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Understanding the inner morphology of intact tissues is one of the most competitive challenges in modern biology. Since the beginning of the twentieth century, optical tissue clearing (OTC) has provided solutions for volumetric imaging, allowing the microscopic visualization of thick sections of tissue, organoids, up to whole organs and organisms (for example, mouse or rat). Recently, tissue clearing has also been introduced in clinical settings to achieve a more accurate diagnosis with the support of 3D imaging. This review aims to give an overview of the most recent developments in OTC and 3D imaging and to illustrate their role in the field of medical diagnosis, with a specific focus on clinical applications. Graphical abstract

open 14 no Breast cancer (BC) is the most commonly diagnosed malignant tumor in women worldwide, and the leading cause of cancer death in the female population. The percentage of patients experiencing poor prognosis along with the risk of developing metastasis remains high, also affecting the resistance to current main therapies. Cancer progression and metastatic development are no longer due entirely to their intrinsic characteristics, but also regulated by signals derived from cells of the tumor microenvironment. Extracellular vesicles (EVs) packed with DNA, RNA, and proteins, are the most attractive targets for both diagnostic and therapeutic applications, and represent a decisive challenge as liquid biopsy-based markers. Here we performed a study based on a multiplexed phenotyping flow cytometric approach to characterize BC-derived EVs from BC patients and cell lines, through the detection of multiple antigens. Our data reveal the expression of EVs-related biomarkers derived from BC patient plasma and cell line supernatants, suggesting that EVs could be exploited for characterizing and monitoring disease progression. open Bandini E.; Rossi T.; Scarpi E.; Gallerani G.; Vannini I.; Salvi S.; Azzali I.; Melloni M.; Salucci S.; Battistelli M.; Serra P.; Maltoni R.; Cho W.C.; Fabbri F. Bandini E.; Rossi T.; Scarpi E.; Gallerani G.; Vannini I.; Salvi S.; Azzali I.; Melloni M.; Salucci S.; Battistelli M.; Serra P.; Maltoni R.; Cho W.C.; Fabbri F.