According to the Central Brain Tumor Registry of the United States (CBTRUS), malignant primary brain tumors cause more than 15,000 deaths per year. The annual incidence is approximately 7 per 100,000 individuals and increases with age. The five-year survival rate is approximately 36%. Gliomas are the most prevalent and aggressive adult-type primary tumors of the central nervous system (CNS). These tumors range from lower-grade gliomas (LGGs) to the highly malignant glioblastoma multiforme (GBM), defined as isocitrate dehydrogenase (IDH)-wild-type diffuse glioma. Anatomic magnetic resonance imaging (MRI) and fluid-attenuated inversion recovery (FLAIR) are the gold standards for diagnosing and following up on GBM, providing macrostructural anatomical information. Despite many therapeutic strategies involving maximal surgical resection, radiotherapy, and alkylating chemotherapy, the prognosis remains poor. Gliomas are very low-immunogenic tumors, called “immune-cold,” located in a T-cell and other immune cell-poor CNS compartment, due to poor infiltration of cytotoxic T lymphocytes (CTLs) and the dominance of immunosuppressive myeloid populations. The extracellular matrix (ECM) and the blood-brain barrier (BBB) play a relevant role in maintaining the immunosuppressive tumor microenvironment (TME). It predominantly consists of microglia and macrophages, but it also includes other myeloid cells, such as bone marrow-derived macrophages (BMDMs), neutrophils, and myeloid-derived suppressor cells (MDSCs). Immunotherapy for CNS tumors, along with trials involving immune checkpoint inhibitors (ICIs), therapeutic vaccines, adoptive cell therapy, monoclonal antibodies, oncolytic viruses, and other molecules, promises to be an exciting avenue of research; however, novel approaches are needed. Classical immunohistochemical techniques or those combined with confocal microscopy can open up new frontiers in diagnostics and immunotherapy planning.

Background and Clinical Significance: The paper investigates the use of the Histolog^® Scanner, a confocal microscopy–based device, as a potential tool for intraoperative neuropathological diagnosis of brain tumors. Traditional intraoperative diagnosis, relying on frozen sections and squash preparations, can introduce artifacts and consume valuable tissue. The Histolog^® Scanner offers a plug-and-play solution capable of acquiring high-resolution images of fresh tissue surfaces in minutes while preserving tissue for further histological or molecular analyses. Cases Presentation: Three clinical cases—two women and one-man, mean age 57.3 years—undergoing neurosurgery for distinct brain lesions were included. Tissue samples were immersed in fluorescent dye, rinsed, and immediately analyzed with the Histolog^® Scanner before standard intraoperative histopathology. In the first case, a glioblastoma wild-type, traditional methods struggled to define tumor margins, whereas the device provided rapid, detailed imaging to guide resection. In the second case, a meningioma, the scanner confirmed lesion identity quickly, eliminating the need for a cryostat and reducing artifacts. In the third case, a brain metastasis, integration with cytological apposition allowed simultaneous assessment of lesion margins and nature without freezing the tissue. Conclusions: The Histolog^® Scanner demonstrated multiple advantages: rapid intraoperative use, clear margin visualization, preservation of tissue for subsequent analyses, reduce unnecessary resection, thereby helping to lower the risk of recurrence. This device may complement standard intraoperative methods, enhancing diagnostic accuracy and influencing postoperative treatment planning. Overall, the Histolog^® Scanner represents an innovative tool combining speed, precision, and tissue preservation, suggesting a promising role in establishing a new standard for intraoperative neurosurgical diagnosis.