Fluorescence molecular imaging (FMI) and endoscopy (FME) hold strong potential to guide interventions and enable earlier, more personalized cancer diagnosis. Alongside novel tracer development, numerous clinical trials are ongoing, and recent FDA approvals of similar to 20 imaging systems and three tracers (5-ALA, hexaminolevulinate, and pafolacianine) mark significant progress. However, FMI and FME face challenges that hinder consistent clinical interpretation, including variability across systems, which limits repeatability, unbiased readouts, and broad adoption. Standardization efforts are emerging in the literature, with several groups proposing initial guidelines. Here, we present current standardization initiatives and our group's work on developing multi-parametric, composite standards for quality control and performance assessment. We also outline test designs linked to acquisition parameters and analysis methods that ensure objective system quantification. This framework may support clinical translation and foster integration into robotic-assisted interventions, where repeated sterilization cycles risk degrading system performance.

Significance: Fluorescence molecular imaging (FMI) has been approved for surgical guidance in several procedures. However, its interpretation still requires substantial clinician expertise due to variable signal levels. For FMI to gain wider clinical acceptance, image quality should be assessed automatically, thereby prompting clinicians to retake low-quality images when necessary. Yet, no user-friendly fidelity assessment tools currently address this need. Aim: Herein, we introduce a real-time, frame-by-frame FMI fidelity assessment method, termed no-reference fidelity assessment of fluorescence molecular imaging (NRFA-FMI). Approach: NRFA-FMI quantifies image fidelity using a combination of histogram-based and textural metrics. A central advantage of NRFA-FMI is that it operates without reference images, making it particularly suitable for clinical FMI workflows. Results: We show that NRFA-FMI outperforms two state-of-the-art no-reference image fidelity assessment methods, the blind/referenceless image spatial quality evaluator (BRISQUE) and the natural image quality evaluator (NIQE). Conclusions: The introduction of NRFA-FMI enables real-time FMI fidelity assessment, an essential step toward reducing data misinterpretation and preventing incorrect diagnosis. This approach provides clinicians with an intuitive, automated indicator of image reliability, supporting more consistent and accurate intraoperative decision-making.

Supplementary Table S2. Tissue Microarray Metadata (merged data from 4 TMAs used in the study): Impact of stage, T-stage, N-stage, and tumor grade on c-MET positivity and H-score of EAC samples.

Supplementary Figure S5. Gross anatomy of mouse stomach.

Supplementary Figure S4. Fluorescence molecular endoscopy study schema.

Ex vivo fluorescence imaging of L2-IL1β mice: A, Representative white light and fluorescence images of the excised stomach taken from L2-IL1β mice. The white dotted line shows the SCJ, which is within the stomach in mice. A protruding, irregular lesion is visible at the EGJ and SCJ in the stomach taken from a high-score group mouse, with the corresponding fluorescence image below. B, Upregulation of c-MET in dysplastic lesions from the low-, intermediate-, and high-score groups. C, Correlation between dysplasia score and level of c-MET expression. D, Box-whisker plot of ROI analysis, which shows increasing fluorescence from low- to intermediate- to high-score lesions. E, Quantification of TBR in ex vivo IVIS fluorescence imaging. Range of box-whisker plots indicate minimum to maximum. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Eso, esophagus; H&E, hematoxylin and eosin; Max, maximum; Min, minimum; ns, not significant; Sbkg, signal intensities of the background; SJC, squamocolumnar junction; Slesion, signal intensities of the lesion.

Abstract Background Treatment of inflammatory bowel disease (IBD) with biologicals, such as adalimumab, is hampered by high non-response rates and lack of reliable response prediction tools. Therefore, patients are potentially exposed to ineffective treatment and side effects, while clinical deterioration continues. Moreover, it is unclear whether the drug reaches its target site in adequate concentrations to achieve treatment response. We aim to visualise adalimumab distribution and detect adalimumab target cells using quantified fluorescence molecular endoscopy (qFME). Methods Adalimumab was labelled with IRDye 680LT under cGMP conditions, resulting in a fluorescent tracer suitable for human use. This tracer is used in an ongoing, non-randomized, non-blinded, prospective feasibility study. A total of 21 IBD patients scheduled for an endoscopy will be included. Up until now, eight patients received adalimumab-680LT (4.5 mg: n=3, 15 mg: n=3, 25 mg: n=2), and three patients were included as a control. Two to four days after tracer administration, qFME was performed to gather in vivo fluorescence data of inflamed and non-inflamed tissue. Fluorescent signals were quantified by multi-diameter single fibre reflectance/single fibre fluorescence (MDSFR/SFF) spectroscopy. Furthermore, biopsies were taken from inflamed and non-inflamed mucosa for ex vivo analysis. Results To date, 11 patients were included. Tracer administration was well tolerated and no adverse events occurred in any dose group. Real-time in vivo macroscopic imaging showed clear uptake of adalimumab-680LT in inflamed tissue compared to non-inflamed tissue (figure 1A). Spectroscopy revealed a dose-dependent increase in fluorescent signal for adalimumab-680LT in inflamed tissue, with a significant difference between 4.5 and 15 mg (0.016 [0.011-0.021] vs 0.033 [0.021-0.043] (p=0.036)) (figure 1B). The difference between inflamed and non-inflamed tissue is most noticeable in the 25 mg group. Ex vivo Mean Fluorescent Intensity (MFI) quantification of all biopsies showed a significant increase of fluorescent signal in the 15 and 25 mg dose groups compared to control in inflamed tissue (65.0 [54.0-75.7] vs 32.4 [27.1-38.2] (p=0.0040), and 62.1 [44.1-82.8] vs 32.4 [27.1-38.2] (p=0.0286), respectively) (figure 1C). Conclusion Preliminary results show adalimumab-680LT is safe for visualising adalimumab distribution. Doses of at least 15 mg are sufficient for visualisation and quantification of fluorescent signal in vivo. Furthermore, higher MFI’s and spectroscopy results were measured in inflamed tissue compared to healthy tissue, indicating targeting of the tracer to inflamed tissue. Future ex vivo experiments are ongoing to visualise adalimumab target cells.

To boost translation of fluorescence molecular endoscopy (FME), we developed a standardization methodology employing composite phantoms to assess FME systems performance. Additionally, we showed a need for the precise definition of contract and signal-to-noise ratio. (c) 2025 The Author(s)

We demonstrate a hybrid system combining Raman spectroscopy and partial wave spectroscopy, improving tissue classification accuracy and detecting variation in tissues from an intestinal tumorigenesis mouse model, showcasing the system's potential for field cancerization studies. (c) 2025 The Author(s)

Supplementary Figure S3. Representative time course fluorescence imaging of the dual xenograft mouse model after EMI-137 injection.

we present developments in Near-Infrared Fluorescence and Intravascular Ultrasound (NIRF-IVUS) imaging. By correcting blood attenuation on the fluorescence signal, we enhance the detection of the NIRF, improving the pathobiological state assessment of atherosclerotic tissue. (c) 2025 The Author(s)

Supplementary Information S1. Details of the Fluorescence Molecular Endoscopy (FME) System; Representative videos with screenshot of FME in L2-IL1b transgenic mice

Field cancerization (FC) refers to spatially distributed premalignant tissue changes that lead to the appearance of local malignancy, and its detection can improve cancer screening. In this work, we employ combined Raman and partial wave spectroscopy (RS-PWS) to detect FC in gastroesophageal (L2-IL1B) and intestinal (Villin-Cre, Apcfl/wt) tumor mouse models. Using a hybrid RS-PWS microscope, we acquire both molecular and morphological information from macroscopically normal tumor-adjacent tissue and investigate the individual and combined performance of each modality. For data analysis, we use partial least-squares discriminant analysis (PLS-DA). In the normal tissue of L2-IL1B mice, we demonstrate a statistically significant increase (p < 0.001) in Raman band intensities associated with free amino acids and a decrease in bands associated with lipids (p < 0.005) and carotenoids (p < 0.001) compared to healthy controls. Similarly, in the normal mucosa of Villin-Cre, Apcfl/wt mice, the intensities of RS bands associated with amino acids increase significantly (p < 0.05) compared to controls, while the intensities of lipid-associated bands decrease significantly (p < 0.05). Transcriptomic profiling using RNA-sequencing analysis on these samples identified a significant correlation between gene expression and optical findings. Moreover, we demonstrate that combining RS and PWS data further improves the significance of our classification results. When macroscopically normal tumor-adjacent tissue is compared with tissue from healthy controls, we observe that PWS increases the R2 of RS results by ∼9% in L2-IL1B mice and ∼5% in Villin-Cre, Apcfl/wt mice. Combining molecular RS with structural PWS information enhances the ability to detect precancerous changes and provides insights into tissue alterations during cancer development.

Fluorescence molecular imaging (FMI) is becoming a powerful tool to improve surgical precision and diagnostics. Although significant advances have been made in imaging technology and contrast agents, debate continues regarding suitable standards for FMI. Standardizing all FMI aspects is crucial for adoption in clinical procedures and routine practice. Currently, the lack of clear-cut guidelines on FMI study reporting complicates comparison between studies and impedes standardization. This work presents community-driven REFLECT guidelines for FMI study reporting for fluorescence-guided surgery and interventions, covering preclinical, translational, and clinical studies. It aims to improve quality of reporting, enable comparison between studies, and enhance interpretation of results. The resulting structured checklist encompasses all essential details for reporting on the contrast agent, imaging device, imaging protocol, and image processing and analysis methods. Adoption of this framework is encouraged to enhance reproducibility and establish alignment with standardization efforts in the field - thus fostering advancements in FMI.

AbstractPurpose: Esophageal cancer carries a poor prognosis with a 5-year overall survival of less than 20%. Barrett’s esophagus increases the risk of esophageal adenocarcinoma. The aim of this study was to investigate the ability of EMI-137, a mesenchymal–epithelial transition factor (c-MET)-targeting optical imaging tracer, to detect dysplasia in Barrett’s esophagus. Experimental Design: c-MET expression in human esophageal tissue was investigated using Gene Expression Omnibus datasets, tissue microarrays, and Barrett’s esophagus biopsies. EMI-137 was tested in a dual xenograft mouse model bearing OE33 (c-MET high expression) and FLO-1 (c-MET low expression) tumors. Fluorescence molecular endoscopy was performed in a mouse model of Barrett’s-like metaplasia and dysplasia (L2-IL1β). Tumors and organs of interest were evaluated through ex vivo fluorescence imaging. Results: MET mRNA expression analyses and c-MET immunostaining confirmed upregulation of c-MET in Barrett’s esophagus and esophageal adenocarcinoma compared with normal epithelium. There was strong accumulation of EMI-137 in OE33 xenografts 3 hours after injection, decreasing by more than 50% on coinjection of a 10-fold molar excess of unlabeled EMI-137. The target-to-background ratio at 3 hours after injection for OE33 and FLO-1 tumors was 10.08 and 1.42, respectively. Fluorescence molecular endoscopy of L2-IL1β mice showed uptake of EMI-137 in dysplastic lesions within Barrett’s esophagus with a target-to-background ratio of 1.9 in vivo and greater than 2 in ex vivo fluorescence imaging. Conclusions: EMI-137 accumulates in dysplastic lesions within Barrett’s esophagus and also in c-MET–positive esophageal adenocarcinoma. EMI-137 imaging has potential as a screening and surveillance tool for patients with Barrett’s esophagus and as a means to detecting dysplasia and esophageal adenocarcinoma.

Details of L2-IL1β ex vivo IVIS experiment. Average radiant efficiencies of ROIs were calculated from ex vivo IVIS images: left and right squamocolumnar junctions, the junction between the forestomach and the cardiac/corpus of the stomach, and EGJ, with the corresponding background regions located in the corpus of the stomach.

c-MET is upregulated in Barrett’s esophagus and esophageal adenocarcinoma compared with normal epithelium: A and B, Upregulation of MET mRNA in Barrett’s esophagus and esophageal adenocarcinoma in two GEO databases in comparison with NSE. C, Representative immunostaining of normal epithelium, inflammation, hyperplasia, and esophageal adenocarcinoma. D, Analysis of human TMAs confirmed upregulation of c-MET protein in esophageal adenocarcinoma compared with NSE, inflammation, and hyperplasia (all P < 0.0001). E and F, Immunostaining of endoscopic human biopsy samples of NSE (n = 6) and Barrett’s esophagus (n = 5) showed an approximately 2-fold greater average H-score and percentage of c-MET positivity in Barrett’s esophagus compared with NSE. **, P < 0.01; ****, P < 0.0001.

Near-infrared fluorescence molecular endoscopy (NIR-FME) is a promising method for earlier, faster, and personalized esophageal cancer (EC) detection. A recent clinical study in Barrett's Esophagus patients highlights its potential in improving early EC diagnosis. Full-text article not available; see video presentation

Supplementary Figure S2. Establishment of a dual xenograft mouse model for testing EMI-137.