Clinical fluorescent contrast agents for image-guided surgery

Jingyao Wang: Writing – review & editing, Writing – original draft, Data curation, Conceptualization. Yu-Si Liao: Writing – review & editing, Writing – original draft, Data curation, Conceptualization. Jian-Xiao Liang: Writing – review & editing, Validation. Ni-Yuan Zhang: Conceptualization. Hong-Wei An: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. Hao Wang: Supervision, Project administration, Funding acquisition, Conceptualization.

LUM015, also known as Lumicell, is a novel small-molecule optical imaging probe. It comprises four key components: a fluorescent bursting agent molecule (QSY21), a GGRK peptide, a 20 kD polyethylene glycol (PEG), and a Cy5 fluorophore. In its intact molecular state, unaltered by histone enzymes, the fluorescence emission of Cy5 is absorbed by the quencher due to fluorescence resonance energy transfer (FRET), resulting in no fluorescence signal [ 53 ]. However, upon cleavage by histone protease, the quencher is liberated, generating an active fragment with a detectable fluorescence signal. Cy5 functions as the primary fluorophore, exhibiting peak absorption at 650 nm and peak emission at 675 nm [ 54 ]. This probe has demonstrated a unique capacity for evaluating colorectal tumors. In colorectal cancer, tissue proteases exhibit overexpression, and the real-time feedback provided by dual-channel colonoscopy allows optical molecular imaging to assume a more significant role in diagnosis and treatment [ 55 ]. Beyond colorectal cancer, LUM015’s feasibility clinical trials have been extended to other cancer types, including breast, brain, prostate, and peritoneal surface malignancies, as well as gastrointestinal tumors such as colon, esophageal, and pancreatic cancers [ 56 ]. These trials aim to further validate LUM015’s potential for a broad spectrum of applications in cancer diagnosis and treatment. In one study, the aim was to reduce residual disease in the tumor bed and to evaluate the advantages of the system in assessing positive margins for tumor subtypes [ 57 ]. In another study, LUM015 was used in conjunction with the LUM Imaging System in women undergoing breast cancer surgery with the aim of rapidly identifying residual tumor in the lumpectomy cavity of the breast tumor. The results of the study showed that the system demonstrated a high tumor detection sensitivity of 84%, a specificity of 73%, and a mean tumor-to-background ratio (TBR) of 3.52–5.69 [ 58 ]. Two pivotal multicenter Phase III studies are currently underway, with patient recruitment phase. The primary objectives are to develop distinct algorithms to evaluate the impact of LUM015 on residual tumor resection based on tissue histology and to assess its benefits in reducing positive margins [ 59 ]. As research progresses, LUM015 provides crucial support for cancer diagnosis and treatment, further advancing the clinical translation of optical molecular imaging. Biotechnology company Lumicell has pioneered the confirmation of LUM015’s safety and efficacy in detecting residual cancer cells in the surgical excision site of female breast cancer patients post-surgery through a Phase III clinical trial ( NCT03686215 ). In May 2023, the FDA announced its acceptance and prioritization of the review process for the LUMISIGHTTM New Drug Application, an optical agent designed for breast cancer imaging. SGM-101, a pioneering drug that ingeniously amalgamates an antibody with a fluorescent dye, presents a promising avenue for cancer diagnosis and treatment. The pivotal component of this drug, the BM104 fluorescent dye, exhibits a distinctive absorbance band at a wavelength as accurate as 700 nm. Notably, it exhibits robust binding affinity to a chimeric monoclonal antibody (mAb) targeting carcinoembryonic antigen (CEA) [ 60 ]. Research indicates that over 95% of colorectal cancer cells exhibit surface expression of CEA, and this specific antibody effectively identifies these cells. During surgical procedures, tumor tissue exhibiting overexpression of CEA becomes conspicuously visible. Phase I/II study was conducted to ascertain the optimal imaging dose of SGM-101 for intraoperative fluorescence imaging of primary and recurrent colorectal cancer (CRC). The findings revealed that the peak intraoperative mean tumor-to-background ratio (TBR) of 1.9 ( p = 0.019) was achieved at a dose of 10 mg, exhibiting 96% sensitivity and 63% specificity, along with a 94% negative predictive value [ 61 ]. Notably, SGM-101 demonstrates significant feasibility for intraoperative tumor detection under CEA-targeted fluorescence guidance in both colorectal cancer and pancreatic liver metastasis [ 62 ]. Furthermore, the data from this study indicated that in patients with peritoneal metastatic colorectal cancer, SGM-101 exhibited a sensitivity of 98.5%, a specificity of 62.2%, a positive predictive value of 82.3%, and a negative predictive value of 95.8% [ 63 ]. The existing preclinical data robustly support SGM-101 as a promising candidate for FIGS in CEA-expressing tumors, and it has successfully progressed to the evaluation phase of Phase III clinical trials, with the evaluation numbers NCT03659448 and NCT04642924 . Dr. Vahrmeijer’s ongoing Phase III investigation of SGM-101 is a comprehensive, multinational, multi-institutional study encompassing research centers in Italy, Germany, the Netherlands, and the United States. The research will employ contrast agents to accurately identify primary and recurrent colorectal tumors, as well as to detect infiltrating lymph nodes, liver metastases, and peritoneal metastases [ 59 ]. Intraoperative ureteral injury (IUI) presents a significant risk during intricate abdominal and pelvic procedures. This injury may result in various long-term complications, such as ureteral stricture, obstruction, ureterovaginal fistula, and acute or chronic renal failure. Consequently, intraoperative ureteral visualization has emerged as a crucial requirement. An optimal solution for this is ASP5354 (Pudexacianinium, TK-1), an innovative compound co-developed by Mie and Nagoya Universities, under exclusive global development and marketing rights held by Astellas. As an ICG derivative, ASP5354 emits NIRF when exposed to specific NIR light (wavelength 780 nm) at a wavelength of 820 nm [ 64 ]. Owing to its unique molecular size and hydrophilicity, the compound can be excreted into the urine, resulting in a distinctive green color ( Fig. 8 , Fig. 9 ). This characteristic makes ASP5354 an ideal candidate for ureteral imaging. Fig. 8 ASP5354 for imaging-guided surgery. (a) Chemical structure of ASP5354. (b) Ureter visualization in minimally invasive colorectal surgery using pudexacianinium at doses of 0.3 mg, 1.0 mg, and 3.0 mg, assessed 30 min post-administration and at the end of surgery. Adapted from Albert et al. (2023) with permission. (c) Fluorescence intensity of the index ureter at various time points during minimally invasive colorectal surgery with pudexacianinium doses of 0.3 mg, 1.0 mg, and 3.0 mg. Adapted from Albert et al. (2023) with permission. (d) Correlation between qualitative (surgeon-assessed) and quantitative (video-recorded) fluorescence intensity of the ureter in minimally invasive colorectal surgery using pudexacianinium (0.3 mg, 1.0 mg, 3.0 mg). The contrast enhancement factor (CEF) threshold is set at 1.5. Adapted from Albert et al. (2023) with permission. Fig 8 dummy alt text Fig. 9 Elucidation of the mechanism of SA-FCA and its application in fluorescence imaging of human tumor tissues in vitro. (a) Chemical structure of SA-FCA. (b) NIRF spectrum of SA-FCA. (c–g) High-performance tumor imaging of SA-FCA in human tumor-bearing bladder. Schematic diagram of the experimental design. SA-FCA (50 µM, 50 mL PBS) was instilled into the bladder for 60 min, followed by three washes with PBS. (d) Representative NIR fluorescence images of urothelial carcinoma detected by SA-FCA, confirmed by H&E histological evaluation. Scale bar: 50 µm. (e) Representative NIR fluorescence images of micro-tumors detected by SA-FCA, confirmed by H&E staining. Scale bar: 50 µm. (f–g) Representative NIR fluorescence images of inflammation and necrosis, confirmed by H&E staining. Scale bar: 50 µm. (h–i) Sensitivity and specificity of SA-FCA imaging in human bladder specimens. (h) Quantitative analysis of fluorescence intensity in biopsy specimens, including urothelium ( N = 60), inflammation ( N = 20), necrosis ( N = 18), urothelial carcinoma ( N = 160), squamous carcinoma ( N = 20), adenocarcinoma ( N = 30), carcinoma in situ ( N = 10), and other lesions ( N = 20). *** p < 0.001, NS: no significance. (i) sensitivity and specificity of SA-FCA in the diagnosis of 338 human bladder biopsy specimens. (sensitivity: 99.6%, specificity: 97.9%). Adapted from Hong-Wei An et al. (2024) with permission. Fig 9 dummy alt text ASP5354 for imaging-guided surgery. (a) Chemical structure of ASP5354. (b) Ureter visualization in minimally invasive colorectal surgery using pudexacianinium at doses of 0.3 mg, 1.0 mg, and 3.0 mg, assessed 30 min post-administration and at the end of surgery. Adapted from Albert et al. (2023) with permission. (c) Fluorescence intensity of the index ureter at various time points during minimally invasive colorectal surgery with pudexacianinium doses of 0.3 mg, 1.0 mg, and 3.0 mg. Adapted from Albert et al. (2023) with permission. (d) Correlation between qualitative (surgeon-assessed) and quantitative (video-recorded) fluorescence intensity of the ureter in minimally invasive colorectal surgery using pudexacianinium (0.3 mg, 1.0 mg, 3.0 mg). The contrast enhancement factor (CEF) threshold is set at 1.5. Adapted from Albert et al. (2023) with permission. Elucidation of the mechanism of SA-FCA and its application in fluorescence imaging of human tumor tissues in vitro. (a) Chemical structure of SA-FCA. (b) NIRF spectrum of SA-FCA. (c–g) High-performance tumor imaging of SA-FCA in human tumor-bearing bladder. Schematic diagram of the experimental design. SA-FCA (50 µM, 50 mL PBS) was instilled into the bladder for 60 min, followed by three washes with PBS. (d) Representative NIR fluorescence images of urothelial carcinoma detected by SA-FCA, confirmed by H&E histological evaluation. Scale bar: 50 µm. (e) Representative NIR fluorescence images of micro-tumors detected by SA-FCA, confirmed by H&E staining. Scale bar: 50 µm. (f–g) Representative NIR fluorescence images of inflammation and necrosis, confirmed by H&E staining. Scale bar: 50 µm. (h–i) Sensitivity and specificity of SA-FCA imaging in human bladder specimens. (h) Quantitative analysis of fluorescence intensity in biopsy specimens, including urothelium ( N = 60), inflammation ( N = 20), necrosis ( N = 18), urothelial carcinoma ( N = 160), squamous carcinoma ( N = 20), adenocarcinoma ( N = 30), carcinoma in situ ( N = 10), and other lesions ( N = 20). *** p < 0.001, NS: no significance. (i) sensitivity and specificity of SA-FCA in the diagnosis of 338 human bladder biopsy specimens. (sensitivity: 99.6%, specificity: 97.9%). Adapted from Hong-Wei An et al. (2024) with permission. In a completed Phase II clinical trial ( NCT04238481 ), it was observed that ASP5354, at doses of 1.0 and 3.0 mg, effectively visualized the ureter during minimally invasive laparoscopic colorectal surgery, exhibiting a favorable safety and tolerability profile ( Fig. 8b – d ) [ 65 ]. Currently, two Phase III clinical trials are underway: NCT05754333 and NCT05999747 . The primary focus of these studies is to assess the efficacy of ASP5354 in ureteral visualization during surgical procedures for adults with normal kidneys or mild complications. Beyond its utility in ureteral identification, ASP5354 has demonstrated significant potential in bladder cancer diagnosis. In a mouse model of bladder cancer, it was established that when administered intravenously, ASP5354 is rapidly cleared by the kidneys and significantly accumulates in the bladder. This characteristic renders ASP5354 an ideal candidate for non-invasive, real-time imaging of bladder cancer [ 66 ]. When paired with the NIRF imaging device, this innovative imaging technology holds the promise of facilitating real-time, highly sensitive detection of bladder cancer in both clinical diagnostics and surgical procedures. ALM-488 is an innovative neuro-specific fluorescent peptide-dye conjugate developed by Alume Biosciences. As an intravenously injectable fluorescently labelled peptide, this molecule comprises a specific amino acid sequence. Its unique property lies in its ability to bind directly to the extracellular matrix of nerve cells without relying on myelin sheaths, thereby enabling immediate localization of nerve imaging [ 67 ]. During surgery, the ALM-488 enables clear and precise delineation of neural structures through fluorescence imaging equipment such as imaging systems with compatible filters, portable handheld devices, microscopes, or laparoscopes. It can label multiple types of nerves, including motor, sensory, autonomic, and even degenerative nerves [ 68 ], making it of critical application value in high-risk neurological surgery areas such as skull base surgery, head and neck surgery, spinal surgery, and nerve-sparing prostatectomy. Furthermore, based on its pharmacokinetic characteristics, ALM-488 is also suitable for ureteric marking in urological, gynecological, and lower abdominal surgery. ALM-488 is currently in the clinical trial phase. In 2021, based on preliminary data from its Phase I/II clinical trial in head and neck surgery patients, the FDA granted ALM-488 Fast Track Designation (FTD). Subsequently, on August 23, 2022, Alume Biosciences announced that the first patient in the pivotal Phase III clinical trial of ALM-488 had completed dosing. This global, multi-center investigation is anticipated to conclude in 2023. Although ALM-488 has demonstrated promising clinical potential, its safety profile and potential adverse reactions warrant further investigation. Panitumumab-coupled IRDye800 is a sophisticated fluorescent imaging agent developed for in vivo imaging applications. Owing to its exceptional optical properties, with emission wavelengths ranging from approximately 780 to 820 nm within the NIR spectral region, IRDye800 is ideally suited for deep tissue imaging. When integrated with panitumumab—an anti-EGFR antibody offering substantial benefits in head and neck therapy—the imaging agent can precisely bind to tumor cells, thereby facilitating clear tumor signals via fluorescence imaging. Compared to normal tissues, EGFR receptors exhibit higher expression levels in tumors. Therefore, upon binding to the cell surface, this contrast agent rapidly accumulates within tumors [ 69 ]. This fluorescent probe offers advantages including straightforward preparation, strong cancer specificity, and favorable safety profiles [ 70 ]. Currently, multiple Phase II clinical trials are evaluating its application value in surgeries for various cancer types. The NCT04511078 study aims to validate the identification capability and safety of panitumumab-IRDye800CW for head and neck cancer lesions; the NCT04085887 study focuses specifically on the performance of this contrast agent in pediatric brain surgery. Additional trials are exploring its efficacy and safety in pancreatic cancer, malignant glioma, lung cancer, and head and neck cancers with lymph node metastasis. ONM-100 can form nanomicelles through polymer self-assembly, whose hydrophobic core encapsulates fluorophores such as ICG to achieve fluorescence quenching. When the tumor microenvironment pH drops below a specific threshold, the nanomicelles dissociate and release fluorescent monomers, restoring the fluorescence signal. This pH-responsive mechanism ensures that the fluorescent probe is only transiently and fully activated when the tumor microenvironment reaches a critical acidic threshold. Over time, the fluorescent signal within acidic regions accumulates continuously and becomes persistently enriched at the tumor site [ 71 ]. This characteristic provides crucial evidence for intraoperative identification of tumor margins, thereby enhancing surgical precision and therapeutic efficacy while mitigating surgical risks. Significantly reduced pH levels are observed within the microenvironments of four solid tumors: head and neck squamous cell carcinoma, breast cancer, oesophageal cancer, and colorectal cancer. As a pH-sensitive fluorescent imaging agent, ONM-100 undergoes specific activation within such acidic tumor microenvironments while exhibiting favorable safety and tolerability. This enables clear differentiation and localization of tumor tissue from benign tissue. In a Phase I clinical trial, ONM-100 successfully delineated tumor contours in 30 subjects, providing crucial evidence for identifying occult lesions. However, the agent retains certain limitations, including potential activation in non-tumoric acidic environments such as inflammation, which may reduce its specificity [ 72 ]. Ongoing Phase II clinical trials are evaluating ONM-100’s efficacy in lung cancer surgery patients. Findings indicate it effectively enables real-time intraoperative localization of lung adenocarcinoma and squamous cell carcinoma, achieving tumor-to-background signal ratios of 2.7 and 2.4, respectively. This demonstrates ONM-100’s clinical potential and significant value in enhancing surgical outcomes [ 72 ]. Furthermore, studies have confirmed that ONM-100 facilitates real-time intraoperative delineation of tumor margins [ 73 ]. In conclusion, ONM-100 demonstrates significant clinical potential as a pH-sensitive fluorescent contrast agent. Its unique pH-responsive mechanism provides crucial information for intraoperative tumor localization. Although certain limitations remain, ongoing research and clinical trials hold promise for delivering important breakthroughs in tumor diagnosis. The overexpression of the EGFR in various tumor types, notably malignant gliomas, head and neck cancers, and soft tissue sarcomas, has garnered significant attention in both scientific research and clinical medicine. This trend is especially evident in patients with an incidence rate ranging from 50% to 90%, underscoring the therapeutic potential of EGFR as a target. To date, several anti-EGFR drugs have been developed, among which Affibody molecules stand out. These non-immunoglobulin structures are based on a 58-amino acid, three-alpha helix scaffold, facilitating their production through recombinant or protein synthesis methods and enabling functionalization to cater to diverse research requirements. Notably, by incorporating fluorescent or radiolabeled groups, such as modifications with a single C-terminal cysteine, Affibody molecules exhibit promising diagnostic and therapeutic attributes. ABY-029 is an antibody-conjugated probe fused with an anti-EGFR affinity antibody and IRDye800CW fluorescent dye, enabling highly specific binding and localization to EGFR-overexpressing tumor cells [ 74 ]. Currently, ABY-029 holds FDA approval as an Investigational New Drug (IND application number: eIND Application 122681). Having undergone rat toxicity and safety testing, this molecule is presently being evaluated in Phase 0 clinical trials across multiple cancer types at minimal dosages [ 75 ]. Regarding potential applications of ABY-029 in other cancers, researchers have employed it for preclinical imaging of epidermal growth factor receptors in soft tissue sarcomas. Results confirmed ABY-029’s efficacy in preclinical imaging of EGFR in soft tissue sarcomas, suggesting significant imaging and therapeutic potential for ABY-029 in other cancer types. Cetuximab-IRDye800CW is similar to ABY-029 in both design and application [ 76 ]. Antibody-fluorescent probe conjugates not only enhance therapeutic specificity but also provide powerful tools for real-time monitoring and evaluation of treatment efficacy. Specifically, cetuximab-IRDye800CW is synthesized by conjugating the anti-EGFR monoclonal antibody cetuximab with the IRDye800CW fluorescent dye. This enables specific recognition and binding to overexpressed EGFR, facilitating precise targeting of tumor cells. Concurrently, the IRDye800CW fluorescent dye confers NIRF imaging capability, facilitating real-time in vivo tracking and monitoring. Notably, cetuximab-IRDye800CW exhibits excitation and emission wavelengths of 778 nm and 795 nm, respectively, enabling highly efficient fluorescence imaging within the NIR spectrum [ 77 ]. By leveraging NIR light’s exceptional tissue penetration properties, cetuximab-IRDye800CW significantly enhances in vivo detection and imaging capabilities for deep-seated tumors. Currently, it has been widely applied in head and neck cancer (NCT0313484, NCT01987375 ), oesophageal cancer ( NCT04161560 ), pancreatic cancer ( NCT02736578 ), malignant brain tumors ( NCT02855086 ), and rectal cancer ( NCT04638036 ). Particularly in head and neck cancers, significant breakthroughs have been achieved in surgery through the use of cetuximab-IRDye800CW [ 76 ]. A landmark study showed that by combining cetuximab-IRDye00CW with multi-diameter single-fiber reflectance spectroscopy and single-fiber fluorescence spectroscopy (MDSFR/SFF) technology, intrinsic fluorescence values were successfully quantified, thereby determining the optimal drug dosage. Under conditions ensuring patient safety, the use of lower doses of cetuximab-IRDye800CW enables highly sensitive detection of head and neck cancer tumors. This holds significant importance for enhancing surgical outcomes and minimizing unnecessary tissue damage. Concurrently, the research conducted by Nagengast’s team corroborated the viability of cetuximab-IRDye800CW for the preliminary detection of EAC. Their findings revealed that a significant proportion, up to 67%, of dysplastic tissues exhibited high EGFR expression. This underscores the promising potential of this fluorescent probe in cancer diagnosis [ 78 ]. Furthermore, a study aimed to investigate the sensitivity, specificity, and positive predictive value of cetuximab-IRDye800CW in detecting tumor-positive margins during intraoperative assessment in patients with oral cancer. Results demonstrated that cetuximab-IRDye800CW was well tolerated in 65 patients encompassing 66 tumors, and enhanced the safety of intraoperative adjustments for weakly fluorescent foci with an SBR ≥ 1.5. This outcome underscores the practical value of cetuximab-IRDye800CW in the real-time intraoperative assessment of tumor margins [ 79 ]. BLZ-100, a pioneering optical imaging agent, is inaugurating a new era in tumor surgery. Its distinctive combination mode—the meticulous amalgamation of the targeting peptide and the NIR fluorescent dye, ICG—enables cancer cells to be visualized in real time with high resolution by surgeons during operations. This imaging method not only offers precise visualization of tumor boundaries for surgical procedures but also significantly enhances surgical precision and patient safety. At the heart of BLZ-100 is its modified cyanotoxin (CTX) peptide component [ 80 ]. This peptide, originally sourced from scorpion venom, has been ingeniously optimized and modified by researchers to form a strong covalent bond with the ICG dye. This modification substantially augments the targeting ability of the fluorescent dye towards tumor cells and the intensity of the fluorescence signal. Its unique excitation and absorption wavelengths (774 nm/690 nm) provide imaging technology with unparalleled precision, ensuring accurate identification of tumor cells even in complex surgical environments. The broad applicability of CTX peptides, which can specifically bind to a range of tumor cells, is noteworthy. This suggests that the potential applications of BLZ-100 extend beyond image-guided surgery of gliomas and other brain tumors. It is anticipated that BLZ-100 will also prove effective in the surgical treatment of prostate cancer, colorectal cancer, lung cancer, breast cancer, head and neck cancer, as well as sarcoma and other forms of cancer. Significantly, the CTX peptide possesses a distinct capability to facilitate the penetration of nanoparticles through the BBB. This discovery introduces a novel realm for the utilization of BLZ-100 in brain tumor surgeries. Consequently, it enables access to regions of the brain tumor where the functionality of the BBB remains unaffected, offering potential relief to an increased number of patients [ 81 ]. The innovative and practical application of BLZ-100 has garnered recognition from esteemed institutions. It has been awarded the Fast Track designation by the US FDA, particularly in the realm of imaging for pediatric brain tumors. This accolade is poised to expedite both the development process and market penetration of this drug, thereby enabling a wider range of physicians and patients to reap the benefits of this remarkable achievement promptly. In conclusion, BLZ-100 is a highly efficient and precise tumor-targeting fluorescent probe. Beyond its outstanding performance in image-guided surgery, it also demonstrates potential for application in cancer therapy. SA-FCA is a novel peptide-conjugated fluorescent probe targeting X-linked inhibitor of apoptosis protein (XIAP). Its structure (AVPIAQKDEVDKLVFFAEC(Cy)G) employs a modular design ( Fig. 9a ), comprising four functional motifs: (i) AVPIAQK, which specifically recognizes XIAP in tumor cells, (ii) DEVD, a procaspinase-3-specific cleavage motif, (iii) KLVFFAECG, a self-assembly motif, and (iv) MHI-148, a fluorescence signal motif [ 82 ]. The excitation and emission wavelengths of this probe are 804 nm and 833 nm, respectively, exhibiting spectral characteristics highly consistent with the conjugated fluorescent probe ( Fig. 9b ). The imaging mechanism of SA-FCA involves the specific recognition of XIAP. Within tumor microenvironments exhibiting high XIAP expression, SA-FCA undergoes cleavage to activate the downstream caspase-3 pathway. This subsequently triggers secondary cleavage, inducing molecular self-assembly into nanofibers rich in β-sheet structures. This in situ self-assembly strategy enhances the stability and persistence of the fluorescent signal. Compared to conventional fluorescent probes, SA-FCA’s novel mechanism of “Tumor-selective cascade activation and in situ self-assembly” not only substantially enhances precise tumor recognition but also markedly improves the probe’s enrichment and retention capacity within tumor tissue. The fluorescence intensity of SA-FCA is approximately tenfold greater than that of the FDA-approved Cytalux. Furthermore, SA-FCA effectively overcomes variations in targeting capability caused by tumor heterogeneity, thereby substantially reducing the risk of false negatives. These characteristics position it as a tool with greater application potential in precision tumor diagnosis and image-guided surgery. In a trial involving 338 tissue samples from bladder cancer patients, researchers evaluated SA-FCA using in vitro fluorescence imaging with a real-time NIR imaging system ( Fig. 9c – d ) [ 83 ]. The results demonstrated that SA-FCA could clearly distinguish malignant from benign samples, achieving a diagnostic specificity of 97.96% and a sensitivity of 99.58% ( Fig. 9h – i ). In both brightfield and fluorescence images, tumor tissue exhibits distinct fluorescence signals with clear boundaries. Even for minute satellite lesions measuring approximately 1 mm in diameter, this probe enables precise identification, fully demonstrating its exceptional imaging sensitivity. Furthermore, SA-FCA exhibits no discernible fluorescence signal in inflammatory tissues, demonstrating high tissue specificity. Compared to traditional fluorescent probes, SA-FCA offers significant advantages in distinguishing tumor from non-tumor tissue, indicating broad application potential in the precise diagnosis of bladder cancer and intraoperative imaging guidance. Currently, fluorescent contrast agent iSAP-0909 with the same design concept has received investigational new drug application (IND) approval from the U.S. FDA and the China’s National Medical Products Administration (NMPA), officially entering the clinical development phase.

The tables below present a concise summary of imaging agents that are currently FDA-approved, as well as those undergoing clinical trials ( Table 1 , Table 2 ). In selecting an appropriate imaging agent, multiple factors must be considered, including specificity, safety, clinical efficacy, and patient tolerance. To ensure optimal outcomes, it is recommended that patients engage in thorough discussions with their healthcare provider or medical imaging specialist to identify the most suitable approach tailored to their individual medical condition. Table 1 Characteristics of fluorescent contrast agents approved by the FDA (Test results in pure water) . Table 1 dummy alt text Chemical structure Excitation wavelength (nm) Emission wavelength (nm) Molar extinciton coefficient (εmax/M⁻¹·cm⁻¹) Fluorescence lifetime (ns) Quantum yield (Φ) Ref. ICG Image, table 1 dummy alt text 785–788 820–822 156,000 0.64–0.66 0.0029 [ 93 , 94 ] Fluorescein Sodium Image, table 1 dummy alt text 460–500 560–690 69,600 3.775–4.11 0.85–0.97 [ 22 , 95 ] Cysview a Image, table 1 dummy alt text 360–375 610–650 40,603 2.45–2.55 0.087 [ 96 ] 5-ALA a Image, table 1 dummy alt text 360–375 610–650 40,603 2.45–2.55 0.087 [ 96 ] MB Image, table 1 dummy alt text 660–670 710–730 75,000–95,000 0.59–1.37 0.02–0.04 [ 97 , 98 ] Cytalux a Image, table 1 dummy alt text 760–785 790–805 272,000 / 0.033 [ 47 ] Akalux a Image, table 1 dummy alt text 680–690 699–702 / 4 0.10–0.14 [ 99 , 100 ] a All molar extinction coefficients, fluorescence lifetimes, and quantum yields reported for Cysview, 5-ALA, Cytalux and Akalux are derived from studies on their respective fluorophores (PpIX for Cysview and 5-ALA; S0456 for Cytalux; IRDye700DX for Akalux). Table 2 FDA-approved clinical fluorescent imaging agents . Table 2 dummy alt text Compounds Indications Clinical stages Sample Pre-operative injection time Clinical effects Registered NCT ICG Laparoscopic radical gastric cancer surgery III 639 24 h preoperative ICG and NIRF imaging enhance lymph node detection, biliary dissection, and surgical precision by expediting biliary structure identification, assessing gastric tube perfusion, predicting complications, and improving hepatic and gastric cancer surgery outcomes, despite limitations in sensitivity for perfusion assessment, highlighting their significant clinical value. NCT03050879 , NCT02702843 , ChiCTR2200057933, ChiCTR2200064726, NCT03811704 Lung tumor metastatic nodule I 30 24 h preoperative ICG ensures negative margins and preserves lung parenchyma in segmental resections, aiding pediatric tumor localization. However, it may miss solid tumors or deep nodules, as NIR imaging is limited to lesions near the pleural surface. NCT02280954 , NCT04084067 Colorectal cancer / 839 Intraoperative ICG fluorescence imaging reduces anastomotic fistulae and aids intraoperative blood flow assessment. Though some results fall short of expectations, it remains a safe tool with potential to improve bowel resection outcomes. NCT03390517 , UMIN000009731, NCT02662946 , NCT02205307 , NCT04226781 , NCT02626091 Cervical, uterine and ovarian lesions I/II 51 24 h preoperative ICG aids in managing deeply infiltrating endometriosis, accurately identifying tumors and pelvic nerves, thus reducing nerve injury. Though sensitive in detecting ovarian lymph nodes, its specificity is limited, yet it supports pathological analysis. NCT04224467 , NCT03850158 , NCT04224467 , NCT01834469 Breast cancer II 414 Preoperative/Intraoperative ICG enhances lymphedema staging and reduces resection volume in breast cancer surgery. While sensitive, its specificity in distinguishing benign from malignant tissue is limited. ICG-based lymphatic mapping matches Tc performance, and combined with SLN biopsy, proves effective for patients receiving neoadjuvant chemotherapy. NCT02027818 , No. NCC2016–0071, NCT02032563 Kidney transplantatio -n, resection II 128 Intraoperative ICG reduces renal artery branch clamping, shortens ischemia time, and preserves renal function, ensuring surgical outcomes. It also quantifies graft microperfusion intraoperatively, aiding in predicting delayed graft function. / Oral squamous cell carcinoma / 20 6–8 h preoperative ICG can be utilized to determine surgical margins. / Pancreatic / 20 24 h preoperative Second-window ICG reliably provides real-time feedback during pancreatectomy. NIR imaging may be useful in assessing response to neoadjuvant therapy / Head and neck cancer / 56 (intracranial) 24 h preoperative, (neck) Intraoperative ICG can effectively improve the localization accuracy of head and neck cancer surgery and contribute to surgical safety. / Cysview Cystoscopy II/III 304 Perfusion for 1 h BLFC outperforms WLC in detecting non-muscle invasive bladder cancer, reducing recurrence risk and extending recurrence intervals. It enhances detection rates without harming patients, complies with FDA standards, and shows superior lesion detection in high-risk cases. NCT02560584 5-ALA Glioma I/II/III 322 2–4 h preoperative 5-ALA fluorescence enhances tumor resection completeness and progression-free survival, with a biopsy PPV over 96% and sensitivity above 67%. High-dose 5-ALA reduces tumor residues but needs further validation. The procedure is safe, flexible, though false-negative rates and specificity vary across tumor types. NCT00241670 Lymphoma II 54 2–4 h preoperative Intraoperative Visualization of 5-ALA Becomes Standard Therapy for Occult Cancer Outside Neurosurgery; Novel Handheld Fluorescent Imaging Device Helps Expand to Other Cancer Surgeries; Marginal Evaluation and Image-Guided Resection Are Valuable. NCT01837225 Malignant diseases of endometrium / 34 3 h preoperative oral 5-ALA shows high sensitivity (93.8%) and moderate specificity (51.9%) in diagnosing uterine malignant tumors, effectively detects microscopic endometrial lesions, and is safe for oral use, making it a promising diagnostic tool. / Oral cancer / 30 3 h preoperatively in 3 doses 5-ALA enables fluorescence imaging and photodynamic therapy, achieving 76% complete remission in T1N0M0 buccal mucosal cancer after one treatment, with no recurrence at 50 weeks. Efficacy correlates with tumor size and light, offering potential to reduce oral cancer incidence, mortality, and costs. / Bladder cancer / 328 Intraoperative No significant difference in recurrence rates / MB Colorectal cancer III 1346 Administered in eight doses 4 h before surgery There was an absolute increase of 8.5% in adenoma detection at colonoscopy. NCT01694966 Hepatobiliary cancer I 18 Intraoperative 94% of patients Successful visualization of target objects as fluorescent by MB fluorescence imaging, including 100% of neuroendocrine tumors and peripancreatic vessels. / Breast cancer / / Intraoperative MB-mediated NIR fluorescence imaging facilitates identification of lymphatic vessels, location of sentinel lymph nodes, and patterns of mammary lymphatic flow. / Sentinel lymph node / 103 Intraoperative Bilateral SLNs were resected in 82.5% of women, with a bilateral detection rate of 83%. / Gastric cancer anastomotic fistula / 148 Intraoperative Effective prevention of anastomotic complications associated with technical defects in gastric cancer patients undergoing gastrectomy. NCT04292496 Breast axillary lymph node dissection (balnd) / 454 Intraoperative Mammary lymphatics and subsequent lymph nodes were blue after MB injection in 89.0% of patients, and 57.8% of patients had fewer than four metastatic lymph nodes. / Preoperative localization of lung nodules and masses / 146 Intraoperative MB mixed with autologous blood enables precise, low-cost, and low-complication CT-guided lung localization. However, its recognition is limited in cases with severe lung pigmentation or deep pleural punctures. / Melanoma / 20 Intraoperative This study demonstrates the usefulness of fluorescent sentinel lymph node biopsy for patients with melanoma using MB as a fluorescent moiety. / Thyroid cancer and parathyroid adenoma / 14 0.5 h preoperative MB dye injection effectively guides thyroid and parathyroid surgeries, aids pathology testing, and safely identifies recurrent tumors and adenomas, ensuring high preoperative success and intraoperative efficacy. / Neuroendocri -ne tumors of the small intestine / 17 Intraoperative NIR fluorescence imaging of MB has a high detection rate in the intraoperative detection of small intestinal neuroendocrine tumors, but is unable to detect primary and occult multiple primary tumors. / Hepatic bile ducts / 1 Intraoperative MB in combination with double-knife fistulotomy for hepatobiliary surgery is low-risk and effective, and is a viable method for achieving single-tube intubation during difficult endoscopic retrograde cholangiopancreatography. / Breast cancer axillary lymph nodes / 170 1 h preoperative MB detection of axillary lymph nodes in breast cancer has low sensitivity but high specificity and high false negative rate. The use of MB staining improves identification accuracy and aids in lymph node identification. / Fluorescein Sodium Peripheral nerve / 5 Intraoperative Intraoperative Fluorescein Sodium imaging helps to accurately depict the most severely affected nerve bundles, identify biopsy targets, and enhance the diagnostic accuracy of nerve biopsies. / Paranasal sinus / 40 / Fluorescein staining scores were higher in the nasal rinse group than the spray group, notably in the maxillary and anterior ethmoid sinuses, while frontal and pterygoid sinuses showed minimal staining. / Gastric / 33 3 min preoperative Endoscopic ultrasound-guided needle confocal laser endoscopy is feasible and safe for accurate diagnosis of gastric subepithelial tumors. / Neck of uterus / 70 Intraoperative The strong fluorescent properties of Fluorescein Sodium are highly specific, cost-effective and easy to perform, and are expected to aid in the diagnosis of high-grade cervical intraepithelial neoplasia. / Retinal nerve / 95 Intraoperative Fluorescein angiography objectively assesses vessel areas and guides laser treatment but cannot fully replace indocyanine green angiography, requiring additional tests for diagnostic confirmation. / Brain / 142 Intraoperative Fluorescein Sodium enhances tumor diagnosis, localization, and resection by improving visibility and boundary identification, thereby optimizing surgical outcomes and prognosis, though its use requires integration with other clinical factors. / OTL38 Pituitary adenoma / 39 2–4 h preoperative Cytalux fluorophores offer high sensitivity and specificity in detecting nonfunctioning adenomas, especially FRα-overexpressing types, enhancing adenoma resection rates. / Lung nodule II 110 2–6 h preoperative Cytalux imaging enhanced lung nodule detection and improved prognosis in 26% of patients by identifying positive cutting edges missed during surgery. / Ovarian cancer II / / / / Bladder cancer, stomach cancer / / / / / Characteristics of fluorescent contrast agents approved by the FDA (Test results in pure water) . All molar extinction coefficients, fluorescence lifetimes, and quantum yields reported for Cysview, 5-ALA, Cytalux and Akalux are derived from studies on their respective fluorophores (PpIX for Cysview and 5-ALA; S0456 for Cytalux; IRDye700DX for Akalux). FDA-approved clinical fluorescent imaging agents . This review provides a comprehensive summary of clinical contrast agents, those in preclinical phases (I–III), and those with potential for clinical translation, covering various aspects such as molecular structure, physicochemical properties, indications, and clinical trial outcomes ( Table 3 ). The findings demonstrate that the development and application of contrast agents have significantly enhanced the precision and completeness of surgical procedures, thereby providing critical support for the long-term survival of patients post-operation. Since the approval of the first contrast agent, ICG, in 1959, a span of over half a century has seen the approval of seven contrast agents for clinical use. Table 3 Fluorescent imaging agents in clinical phases I–III . Table 3 dummy alt text Compounds Indications Clinical stages Pre-operative injection time Clinical effects Registered NCT SGM-101 Pancreatic, colorectal cancer III Intravenous injection within 48 h prior to surgery For colorectal cancer, the sensitivity was 98%, the specificity was 62%, and the fluorescence intensity accuracy was 84%. The predictive positive values for SGM-101 ranged from 99.04% to 90.24%. NCT03659448 , NCT04642924 , NCT02973672 , NCT05965817 , NCT04737213 , NCT04755920 LUM015 Chondrosarcoma, breast cancer, glioblastoma, rectal cancer III Intravenous injection 2–6 days prior to surgery It can effectively differentiate between cancerous and non-cancerous tissues, with a 100% negative prediction rate and 72–73% specificity. NCT01626066 , NCT02438358 , NCT02584244 , NCT03321929 , NCT03441464 , NCT03717142 , NCT03834272 , NCT03686215 , NCT04276909 , NCT04440982 ASP5354 Ureter III Intravenous 2 to 6 h before surgery Can be used to show ureters in real time during surgery to reduce the risk of ureteral injury. passes through the urine almost completely unaltered within 24 h. NCT05457842 , NCT04878471 , NCT05999747 , NCT05754333 , NCT05495581 , NCT04238481 , NCT03698305 ALM-488 Nape III 1–5 h prior to surgery / NCT04420689 , NCT05377554 , NCT06227585 Panitumumab-IRDye800 Brain cancer, glioma, head and neck cancer, breast cancer, skin cancer, colorectal cancer (tumors with high EGFR expression) II Intravenous 1–5 days prior to surgery In tissue sections, panitumumab-IRDye800 was highly sensitive (95%) and specific (96%) for pathologically confirmed tumor-containing tissue. Intraoperative fluorescence improved the optical contrast of tumor tissue inside and outside the T1 contrast-enhanced margins with contrast-to-noise ratios of 9.5 ± 2.1 and 3.6 ± 1.1, respectively. NCT04511078 , NCT03405142 , NCT02415881 , NCT03582124 , NCT04085887 , NCT03733210 , NCT03384238 , NCT03510208 , NCT05945875 TumorGlow Brain tumor, head and neck tumor, breast cancer, malignant pleural mesothelioma II 5 days before surgery Often used when it is difficult to distinguish between tumor and normal tissue, sensitivity and positive predictive value both around 85%,visualize tumors through the dura to a depth of 13 mm. NCT04723810 , NCT02280954 ONM-100 Head and neck cancer, breast cancer, colorectal cancer, esophageal cancer II Intravenous administration 24 ± 8 h before surgery HNSCC sensitivity was 100%, specificity 57% NCT03735680 TumorGlow second window Tumors of the central nervous system I Approximately 1–4 h before surgery / NCT03262636 , NCT05746104 ABY-029 Head and neck cancer, glioma, breast cancer, colorectal cancer (high EGFR expression) I Administered as a single intravenous injection approximately 1–3 h prior to surgery Have excellent localization to various extremity soft-tissue sarcomas with an average TBR of 3.25. NCT03282461 , NCT03154411 , NCT02901925 , NCT0290925 Cetuximab-IRDye 800CW Head and neck cancer, colorectal cancer, malignant glioma, pancreatic cancer I Administered as a single intravenous injection approximately 1–3 h prior to surgery An ex vivo fluorescence molecular imaging approach using cetuximab-800CW in squamous cell carcinoma surgery not only detects tumor-positive margins with 100% sensitivity, but also most of the proximal margins. NCT03134846 , NCT02736578 , NCT02855086 , NCT05929456 , NCT04638036 , NCT01987375 , NCT05376202 , NCT06101394 , NCT04161560 , NCT05499065 BLZ-100 Glioma, brain tumor, skin cancer, soft tissue sarcoma I Intravenous 2–3 days prior to surgery / NCT02464332 , NCT02234297 , NCT02462629 , NCT02496065 , NCT02097875 , NCT04343274 , NCT05316688 , NCT03579602 Fluorescent imaging agents in clinical phases I–III . Historically, the evolution of contrast agents has shifted from nonspecific probes to targeted fluorescence agents. In the last decade alone, four specific contrast agents have been approved, highlighting a major advancement in the field. These agents not only mark the transition from general imaging tools to highly specialized, targeted molecular probes but also signify a leap forward in clinical diagnostics and therapeutic outcomes. From a clinical efficacy perspective, the use of targeted fluorescent contrast agents has contributed to a remarkable improvement in imaging specificity and sensitivity, delivering tangible benefits to patients. The targeted properties of these agents enable more precise identification and localization of pathological regions, thereby reducing misdiagnosis and missed diagnoses while improving treatment outcomes. Consequently, targeted contrast agents demonstrate broad prospects across multiple medical fields such as oncology and vascular surgery, laying the foundation for the future development of precision medicine. In short, with the continuous advancement of contrast agent technology, more contrast agents with clinical translation potential will be optimized and developed in the future. These agents will provide robust support for clinical cancer treatment and drive the realization of precision medicine.

The fluorescent contrast agent ICG was uniformly distributed in well or moderately differentiated tumors but exhibited partial type and marginal type distribution in poorly differentiated hepatocellular carcinoma. The development of smart responsive contrast agents has injected new momentum into the field of precision medicine [ 56 , 84 , 85 ]. With ongoing advancements in molecular probe technology, an increasing number of structurally innovative and functionally superior contrast agents are expected to emerge and find clinical application. Fluorescent probes, with their high sensitivity, excellent selectivity, and non-invasive imaging capabilities, demonstrate tremendous potential in both basic research and clinical practice [ 69 , 86 , 87 ]. Currently, commonly used fluorescent contrast agents such as ICG and MB emit in the NIR region, providing excellent tissue penetration and enabling imaging depths of approximately 5–10 mm. These properties make them particularly well-suited for in vivo imaging and real-time image-guided surgery of tumors, blood vessels, and lymphatic structures. However, traditional NIR-I region fluorescent dyes (such as ICG and MB) still have several limitations. One of the most prominent issues is the photobleaching effect. This not only limits the stability of imaging but also leads to a decrease in signal-to-noise ratio and a reduction in spatial resolution during image-guided surgery. In addition, some contrast agents undergo rapid metabolism in vivo, which easily causes signal attenuation and background interference, thereby limiting the stable imaging window. With the advancement and upgrading of probes, in the experimental phase, a range of fluorescent contrast agents is emerging with the potential to offer real-time visual assistance during surgical procedures. This could enhance precision in surgical operations and improve treatment outcomes. Among these agents, Cy7 stands out as an exceptional cyanine dye. As an NIR molecule, Cy7 facilitates easy modification with other molecules. Its excitation absorption typically ranges from 745 to 772 nm, while emission peaks between 792 and 810 nm, making it particularly suitable for precise tumor imaging. In peptide-conjugated drug research, scholars innovatively employed an in situ self-assembly strategy to integrate Cy7 with peptide chains. This approach not only significantly enhances the accuracy of imaging navigation during surgery but also capitalizes on the unique self-assembly properties of peptides. These peptides can autonomously form stable and orderly structures, thereby extending the residence time of peptide-conjugated drugs in vivo. Consequently, the drug has more opportunities to accumulate within the target tissue while effectively minimizing nonspecific effects on nontarget tissues. Several next-generation fluorescent probes with enhanced targeting capabilities and stimuli responsiveness—such as FA-SCA and ABY-029—have entered clinical trials, demonstrating great potential in intraoperative guidance and precise tumor resection. The ongoing optimization of fluorescent probes not only improves imaging quality and diagnostic accuracy but also supports the development of personalized treatment regimens, thereby accelerating the translation of precision medicine from research into clinical practice. A recent study introduced a novel peptide nanoprobe designed to target bladder cancer cells by constructing a tumor-selective cascade activatable self-closing system (TCASS). This probe design integrates a tumor-specific recognition motif, an enzyme-cleavable linker, a self-assembly motif, and the functional molecule Cy7. It enables specific binding to bladder cancer cells under specific conditions and releases the cyanine dye. Experimental results reveal that this peptide probe precisely localizes tumor tissue, notably enhances tumor boundary clarity, and exhibits a high signal-to-noise ratio, demonstrating its potential for application in cancer imaging. Furthermore, Liao’s group pioneered the use of CD47 as a molecular imaging agent for bladder cancer identification. They employed a fluorescently labeled CD47 antibody (anti-CD47), in conjunction with advanced clinical-grade imaging systems and endoscopic technology, to develop an efficient method for bladder cancer detection. With the aid of blue light cystoscopy, this method demonstrated remarkable performance, achieving a sensitivity of 82.9% and a specificity of 90.5%, fully validating the potential and effectiveness of CD47 as a molecular imaging agent for bladder cancer. Notably, this method also successfully detected some bladder cancer variants that were challenging to detect using traditional diagnostic methods, providing robust support for early detection and treatment of bladder cancer. Compared to NIR-I probes, NIR-II probes feature longer wavelengths, possess longer wavelengths, exhibiting a scattering coefficient merely one-tenth to one-fifteenth that of NIR-I probes. Furthermore, absorption interference caused by water and fat is significantly reduced, enabling imaging penetration depths of 1–3 cm [ 88 ]. This facilitates high-precision detection of deep-seated organs orlesions. Notably, when first applied during primary liver cancer surgery in 2020, NIR-II imaging successfully detected five micro-lesions (0.3–0.8 cm diameter) missed by NIR-II imaging, thereby increasing complete tumor resection rates by approximately 15%–20% [ 89 ]. Despite demonstrating significant technical advantages, the clinical translation of NIRF probes remains in its early stages, with only a limited number of Phase I/II clinical trials currently underway. Existing research and preclinical data indicate that potential biosafety risks associated with NIR-II probes primarily involve material-intrinsic toxicity, in vivo accumulation and metabolic disorders, as well as on-target/off-target effects. In the future, with further optimization of optical performance and refinement of systematic safety assessments, NIR-II probes will achieve clinical translation for use in image-guided surgery. To optimize intraoperative optical imaging performance, it is essential not only to employ high-performance imaging equipment but also to significantly enhance its sensitivity and spatial resolution. In recent years, optical imaging systems have expanded their applications across diverse clinical scenarios, including open surgery and minimally invasive procedures [ 90 ]. Currently, intraoperative fluorescence imaging devices in clinical use are primarily categorized into single-channel and multi-channel systems. The Photodynamic Eye (PDE), developed by Hamamatsu Photonics K.K. of Japan, represents a typical example of a single-channel system. This system is chiefly employed for fluorescence navigation based on ICG during hepatocellular carcinoma surgery, enabling basic identification of tumor boundaries and vascular visualization [ 91 ]. Additionally, multi-channel systems can fuse white-light anatomical images with NIR fluorescence signals, enabling real-time image-guided surgery through “image overlay”. For instance, the Fluorescence-Assisted Resection and Exploration (FLARE) system simultaneously identifies tumors, lymph nodes, and vascular structures. It has been extensively applied in procedures such as sentinel lymph node mapping for breast cancer and resection of liver metastases from colorectal cancer, significantly enhancing intraoperative visualization accuracy and surgical safety. With ongoing improvements in resolution and sensitivity, certain systems now achieve 4K high-definition imaging. During pediatric laparoscopic common bile duct cyst repair, high-resolution systems clearly display minute bile duct structures, effectively reducing intraoperative injury risks. Furthermore, a domestically developed multispectral NIR-I/II imaging system (ChiCTR1900022453) has demonstrated superior detection capabilities compared to conventional NIR-I systems during hepatocellular carcinoma surgery [ 89 ]. This system increased tumor detection rates by 10.26% (from 46.15% to 56.41%) and can identify < 5 mm intrahepatic micrometastases undetectable by conventional NIR-I systems, thereby reducing postoperative recurrence risk [ 89 ]. All currently clinically deployed intraoperative imaging instruments now achieve video-grade frame rates (25–30 fps), meeting real-time image-guided surgery requirements. Notably, the NIR-II imaging system achieves a dynamic range of 120 dB, enabling simultaneous differentiation of highly fluorescent vessels and low-fluorescence microtumors (< 3 mm) within the same field of view [ 92 ]. This effectively avoids the problems of overexposure or signal loss commonly found in traditional systems. To optimize imaging contrast agents, augmenting the sensitivity of optical imaging instruments is pivotal for enhancing intraoperative imaging. Technological advancements can bolster the instrument’s capacity to detect dye signals, leading to sharper and more precise images during surgical procedures. Tian et al. underscored the significance of elevating the sensitivity of optical imaging instruments via technological enhancements. They introduced an integrated visible and NIR multispectral imaging apparatus that facilitates concurrent visible, NIR-I, and NIR-II imaging for highly assisted fluorescence-guided liver cancer surgeries. Their findings revealed that, in comparison to NIR-I imaging, intraoperative NIR-II imaging offered superior tumor detection sensitivity, a greater tumor-to-normal tissue signal ratio, and an elevated tumor detection rate. Merging NIR-I and NIR-II spectral windows with suitable fluorescent probes could markedly enhance the efficacy of image-guided surgeries in clinical settings. These findings hold critical implications for the ongoing optimization of intraoperative imaging and surgical navigation. Enhanced sensitivity in optical imaging systems, coupled with the application of multispectral imaging, can yield sharper and more precise images. This helps surgeons accurately identify tumor tissue boundaries during surgery, thereby improving surgical precision and efficacy. However, it is imperative to note that while these research outcomes are highly promising, they necessitate further investigation and clinical validation. The safety and effectiveness of novel imaging techniques and fluorescent probes remain paramount considerations. Consequently, future research endeavors and trials are essential to ascertain the feasibility and tangible impacts of these technologies in clinical settings.

In the domain of precision medicine, targeted contrast agents for image-guided surgery are pivotal factors, with their significant advantages positioning them as the preferred solution for future healthcare system development. The importance of these probes is increasingly evident in fields such as tumor treatment, neuroscience, and cardiovascular research. They enable precise localization of tumors, inflammation, or injury sites, providing real-time imaging data to assist physicians in accurately identifying pathological areas. This enhances surgical precision and treatment efficacy. Consequently, targeted probes are regarded as one of the pioneering technologies in future precision medicine, aligning closely with the developmental trends of healthcare systems. However, the specificity and sensitivity of targeted contrast agents remain challenges in current image-guided surgery. Furthermore, the stability of targeted probes is particularly critical. Due to the complexity and unpredictability of the in vivo environment, probes may be affected by multiple factors during use, leading to reduced stability. To address these issues, researchers are actively exploring various methods and technologies, including structural modulation and nanomaterials. These explorations will provide new perspectives and directions for optimizing and advancing targeted probes for image-guided surgery. In the evolution of fluorescent contrast agents for tumor detection, the development of NIR-II probes holds paramount importance. NIR-II fluorescence imaging technology enables penetration through thick tissue layers, granting clinicians deeper perspectives into tumor structures. This technique significantly enhances image clarity by minimizing interference from tissue autofluorescence. It also boasts exceptional biocompatibility and biosafety, effectively mitigating potential risks. Future advancements in tumor fluorescent probes increasingly emphasize the integration of multimodal imaging technologies. By combining multiple imaging modalities, a more comprehensive understanding of tumors can be achieved, thereby enhancing diagnostic accuracy and reliability. For instance, optimizing the fusion of NIR-II fluorescence imaging with medical imaging techniques such as ultrasound, MRI, and CT leverages the complementary strengths of each modality. This multimodal imaging approach holds the promise of revolutionizing tumor diagnosis and treatment. Additionally, AI plays a crucial role in image-guided surgery for tumor diagnosis. Its core value lies in deeply analyzing contrast-enhanced imaging results, providing clinicians with objective, quantitative support for image interpretation. Utilizing AI tools eliminates subjective judgment variations inherent in human recognition, yielding more objective outcomes [ 101 ]. In summary, the future landscape of tumor fluorescent contrast agents will encompass the convergence of NIR-II fluorescence imaging, multimodal imaging technologies, AI, and other related fields. The integration of these technologies holds promise for delivering more precise and reliable solutions in tumor diagnosis and treatment. This advancement will enhance the patient treatment experience and considerably improve their quality of life. As research deepens and technology continues to evolve, these innovative approaches and directions are anticipated to gain widespread adoption and promotion in the future, thereby making significant contributions to human health.

Malignant tumors have emerged as the second leading cause of mortality globally, with a projected 45% increase in incidence from 2010 to 2030 [ 1 ]. Despite significant advancements in non-invasive treatments, surgery remains a primary therapeutic modality for solid tumors [ 2 ]. For surgeons, achieving complete tumor resection is paramount; however, this objective often encounters numerous obstacles. Patients frequently present with concurrent lesions undetected on preoperative imaging, and the tumors’ large size and invasiveness into critical structures significantly complicate complete resection. Traditional surgical methods predominantly relied on tactile and visual assessments by the surgeon. However, the advent of minimally invasive techniques has further constrained the ability to detect lesions through palpation [ 3 ]. During surgical procedures, ensuring complete tumor removal is crucial for prognostication [ 4 ]. The challenge lies in detecting and eliminating minute residual tumor cells to mitigate the risk of recurrence and metastasis. To address this concern, physicians employ adjuvant treatments such as radiotherapy or chemotherapy. Concurrently, innovative therapies like immunotherapy and targeted therapy have shown promise in clinical trials. In recent years, image-guided technology has been widely adopted in surgical procedures. This innovative technique utilizes specific fluorescent contrast agents to locate tumor positions and boundaries, thereby facilitating easier identification of tumor tissue during surgery. When the fluorescent contrast agent is excited at a specific wavelength, it emits fluorescence. Its high signal-to-noise ratio significantly enhances the visualization of tumor tissue, hence reducing the rate of positive surgical margins [ 5 ]. This image-guided surgical technique not only enhances surgical precision but also reduces trauma and associated complications. The advantages of fluorescent contrast agents lie in the high sensitivity, real-time monitoring capabilities, and non-invasive, pain-free characteristics [ 6 ]. By binding to specific biomolecules, these contrast agents enable molecular-level detection of lesions, thereby enhancing diagnostic sensitivity. During surgery, doctors can use fluorescent contrast agents to monitor lesions in real time, ensuring timely identification and treatment of affected tissue. Furthermore, since fluorescent contrast agents do not require invasive procedures, they significantly reduce patient discomfort. The inception of fluorescent contrast agents can be traced back to the early 20th century, during which the exploration of fluorescent substances as diagnostic agents was initiated. As technological advancements persist, a myriad of substances exhibiting diverse fluorescent properties have been unearthed and subjected to rigorous research and application [ 7 ]. A number of these fluorescent contrast agents have received FDA approval and are extensively employed in clinical settings, including ICG, fluorescein sodium, and 5-ALA, among others [ 5 ] ( Fig. 1a - c ). With the ongoing evolution of medical technology, there is a constant innovation and refinement in fluorescent contrast agents. Novel agents can bind with specific biomolecules, thereby enhancing both the sensitivity and specificity of diagnosis. The trajectory of future development for fluorescent contrast agents will be shaped by a multitude of factors. As health concerns escalate, the demand for prompt diagnosis and treatment of diseases is projected to rise, propelling the further expansion of the fluorescent contrast agent market. Furthermore, policy support coupled with technological advancements will provide robust impetus for the progression of fluorescent contrast agents. In summary, while malignant tumors remain a major challenge for the biomedical community, our understanding and approaches to their treatment continue to evolve through sustained research and innovation. From surgical interventions to the application of fluorescent contrast agents, each advancement has driven the optimization of therapeutic methods, significantly improving patient survival rates. Looking ahead, we expect more innovative research findings to be integrated into clinical practice, thereby achieving superior treatment outcomes and tangibly enhancing patients’ quality of life ( Scheme 1 ). Scheme 1 Fluorescent contrast agents are used for image-guided tumor surgery . Scheme 1 dummy alt text Fluorescent contrast agents are used for image-guided tumor surgery .

The authors declare that they have no conflicts of interest in this work.