Fluorescence Imaging Applications Research Articles

Research progress of near-infrared fluorescence imaging in accurate theranostics in bladder cancer.

With the highest 5-year recurrence rate among malignancies, bladder cancer is a relatively common type of cancer that typically originates from the urothelial cells lining the bladder. Additionally, bladder cancer is one of the most financially burdensome neoplasms to medical institutions in terms of management. Hence, prompt identification and accurate handling of bladder cancer are pivotal for enhancing patient prognosis. Optical imaging has experienced remarkable advancements in fundamental medical research owing to its cost-effectiveness and capacity for real-time imaging. The utilization of near-infrared imaging techniques has also become a prominent area of research in recent times. By effectively decreasing the adverse effects of light scattering and tissue autofluorescence, this technique offers a deeper penetration depth, a better signal-to-noise ratio of images, and a clear resolution for imaging. Thus, this article introduces the application of near-infrared fluorescence imaging in diagnosing and treating bladder cancer. Furthermore, the paper delves into the field's obstacles, possibilities, and upcoming prospects. Near-infrared fluorescence has advantages over white or blue light in theory and in most articles. However, the lack of penetration depth of NIR fluorescence imaging is still a challenge. Despite notable improvements in the depth of near-infrared fluorescence imaging, the penetration of deeper tissues remains a barrier. It is our hope and pursuit that NIR fluorescence imaging technology can achieve good depth and precision in surgery.

Visualization of Hg2+ Stress on Plant Health at the Subcellular Level Revealed by a Highly Sensitive Fluorescent Sensor.

The presence of Hg2+ causes substantial stress to plants, adversely affecting growth and health by disrupting cell cycle divisions, photosynthesis, and ionic homeostasis. Accurate visualization of the spatiotemporal distribution of Hg2+ in plant tissues is crucial for the management of Hg pollution; however, the related research is still at its early stage. Herein, a small-molecule amphiphilic fluorescent probe (termed LJTP2) was developed for the specific detection of Hg2+ with a high sensitivity (~16 nM). Fluorescent imaging applications with LJTP2 not only detected the dynamic distribution of Hg2+ within plant cells at the subcellular level but also enabled the understanding of cell membrane health under Hg2+ stress. This study introduces a valuable imaging tool for elucidating the molecular mechanism of Hg2+ stress in plants, demonstrating the potential of the application of small-molecule fluorescent probes in plant science.

Multiphoton Excited Fluorescence Imaging over Metal-Organic Frameworks.

Multiphoton excited fluorescence (MPEF) imaging has emerged as a powerful tool for visualizing biological processes with high spatial and temporal resolution. Metal-organic frameworks (MOFs), a class of porous materials composed of metal ions or clusters coordinated with organic ligands, have recently gained attention for their unique optical properties and potential applications in MPEF imaging. This review provides a comprehensive overview of the design, synthesis, and applications of multiphoton excited fluorescence imaging using MOFs. We discuss the principles behind the fluorescence behavior of MOFs, explore strategies to enhance their photophysical properties, and showcase their applications in bioimaging. Additionally, we address the current challenges and future prospects in this rapidly evolving field, highlighting the potential of multiphoton excited fluorescence imaging by MOFs for advancing our understanding of complex biological processes.

QTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging.

Endogenous tagging enables the study of proteins within their native regulatory context, typically using CRISPR to insert tag sequences directly into the gene sequence. Here, we introduce qTAG, a collection of repair cassettes that makes endogenous tagging more accessible. The cassettes support N- and C-terminal tagging with commonly used selectable markers and feature restriction sites for easy modification. Lox sites also enable the removal of the marker gene after successful integration. We demonstrate the utility of qTAG with a range of diverse tags for applications in fluorescence imaging, proximity labeling, epitope tagging, and targeted protein degradation. The system includes novel tags like mStayGold, offering enhanced brightness and photostability for live-cell imaging of native protein dynamics. Additionally, we explore alternative cassette designs for conditional expression tagging, selectable knockout tagging, and safe-harbor expression. The plasmid collection is available through Addgene, featuring ready-to-use constructs for common subcellular markers and tagging cassettes to target genes of interest. The qTAG system will serve as an open resource for researchers to adapt and tailor their own experiments.

Thienothiophene-Benzopyran Derivative and AQ4N-Assembled Liposomes for Near-Infrared II Fluorescence Imaging-Guided Phototherapy, Chemotherapy, and Immune Activation.

Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), has attracted wide attention in tumor treatment. However, the hypoxic tumor microenvironment and the heat shock proteins produced by tumor cells significantly reduce their efficacy. Developing effective phototherapy agents that have high reactive oxygen species generation efficiency and photothermal conversion efficiency (PCE) while simultaneously utilizing the hypoxic tumor microenvironment is of great importance. Here, a thienothiophene-benzopyran derivative, BTPIC4F-C10 is designed and synthesized, with near-infrared (NIR) absorption and fluorescence. Then the lipid nanoparticles (LipBFCA NPs) which encapsulated BTPIC4F-C10 in a phospholipid bilayer together with hypoxia-activated prodrug banoxanthrone (AQ4N) are constructed for NIR-II fluorescence imaging-guided synergistic PDT/PTT/chemotherapy and immune activation. Under 808nm laser irradiation, LipBFCA NPs is a high singlet oxygen quantum yield of 20.2% and PCE of 78.8%. With ultra-high photon energy utilization efficiency of 99%, LipBFCA NPs is an excellent phototherapy effect. The hypoxic environment caused by phototherapy can further activate AQ4N to transform into chemically toxic AQ4 radicals to kill tumor cells. Moreover, phototherapy can induce immunogenic cell death, release tumor-associated antigens, and activate immune responses. This work provides a new way for the clinical application of fluorescence imaging in guiding tumor diagnosis and treatment.

Initial Application of Fluorescence Imaging for Intraoperative Localization of Small Neuroendocrine Tumors in the Pancreas: Case Report and Review of the Literature.

Indocyanine green is commonly used for laparoscopic hepatectomy but remains uncommon in pancreatic surgery. Given the increasing number of small neuroendocrine tumors found in the pancreas and the heavy reliance on laparoscopic ultrasound for intraoperative localization, we attempted to use indocyanine green for these tumors. Our results show good localization and have the potential to provide a valuable clinical aid. This case report details five patients with preoperative diagnosis of pancreatic neuroendocrine tumors of small endocrine tumors, intraoperative successful localization, and successful completion of laparoscopic partial resection of pancreatic tumors by indocyanine green fluorescence staining; none of the patients experienced serious complications after surgery and were discharged from the hospital, and routine pathology confirmed that four cases were pancreatic neuroendocrine tumors of G1 stage, and one case was pancreatic neuroendocrine cell hyperplasia. Fluorescence imaging technology safely aids in the intraoperative localization of small pancreatic neuroendocrine tumors.

Water-Soluble Bimodal Magnetic-Fluorescent Radical Dendrimers as Potential MRI-FI Imaging Probes.

Dual or multimodal imaging probes have become potent tools for enhancing detection sensitivity and accuracy in disease diagnosis. In this context, we present a bimodal imaging dendrimer-based structure that integrates magnetic and fluorescent imaging probes for potential applications in magnetic resonance imaging and fluorescence imaging. It stands out as one of the rare examples where bimodal imaging probes use organic radicals as the magnetic source, despite their tendency to entirely quench fluorophore fluorescence. Opting for organic radicals over metal-based contrast agents like gadolinium (Gd3+)-chelates is crucial to mitigate associated toxicity concerns. We utilized an amino-terminated polyamide dendrimer containing a 1,8-naphthalimide (Naft) fluorescent group, amino acid derivatives as linkers to enhance water solubility, and TEMPO organic radicals as terminal groups. The same dendrimer structure, featuring an equivalent number of branches but lacking the fluorophore group, was also functionalized with amino acid and terminal radicals to serve as a reference. Remarkably, we achieved a fully water-soluble dendrimer-based structure exhibiting both magnetic and fluorescent properties simultaneously. The fluorescence of the Naft group in the final structure is somewhat quenched by the organic radicals, likely due to photoinduced electron transfer with the nitroxyl radical acting as an electron acceptor, which has been supported by density functional theory calculations. Molecular dynamics simulations are employed to investigate how the dendrimers' structure influences the electron paramagnetic resonance characteristics, relaxivity, and fluorescence. In summary, despite the influence of the radicals-fluorophore interactions on fluorescence, this bimodal dendrimer demonstrates significant fluorescent properties and effective r1 relaxivity of 1.3 mM-1 s-1. These properties have proven effective in staining the live mesenchymal stem cells without affecting the cell nucleus.

Investigation of the Structure and Optical Properties of Polymethine-Based NIR-II Fluorophores Using Many-Body Perturbation Theory: GW-BSE Approaches.

Fluorescence imaging is a widely used technique for detecting pathophysiological microenvironments and guiding fluorescence-guided therapy owing to its noninvasiveness, high spatiotemporal resolution, ease of operation, and real-time monitoring capabilities. In particular, NIR-II materials are promising for fluorescence imaging applications because they exhibit reduced light scattering and absorption by biological tissues, enabling deeper imaging with improved spatial resolution and contrast compared to visible or first near-infrared imaging. NIR-II materials refer to those that emit in the second near-infrared region of the electromagnetic spectrum, spanning wavelengths from approximately 1000 to 1700 nm. The emission peaks of organic fluorophores within the NIR-II window are of particular interest due to their minimal biotoxicity, in vivo biocompatibility, and biodegradability. In this study, we investigated a new series of NIR-II fluorescent polymethine-based dyes and their NIR-II absorption properties using density functional theory and the GW-BSE approximation. Our calculated maximum absorption peak under the GW-BSE approximation showed good agreement with experimental results, demonstrating the potential of these dyes for NIR-II fluorescence imaging applications.

High Quantum Yields and Biomedical Fluorescent Imaging Applications of Photosensitized Trivalent Lanthanide Ion-Based Nanoparticles.

In recent years, significant advances in enhancing the quantum yield (QY) of trivalent lanthanide (Ln3+) ion-based nanoparticles have been achieved through photosensitization, using host matrices or capping organic ligands as photosensitizers to absorb incoming photons and transfer energy to the Ln3+ ions. The Ln3+ ion-based nanoparticles possess several excellent fluorescent properties, such as nearly constant transition energies, atomic-like sharp transitions, long emission lifetimes, large Stokes shifts, high photostability, and resistance to photobleaching; these properties make them more promising candidates as next-generation fluorescence probes in the visible region, compared with other traditional materials such as organic dyes and quantum dots. However, their QYs are generally low and thus need to be improved to facilitate and extend their applications. Considerable efforts have been made to improve the QYs of Ln3+ ion-based nanoparticles through photosensitization. These efforts include the doping of Ln3+ ions into host matrices or capping the nanoparticles with organic ligands. Among the Ln3+ ion-based nanoparticles investigated in previous studies, this review focuses on those containing Eu3+, Tb3+, and Dy3+ ions with red, green, and yellow emission colors, respectively. The emission intensities of Eu3+ and Tb3+ ions are stronger than those of other Ln3+ ions; therefore, the majority of the reported studies focused on Eu3+ and Tb3+ ion-based nanoparticles. This review discusses the principles of photosensitization, several examples of photosensitized Ln3+ ion-based nanoparticles, and in vitro and in vivo biomedical fluorescent imaging (FI) applications. This information provides valuable insight into the development of Ln3+ ion-based nanoparticles with high QYs through photosensitization, with future potential applications in biomedical FI.

The Influence of the Functional Group on the Physicochemical and Biological Properties of New Phenanthro[9,10-d]-Imidazole Derivatives.

The search for safe, cheap, and repeatable diagnostic methods is a fundamental research goal. Currently, great hope is placed on fluorescence imaging. However, the development of this method mainly depends on efficient fluorescent probes. Designing and obtaining new probes with potential applications in fluorescence imaging is very difficult because compounds of this type must meet several requirements related to their properties. Therefore, this article attempted to obtain and study new phenanthro[9,10-d]-imidazole derivatives (PK1-PK3) with potential application as fluorescent probes for fluorescence imaging. The main goal of the work was to assess the effect of two functional groups (such as the formyl group (PK2) and rhodanine-3-acetic acid (PK3)) on selected physicochemical properties and possibilities of practical application of the considered compounds. The conducted studies proved that the influence of the functional group is significant, as it causes a bathochromic shift in both absorption and emission results (by the order PK1 < PK2 < PK3). Moreover, all compounds could stain live cells cultured in vitro. The staining efficiency was not affected by the cell line, thanks to which we obtained the correct staining of both mouse and human cell lines. PK3 was the most attractive of the tested compounds due to its staining potential of live cells and retention after fixation. Our results also showed some antibacterial and antifungal activity of the newly synthesized compounds (PK1-PK3). Among them, PK3 showed the highest antimicrobial effect, especially against Gram-positive bacteria.

Optimization of GEM detectors for applications in X-ray fluorescence imaging

In this work a set of simulations that aim at the optimization of Micropattern Gaseous Detectors (MPGD) for applications in X-ray fluorescence imaging in the energy range of 3–30keV is presented. By studying the statistical distribution of electrons from interactions of X-rays with gases, the energy resolution limits after charge multiplication for 6keV X-ray photons in Ar/CO2(70/30) and Kr/CO2(90/10) were calculated, resulting in energy resolutions of 15.4(4)% and 14.6(2)% respectively. These two mixtures were studied in simulations to evaluate the advantages of using krypton-based mixtures to reduce the presence of escape peaks in fluorescence spectra. A model to evaluate the X-ray fluorescence from the conductive materials inside the detectors was implemented, serving as a tool to estimate the extent of contamination of fluorescence spectra when using copper or aluminum layers in the material composition of MPGDs.

A simple benzothiazolium-based sensor for cyanide detection: Applications in environmental analysis and bioimaging

A new sensor based on Ethylbenzothiazolium-2-hydroxynaphthaldehyde conjugate-based fluorescent sensor, (E)-3-ethyl-2-(2-(2-hydroxynaphthalen-1-yl) vinyl) benzo[d]thiazol-3-ium iodide (SU-1) was designed and synthesized. The structure of SU-1 was confirmed by 1H NMR, 13C NMR, HRMS, and single crystal XRD spectral analysis. SU-1 displayed a colorimetric and fluorometric response in a DMSO:H2O (1:1,v/v) matrix, changing color from pale yellow to colorless visible to the naked eye, accompanied by a ∼ 120 nm red-shift in the absorption spectra upon CN− addition. This shift, due to formation of deprotonation followed by the nucleophilic attack on the benzothiazolium ring’s double bond, disrupts π-conjugation, blocking intramolecular charge transfer within SU-1. However, competitive anions showed negligible interference while detecting CN−. The Limit of detection for CN− was determined to be 0.27 nM, significantly below the WHO’s permissible CN− concentration in drinking water (1.9 μM). Job’s plot analysis shows that the binding stoichiometry of SU-1 to CN− is a 1:1, with a stability constant (Ka) of 1.58 x 104 M−1. The sensor demonstrated practical applications in environmental water samples and fluorescence imaging of intracellular CN− in CAD cell line.

Red-Emitting Coumarin-Derived fluorescent probe for thiol detection and indirect recognition of β-Lactamase

Biothiols, comprising cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play a pivotal role in maintaining the delicate redox balance essential for life processes, intricately intertwined with the well-being of living organisms. Deviations in their concentrations can precipitate a range of diseases. Herein, we presented a novel fluorescent probe, DIcou-DNBS, derived from coumarin, which possessed red emission and a substantial Stokes shift (122 nm), tailored for biothiol detection. DIcou-DNBS demonstrated rapid-responsiveness (6 min) towards Cys, Hcy and GSH with detection limits of 0.015 µM, 0.032 µM and 0.059 µM, respectively. Furthermore, its excellent biocompatibility enabled successful applications in biothiol fluorescence imaging of living cells and zebrafish. Beyond biothiol sensing, DIcou-DNBS leveraged its unique property to indirectly detect β-lactamases, making it a valuable tool in inhibitor screening and susceptibility testing of both sensitive (Staphylococcus aureus ATCC 29213) and resistant (Enterobacter cloacae ATCC 13047) bacterial strains. This assay system anticipates to furnish a potent instrument for visualization studies across biological and medical domains.

Use of intra-operative fluorescence imaging in periprosthetic joint infection: State of the art and future perspectives.

In periprosthetic joint infections (PJIs), the surgeon's role becomes pivotal in addressing the infection locally, necessitating the surgical removal of infected and necrotic tissue. Opportunity to enhance the visualization of infected tissue during surgery could represent a game-changing innovation. The aim of this narrative review is to delineate the application of intraoperative fluorescence imaging for targeting infected tissues in PJIs. A systematic review, adhering to the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines, was carried out. The search included multiple online database; MEDLINE, Scopus, and Web of Science. For data extraction the following were evaluated: (i) diagnosis of musculoskeletal infection; (ii) use of intraoperative fluorescence imaging; (iii) infected or necrotic tissues as target. Initially, 116 studies were identified through online database searches and reference investigations. The search was narrowed down to a final list of 5 papers for in-depth analysis at the full-text level. Subsequently, 2 studies were included in the review. The study included a total of 13 patients, focusing on cases of fracture-related infections of the lower limbs. The primary and crucial role for orthopedic surgeons in PJIs is the surgical debridement and precise removal of necrotic and infected tissue. Technologies that enable clear and accurate visualization of the tissue to be removed can enhance the eradication of infections, thereby promoting healing. A promising avenue for the future involves the potential application of intraoperative fluorescence imaging in pursuit of this objective.

The Usefulness of Fluorescence Imaging in Laparoscopic Liver Resection

AbstractIndocyanine green (ICG) has been used extensively in medical applications after being approved by the Food and Drug Administration (FDA) in 1954. In the beginning, this substance was originally employed for evaluating cardiac output and quantifying liver function. The widespread acceptance of the technique was delayed until the early 2000s, when advancements in digital imaging resolution provided more satisfactory images compared with those of film-based photography. ICG-based fluorescence imaging has since been used in various aspects of liver surgery. In 2008, Aoki et al first reported an intraoperative technique for subsegmental and segmental fluorescent staining during open hepatectomy, achieving a success rate of 93% in identifying the anatomical plane. At about the same time, laparoscopic liver resections (LLRs) were increasingly used for liver malignancies but were still in a developmental phase with yet to be standardized surgical protocols. Combined ICG fluorescence imaging in LLR has since been developed to improve the quality and precision of anatomical liver resections. This article is a case presentation and review of the clinical applications of ICG fluorescence imaging in liver surgery.

Extending the emission peak tail of indole cyanine for deep-near-infrared bioimaging

We propose a novel strategy for tailoring the structure of fluorescent molecules to achieve emission at the tail end of the NIR-II window. The favorable spectroscopic properties and low cytotoxicity of YNs make them powerful tools for bioimaging. Notably, YN-4 exhibits a brightness 2.5 times greater than YN-3, 6 times that of IR-783, and 5 times that of ICG. This enhanced brightness enabled high-resolution imaging of mouse thoracic and abdominal cavities, tumor vasculature, and real-time monitoring of gastrointestinal motility using YN-4. Furthermore, covalent grafting of glucose onto the YN-Glu scaffold significantly improved tumor-targeting capability and facilitated tracking of glucose metabolism. This work aims to extend the application of fluorescent molecule imaging beyond the NIR-IIa window.

Initial application of near-infrared fluorescence imaging using indocyanine green in urethral reconstructive surgery

ABSTRACT Introduction: Near-infrared fluorescence (NIRF) imaging using indocyanine green (ICG) is increasingly being explored in various urological procedures, including urethral reconstructive surgery. This real-time technology assists surgeons in the visualization of critical anatomical structures, thereby potentially improving surgical precision and patient outcomes. Objective: This study aimed to report our preliminary experience using ICG technology in urethral reconstructive surgery using the SPY system by Novadaq. Materials and Methods: ICG technology was adopted in seven urethral reconstructive procedures performed in Saiful Anwar Hospital over 1 month in 2018. The procedures were performed by two surgeons, consisting of five excisions and primary anastomosis (EPA) and two substitution urethroplasties with real-time evaluation of ICG expression in corpus spongiosum intraoperatively. Result: The ICG solution was injected intravenously in all urethral reconstructive procedures. The ICG injection was allowed to visualize the corpus spongiosum in a matter of 30–60 s. The dosage of ICG used was 5 mg diluted in 10 mL of normal saline. One procedure had been converted from EPA to vascular-sparring anastomotic urethroplasty because of poor perfusion of the corpus spongiosum. All the procedures had good outcomes. No adverse and allergic reactions to ICG and other complications occurred postoperatively. Conclusion: Our preliminary experience confirmed the safety and efficacy of ICG technology in urethral reconstructive surgery. The main advantage of using NIRF imaging is allowing the surgeon to real-time evaluate corpus spongiosum qualitatively and quantitatively. The limitation is specific equipment needed like the SPY system by Novadaq.

Monitoring of three-dimensional live-cell cultures using a multimode, multiscale imaging system combining confocal fluorescence microscopy and optical coherence microscopy

Live-cell monitoring involves long-term observation and analysis of living cells in tissue cultures, which develop cells or tissues in an artificial environment and are essential for tissue engineering. Fluorescence microscopy (FM) is widely used as a monitoring tool owing to its powerful ability to visualize protein distribution within cells and tissues, thereby providing information on their functions in biological processes. To reduce the photobleaching effect, which is a major limiting factor in FM, FM is generally combined with other imaging modalities, such as phase contrast and differential interference contrast microscopy, which are nondestructive to fluorochromes, to selectively use fluorescence signals only in specific areas of interest within a sample. However, these methods are restricted to thin samples and cannot produce depth-resolved images. Here, we propose a method to determine three-dimensional (3D) positions in volumetric samples for application in high-resolution 3D fluorescence imaging using a multimode and multiscale imaging system that combines optical coherence microscopy (OCM) and line confocal FM (LC–FM). We also demonstrate the benefits of multimodal imaging in 3D cell culture monitoring. To evaluate the performance of the proposed system and method, we rapidly and accurately located the regions of interest in 3D tissue culture models, such as tumor spheroids and microvascular bed, and instantaneously acquired volumetric high-resolution fluorescence images. We also used an integrated system to monitor tumor spheroids mixed with extracellular matrix (ECM) over five days of the culture process, and the FM–OCM integrated technique successfully imaged the overall 3D structures, such as the distribution and boundaries of the spheroids and ECM as well as stained tumor cells. This complementary information is useful for understanding the relationship between cellular behaviors, such as the proliferation and migration of tumor cells, and microenvironments, such as the ECM.

TMVP1448, a novel peptide improves detection of primary tumors and metastases by specifically targeting VEGFR-3

Lymphangiogenesis at primary tumor and draining lymph nodes plays a pivotal role in tumor metastasis, which has been demonstrated to be regulated by the vascular endothelial growth factor receptor 3 (VEGFR-3) pathway. However, the effect of molecular imaging peptides, which specifically bind VEGFR-3, in tracing tumors remains unclear. We prepared a novel peptide, TMVP1448, with high-affinity to VEGFR-3. The dissociation constant (KD) of TMVP1448 with VEGFR-3 was 7.07 ×10‐7 M. In vitro cellular assay showed that TMVP1448 could bind specifically with VEGFR-3. Near infrared imaging results showed that Cy7-TMVP1448 was able to accurately trace primary and metastatic cancers, and PET/CT results showed that [68Ga]Ga-DOTA-TMVP1448 was superior to commonly used radiotracers 18F-FDG. Additionally, no significant negative effect of TMVP1448 was found in mice. Our results suggested that TMVP1448 had great potential for future clinical applications in fluorescence imaging and nuclear imaging of tumors.

Application of indocyanine green fluorescence imaging in hepatobiliary surgery.

Indocyanine green (ICG) is a fluorescent dye with an emission wavelength of about 840nm, which is selectively absorbed by the liver after intravenous or bile duct injection, and then it is excreted into the intestines through the biliary system. With the rapid development of fluorescence laparoscopy, ICG fluorescence imaging is safe, feasible, and widely used in hepatobiliary surgery. ICG fluorescence imaging is of great significance in precise preoperative and intraoperative localization of liver lesions, real-time visualization of hepatic segmental anatomy, intrahepatic and extrahepatic biliary tract visualization, and liver transplantation. ICG fluorescence imaging facilitates efficient intraoperative hepatobiliary decision-making and improves the safety of minimally invasive hepatobiliary surgery. Advances in imaging systems will increase the use of fluorescence imaging as an intraoperative navigation tool, improving the safety and accuracy of open and laparoscopic/robotic hepatobiliary surgery. Herin, we have reviewed the status of ICG applications in hepatobiliary surgery, aiming to provide new insights for the development of hepatobiliary surgery.