Photonic Pathways FOCUS of individual cells within the tumor stroma. The image reveals a heterogeneous immune infiltrate consisting of lymphocytes, eosinophils, and macrophages. Lymphocytes appear as small, round cells with minimal cytoplasm, often clustered near stromal interfaces or blood vessels. Eosinophils are identifiable by their bright, granular cytoplasm and lobed nuclei, whereas macrophages are larger, irregularly shaped cells with variable internal contrast.
Figure 5 shows numerous elastin and collagen fibers to form a complex extracellular network interspersed with tumor cells and lymphocytes. The magnified view highlights the detailed cellular morphology of tumor cells: HHG imaging clearly resolves nuclei as ringshaped structures and nucleoli as bright, dot-like features within them. Between the elastin fibers, white – purple structures correspond to macrophages laden with anthracotic pigment( carbon particles), readily distinguishable by their dark inclusions.
Together, these images demonstrate the capability of HHG microscopy to visualize both structural and immune components of the tumor microenvironment in situ, without the need for labeling or staining. This intrinsic contrast provides a powerful means to study tumor – stroma organization and tumor – immune cell interactions at subcellular resolution.
CONCLUSION The convergence of compact femtosecond lasers, label-free nonlinear imaging, and deep learning is opening a new frontier in digital pathology. Higher Harmonic Generation Microscopy( HHGM) generates information-rich, multicolored images that capture the architectural fidelity of histology with the speed and immediacy of cytology, while enabling fully automated analysis, all without staining or sectioning. HHGM can be seamlessly integrated into clinical workflows, delivering diagnostic feedback within minutes, and importantly, the imaged tissue remains intact for subsequent molecular or histopathological evaluation. The relative simplicity of the technique, requiring, for instance, only a single laser source, renders the machine robust, compact, and cost-effective, facilitating translation to the clinic.
ACKNOWLEDGEMENTS We thank our clinical collaborators in the Prinses Máxima Centrum for Pediatric Oncology: Prof. Dr. R. R. de Krijger and Prof. Dr. P. Wesseling and colleagues; and from the Department of Pulmonary Medicine and Cardiothoracic Surgery, Amsterdam UMC, Amsterdam, The Netherlands: Prof. Dr. J. T. Annema, Dr. C. Dickhoff and colleagues. MLG discloses an indirect interest in Flash Pathology BV.
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