Open AccessSystematic Review Confocal Laser Endomicroscopy in Brain Metastasis Surgery: A Systematic Review of the Evidence at the Tumor–Brain Interface 1 Department of Neurosurgery, Fondazione IRCCS Istituto Neurologico Carlo Besta, 20133 Milan, Italy 2 Institute of Neuroscience, Faculty of Medicine, El Bosque University, Bogota 111321, Colombia 3 Department of Neurosurgery, University of Jena Hospital, 07747 Jena, Germany 4 Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, 56126 Pisa, Italy 5 Department of Neurosurgery, Azienda Ospedaliero Universitaria Pisana, 56124 Pisa, Italy * Author to whom correspondence should be addressed. † These authors contributed equally to this work. J. Clin. Med. 2026, 15(12), 4420; https://doi.org/10.3390/jcm15124420 (registering DOI) Submission received: 23 April 2026 / Revised: 27 May 2026 / Accepted: 4 June 2026 / Published: 7 June 2026 Abstract Background: Brain metastases are the most common intracranial tumors in adults and are traditionally considered well-demarcated lesions amenable to complete surgical resection. Nonetheless, increasing histopathological evidence demonstrates that metastatic cells may infiltrate beyond the contrast-enhancing margin into surrounding brain parenchyma, challenging the reliability of conventional imaging for defining true tumor boundaries. Confocal laser endomicroscopy (CLE) using Sodium Fluorescein (SF) has emerged as a novel intraoperative imaging modality capable of providing real-time, high-resolution optical biopsies, potentially improving margin assessment during metastasis surgery. Methods: A systematic literature search was performed according to PRISMA guidelines across PubMed, Embase, Scopus, Cochrane Library, and Google Scholar up to 3 March 2026. Studies evaluating intraoperative CLE with SF in adult patients with brain metastases were included. Data regarding study design, patient population, CLE system, imaging characteristics, and diagnostic performance were extracted. Risk of bias was assessed using the QUADAS-2 tool. Results: Ten studies met the inclusion criteria for qualitative synthesis, comprising over 650 patients; however, most studies included heterogeneous intracranial tumor populations, with only a subset specifically involving brain metastases. CLE enabled real-time visualization of tumor microarchitecture and demonstrated high sensitivity for tumor detection, frequently exceeding 90% in prospective studies. Specificity varied across studies, reflecting challenges in distinguishing tumor infiltration from reactive tissue at the tumor–brain interface. The MetInfilt trial highlighted that infiltrative growth patterns are common in brain metastases and can be visualized intraoperatively using CLE. Additional studies demonstrated that fluorescein-based CLE allows differentiation of tumor zones and may facilitate targeted margin assessment; however, evidence demonstrating improvement in clinically meaningful outcomes such as extent of resection, local recurrence, progression-free survival, or overall survival remains limited. Conclusions: Confocal laser endomicroscopy using SF represents a promising intraoperative adjunct for assessing tumor margins in brain metastasis surgery. By enabling real-time microscopic visualization of the metastasis–brain interface, CLE may support a more biologically informed surgical strategy. 1. Introduction Brain metastases (BM) represent the most common intracranial tumors in adults and occur in up to 20–40% of patients with systemic cancer, frequently originating from lung, breast, skin, renal, or colorectal malignancies [ 1] Surgical resection remains a milestone of the management of large or symptomatic BM, particularly when rapid neurological deterioration, mass effect, or diagnostic uncertainty is present [ 2]. Prospective clinical studies have demonstrated that CLE using SF can achieve high diagnostic accuracy when compared with conventional histopathology for identifying tumor tissue during brain tumor surgery [ 8, 10]. The potential role of CLE for assessing the metastasis–brain parenchyma interface (MBPI) and detecting tumor infiltration at surgical margins remains insufficiently characterized. Therefore, a systematic evaluation of the current clinical evidence is required to clarify the role of fluorescein-guided confocal laser endomicroscopy in metastasis surgery. The objective of this systematic review is to assess the available literature regarding the intraoperative use of confocal laser endomicroscopy with SF for identifying tumor tissue and evaluating the tumor–brain interface during brain metastasis surgery. 2. Methods 2.1. Search Strategy A comprehensive literature search was conducted to identify all studies evaluating the intraoperative use of CLE with SF for tumor detection and margin assessment during BM surgery. The following electronic databases were systematically searched: PubMed (n = 28), Ovid (n = 0), Scopus (n = 1), Embase (n = 183), Cochrane Library (n = 1), and Google Scholar (n = 2860). The search included studies published up to 3 March 2026. The search strategy combined Medical Subject Headings (MeSH) and free-text terms related to brain metastases, confocal laser endomicroscopy, and fluorescein. The following search query was used: (“Brain Neoplasms” OR “Neoplasm Metastasis” OR brain metastas* OR intracranial metastas* OR metastatic brain tumor*) AND (“Microscopy, Confocal” OR “confocal laser endomicroscopy” OR “confocal endomicroscopy” OR CLE OR CONVIVO ପ୍ପ) AND (“Fluorescein” OR “fluorescein sodium” OR “SF” OR fluorescein). The review protocol was not registered. Additional articles were identified through manual screening of reference lists from relevant publications. The systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. 2.2. Eligibility Criteria Studies were considered eligible if they met the following criteria. 2.3. Inclusion Criteria Adult patients (≥18 years) undergoing surgical treatment for BM; Studies reporting the intraoperative use of CLE with SF; Articles providing clinical outcomes, diagnostic accuracy, or intraoperative imaging findings related to tumor tissue or tumor margins; Original studies including prospective studies, retrospective cohorts, case series, or case reports. 2.4. Exclusion Criteria Studies involving pediatric-only populations; Non-English publications without accessible full-text English translation; Review articles, editorials, letters, conference abstracts without full text, or expert opinions; Animal or preclinical studies; Studies using CLE without SF; Studies limited to ex vivo analysis without intraoperative clinical application. 2.5. Study Selection All records identified through the database search were imported into a reference management software and duplicates were removed. After duplicate removal, 2676 articles remained for screening. Titles and abstracts were independently screened by two reviewers (SACM, NA). Studies that did not meet the eligibility criteria were excluded at this stage. Full texts of potentially relevant studies were then reviewed independently by three reviewers (SACM, NA, FR). A total of 33 studies were assessed for full-text eligibility. Of these, 10 studies met the inclusion criteria and were included in the qualitative systematic review. Any disagreements regarding study eligibility were resolved through discussion and consensus among the reviewers or through consultation with a fourth reviewer (MB). 2.6. Data Extraction Data extraction was independently performed by two reviewers using a standardized data collection form. Extracted variables included: Study characteristics (author, year, country, study design); Sample size; Number of brain metastases included; CLE system used (e.g., CONVIVO ପ୍ପ (Carl Zeiss Meditech, Oberkochen, Germany) or probe-based CLE); Fluorescein sodium usage; Imaging modality (in vivo or ex vivo intraoperative imaging); Location of imaging (tumor core, tumor margin, tumor–brain interface); Reference standard used for comparison (frozen section or permanent histopathology). 2.7. Quality Assessment The methodological quality and risk of bias of the included studies were assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) tool. The methodological quality of included studies is listed in Supplementary Table S2. This tool evaluates studies across four domains: Patient selection; Index test (confocal laser endomicroscopy); Reference standard (histopathological analysis); Flow and timing between the index test and reference standard. Each domain was assessed for risk of bias and concerns regarding applicability and categorized as low, high, or unclear risk of bias. 4. Discussion Brain metastasis has traditionally been regarded as well-demarcated lesions amenable to resection along a clear surgical plane; however, accumulating evidence indicates that this concept is overly simplistic. The prospective MetInfilt trial by Proescholdt et al. demonstrated that metastatic cells frequently extend beyond the contrast-enhancing lesion into surrounding brain parenchyma, with infiltrative growth patterns observed in most cases [ 5]. These findings indicate that the radiological margin does not necessarily correspond to the true biological boundary of disease and that reliance on macroscopic or imaging-based resection alone may result in residual microscopic tumor at the metastasis–brain interface. CLE has emerged as a promising intraoperative imaging adjunct to address this limitation. By combining SF with high-resolution optical imaging, CLE enables real-time visualization of tissue microarchitecture, effectively functioning as an intraoperative optical biopsy. Early feasibility studies by Sanai et al. and Martirosyan et al. established that CLE can reliably visualize cellular and vascular features of brain tumors intraoperatively, demonstrating high concordance with histopathology [ 5, 15]. Furthermore, telepathology-based CLE interpretation has been shown to be feasible, suggesting that intraoperative decision-making could be supported by remote expert analysis [ 9]. These foundational studies were not metastasis-specific but were critical in showing that microscopic assessment can be integrated directly into surgical workflow. More recent studies using clinically integrated systems such as CONVIVO ପ୍ପ have extended these findings into practical intraoperative applications. Restelli et al. demonstrated that CLE can accurately identify pathological tissue at infiltration margins, reporting high diagnostic accuracy across CNS tumors, including metastases ( Figure 2) [ 14]. Similarly, Wagner et al. showed that fluorescein-based CLE allows rapid intraoperative assessment compared with frozen section histology, although with somewhat lower specificity [ 8]. These findings suggest that CLE is best positioned as a complementary tool that provides rapid, spatially targeted information at the resection margin rather than replacing standard pathology. Several studies have specifically emphasized the ability of CLE to interrogate different intraoperative zones. Höhne et al. demonstrated distinct CLE patterns between tumor core, tumor border, and perilesional tissue, supporting its use for intraoperative assessment of the MBPI [ 13]. Likewise, Belykh et al. reported that fluorescein-guided CLE allows visualization of tumor microstructure and architectural disruption in intraoperative samples, reinforcing its role in tissue characterization at the margin [ 10]. These observations underscore a fundamental limitation of conventional imaging: although MRI can suggest infiltrative behavior, it lacks the resolution to reliably detect microscopic tumor extension beyond the contrast-enhancing margin, a phenomenon consistently demonstrated in histopathological analyses of brain metastases [ 5]. An additional metastasis-specific aspect is highlighted by Brielmaier et al., who demonstrated that metastatic tumors exhibit significantly higher intracellular SF accumulation compared with primary brain tumors [ 12]. This finding is particularly relevant for margin assessment, as it suggests that metastatic tumor cells may be more readily identifiable on CLE, potentially improving detection of infiltrative disease at the interface. However, the same study also underscores technical limitations, including non-diagnostic images due to motion artifacts, hemorrhage, or suboptimal contrast, indicating that image quality and operator experience remain critical factors. Taken together, the available evidence supports a consistent biological and technical interpretation. Brain metastases frequently exhibit infiltrative growth at the metastasis–brain interface, and CLE provides a feasible method for visualizing this interface intraoperatively. The ability to identify microscopic tumor infiltration in real time addresses a major limitation of current surgical practice, where margin assessment is otherwise indirect and delayed. In this context, CLE may contribute to a more biologically informed surgical strategy, including the potential application of supramarginal resection in selected cases. Nevertheless, important limitations must be acknowledged. The current evidence base is characterized by relatively small cohorts, heterogeneous study designs, and frequent inclusion of mixed intracranial tumor populations, including gliomas, meningiomas, schwannomas, and metastatic lesions, with only a limited number of studies specifically designed to evaluate brain metastases and the metastasis–brain interface. Even in the most relevant studies, including the MetInfilt trial, the impact of CLE-guided margin assessment on clinically meaningful outcomes such as extent of resection, local recurrence, progression-free survival, or overall survival remains unproven. Consequently, the current literature supports the technical feasibility and biological plausibility of CLE more strongly than its demonstrated impact on oncological outcomes [ 5]. Furthermore, variability in specificity across studies highlights the ongoing challenge of distinguishing tumor infiltration from reactive or treatment-related changes at the periphery. At present, CLE should be considered an adjunctive modality that may assist intraoperative decision-making rather than a stand-alone determinant of surgical strategy. Future prospective studies focusing specifically on brain metastases, with standardized imaging protocols and outcome measures, are required to define its role more precisely. 5. Conclusions The current literature increasingly indicates that BM are often infiltrative beyond the radiological enhancing border. In this setting, CLE may provide additional microscopic information at the resection margin and could potentially support more biologically informed intraoperative decision-making; however, evidence demonstrating improvement in long-term oncological outcomes remains insufficient. Prospective studies specifically designed to test whether CLE-guided margin assessment improves local control are now needed to determine whether this promise can be translated into measurable clinical benefit. Supplementary Materials The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jcm15124420/s1. Table S1: PRISMA 2020 Checklist; Table S2: Risk of bias assessment using the QUADAS-2 tool. Author Contributions Conception of the idea was made by S.A.C.M. and F.R. A comprehensive literature search was conducted by S.A.C.M., P.F. and F.R. Initial screening of articles was performed by S.A.C.M. and N.A. The included studies were analyzed collaboratively by S.A.C.M., N.A. and F.R. Quality assessments were conducted independently by S.A.C.M. and F.R. All authors contributed to the interpretation of the data and discussed the results. The manuscript was written by S.A.C.M. and F.R. All authors have read and agreed to the published version of the manuscript. Funding This research was partially supported by the Associazione Paolo Zorzi per le Neuroscienze ONLUS, and by the Italian Ministry of Health (RRC). Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The raw data supporting the conclusions of this article will be made available by the authors on request. Acknowledgments The authors would like to thank the members of the Neurosurgery Team 2 at the Carlo Besta Neurological Institute for their valuable clinical support and contributions to patient care. We acknowledge Octavian Vatavu, Riccardo Ciocca, Erica Boccardi, Marco Paolo Schiariti, Marco Saini, and Alberto Cusin for their involvement and collaboration. Conflicts of Interest The authors declare no conflict of interest. References Sacks, P.; Rahman, M. Epidemiology of Brain Metastases. Neurosurg. Clin. N. Am. 2020, 31, 481–488. [] [ CrossRef] [ PubMed] Karschnia, P.; Le Rhun, E.; Vogelbaum, M.A.; Van Den Bent, M.; Grau, S.J.; Preusser, M.; Soffietti, R.; Von Baumgarten, L.; Westphal, M.; Weller, M.; et al. The evolving role of neurosurgery for central nervous system metastases in the era of personalized cancer therapy. Eur. J. Cancer 2021, 156, 93–108. [] [ CrossRef] [ PubMed] Berghoff, A.S.; Rajky, O.; Winkler, F.; Bartsch, R.; Furtner, J.; Hainfellner, J.A.; Goodman, S.L.; Weller, M.; Schittenhelm, J.; Preusser, M. Invasion patterns in brain metastases of solid cancers. Neuro-Oncology 2013, 15, 1664–1672. [] [ CrossRef] [ PubMed] Siam, L.; Bleckmann, A.; Chaung, H.-N.; Mohr, A.; Klemm, F.; Barrantes-Freer, A.; Blazquez, R.; Wolff, H.A.; Lüke, F.; Rohde, V.; et al. The metastatic infiltration at the metastasis/brain parenchyma-interface is very heterogeneous and has a significant impact on survival in a prospective study. Oncotarget 2015, 6, 29254–29267. [] [ CrossRef] [ PubMed] Proescholdt, M.A.; Araceli, T.; Schebesch, K.-M.; Doenitz, C.; Wendl, C.; Evert, K.; Noeva, E.; Hoehne, J.; Riemenschneider, M.J.; Hirsch, D.; et al. MetInfilt: A prospective trial highlighting the importance of the histological growth pattern in brain metastases. Transl. Oncol. 2025, 60, 102480. [] [ CrossRef] [ PubMed] Raore, B.; Schniederjan, M.; Prabhu, R.; Brat, D.J.; Shu, H.-K.; Olson, J.J. Metastasis Infiltration: An Investigation of the Postoperative Brain–Tumor Interface. Int. J. Radiat. Oncol. Biol. Phys. 2011, 81, 1075–1080. [] [ CrossRef] [ PubMed] Martirosyan, N.L.; Eschbacher, J.M.; Kalani, M.Y.S.; Turner, J.D.; Belykh, E.; Spetzler, R.F.; Nakaji, P.; Preul, M.C. Prospective evaluation of the utility of intraoperative confocal laser endomicroscopy in patients with brain neoplasms using fluorescein sodium: Experience with 74 cases. Neurosurg. Focus 2016, 40, E11. [] [ CrossRef] [ PubMed] Wagner, A.; Brielmaier, M.C.; Kampf, C.; Baumgart, L.; Aftahy, A.K.; Meyer, H.S.; Kehl, V.; Höhne, J.; Schebesch, K.-M.; Schmidt, N.O.; et al. Fluorescein-stained confocal laser endomicroscopy versus conventional frozen section for intraoperative histopathological assessment of intracranial tumors. Neuro-Oncology 2024, 26, 922–932. [] [ CrossRef] [ PubMed] Abramov, I.; Park, M.T.; Gooldy, T.C.; Xu, Y.; Lawton, M.T.; Little, A.S.; Porter, R.W.; Smith, K.A.; Eschbacher, J.M.; Preul, M.C. Real-time intraoperative surgical telepathology using confocal laser endomicroscopy. Neurosurg. Focus 2022, 52, E9. [] [ CrossRef] [ PubMed] Belykh, E.; Zhao, X.; Ngo, B.; Farhadi, D.S.; Byvaltsev, V.A.; Eschbacher, J.M.; Nakaji, P.; Preul, M.C. Intraoperative Confocal Laser Endomicroscopy Ex Vivo Examination of Tissue Microstructure During Fluorescence-Guided Brain Tumor Surgery. Front. Oncol. 2020, 10, 599250. [] [ CrossRef] [ PubMed] Abramov, I.; Park, M.T.; Belykh, E.; Dru, A.B.; Xu, Y.; Gooldy, T.C.; Scherschinski, L.; Farber, S.H.; Little, A.S.; Porter, R.W.; et al. Intraoperative confocal laser endomicroscopy: Prospective in vivo feasibility study of a clinical-grade system for brain tumors. J. Neurosurg. 2023, 138, 587–597. [] [ CrossRef] [ PubMed] Brielmaier, M.C.; Reifenrath, J.; Ganster, F.; Pensel, N.; Gempt, J.; Meyer, B.; Schlegel, J.; Wagner, A. Fluorescein-distribution in confocal laser endomicroscopy allows for discrimination between primary brain tumours and metastases. Front. Surg. 2025, 12, 1567711. [] [ CrossRef] [ PubMed] Höhne, J.; Schebesch, K.-M.; Zoubaa, S.; Proescholdt, M.; Riemenschneider, M.J.; Schmidt, N.O. Intraoperative imaging of brain tumors with fluorescein: Confocal laser endomicroscopy in neurosurgery. Clinical and user experience. Neurosurg. Focus 2021, 50, E19. [] [ CrossRef] [ PubMed] Restelli, F.; Pollo, B.; Mazzapicchi, E.; Tramacere, I.; Broggi, M.; Falco, J.; Schiariti, M.; Stanziano, M.; Dimeco, F.; Ferroli, P.; et al. Confocal endomicroscopy accuracy in identifying central nervous system tumors tissue at the infiltration margins: Results from a prospective clinical trial. J. Neurosurg. Sci. 2025, 69, 225–235. [] [ CrossRef] [ PubMed] Sanai, N.; Eschbacher, J.; Hattendorf, G.; Coons, S.W.; Preul, M.C.; Smith, K.A.; Nakaji, P.; Spetzler, R.F. Intraoperative Confocal Microscopy for Brain Tumors: A Feasibility Analysis in Humans. Oper. Neurosurg. 2011, 68, ons282–ons290. [] [ CrossRef] [ PubMed] Figure 1. PRISMA flow diagram of study selection. Flow chart illustrating the study identification, screening, eligibility assessment, and final inclusion process according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A total of 3073 records were identified through database searches. After removal of duplicates, 2676 records were screened by title and abstract. Thirty-three full-text articles were assessed for eligibility, of which 10 studies met the inclusion criteria and were included in the qualitative systematic review. Figure 1. PRISMA flow diagram of study selection. Flow chart illustrating the study identification, screening, eligibility assessment, and final inclusion process according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A total of 3073 records were identified through database searches. After removal of duplicates, 2676 records were screened by title and abstract. Thirty-three full-text articles were assessed for eligibility, of which 10 studies met the inclusion criteria and were included in the qualitative systematic review. Figure 2. The CLE probe can be used freehand and placed directly in contact with the brain surface, provided that it is covered with its dedicated sterile sheath ( A). ( B) Preoperative imaging of a left temporal metastasis. The lower-right inset shows the site where the CLE probe was placed to perform the virtual biopsy. ( C) Intraoperative CLE image acquired at the center of the brain lesion, showing a nest of tumor cells with hyperchromatic and irregular nuclei in the lower-left area, corresponding to the finding observed on definitive hematoxylin and eosin histology ( D). Figure 2. The CLE probe can be used freehand and placed directly in contact with the brain surface, provided that it is covered with its dedicated sterile sheath ( A). ( B) Preoperative imaging of a left temporal metastasis. The lower-right inset shows the site where the CLE probe was placed to perform the virtual biopsy. ( C) Intraoperative CLE image acquired at the center of the brain lesion, showing a nest of tumor cells with hyperchromatic and irregular nuclei in the lower-left area, corresponding to the finding observed on definitive hematoxylin and eosin histology ( D). Table 1. Characteristics of the studies included in the systematic review. Summary of study design, patient population, CLE system, fluorescein usage, imaging modality, and reference standards across the included studies evaluating intraoperative confocal laser endomicroscopy for brain tumor surgery with emphasis on brain metastasis cases (BM). Table 1. Characteristics of the studies included in the systematic review. Summary of study design, patient population, CLE system, fluorescein usage, imaging modality, and reference standards across the included studies evaluating intraoperative confocal laser endomicroscopy for brain tumor surgery with emphasis on brain metastasis cases (BM). Study Year Study Design Patients (n) BM (n) CLE System Fluorescein Use Imaging Type Reference Standard Main Outcome Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. © 2026 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license. Share and Cite MDPI and ACS Style Calero Martinez, S.A.; Aboud, N.; Ferroli, P.; Acerbi, F.; Broggi, M.; Restelli, F. Confocal Laser Endomicroscopy in Brain Metastasis Surgery: A Systematic Review of the Evidence at the Tumor–Brain Interface. J. Clin. Med. 2026, 15, 4420. https://doi.org/10.3390/jcm15124420 AMA Style Calero Martinez SA, Aboud N, Ferroli P, Acerbi F, Broggi M, Restelli F. Confocal Laser Endomicroscopy in Brain Metastasis Surgery: A Systematic Review of the Evidence at the Tumor–Brain Interface. Journal of Clinical Medicine. 2026; 15(12):4420. https://doi.org/10.3390/jcm15124420 Chicago/Turabian Style Calero Martinez, Sergio Alexander, Nazeer Aboud, Paolo Ferroli, Francesco Acerbi, Morgan Broggi, and Francesco Restelli. 2026. "Confocal Laser Endomicroscopy in Brain Metastasis Surgery: A Systematic Review of the Evidence at the Tumor–Brain Interface" Journal of Clinical Medicine 15, no. 12: 4420. https://doi.org/10.3390/jcm15124420 APA Style Calero Martinez, S. A., Aboud, N., Ferroli, P., Acerbi, F., Broggi, M., & Restelli, F. (2026). Confocal Laser Endomicroscopy in Brain Metastasis Surgery: A Systematic Review of the Evidence at the Tumor–Brain Interface. Journal of Clinical Medicine, 15(12), 4420. https://doi.org/10.3390/jcm15124420 Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details . Article Metrics Article metric data becomes available approximately 24 hours after publication online.
Confocal Laser Endomicroscopy in Brain Metastasis Surgery: A Systematic Review of the Evidence at the Tumor–Brain Interface
