of GGT. (A) Schematic design of Rho-GGT. (B) In the fibrosis intervention study, silybin was administered orally to model mice, while glutamlhydrine and Rho-GGT were delivered via injection. (C) Representative hepatic tissue sections from normal, fibrotic, and treated groups were subjected to histological examination and fluorescence imaging. The figures were adapted from Wang, K., et al. 2023³⁶ with permission from Elsevier, copyright 2023.

For further accurate detection of GGT, Wang et al.³⁸ developed the probe ETYZE-GGT, which achieved bimodal imaging in far-infrared fluorescence (FL) and photoacoustic (PA) modes in several hepatocellular carcinoma (HCC)-related models. The selection of typical γ-glutamyl recognition groups and benzo-indole-xanthene signalling reporter genes ensured stability and adaptability to a range of conditions. The pattern of GGT mutation differs in cases of hepatocellular injury, liver fibrosis and hepatocellular carcinoma (HCC). In general, GGT levels in the context of liver injury exhibit a rapid increase to a certain value, while in liver fibrosis, they display relatively high fluctuations. In hepatocellular carcinoma, however, they tend to remain at relatively high levels. By conceptualizing the detection of GGT as a dynamic indicator rather than a simple value, it is possible to distinguish between these three processes. Furthermore, the researchers extended the probe's conjugate system to increase the emission wavelength, thereby enhancing the depth of penetration and potentially rendering it suitable for human tissue imaging.

While optical imaging probes show promise for hepatic fibrosis detection, their clinical application is often limited by poor tissue penetration depth, restricting in vivo imaging performance. To overcome this limitation, Miao and colleagues³⁹ engineered a dual-modality probe combining activatable fluorescence and photoacoustic imaging capabilities for specific fibrosis visualization. The molecular architecture incorporates a near-infrared thioxanthene-semicarbocyanine fluorophore conjugated with: a γ-glutamyl transpeptidase (GGT)-cleavable moiety and a cyclic RGD peptide for integrin targeting. This innovative design confers two critical functions: selective accumulation in fibrotic tissue through integrin binding, and subsequent activation of both fluorescent and photoacoustic signals upon GGT-mediated uncaging. Such target-responsive signal amplification enables accurate spatial mapping of fibrotic lesions, demonstrating the probe's potential for non-invasive diagnosis of early-stage hepatic fibrosis. This work establishes a novel design paradigm for developing dual-targeted molecular imaging agents for fibrotic diseases.

2.2.2. Monoamine oxidase-A/B(MAO-A/B)-sensitive probes. In 2020, Qin et al.⁴⁰ reported four near-infrared (NIR) fluorescent probes containing a dihydroxyanthracene backbone for the detection of monoamine oxidase A (MAO-A) in vivo and in vitro. The design of these probes capitalizes on propylamine-induced selective deprotection of MAO-A. One of these is the DHMP2 probe, which demonstrated a 31-fold fluorescence shift in vitro. Furthermore, it has the capacity to effectively concentrate within mitochondria and accurately quantify endogenous MAO-A levels in PC-3 and SH-SY5Y cell lines. The DHMP2 probe enables visualization of MAO-A activity in both zebrafish models and tumor-xenografted murine systems. Notably, this molecular tool was successfully adapted for pioneering studies of monoamine oxidase A dynamics in hepatic fibrosis specimens.

Subsequently, Tang et al.⁴¹ developed a TP fluorescence imaging probe to characterize monoamine oxidase-B (MAO-B) BiPhAA in vivo. The use of TP fluorescence imaging revealed that liver tissue from fibrotic mice exhibited fluorescence at a level 138 times greater than that observed in normal tissue, thereby enabling the differentiation between the two types of mice. The developed BiPhAA probe demonstrates superior sensitivity and precision as a fluorescent imaging agent for early-stage fibrosis detection. Additionally, this molecular tool shows potential for elucidating pathogenic mechanisms involved in hepatic fibrogenesis.

Furthermore, Mingzhao Sun et al.⁴² developed an enzyme-regulated activated fluorescent probe, YXHcy-NH2, for the detection of monoamine oxidase-B activity in living cells and in vivo (Fig. 5). The developed probe demonstrates excellent biocompatibility, target selectivity, and detection sensitivity. Upon activation by MAO-B within 100 minutes, it generates strong fluorescence emission at its characteristic wavelength. Experimental results revealed a 10-fold increase in the fluorescence signal in fibrotic LX-2 cells compared to healthy controls, along with a 5-fold enhancement in liver tissues from fibrotic mice relative to normal specimens. This work establishes a straightforward, rapid, and sensitive approach for in situ MAO-B imaging in living systems, offering promising potential for early-stage liver fibrosis diagnosis.

Fig. 5 The YXHcy-NH2 optical probe enables targeted MAO-B detection in fibrotic liver tissue. (A) Structural composition and operational mechanism of the molecular probe. (B) Comparative in vivo fluorescence imaging of MAO-B expression in mice at varying fibrosis severity levels. The figures were adapted from Sun, M., et al. 2024⁴² with permission from Elsevier, copyright 2024.

2.2.3. Aminopeptidase N (APN)-targeted nanoprobes. Aminopeptidase N (APN/CD13), a ubiquitously expressed transmembrane ectoenzyme, serves critical functions in physiological homeostasis.^43,44 Elevated enzymatic activity of this protein has been correlated with hepatic fibrogenesis and non-alcoholic fatty liver pathogenesis, highlighting the clinical need for non-invasive APN detection methodologies in both diagnostic and research applications. Xiao Sun and colleagues⁴⁵ developed Hcy-APN@MSN as a fluorescent nanoprobe for the selective and sensitive detection of APN changes in NAFLD and liver fibrosis cell/mouse models. Analysis demonstrated that increasing APN expression levels directly corresponded with worsening stages of NAFLD and fibrotic liver damage. The study also established the pivotal involvement of intracellular APN in driving disease progression. Furthermore, the study demonstrated that bestatin was effective in reducing APN activity in cellular and murine models of NAFLD and liver fibrosis. Hcy-APN@MSN was designed to enable real-time monitoring of APN activity fluctuations in both cellular environments and living organisms. This capability facilitates the diagnosis of liver fibrosis and NAFLD (Fig. 6).

Fig. 6 Hcy-APN probe for liver fibrosis detection. (A) Detection mechanism of Hcy-APN@MSN against APN. (B) Imaging of APN levels in mouse models of liver fibrosis. The figures were adapted from Sun, X., et al. 2024⁴⁵ with permission from American Chemical Society, copyright 2024.

2.3. Microenvironment-responsive probes

The liver fibrotic microenvironment is a dynamic interplay of physical and chemical alterations that drive disease progression.^31,46 Physical changes (e.g., increased tissue stiffness and organelle structural remodeling) and chemical imbalances (e.g., oxidative/reductive stress and enzyme dysregulation) collectively serve as hallmarks of fibrosis.^47,48 Probes responsive to these microenvironmental cues not only enhance diagnostic accuracy but also provide mechanistic insights into fibrogenesis (Table 4).

Table 4 Physical microenvironment probes and intracellular targeting probes

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Physical properties of the fibrotic liver, such as elevated viscosity and organelle deformation, are critical indicators of ECM deposition and cellular dysfunction.^49,50 Probes targeting these biomechanical parameters enable non-invasive mapping of fibrosis severity and spatial heterogeneity. Despite their promise, challenges remain in achieving tissue-specific targeting and minimizing interference from non-fibrotic pathologies (e.g., inflammation-induced edema). Below, we discuss three key subcategories of physical microenvironment sensors.

2.3.1. Viscosity-sensitive probes. Cellular viscosity serves as a critical microenvironmental parameter that can provide insights into complex biological processes under both physiological and pathological conditions.^51,52 Building on this concept, Yin's research team created an NIR-emissive molecular probe specifically tailored to measure liver viscosity. The probe's responsiveness stems from restricted rotation of the C–C bond linking phenyl spacers to benzo(a)pyrene salts in high-viscosity microenvironments. This mechanism was confirmed using the LCFM technique, which enabled real-time monitoring of mitochondrial viscosity alterations in living cells following stimulation with H₂O₂ and nitrastatin. Additionally, the probe (LV) successfully visualized viscosity changes in both CCl₄-induced fibrotic liver models and MET-treated specimens, demonstrating its potential as a diagnostic tool for hepatic fibrosis.⁵³

Elevated endoplasmic reticulum viscosity shows significant correlation with liver fibrosis development, suggesting its potential as a clinical biomarker.⁵⁴ The research team has developed HBT-PP, the first viscosity-responsive near-infrared (NIR) fluorescent probe targeting the ER, for use in studying the pathogenesis of liver fibrosis. The probe combines two distinct mechanisms, namely twisted intramolecular charge transfer (TICT) and excited-state intramolecular proton transfer (ESIPT), with a selective response to viscosity (∼62-fold) and an exceptionally large Stokes shift (∼320 nm). The HBT-PP probe demonstrates selective localization to the endoplasmic reticulum, facilitating real-time monitoring of viscosity changes induced by pharmaceutical compounds or ethanol exposure in cellular systems. Notably, compared to healthy liver, this imaging tool has revealed sex-dependent variations in hepatic fibrogenesis and provided visual evidence for estrogen's protective effects against disease progression. Remarkably, fibrotic liver tissue showed differential staining patterns within just 10 minutes of HBT-PP application, suggesting its utility for rapid clinical assessment of fibrosis severity. These findings collectively highlight HBT-PP's significant value as a research tool for studying ER-related pathophysiological mechanisms.⁵⁵

Emerging research suggests that abnormal nitric oxide (NO) concentrations and elevated hepatic viscosity could serve as diagnostic indicators for liver fibrosis.⁵⁶ In response to this finding, Song et al. (2023) designed a fluorescence ratio-based sensing probe, BDP, to simultaneously detect upregulated NO and microenvironmental viscosity for fibrosis assessment. As depicted in the figure, the probe incorporates an aromatic secondary amine with high electron density as the NO-responsive unit, where N-nitrosylation modulates the intramolecular charge transfer (ICT) process for ratiometric imaging (Fig. 7A). At low viscosity, fluorescence quenching occurs due to unrestricted C–C bond rotation between the N-methylaniline moiety and the BODIPY fluorophore. However, in the highly viscous fibrotic liver microenvironment, restricted molecular rotation enhances fluorescence emission. Upon NO binding, the probe undergoes rapid conversion from a secondary amine to an N-nitrosamine, accompanied by a spectral shift from red to orange emission. Comparative imaging analysis revealed that fibrotic liver tissues from murine models exhibited pronounced inflammatory infiltration, along with significantly intensified NO fluorescence signals and higher viscosity than healthy controls (Fig. 7B).⁵⁷

Fig. 7 Dual-parameter detection using the ratiometric probe BDP for nitric oxide and viscosity monitoring in hepatic fibrosis. (A) Working principle of the BDP probe, demonstrating its simultaneous response to NO concentration and microenvironmental viscosity changes. (B) Comparative fluorescence imaging analysis of NO levels in hepatic tissues from healthy control mice and fibrotic model mice, with the corresponding ex vivo organ distribution profiles. The figures were adapted from Han, S., et al. 2023⁵⁷ with permission from Elsevier, copyright 2023.

2.3.2. Interstitial space probes. Lee et al.⁵⁸ established a novel approach for detecting hepatic fibrosis by analyzing the diffusion patterns of fluorescent single-walled carbon nanotubes (SWCNTs) in the extracellular matrix. Their experimental results revealed that SWCNTs exhibited constrained movement through progressively narrower interstitial channels as fibrosis developed in murine models. Notably, these biophysical changes were detectable during early disease stages prior to observable histological alterations.

Key findings include: (1) SWCNT trajectory analysis revealed decreasing exploration distances correlating with disease progression. (2) The restricted nanotube mobility resulted from spatial confinement rather than changes in diffusion kinetics. (3) This methodology demonstrated superior sensitivity for early fibrosis detection compared to conventional histopathology. This nanotechnology-based approach provides new insights into the biophysical microenvironmental changes accompanying fibrogenesis, offering potential for both early diagnosis and fundamental studies of disease mechanisms.⁵⁸

2.4. Intracellular targeting probes

2.4.1. Organelle-targeted probes. As a core organelle for intracellular degradation and signal regulation, lysosomal dysfunction is a key factor in the progression of chronic inflammatory diseases, such as liver fibrosis.^50,59 Studies have shown that the persistent inflammatory microenvironment during liver fibrosis can lead to decreased lysosomal membrane stability, impaired enzyme activity and clearance dysfunction, which in turn exacerbate the accumulation of damage-associated molecular patterns (DAMPs) and the abnormal activation of hepatic stellate cells (HSCs).⁶⁰ In order to accurately resolve lysosomal dynamics, the LysoI probe, which is designed based on the carbazole-BODIPY structure, shows significant advantages: its 15-minute rapid targeting of lysosomes, combined with the large Stokes shift of 180 nm and up to 24-hour stability, thus overcoming the limitations of traditional dyes that are prone to photobleaching and have low signal-to-noise ratios, providing a reliable tool for long-time imaging of living cells (Fig. 8). Utilizing the LysoI probe, researchers observed that under inflammatory stimulation, lysosomes in hepatocytes exhibited aberrant phenotypes, including a reduced number, augmented volume, and diminished movement rate. Furthermore, they ascertained that tissue protease leakage, triggered by lysosomal membrane permeabilization (LMP), activated the caspase cascade reaction, thereby promoting apoptosis and collagen deposition. These results demonstrate that lysosomal impairment serves not merely as a secondary effect of hepatic fibrosis, but rather as a critical component in the self-perpetuating lysosome-inflammation-fibrosis cycle that exacerbates disease advancement. The application of LysoI probes has significantly enhanced comprehension of fibrogenic molecular pathways while simultaneously providing a platform for creating comprehensive lysosome-directed treatment strategies.

Fig. 8 BODIPY carbazole derivative probe for lysosomal imaging. (A) Schematic illustration of BODIPY. (B) Confocal images of LysoI-labeled lysosomes and a commercial nuclear labeling dye (Syto63) co-staining in normal LX2 cells and LX2 cells with liver fibrosis induced by TGF-β. The figures were adapted from Su, L., et al. 2025⁶¹ with permission from American Chemical Society, copyright 2025.

2.4.2. Intracellular redox-sensitive chemical probes. Chemical imbalances in the fibrotic liver—marked by oxidative stress (ROS/RNS overproduction) and disrupted redox homeostasis (GSH depletion)—are both drivers and consequences of fibrosis.^62,63 Probes detecting these chemical signals offer real-time monitoring of disease activity and therapeutic responses. This section reviews probes tailored to oxidative and reductive microenvironments.^49,64
2.4.2.1. Oxidative stress probes. The pathogenesis and progression of hepatic fibrosis are closely linked to the propagation of reactive oxygen species (ROS) cascades. The superoxide anion (O₂⁻), the primary ROS, is generated at mitochondrial and plasma membranes by NADPH oxidase (NOX). This species drives NF-κB pathway activation, inducing the release of inflammatory mediators.⁹ Its downstream metabolite hydrogen peroxide (H₂O₂) acts as a messenger in TGF-β signaling, directly stimulating collagen synthesis in hepatic stellate cells (HSCs).⁶⁵ Within iron-overloaded microenvironments, H₂O₂ undergoes Fenton reactions yielding highly reactive hydroxyl radicals (˙OH), which provoke DNA strand breaks and lipid peroxidation in hepatocytes.⁶⁶ Furthermore, neutrophil-derived hypochlorous acid (HOCl) and inflammation-site peroxynitrite (ONOO⁻) contribute to protein halogenation and cellular necrosis, establishing a self-perpetuating cycle. Recent advances in molecular probe technology enable precise mapping of ROS dynamics, marked by key developments: the genetically encoded probe SOx-FRP⁶⁷ leverages fluorescence resonance energy transfer (FRET) to achieve macrophage-specific O₂⁻ imaging. Its 0.5 μm spatial resolution demonstrated that metformin suppresses O₂⁻ generation by 60% through NOX4 inhibition.

However, clinical translation faces three persistent challenges: (a) Limited specificity: certain chemical probes (e.g., boronate-based) fail to discriminate between H₂O₂, ONOO⁻, and HOCl.⁶⁸ (b) Inadequate dynamic range: pathological ROS concentrations can reach millimolar levels, exceeding the typical probe saturation threshold of 500 μM. (c) Metabolic instability: reduced CYP450 activity in fibrotic patients accelerates degradation of cyanine-benzopyran-based probes.⁶⁹

The research team led by Zhang developed a pair of wavelength-tunable fluorescent dyes by modifying TPQL structures with electron-accepting groups and expanded conjugation systems.^70,71 Among these, TPCO2 shows particular promise for biological imaging applications due to its bright red emission, favorable two-photon characteristics, and good water solubility - features that collectively enable deep-tissue visualization with superior signal discrimination. Based on the excellent photophysical characteristics of TPCO2, two distinct fluorescent probes were developed. Among them, TPCO–NO₂ demonstrates remarkable selectivity and sensitivity toward nitroreductase (NTR), allowing for precise monitoring of NTR expression levels in cellular models under varying hypoxic conditions. Notably, this probe has been effectively utilized for high-contrast imaging of NTR activity in liver fibrosis models, providing a valuable tool for assessing hypoxia severity during fibrotic progression.⁷²

The research team led by Li et al. developed a novel hypoxia-responsive fluorescent probe named Cy-AP, which integrates a semi-cyanine fluorophore with an azo-based recognition moiety. Under normoxic conditions, this probe remains non-fluorescent. However, in hypoxic environments, the azo group undergoes reductive cleavage to form an amino group, triggering strong fluorescence emission. In vitro experiments revealed that Cy-AP displays excellent sensitivity and specificity toward sodium dithionite (Na₂S₂O₄). This probe was successfully applied for hypoxia detection in four distinct cell lines (HepG2, HCT 116, HeLa, and MCF-7) using fluorescence microscopy, flow cytometry, and three-dimensional imaging techniques. Additionally, owing to its near-infrared (NIR) emission properties, Cy-AP enabled real-time monitoring of hypoxic conditions in mouse models of liver fibrosis. Notably, this probe provided the first experimental evidence supporting the functional existence of the “gut-liver axis” in vivo through hypoxia tracking. As the first NIR fluorescent probe specifically designed for hypoxia detection in enterohepatic studies, Cy-AP represents a powerful tool for investigating the pathological mechanisms underlying liver fibrosis and may contribute to the development of improved therapeutic strategies for liver-related diseases.⁷³

In 2023, Yuan and colleagues introduced an innovative strategy for tracking liver fibrosis development through a fluorescent probe specifically targeting hypochlorous acid (HOCl).⁷⁴ The designed probe, based on a phenothiazine-coumarin hybrid structure (PC-Py), displayed outstanding detection capabilities, including high sensitivity and selectivity toward HOCl, robust photostability, and low cellular toxicity. Notably, PC-Py could accurately measure trace amounts of HOCl and monitor minute fluctuations at intracellular HOCl concentrations. These results highlight HOCl's role as a potential diagnostic marker for distinguishing between progressive liver fibrosis and acute drug-induced hepatic damage, offering valuable insights for early-stage fibrosis identification and therapeutic intervention. The probes were predominantly localized within lysosomes, rather than mitochondria. Concurrently, the findings were corroborated by trichrome staining in conjunction with hematoxylin–eosin and Masson, which elucidated the structural alterations and progression of liver fibrosis in tissue sections. The observations indicate that HOCl has the potential to serve as a biomarker for differentiating between liver fibrosis, acute liver injury, and cirrhosis. This makes the probe an appropriate tool for monitoring HOCl-induced cellular alterations and a promising avenue for the diagnosis and treatment of liver-related disorders.

Accumulation of peroxynitrite (ONOO⁻), a potent oxidizing agent, has been demonstrated to contribute to the pathogenesis of hepatic fibrosis, as supported by multiple studies.^75–79 To enable precise detection and evaluation of treatment efficacy in fibrotic liver disease, Zhang Tianao and colleagues⁸⁰ designed a selective ratiometric fluorescence sensor named Golgi-PER, which exhibits remarkable sensitivity in monitoring ONOO⁻ levels (Fig. 9). The probe exhibits favorable biocompatibility, optimal cell membrane permeability and effective Golgi-targeting capability, thereby enabling its utilization for ratiometric monitoring of ONOO⁻ fluctuations within the Golgi apparatus and the identification of polyphenol inhibitors in living cells. Concurrently, the therapeutic response of liver fibrosis to rosmarinic acid (RosA) was meticulously visualized for the first time by overexpression of Golgi ONOO⁻ in liver fibrosis using Golgi-PER as a probe. Consequently, this probe can be an effective and useful tool for investigating the occurrence, development and treatment of liver fibrosis.

Fig. 9 Golgi-PER probe for ONOO⁻ imaging. (A) Detection principle of Golgi-PER, a ratiometric fluorescent probe targeting ONOO⁻ in the Golgi apparatus. (B) Tracking peroxynitrite (ONOO⁻) dynamics in liver fibrosis models using Golgi-PER. The figures were adapted from Zhang, T., et al. 2024⁸⁰ with permission from Elsevier, copyright 2024.

Oxidative stress serves as a core pathological mechanism driving disease progression in hepatic fibrosis, involving an imbalance of multiple reactive oxygen species (ROS). Beyond the previously detailed hypochlorous acid (HOCl) and peroxynitrite (ONOO⁻), other critical ROS—including hydrogen peroxide (H₂O₂), hydroxyl radical (˙OH), superoxide anion (O₂˙⁻), and singlet oxygen (¹O₂)—play significant roles in hepatic stellate cell (HSC) activation, extracellular matrix deposition, and inflammatory cascades.⁸¹ The aberrant accumulation of these ROS exacerbates fibrosis by promoting lipid peroxidation, DNA damage, and collagen cross-linking. Recent advances in highly specific fluorescent probes have significantly enhanced monitoring capabilities for these molecular dynamics, providing new tools for early diagnosis and mechanistic studies of hepatic fibrosis. The following sections systematically elaborate on the design principles, application performance, and limitations of various ROS probes.

H₂O₂, a key signaling molecule in hepatic fibrosis, exhibits elevated concentrations directly correlated with TGF-β pathway activation and HSC stimulation. For instance, the IR-990 probe developed by Tian et al.⁸² achieved triple optimization through molecular engineering: emission of intense NIR-II fluorescence at 990 nm; generation of a large Stokes shift (200 nm) upon reaction with H₂O₂, effectively circumventing tissue autofluorescence; quantitative detection range spanning 2–250 μM with a low detection limit (0.59 μM), enabling precise capture of H₂O₂ fluctuations in pathological microenvironments. In validation studies, this probe not only successfully visualized endogenous H₂O₂ bursts in HepG2 cells but also achieved a high signal-to-noise ratio (11.3 : 1) in an acetaminophen (APAP)-induced liver injury mouse model, representing the first demonstration of noninvasive DILI progression monitoring. Addressing the critical challenge of microenvironment specificity, the Z-1065 probe developed by Chen's team⁸³ employs a mitochondrial targeting strategy. This probe exhibits excellent selectivity (<5% interference from other ROS) and biocompatibility (negligible cytotoxicity). Its innovative value lies in: confirmed response specificity through >80% fluorescence reduction upon treatment with the ROS scavenger N-acetylcysteine (NAC), successful spatiotemporal imaging of mitochondrial H₂O₂ bursts at the subcellular scale in an isoniazid (INH)-induced liver injury model, revealing spatial co-localization patterns between oxidative stress and hepatic damage.

Hydroxyl radicals (˙OH) and superoxide anions (O₂˙⁻) act as central mediators of hepatocyte lipid peroxidation and collagen cross-linking due to their potent oxidizing capacity and ultrashort half-lives (nanosecond scale).^84,85 For ˙OH detection, Yu et al.⁸⁶ reported the novel, highly selective turn-on fluorescent probe BIJ-H. Specific for ˙OH, its oxidative dehydrogenation generates the fluorescent product BIJ, emitting significantly enhanced near-infrared (NIR) fluorescence at 625 nm. BIJ-H exhibits stability under physiological conditions, rapid response kinetics, high selectivity for ˙OH, and a low detection limit (0.1379 μM). Studies demonstrate that BIJ-H enables sensitive, spatiotemporally resolved imaging of both exogenous and endogenous ˙OH in HepG2 cells. It successfully monitored ˙OH level fluctuations in acetaminophen (APAP)-induced liver injury mouse models, demonstrating significant potential for assessing oxidative stress in ˙OH-related pathologies. Targeting O₂˙⁻, Wang et al.⁸⁷ developed the activatable NIR fluorescent probe QL-3F, specifically designed for this key ROS. Its core advantage—a large Stokes shift—enables specific localization and response to site-specific O₂˙⁻ generation. At the cellular level, QL-3F precisely monitored endogenous O₂˙⁻ dynamics within hepatocytes (HepG2 cells), triggered by stimuli such as lipopolysaccharide (LPS), phorbol ester (PMA), and APAP-induced hepatotoxicity.

Singlet oxygen (¹O₂), generated in periductal fibrosis regions via bilirubin photosensitization, promotes myofibroblast differentiation and collagen deposition. The BAD probe,⁸⁸ a BODIPY-anthracene derivative, activates NIR fluorescence (λ_em = 780 nm) upon ¹O₂-triggered [4+2] cycloaddition. It achieved the first spatially resolved imaging of periductal fibrosis, offering a new tool for studying primary sclerosing cholangitis mechanisms. However, future clinical translation requires integration with bile duct-targeted delivery systems to enhance specificity.

2.4.2.2. Reductive stress probes. Reductive stress in hepatic fibrosis manifests as a systemic imbalance of antioxidant molecular networks, with core mechanisms involving three key molecular entities: (a) diminished hydrogen sulfide (H₂S) synthesis: mediated through downregulation of cystathionine γ-lyase (CSE) expression in hepatocytes,⁸⁹ attenuating H₂S-mediated suppression of the TGF-β/Smad3 pathway and accelerating collagen deposition. (b) Cysteine (Cys) depletion driving glutathione (GSH) cycle collapse: reduced Cys concentrations in portal hypertension models disrupt the γ-glutamyl cycle, inducing mitochondrial oxidative phosphorylation uncoupling and hepatocyte apoptosis.⁹⁰ Concomitantly, a positive shift ≥20 mV in the Cys/cystine (CySS) redox potential (Eh) emerges as a biomarker for hepatic stellate cell (HSC) activation. (c) Loss of reduced thioredoxin (Trx) status: decreased Trx-(SH)₂ in NASH models sustains apoptosis signal-regulating kinase 1 (ASK1) activation, promoting fibrosis via the p38-MAPK pathway.⁹¹

Advances in probe technology targeting these pathways focus on precise localization and dynamic monitoring. Li et al.⁹² developed the liver-targeted fluorescent probe series H₂S-YL for in situ specific detection of hepatic hydrogen sulfide (H₂S). Its core innovation integrates a cholic acid (CA) moiety as an active liver-targeting unit. Binding to hepatobiliary-specific transporters drives precise probe accumulation within liver tissue. This hepatobiliary-specific localization enables in situ, high-efficiency monitoring of dynamic H₂S fluctuations in both physiological conditions and drug-induced liver injury (DILI) models, facilitating assessment of its correlation with DILI severity. Validation confirms that H₂S-YL combines rapid response kinetics (<3 min), high sensitivity (LOD = 0.83 μM), and excellent H₂S selectivity, enabling successful site-specific H₂S imaging in both in vitro (hepatocytes) and in vivo (murine liver) settings.

Cui et al. developed ZpSiP, a novel reversible FRET-based biosensor capable of precise glutathione (GSH) quantification in living systems. The research team engineered this probe through strategic modification of phosphate-substituted rhodamine derivatives, optimizing both electronic configuration and steric hindrance to achieve selective reactivity. As shown in Fig. 10, the probe enabled ratiometric fluorescence monitoring of hepatic GSH fluctuations in fibrotic liver models under different experimental conditions, including pirfenidone treatment and graded CCl₄ exposure. Key findings include: (1) an excellent linear response (R² = 0.996) between probe signal intensity and tissue GSH concentrations, (2) successful demonstration of real-time GSH tracking in hepatic tissue, (3) validation of the probe's sensitivity for early detection of oxidative stress-related lesions. This work represents a significant advancement in molecular imaging, providing researchers with a robust tool for quantitative assessment of redox dynamics in biological systems.⁹³

Fig. 10 ZpSiP probes for GSH imaging. (A) Synthetic routes and reaction principles of four ratiometric GSH-responsive probes. (B) Ratiometric fluorescence visualization of hepatic GSH dynamics in murine models. The figures were adapted from Li, N., et al. 2023⁹³ with permission from John Wiley and Sons, copyright 2023.

Jiao et al.⁹⁴ established the thioredoxin/thioredoxin reductase (Trx/TrxR) system as a pivotal molecular target in hepatic fibrosis progression, demonstrating its critical role for the first time.¹ Their study revealed that the compound BS, a specific TrxR inhibitor, exhibited significant therapeutic efficacy in a carbon tetrachloride (CCl₄)-induced mouse model of hepatic fibrosis. The primary cellular target of BS is hepatic stellate cells (HSCs), the central effector cells in fibrosis. BS effectively suppressed HSC activation. At the molecular level, BS acts on the transforming growth factor-β1/Smads (TGF-β1/Smads) signaling pathway. By precisely inhibiting signal transduction through this pathway, BS disrupts downstream profibrotic events, thereby ameliorating fibrotic severity. This study not only validated the Trx/TrxR system as a viable therapeutic target for antifibrotic intervention but also highlighted BS as a multifunctional compound. Its mechanism—targeted TrxR inhibition coupled with blockade of the TGF-β1/Smads pathway—provides a solid theoretical and experimental foundation for developing BS into a novel targeted therapeutic agent for clinical hepatic fibrosis management.

Clinical translation faces three principal challenges: (a) detection interference: hypoalbuminemia (<30 g L⁻¹) increases free cysteine (Cys) concentration, inducing false positives in Cy-OFF-type fluorescence-quenching probes. (b) Insufficient dynamic range: pathological glutathione (GSH) fluctuations (0.1–10 mM) exceed the linear detection range (upper limit: 5 mM) of probes like ZpSiP. (c) Low delivery efficiency: liver-targeting efficiency of H₂S prodrugs (e.g., NaHS) remains below 10%.

2.5. Multifunctional probes and emerging technologies

2.5.1. Multiplexed imaging probes. Peveler and colleagues developed an innovative multiplexed fluorescent sensing platform for liver fibrosis detection using minimal serum volumes.⁹⁵ Their system integrated three fluorescent reporters: pyrene (Py), dapoxy (Dap), and PyMPO dyes, each selected for their distinct photophysical properties. The methodology involved dissolving the polymer sensors in phosphate-buffered saline (PBS) followed by serum sample incubation in 96-well plates. Fluorescence emission profiles were then acquired through spectroscopic analysis. For data interpretation, the researchers implemented a two-tier analytical approach: (1) pattern recognition via linear discriminant analysis (LDA) achieved 80% diagnostic accuracy in differentiating fibrotic from healthy samples, and (2) receiver operating characteristic (ROC) evaluation demonstrated excellent discriminative capacity (AUROC = 0.89), surpassing clinically relevant thresholds. This polymer-based diagnostic platform shows comparable performance to established techniques including elastography and serum biomarker assays while offering potential advantages in throughput and sample requirements.

2.5.2. Genetic encoded probes. Lei Wang et al.⁹⁶ successfully constructed a reporter system for estimating hepatic stellate cell activation based on enhanced green fluorescent protein (EGFP) expression regulated by the collagen type I alpha 1 chain (COL1A1) promoter. The proposed assay provides optical detection of COL1A1 expression fluctuations, serving as an indicator of stellate cell activation in vitro. The model offers a novel cellular system for the screening and investigation of anti-liver fibrosis pharmaceuticals (Fig. 11). As Fig. 11B, across treatment groups, mRNA levels of COL1A1 and EGFP displayed comparable regulation. Relative to the negative control, TGF-β1 induced upregulation of both genes; this induction was attenuated by dihydrotanshinone I or artesunate, with inhibitory effects strengthening as drug concentrations increased.

Fig. 11 pLVX-COL1A1-EGFP reporting system. (A) Schematic illustrating the pLVX-COL1A1-EGFP reporter construct and its functional mechanism. (B) Dose-dependent effects of dihydrotanshinone I and artesunate on COL1A1 and EGFP transcript levels in LX-2-CE cells. The figures were adapted from Wang, L., et al. 2023⁹⁶ with permission from Journal of Peking University (Health Sciences), copyright 2023.

2.5.3. Theranostic probes. Zeng's research team⁹⁷ engineered an innovative theranostic platform (LnNRs@mSiO2-RBS) combining NIR-II ratiometric imaging with controlled nitric oxide release for simultaneous liver fibrosis diagnosis and treatment monitoring. This nanosystem demonstrated enhanced NIR-IIa emission specifically in fibrotic liver tissue. Thus, it can not only accurately detect early disease through noninvasive optical imaging, but also realize the real-time visualization of therapeutic NO distribution. The platform's diagnostic capability stems from its distinct fluorescence signature in pathological microenvironments, offering significant potential for clinical translation in hepatic disease management. In addition, laser-induced NO gas therapy at 980 nm demonstrated notable efficacy in alleviating and reversing liver fibrosis (Fig. 12). It is also noteworthy that the researchers were able to successfully perform in vivo near-infrared-II ratio imaging of the nitrogen oxide treatment process, thereby providing a precise, sensitive, and novel strategy for assessing the impact of nitrogen oxide on hepatic fibrotic progression. Consequently, the findings of this study offer a potential avenue for real-time efficacy prediction and highly specific and sensitive precision treatment of liver fibrosis.

Fig. 12 Dual-functional NIR-II nanoprobe for liver fibrosis theranostics. (A) Design strategy of the NIR-responsive nanoprobe integrating NO gas therapy with real-time fibrosis monitoring through ratiometric fluorescence imaging. (B) In vivo NIR-II fluorescence visualization of therapeutic NO delivery in murine liver fibrosis models. The figures were adapted from Jiang, M., et al. 2023⁹⁷ with permission from Elsevier, copyright 2023.

Facing the persistent clinical challenge of liver fibrosis lacking effective treatments, Ribera et al.⁹⁸ developed an innovative targeted photothermal therapy (PTT) strategy using gold nanorods (GNRs). Their approach capitalizes on the critical role of activated hepatic stellate cells (HSCs) and the overexpression of PDGFRβ on their surface during fibrosis progression. The researchers engineered anti-PDGFRβ antibody-coated GNRs designed to specifically bind activated HSCs. Utilizing the natural liver tropism of nanoparticles combined with this active targeting, the constructs selectively accumulated on target cells. Upon exposure to near-infrared (NIR) light, the plasmonic properties of the GNRs generated localized heat, inducing photothermal ablation specifically within targeted HSCs. In a CCl₄-induced mouse model of liver fibrosis, this nanoplatform-mediated PTT demonstrated significant therapeutic efficacy: it successfully reduced established fibrosis, diminished liver inflammation, and alleviated hepatocyte damage. The authors highlight this strategy's strength in targeting the core pathological process of fibrosis (activated HSC proliferation) irrespective of the underlying disease etiology, presenting it as a promising baseline for developing novel, pathology-focused antifibrotic therapies applicable potentially to fibrosis in other organs. This work exemplifies the potential of nanotechnology to fundamentally alter therapeutic strategies for complex chronic diseases.

Gold nanorods (Au NRs) enable combined photoacoustic imaging and photothermal therapy.⁹⁹ Xiang et al.¹⁰⁰ developed a gold nanostar (GNS)-based probe (GLTTs) engineered for specific targeting of receptors highly expressed on hepatocyte membranes. This active targeting was achieved by modifying the nanostars with glycyrrhetinic acid (GA), a ligand known to bind specific receptors abundant on the surface of liver parenchymal cells. Leveraging this mechanism, GLTTs demonstrated significantly enhanced accumulation in liver tissue (12.85-fold higher signal intensity) compared to unmodified GNS. This efficient hepatic parenchymal cell targeting proved foundational for the successful application of surface-enhanced Raman scattering (SERS) technology, enabling in situ detection of early molecular changes associated with fibrosis and the identification of 10 diagnostic biomarker peaks. This strategy establishes a crucial tool for non-invasive early diagnosis.

2.5.4. Quantum dot or carbon nanoparticle probes. Quantum dots (QDs) and carbon nanoparticles (CNPs) have shown unique potential in the diagnosis and treatment of liver fibrosis, but their design must balance optical performance, targeting efficiency, and biosafety. QDs, with their high fluorescence quantum yield and tunable emission wavelengths, are widely used for highly sensitive imaging of liver fibrosis. For example, Moreno-Lanceta et al. designed dendrimer–graphene carbon nanoparticles (DGNP) to specifically target CD11b⁺ macrophages for delivering the RNF41 gene, utilizing their quantum dot optical properties to achieve high-sensitivity fluorescence imaging in lesion areas, while inducing insulin-like growth factor 1 (IGF-1) to reverse fibrotic progression and promote liver regeneration.¹⁰¹ Near-infrared QDs (e.g., Ag₂S QDs) can integrate with photoacoustic imaging technology to overcome the penetration depth limitations of conventional optical imaging, improving the spatial resolution for staging liver fibrosis.¹⁰²

However, both materials face challenges in clinical translation. The heavy metal toxicity of cadmium-based QDs has spurred the development of cadmium-free alternatives (e.g., InP/ZnS QDs),¹⁰³ while the long-term retention risks of carbon nanoparticles require size optimization and surface modifications to improve renal clearance.¹⁰⁴ Furthermore, the heterogeneity of fibrotic liver tissues (e.g., necrotic areas or steatosis) may interfere with nanoparticle optical signals, necessitating the use of targeting ligands (e.g., collagen-binding peptides) or dual-modal imaging strategies to enhance detection specificity.

2.6. Technical hurdles in probe synthesis

The preparation of fibrosis-targeted fluorescent probes involves multi-step processes with critical technical bottlenecks: (1) ligand conjugation efficiency: covalent coupling of targeting moieties (e.g., aptamers and antibodies) to fluorophores often yields <30% conjugation efficiency due to steric hindrance, necessitating costly HPLC purification (e.g., ZY-1 aptamer probes require 5-step purification, doubling production costs). (2) Nanoparticle batch consistency: colloidal probes (e.g., Ag@MSN) exhibit ±15% size variability across batches when synthesized via traditional sol–gel methods, impairing biodistribution reproducibility. Microfluidic synthesis could reduce this to <5% but requires substantial capital investment. (3) Long-term stability: lyophilized probes (e.g., protein-based Col1-TRAP) lose 40% activity after 6-month storage at −80 °C, highlighting the need for cryoprotectant optimization.

3. Clinical challenges

At present, the translation of these promising laboratory findings into validated clinical tools remains in its nascent stages. Despite the wealth of preclinical data, the number of fluorescent probes that have progressed to formal human clinical trials for liver fibrosis diagnosis is remarkably limited. A pivotal study conducted by de Lédinghen et al.¹⁰⁵ exemplifies clinical utility: among 286 patients, a sequential protocol (M probe first, XL probe if M fails) achieved reliable liver stiffness measurements (LSM) in 91.2% of cases with comparable diagnostic accuracy, proving particularly critical for obese/BMI > 30 kg m⁻² cohorts where conventional methods fail. To date, the vast majority of these innovative probes exist primarily as research tools, with only a handful venturing into early-phase human studies.

This stark gap underscores the critical hurdles impeding clinical integration. Successfully deploying these probes requires overcoming key challenges: long-term biocompatibility/toxicity concerns, limited imaging depth, suboptimal pharmacokinetics/targeting efficiency, and the urgent need for standardized validation across diverse patient populations and liver disease etiologies.

3.1. Biocompatibility and long-term toxicity

The biocompatibility of fluorescent probes, along with their long-term toxicity, is one of the central barriers to their movement from the laboratory to clinical applications. Although many probes have demonstrated good biosafety in short-term experiments, e.g., no acute inflammation or organ damage was induced after intravenous injection, the potential risks of these probes in long-term, repeated use remain a major blind spot. At present, we cannot know whether the probes interfere with the normal function of immune cells or induce chronic inflammation, and whether the various degradation products of the probes have insidious effects on long-term liver and kidney functions. These issues still need to be systematically evaluated in future studies.

Beyond acute safety assessments, clinical limitations persist in probe biocompatibility. For instance, antibody-conjugated probes (e.g., anti-APOL2 probes) may trigger immune reactions (e.g., ADA formation) upon repeated administration, compromising both imaging accuracy and patient safety. Critically, fluorophore degradation products (e.g., cyanine dye-derived indoles) exhibit unpredictable hepatic accumulation in cirrhotic patients with impaired detoxification pathways, posing risks of secondary toxicity.

3.2. Imaging depth

The optical performance of existing probes is still limited by the maximum emission wavelength, resulting in suboptimal imaging depth and resolution. The tissue penetration ability of many of the current probes is insufficient, and the fluorescent signals are not able to reach the deep tissues of the liver and clearly outline the subtle changes in the fibrotic areas of the liver, thus failing to present a comprehensive, three-dimensional picture of the temporal and spatial progression of the disease.

Clinical applicability of optical probes is constrained by inherent physical limitations. While NIR-II probes (e.g., Ag₂S quantum dots) achieve deeper tissue penetration (∼5 mm), their signals remain insufficient for visualizing posterior liver segments in obese patients (BMI >30), where adipose layers exceed 4 cm. This necessitates complementary modalities like intraoperative laparoscopic imaging, increasing procedural invasiveness.

3.3. Pharmacokinetics and targeting efficiency

The in vivo distribution, clearance rate and targeting efficiency of probes directly affect the imaging signal-to-noise ratio. Currently, there is a common problem of non-specific enrichment or too fast renal clearance of probes. These problems greatly affect the accuracy and sensitivity of the diagnosis.

3.4. Standardization and clinical validation

There is a lack of standardized criteria for the evaluation of probe performance, including sensitivity thresholds, specificity validation methods, and stability indicators. In addition, most of the existing studies are limited to mouse models, and large-scale multicenter clinical trials are urgently needed to verify their reliability in human liver fibrosis.

Clinical translation further requires addressing cost-effectiveness and scalability. Current probe synthesis often relies on multi-step organic reactions or recombinant protein expression, which are resource-intensive and hinder mass production. For example, collagen-targeting protein probes (e.g., Col1-TRAP) necessitate mammalian cell culture systems, raising manufacturing costs by ∼30% compared to small-molecule probes. Additionally, regulatory hurdles, such as good manufacturing practice (GMP) compliance for clinical-grade probes, demand standardized protocols for purification, sterilization, and stability testing—areas still underdeveloped for most experimental probes.

4. Future directions

To overcome the above challenges, future research can focus on the following directions:

4.1. Important directions for technological innovation

In the future, fluorescent probes for liver fibrosis can focus on three directions: improving probe sensitivity and precision, probe function integration, and clinical utility.

4.1.1. Improvement of probe sensitivity and accuracy. First, researchers need to develop more sensitive probes for liver fibrosis; this includes pushing the boundaries of imaging depth and resolution. Second, probe design needs to break through the limitations of existing markers and move from “broad-spectrum targeting” to “precise identification”. This involves developing probes with higher molecular specificity and affinity for validated targets (e.g., optimized PDGFR-β probes like destruxin A5 derivatives) and exploring novel biomarker classes. For example, the development of probes capable of monitoring epigenetic changes (e.g., DNA methylation dynamics or specific non-coding RNA expression) specific to the very early stages of liver fibrosis is expected to capture molecular abnormalities prior to histological changes, thus achieving the ultimate goal of genuine early diagnosis.

4.1.2. Functional integration of probes. Single-function probes should gradually shift to diagnostic and therapeutic integrated probes. For example, the probe can directly ablate activated hepatic stellate cells through photothermal or photodynamic effects (potentially activated by NIR-II light for deeper penetration) after completing lesion imaging, realizing “immediate diagnosis and treatment”. Alternatively, drugs can be released in a spatiotemporally controlled manner when abnormalities in the microenvironment (e.g., specific enzyme activity, pH, and redox state) are detected, allowing for precise, on-demand treatment. This “sense-and-treat” paradigm can not only shorten the diagnosis and treatment process but also significantly reduce the burden of invasive operations on patients.

4.1.3. Clinical utilization. Transitioning molecular imaging probes from laboratory research to clinical applications demands careful balancing of sensitivity, specificity, and safety, alongside improvements in practicality and cost reduction. To advance in this direction, researchers should firstly simplify probe synthesis by developing robust, scalable, and cost-effective methods, such as microfluidics or optimized recombinant expression, to streamline production and purification. Secondly, optimizing in vivo performance is crucial: probes require engineered pharmacokinetics (e.g., extended circulation for enhanced target accumulation and rapid clearance of unbound agents), reduced immunogenicity, and improved biocompatibility to enable repeated use, underpinned by rigorous long-term toxicity studies. Thirdly, developing user-friendly imaging systems—like portable, affordable devices leveraging smartphone integration or miniaturized NIR-II cameras—will make the technology accessible for clinical settings and point-of-care diagnostics. Standardizing probe administration, image acquisition, and signal quantification protocols across different platforms and patient groups is crucial for reproducibility and reliable clinical applications.

4.2. Interdisciplinary integration

The design of novel probes can be based on multidisciplinary integration, and AI can accelerate the virtual screening of probe molecular libraries to predict the best combinations of targeting moieties and fluorophores, dramatically shortening the development cycle. For example, generative AI models can design novel probe backbones that combine high affinity and low toxicity. Secondly, materials science can be integrated into probe development, such as biocompatible materials to improve the in vivo distribution and metabolic efficiency of probes, and microfluidic organ-chip technology to simulate the microenvironment of liver fibrosis in a high-throughput manner, accelerating the validation of probe performance.

Recent advances in interdisciplinary research have significantly enhanced the diagnostic and therapeutic potential of fluorescent probes for liver fibrosis. Compared to traditional imaging modalities, fluorescence-based strategies now synergize with emerging technologies to address critical limitations. For instance, photoacoustic imaging (PAI) combines the molecular specificity of fluorescent probes with deep-tissue penetration (up to 5 cm), enabling quantitative mapping of collagen deposition in cirrhotic livers with submillimeter resolution. Meanwhile, CRISPR-based biosensors have been engineered to detect fibrosis-associated microRNAs in serum, achieving attomolar sensitivity through fluorophore-tagged sgRNA systems.

Compared with existing technologies, fluorescent probes achieve 10× higher molecular specificity for early-stage fibrosis, while MRI provides whole-organ stiffness mapping but lacks biomarker resolution. Also, surface-enhanced Raman scattering (SERS) probes enable multiplexed collagen typing but require complex instrumentation, whereas fluorescence imaging offers real-time bedside applicability. This interdisciplinary convergence not only refines diagnostic accuracy but also paves the way for personalized antifibrotic therapies.

4.3. Establishment of standards related to probe design and applications

To robustly facilitate the clinical translation of molecular imaging probes, establishing a comprehensive standardization framework is essential. This begins with defining unified performance metrics: internationally recognized consensus standards must be developed to evaluate core indicators such as sensitivity, specificity, limit of detection (LOD), dynamic range, photostability, batch-to-batch consistency, shelf-life stability, and biocompatibility/toxicity profiles. Building on these metrics, standardized validation protocols are critical, requiring rigorous procedures to validate probe performance across diverse model systems—from cell lines and primary cells to animal models—and ultimately in human samples, including standardized methods for correlating probe signals with gold-standard diagnostics like histopathology. Furthermore, the creation of well-characterized reference sample banks containing human liver tissues (normal and across various fibrosis stages and etiologies) and biofluids (serum, plasma) is vital, providing essential materials for probe calibration, validation, and reliable inter-laboratory comparisons. Open science practices, notably sharing probe characterization data, imaging protocols, and validation results, are fundamental for reproducibility and trust-building. In this context, clinically relevant platforms (e.g., humanized organoids and advanced in vitro models) play a pivotal role: they permit standardized, controlled testing and personalized probe development, thereby ensuring consistent clinical translation.

5. Conclusion

In the biomedical domain, fluorescence imaging has emerged as a powerful tool for investigating hepatic fibrosis, offering novel insights into its pathological mechanisms. Notably, the advancement of organic small-molecule fluorescent probes has become a key breakthrough in this research area, enabling more precise visualization and analysis of disease progression. This technological progress has substantially enhanced our comprehension of fibrotic liver disorders at the molecular level. Due to their precise localization and sensitive feedback, these probes are capable of closely tracking a wide range of biologically active substances and cells that are closely related to the pathological evolution of liver fibrosis. These include collagen, hepatic stellate cells and other core markers, which have greatly advanced the diagnostic research of liver fibrosis. Despite the impressive results, the road ahead is still challenging, and numerous challenges remain to be addressed. The transition from preclinical success to clinical utility demands urgent resolution of two bottlenecks: (1) clinical limitations rooted in human biological complexity (e.g., immune reactivity and anatomical variability) and (2) scalable probe synthesis with batch-to-batch consistency. Industrial partnerships must prioritize GMP-compliant production lines for ligand-functionalized nanoparticles, reducing costs by >50% through microfluidic synthesis platforms.

In conclusion, in order to transform the cutting-edge laboratory findings into efficacious clinical tools and illuminate the path to recovery for the majority of liver disease patients, researchers must cultivate the field of molecular design, dismantle the disciplinary silos, and harness the synergies of multiple fields to collectively overcome the existing challenges and accelerate the development of liver fibrosis diagnosis and treatment.

Author contributions

Conceptualization: Fu Wang; methodology: Yutong Lv, Zhe Ma, Yue Chong; formal analysis and investigation: Yutong Lv, Zhe Ma, Yue Chong; software: Yutong Lv, Zhe Ma, Yue Chong; writing – original draft preparation: Yutong Lv, Zhe Ma; writing – review and editing: Fu Wang; funding acquisition: Fu Wang; supervision: Fu Wang, Li Xue, Zhenlong Wang. All authors reviewed the manuscript.

Conflicts of interest

No potential conflicts of interest were disclosed.

Data availability

No primary research results, software or code have been included and no new data were generated or analyzed as part of this review.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 32271512 and 82572281) and Natural Science Basic Research Program of Shaanxi (no. 2022JC-56 and 2023-JC-ZD-43).

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Footnote

† These authors contributed equally to this work.

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