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Figure 5. HHG( SHG-THG-2PEF) image of a lung tumor( adenocarcinoma) with close-up of the cellular details of the tumor cells.
and discrimination of tumor and healthy tissue for brain and lung tissue. Our foundation model, Cell- SAM, trained on HHGM data, achieved near-perfect segmentation of tumor, immune, and stromal cells in brain tissue( precision 99.4 %, recall 98.7 %)( 6).
In the following, we present examples of higher harmonic generation( HHG) microscopy images from various tissue types.
Figure 2 compares HHG microscopy images of healthy tissues with corresponding standard hematoxylin and eosin( H & E) histology images. In HHG microscopy, cellular and most tissue structures are visualized via the third harmonic generation( THG) signal( displayed in green), while collagen fibers generate second harmonic generation
( SHG) contrast( shown in red). In addition, two-photon excited fluorescence( 2PEF), displayed in blue, provides complementary information. For reference, H & E staining uses two dyes to visualize tissue architecture: hematoxylin stains cell nuclei blue to purple, and eosin stains cytoplasmic and extracellular components pink to red. The overall tissue architecture in HHG images closely resembles that seen in H & E histology.
In skin tissue, the distinct epidermal and dermal layers are clearly resolved. In HHG images, nuclei appear as dark voids within the cellular structures. Lymphatic tissue exhibits high cellular density, with small cells containing minimal cytoplasm. Adipose tissue consists of large adipocytes that appear empty in H & E images due to lipid loss during sample processing. Hyaline cartilage shows a homogeneous, glass-like background with embedded chondrocytes. In muscle tissue, the HHG image reveals cross-sections of skeletal muscle fascicles. Finally, brain tissue displays the grey matter region with several identifiable neurons( circled) and a few myelinated axons, visible as bright green linear structures in the HHG image.
Figure 3 shows an HHGM image of a lung tumor biopsy( adenocarcinoma). Collagen fibers appear in red, elastin fibers in blue, and cells in green. In the lower left region, remnants of alveolar tissue are visible, displaying the characteristic fine collagen and elastin network interspersed with macrophages. Toward the upper right, this orderly structure gives way to densely packed, enlarged cells consistent with malignant transformation. HHGM thus readily distinguishes normal from tumor regions within a single field of view. Feedback on the representativity of the biopsy was available to the endoscopist within five minutes after extraction, allowing rapid recognition of tumor type based on cellular morphology and growth pattern.
The composition of the tumor microenvironment( TME) and the types of immune cells it contains are critical determinants of tumor progression, therapeutic response, and clinical outcome. Figures 4 and 5 present HHG microscopy images of adenocarcinoma tissue, illustrating the cellular and extracellular complexity of the TME.
In Figure 4, the second harmonic generation signal highlights collagen fibers, revealing the dense and often disorganized stromal matrix characteristic of epithelial tumors. Elastin fibers contribute additional structural contrast in the two-photon excited fluorescence channel, while the third harmonic generation signal enables label-free visualization
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